M

Mammoth and Woolly Rhinoceros, primigenius) and woolly rhinoceros Metagenomics of (Coelodonta antiquitatis). The microbiome of an intestinal tract plays important role in the Nikolai V. Ravin1, Egor B. Prokhortchouk2 and animal nutrition and overall health. In particular, Konstantin G. Skryabin2 herbivores have evolved to maintain microbial 1Centre of Bioengineering, Russian Academy of consortia that coordinate relatively rapid rates of Sciences, Moscow, Russia degradation of complex plant carbohydrates 2Centre “Bioengineering” of the Russian under anaerobic conditions (Flint 1997). The Academy of Sciences, Moscow, Russia coevolution of herbivorous mammalian lineages and their gut microbes involved enlargement of the foregut or hindgut to increase the gut reten- Synonyms tion times required for fermentation and selection of appropriate microbial communities (Ley Microbiome of Mammoth and Woolly rhinoceros et al. 2008). Comparison of the intestinal microbiomes of the present-day animals and their extinct relatives may provide information Definition about the diet of the latter and the evolution of microbial communities. Metagenomics is the study of metagenome, Permafrost-preserved animals provide unique a composite of the animal genes and genes pre- opportunity to study the genomes and sent in the genomes of microorganisms coloniz- microbiomes of extinct species. Several studies ing their bodies. More narrow definition of the were focused on nuclear and mitochondrial term metagenome is limited to genomes of only genomes of woolly mammoth and woolly rhi- microbial community (microbiome). noceros (e.g., Poinar et al. 2006; Miller et al. 2008); they are out of scope of this entry. The discoveries of permafrost-preserved mam- Introduction mals, especially the ones with intact intestines protected from the environment, are exceedingly The subject of this entry is to outline the infor- rare events, and, to the best of our knowledge, mation available on the composition of the gut only a single molecular study of their endoge- microbiomes of two extinct herbivorous ani- nous microbiomes has been reported (Mardanov mals – woolly mammoth (Mammuthus et al. 2012).

S.K. Highlander et al. (eds.), Environmental Metagenomics, DOI 10.1007/978-1-4899-7475-4, # Springer Science+Business Media New York 2015 M 302 Mammoth and Woolly Rhinoceros, Metagenomics of

Mammoth and Woolly Rhinoceros, Metagenomics of, Fig. 1 The percentage of 16S rRNA sequences from intestinal samples of the woolly rhinoceros (a) and mammoth Lyuba (b) assigned to different bacterial taxa

Composition of the Gut Microbiomes of Archaeal 16S rRNA sequences were not found. Mammoth and Woolly Rhinoceros The phylum Firmicutes was primary represented by different lineages of clostridia. The most The mammoth Lyuba, who died at the age of numerous group comprising more than a half 1 month, was found in Western Siberia (Russia) of all sequences assigned to the family in 2007 and studied by the team of Dr. Tikhonov Clostridiaceae is related to the ruminal cellulo- at Zoological Institute of the Russian Academy of lytic fermenter Clostridium longisporum. Sciences and his collaborators (Kosintsev Another saccharolytic lineage, which is close to et al. 2010; Van Geel et al. 2011; Fisher Clostridium beijerinckii, includes about 8 % of et al. 2012). Although its geological age is around the sequences. About 25 % of clostridial 40,000 years, it is the best preserved mammoth sequences clustered with Clostridium limosum – found to date. Lyuba is an unweaned calf; its proteolytic bacteria found in soil but also associ- intestinal tract was intact and contained milk ated with a variety of infections in animals. and fecal matter, presumably of her mother’s Notably, Ruminicoccus and Selenomonas spp., (Van Geel et al. 2011). The 39,000-year-old most commonly isolated cellulolytic bacteria woolly rhinoceros is an adult female found in from both ruminants and nonruminant herbivores 2007 near the settlement of Cherskiy, Eastern (Nelson et al. 2003; Larue et al. 2005), were Siberia (Boeskorov et al. 2009). Her intestines completely absent. Another particular characteris- contained the partly undigested composite plant tic of the woolly rhinoceros microbiome is the low material. The above intestinal samples were used abundance of representatives of Bacteroidetes, for collection of bacterial fraction and isolation of typically the second after the Firmicutes major microbial community DNA. bacterial phylum in microbiomes of herbivores, Analysis of the microbial communities was including the present-day black and Indian rhinoc- based on pyrosequencing of 16S rRNA gene eroses (Ley et al. 2008). Relatively high fraction of fragments on GS FLX (Roche). The microorgan- TM7 bacteria is also unusual, and the closest isms of the woolly rhinoceros intestinal homologs of 16S rRNA sequences assigned to microbiome were assigned to five bacterial this phylum were identified in environmental phyla (Fig. 1), the most numerous being clones isolated from cellulosic wastes (Field Firmicutes (68 % of all clones), followed by et al. 2010), suggesting that TM7 bacteria may Proteobacteria (19.2 %), Actinobacteria (5.7 %), play an important role in carbohydrate decompo- TM7 (4.3 %), and Bacteroidetes (0.2 %). sition in animal intestines. Mammoth and Woolly Rhinoceros, Metagenomics of 303 M

The intestinal microbiome of the mammoth proportion of Pseudomonas spp. in the intestinal Lyuba had a completely different content. Rep- microbiome of mammoth Lyuba. resentatives of two bacterial phyla were identi- fied – Proteobacteria (81 % of all clones) and Actinobacteria (18 %) – while less than 0.5 % of Possible Technical Problems Related to sequences were assigned to Firmicutes, TM7, Analysis of the Ancient Microbiomes and Bacteroidetes (Fig. 1). The majority of microorganisms (71 %) represented the family The major problem in analysis of the ancient Pseudomonadaceae, widely distributed and microbiomes is the possible ancient and/or mod- metabolically diverse group of Gammaproteo- ern microbial contamination of the permafrost- bacteria. Although the Pseudomonas spp. are preserved samples from the environment material usually not the major components of the animal such as soil and water as well as human-related intestinal microbiomes (Ley et al. 2008), some contamination in course of sample processing. species are common bacterial contributors to Contamination issues are particularly important spoilage of fluid milk products (Cousin 1982). for ancient samples since most DNA extracted The presence of indigested mother’s milk in from fossil remains is truncated into fragments of Lyuba’s stomach and intestines may explain very short length (usually 80–120 nucleotides) the prevalence of pseudomonads. Another par- from the hydrolysis of the DNA backbone. This ticular characteristic of Lyuba’s intestinal is much shorter than the length of 16S rRNA microbiome is almost complete absence of amplicons used for identification of microorgan- Firmicutes, while this phylum dominates the isms. For instance, the study of Mardanov rhino’s microbiome. It may reflect the differ- et al. (2012) involved analysis of about 500 bp- ences in the diets of the animals (milk vs plant long 16S fragment. Therefore, PCR amplification biomass). may enrich the amplicons derived from the lon- M Overall, the data presented by (Mardanov ger fragments of modern contaminating DNA. It et al. 2012) provides the first insight into the is always difficult to prove the indigenous nature composition of the intestinal microbiomes of the of the revealed microbial community, and the representatives of the Pleistocene megafauna. appearance of known environmental or human- Some bacterial lineages found in microbiomes related microorganisms would be an alarming of present-day herbivores were also found in indication. the woolly rhinoceros microbiome, while some Taxonomic assignment of 16S reads may be important groups (e.g., Ruminococcus spp.) also confounded by nucleotide transitions that were absent. It is possible that such specialized normally happen in ancient DNA. Most frequent microorganisms appeared and became wide- among such pitfalls is the deamination of cyto- spread late in the evolution of herbivores sine to uracil resulting in C-to-T and G-to-A sub- and/or was associated with human activities. It stitutions in sequencing data (Hofreiter is also possible that their absence in the particu- et al. 2001). The rate of deamination is consider- lar sample reflects the specific diet of this woolly ably higher for single-stranded DNA than for rhinoceros. For instance, the fraction of double-stranded. For highly degraded ancient Ruminococcus spp. is higher in sheep consuming DNA samples, local unpairing of double strand the starch-containing diet than in consuming near the breaks may create a lot of C-to-T transi- grass (Larue et al. 2005). Analysis of a larger tions (up to 40 %) within the nearest 5–7 nucle- number of microbiome samples of permafrost- otides with a significant decrease of the preserved animals is required to clarify these transitions (to less than 5–7 %) in the “body” of issues. Another factor that should be taken into DNA fragment (Overballe-Petersen et al. 2012). account is the possibility of selective prolifera- The deamination of cytosine may result in under- tion of particular microorganisms after the death estimation of similarity between the amplicons of the animal, which might explain the high and reference 16S sequences. Therefore M 304 Mammoth and Woolly Rhinoceros, Metagenomics of taxonomic assignment of the 16S reads may be were present in minor amounts. The presence of complicated, especially at lower taxonomic undigested mother’s milk in mammoth stomach ranks. On the other hand, the prevalence of and intestines may explain the prevalence of C-to-T and G-to-A transitions over other types pseudomonads, since some species of this group of dissimilarities between the 16S reads and the are common bacterial contributors to spoilage of closely homologues reference sequence (derived fluid milk products. This data provides the first from present day samples) could be a good indi- insight into the composition of the intestinal cation of an ancient origin of the reads. Such microbiomes of the representatives of the dissimilarity patterns have been reported for the Pleistocene “megafauna.” mammoth and elephant genomes (Poinar et al. 2006). The study of the gut microbiome of the mammoth (Mardanov et al. 2012) also Cross-References revealed the bias toward C-to-T and G-to-A tran- sitions in 16S rRNA sequences closely related to ▶ Diversity of Microbiomes in Beef Cattle Pseudomonas (Ravin et al., unpublished data). ▶ Human Intestinal Microbiome However, the frequencies of C-to-T transitions ▶ Terrestrial Vertebrate Animal Metagenomics, in both studies were quite low (less than 1 %) Wild Ruminants and thus could not impair taxonomic assignment of 16S sequences. References

Summary Boeskorov GG, Lazarev PA, Bakulina NT, Shchelchkova MV, Davydov SP, Solomonov NG. Preliminary study The herbivorous animals have evolved to main- of a mummified woolly rhinoceros from the lower tain microbial consortia that coordinate relatively reaches of the Kolyma River. Dokl Biol Sci. 2009;424:53–6. rapid rates of degradation of complex plant Cousin MA. Presence and activity of psychrotrophic carbohydrates under anaerobic conditions. microorganisms in milk and dairy products: a review. Comparison of the intestinal microbiomes of the J Food Prot. 1982;45:172–207. present-day animals and their extinct relatives Field EK, D’Imperio S, Miller AR, Van Engelen MR, Gerlach R, Lee BD, Apel WA, Peyton BM. Applica- may provide information about the diet of the tion of molecular techniques to elucidate the influence latter and the evolution of microbial communi- of cellulosic waste on the bacterial community struc- ties. The intestinal microbiomes of two ture at a simulated low-level-radioactive-waste site. permafrost-preserved extinct animals, unweaned Appl Environ Microbiol. 2010;76:3106–15. Fisher DC, Tikhonov AN, Kosintsev PA, Rountrey AN, calf woolly mammoth and adult woolly rhinoc- Buigues B, Van Der Plicht J. Anatomy, death, and eros, were analyzed by pyrosequencing of 16S preservation of a woolly mammoth (Mammuthus rRNA genes. The microbiome of the woolly primigenius) calf, Yamal Peninsula, northwest Siberia. rhinoceros was dominated by polysaccharide- Quat Int. 2012;255:94–105. Flint HJ. The rumen microbial ecosystem – some recent degrading lineages of Clostridia, also found in developments. Trends Microbiol. 1997;5:483–8. the present-day herbivores, while Ruminococcus Hofreiter M, Jaenicke V, Serre D, Haeseler Av A, Paabo spp., most commonly isolated cellulolytic bacte- S. DNA sequences from multiple amplifications reveal ria from herbivores intestines, were absent. Rep- artifacts induced by cytosine deamination in ancient DNA. Nucleic Acids Res. 2001;29:4793–9. resentatives of Bacteroidetes, typically the Kosintsev PA, Lapteva EG, Trofimova SS, Zanina OG, second after the Firmicutes major bacterial phy- Tikhonov AN, van der Plicht J. The content of the lum in microbiomes of herbivores, were found in intestines of mammoth calf (Mammuthus primigenius minor amounts. The intestinal microbiome of the Blumenbach, 1799) from the Yuribei River (Yamal Peninsula). Dokl Biol Sci. 2010;432:556–8. young mammoth, unweaned calf, comprised LarueR,YuZ,ParisiVA,EganAR,MorrisonM.Novel mainly bacteria of the family Pseudomo- microbial diversity adherent to plant biomass in the nadaceae, while Firmicutes and Bacteroidetes herbivore gastrointestinal tract, as revealed by Marine Bacterial, Archaeal, and Protistan Association Networks 305 M

ribosomal intergenic spacer analysis and rrs gene Definition sequencing. Environ Microbiol. 2005;7:530–43. Ley RE, Hamady M, Lozupone C, Turnbaugh PJ, Ramey RR, Bircher JS, Schlegel ML, Tucker TA, Schrenzel The study of the covariation of many taxonomic MD, Knight R, Gordon JI. Evolution of mammals and groups across all domains of life within a set of their gut microbes. Science. 2008;320:1647–51. samples using a family of techniques, all of Mardanov AV, Bulygina ES, Nedoluzhko AV, Kadnikov whichidentifymanystatistical associations and VV, Beletskii AV, Tsygankova SV, Tikhonov AN, Ravin NV, Prokhorchuk EB, Skryabin KG. Molecular allow those associations to be subsequently analysis of the intestinal microbiome composition of visualized. mammoth and woolly rhinoceros. Dokl Biochem Biophys. 2012;445:203–6. Miller W, Drautz DI, Ratan A, Pusey B, Qi J, Lesk AM, Tomsho LP, Packard MD, Zhao F, Sher A, Background Tikhonov A, Raney B, Patterson N, Lindblad-Toh K, Lander ES, Knight JR, Irzyk GP, Fredrikson KM, Molecular techniques used to describe marine Harkins TT, Sheridan S, Pringle T, Schuster microbial communities are constantly improving SC. Sequencing the nuclear genome of the extinct woolly mammoth. Nature. 2008;456:387–90. and are describing the structure of marine micro- Nelson KE, Zinder SH, Hance I, Burr P, Odongo D, bial communities in ways that are progressively Wasawo D, Odenyo A, Bishop R. Phylogenetic anal- more detailed. At the same time, large-scale ysis of the microbial populations in the wild herbivore efforts sample many locations of the ocean, gastrointestinal tract: insights into an unexplored niche. Environ Microbiol. 2003;5:1212–20. while others regularly sample the same locations Overballe-Petersen S, Orlando L, Willerslev E. Next- many times in the ocean. Together these efforts generation sequencing offers new insights into DNA promise to increase the resolution of data sets that degradation. Trends Biotechnol. 2012;30:364–8. describe the spatiotemporal distributions of Poinar HN, Schwarz C, Qi J, Shapiro B, Macphee RD, Buigues B, Tikhonov A, Huson DH, Tomsho LP, marine microorganisms including bacteria, archaea, protists, and marine viruses. These Auch A, Rampp M, Miller W, Schuster SC. M Metagenomics to paleogenomics: large-scale sequenc- large data sets present challenges in identifying ing of mammoth DNA. Science. 2006;311(5759):392–4. trends and patterns due to the data’s complexity. Van Geel B, Fisher DC, Rountrey AN, Van Arkel J, Duivenvoorden JF, Nieman AM, Van Reenen GBA, Network association analysis is a tool for Tikhonov AN, Buiguese B, Gravendeel B. Palaeo- performing statistical comparisons of most or environmental and dietary analysis of intestinal con- every pair of parameters in a complex data set tents of a mammoth calf (Yamal Peninsula, northwest and then allowing these comparisons to be visu- Siberia). Quat Sci Rev. 2011;30:3935–46. alized in such a way that one can identify patterns within those associations. In microbial data sets, networks provide an overview that allows Marine Bacterial, Archaeal, and researchers to observe overlying patterns in sta- Protistan Association Networks tistical associations that describe the data set (Fuhrman and Steele 2008) and allow researchers Jacob Cram, Fengzhu Sun and Jed A. Fuhrman to generate hypotheses about ecologically mean- Department of Biological Sciences, University of ingful relationships. Southern California, Dana and David Dornsife College of Letters, Arts and Sciences, Los Angeles, CA, USA Methodology

Suitable Data Synonyms Any data sets with multiple samples and multiple parameters that can be cross-compared are suit- Correlation networks; Co-occurrence networks; able for network analysis (Faust and Raes 2012). Local similarity analysis; Microbial association Microbial association networks, by definition, networks; Network analysis contain data about the microbial community M 306 Marine Bacterial, Archaeal, and Protistan Association Networks structure. Community structure refers to the topology (Deng et al. 2012; Faust and Raes 2012; kinds of organisms found in a given sample and Steele et al. 2011). One can also “filter” the their proportions and can describe any group of network to look at particular subsets of an associ- organisms. Here, we refer primarily to descrip- ation network in order to ask targeted questions. tions of microscopic bacteria, archaea, and eukaryotes. These domains of microscopic Association Analysis Methods organisms can be investigated together or Given a data matrix, there are a number of separately. approaches that can be used to generate an asso- Molecular tools that generate community struc- ciation network. Faust and Raes (2012)summa- ture data include high-throughput sequencing rize several methods that could be applied to techniques, DNA fingerprinting approaches, and microbial data sets to generate networks. They microarray data. Additionally, “classical” commu- divide their analysis methods into the pairwise nity structure information from flow cytometry and regression-based approaches. Pairwise anal- and microscopy has previously been incorporated. ysis, at its most basic, includes the simple appli- These metrics may present data in the form of cation of Spearman or Pearson correlations for estimates of relative abundance (proportions of abundance data sets (Arumugam et al. 2011; a community made up by a given organism or Barbera´netal.2011;Zhouetal.2010), taxonomic group), counts of organisms per unit hypergeometric distributions for presence- volume, or as presence-absence data. Data sets absencedata(Chaffronetal.2010; Freilich may also contain data about the surrounding envi- et al. 2010), and local similarity analysis (LSA) ronment including physical properties of the water (Ruan et al. 2006) method for time-series data (temperature, light, depth of mixing), water chem- where information about temporary or time- istry (salinity, nutrient concentrations), and biotic lagged associations may be valuable. An addi- parameters (cell growth rates, chlorophyll concen- tional pairwise metric, the maximal information trations). Data are generally compiled as a single coefficient (MIC) can generate a variety of matrix with columns representing data about spe- nonlinear associations that may be important in cific species or environmental parameters and some environments, including multiple lines, rows representing the sample under investigation, parabolic distributions, sinusoids and for example. non-coexistence, and combinations of the above that might not be detected by correlation Network Generation Overview or regression alone (Reshef et al. 2011). Faust Generating an association network may utilize et al. (2012) employ an “ensemble approach” in several techniques, but in all cases network ana- which several of these statistics were combined lyses rely on several common steps. The first step to investigate the human microbiome data set. is to generate a matrix summarizing the associa- The second category described by Faust and tions between the variables, usually through Raes (2012) involves regression and “rule-based” some form of pairwise analysis. The second step techniques in which several parameters predict is to apply permutation tests to determine the the abundance of each parameter of interest statistical significance of these associations and (Agrawal et al. 1993; Borgelt and Kruse 2002; to filter the associations according to selected Faust and Raes 2012). criteria so that only significant associations are analyzed and visualized via network analysis. Extended Local Similarity Analysis The third step is to render the network: generally While there are a number of possible ways of variables are represented as nodes (shapes) generating association networks, published stud- and associations between variables as edges ies of marine microbial association networks (lines) connecting nodes (Faust and Raes 2012) (Fuhrman and Steele 2008; Gilbert et al. 2012; (Fig. 1). From here, one can ask questions about Steele et al. 2011) have been time-series studies overall properties of the network, known as that have utilized LSA. aieBceil rhel n rtsa soito ewrs307 Networks Association Protistan and Archaeal, Bacterial, Marine

Marine Bacterial, Archaeal, and Protistan Association Networks, local similarity score, is calculated. Alternatively, several variables can be used to Fig. 1 Overview of common elements in network analysis, from Faust and Raes predict each taxon through multivariate regression analysis. (c) Permutation tests or (2012). (a) All network analyses start with data sets containing presence-absence or theoretical distributions of the statistics are applied on the pairwise tests to determine relative abundance data of microbial taxa, and sometimes other environmental param- statistical significance and nonsignificant results are filtered out. (d) Finally, networks eters. (b) For every pair of taxa or variables, a score, such as Spearman correlation or are generated from variable nodes and statistically significant edges M M M 308 Marine Bacterial, Archaeal, and Protistan Association Networks

Marine Bacterial, Archaeal, and Protistan Associa- represents transformed similarity, as this would depend on tion Networks, Fig. 2 Example of two variables that parameters, no units are shown. (b) The dashed line var- are related to each other in a time-lagged fashion. (a) The iable is time-shifted by 2 months at which point the two x-axis indicates the sample number (in chronological variables are correlated order) from which the variables were taken. The y-axis

LSA identifies “local” and “time-lagged” rela- vs. not, can be represented by the properties of tionships between parameters (Ruan et al. 2006; the line used to connect the node. Furthermore, Xia et al. 2011, 2012). Local relationships are characteristics about the parameters themselves those that occur over only a particular window can also be incorporated into the properties of the of time of the data set. Time-lagged relationships nodes. For instance, shape could indicate the are those in which a change in one of the variables domain an organism comes from, while node leads to the change in the other variable (Ruan size could indicate mean relative abundance. et al. 2006) (Fig. 2). Together these analyses may After network construction, it is often useful to identify relationships that are meaningful in time- “filter” a network. Filtering is the process of series data that would be missed by other ana- removing edges and nodes and leaving only lyses. Users may be interested in time-lagged those that meet user or statistically defined relationships that are nonlocal in which case criteria. Almost all networks involve extended local similarity analysis allows users a preliminary round of filtering in order to leave to identify lagged “global” associations (Xia only those edges that are statistically significant. et al. 2011, 2012). Edges are often filtered by Q value to ensure that fewer than a given fraction of the edges in the data Network Visualization set are due to random chance. It is also common to Any of the aforementioned methods generate look only at the strongest associations in a data at least one measure of association and set. Further filtering can be used to look at specific corresponding significance values for every com- subsets in a network. For instance, a researcher bination of variables, which for large data sets is could examine only one group of organisms and a large number of association measures, specifi- the organisms and parameters that are related to cally n*(nÀ1)/2, where n is the number of vari- that group. This generates simpler and easier to ables being studied. Networks are generally follow diagrams which can be used to make visualized as a map of edges and nodes. Nodes targeted observations about data sets. are represented as shapes which indicate the vari- ables that were measured. Edges are lines that A Network Association Example connect the nodes and represent statistically sig- An example of time-lagged local association net- nificant associations between nodes. Different work from the San Pedro Ocean Time Series that kinds of associations, for instance, positive meets a threshold p-value can be seen in Fig. 3. vs. negative, strong vs. weak, or time lagged From this network, it is apparent that the most Marine Bacterial, Archaeal, and Protistan Association Networks 309 M

M Marine Bacterial, Archaeal, and Protistan Associa- diamonds are Eukaryotes, triangles are Archaea, squares tion Networks, Fig. 3 A network of all bacterial, are biotic environmental variables and hexagons are abi- archaeal, and eukaryotic operational taxonomic units otic environmental variables. Sizes of the bacterial and (OTUs) and environmental variables, observed in the Eukaryotic nodes indicate the average abundance of the Steele et al. (2011) analysis of the San Pedro Ocean OTUs as measured by ARISA and TRFLP, respectively. Time Series. Variables are organized from most-to-least Solid lines show a positive correlation, dashed lines show connected. Shapes represent variables, while edges repre- a negative correlation, arrows indicate a 1-month shift in sent statistically significant interactions (P < 0.01, the correlation” q < 0.063). From Steele et al. 2011:“Circles are Bacteria, highly connected nodes tend to be less-abundant Topology OTUs and that many environmental parameters do Once association networks have been generated, not appear to be connected to OTUs. This network there are a variety of options of statistics that can was then filtered to generate “hub-and-spoke-like” be applied to analyze the topology of these net- subnetworks. This technique involves filtering to works (Deng et al. 2012; Faust and Raes 2012; look only at connections to specific variables. Steele et al. 2011). These measurements quantify Figure 4, for instance, shows only at bacteria and patterns found within the network and can iden- protists that associate with alveolates (Steele tify, for instance, the degree to which a network et al. 2011). In this filtered network, it appears can be characterized as clusters of nodes rather that three of the alveolates are connected to each than as a diffuse web. Topological parameters other and a set of unidentified eukaryotic OTUs. also allow researchers to identify key groups or On closer examination, it appeared that these “modules.” Generally, these topological parame- nodes formed a tightly connected group that covar- ters are best compared to random networks to ied (Steele et al. 2011). determine whether the topology reflects some M 1 aieBceil rhel n rtsa soito Networks Association Protistan and Archaeal, Bacterial, Marine 310

Marine Bacterial, Archaeal, and Protistan Association Networks, Fig. 4 A subset of the network in Fig. 3, in which only parameters correlated with alveolate OTUs are shown. Shapes and edge types are the same as described in the legend for Fig. 3 Marine Bacterial, Archaeal, and Protistan Association Networks 311 M underlying aspect of the community rather than Driving Variables random chance. Deng et al. (2012) (Kara An important consideration is the possibility that et al. 2012; Steele et al. 2011) have described a few parameters influence large fractions of microbial networks as having “modular,” “small community variability. A classic example is world,” and “scale-free” properties. Modular net- depth. It is known that surface communities are works are those that can be divided into several very different from, e.g., bathypelagic commu- clusters. Small world networks are those in nities. Performing a network analysis on sam- which, while most nodes are not connected to ples from different depths, even if depth many other nodes, each node is only a few steps is included as a parameter, is likely to be away from most other nodes. Scale-free networks uninformative about the relationships that are are those in which there are a few nodes that are not driven by depth because depth-related asso- highly connected to other nodes (Deng ciations would drown out the patterns that occur et al. 2012; Faust and Raes 2012). within depths. To avoid this problem, networks can be done on sets of samples from within one Statistical Considerations given depth. Season, in some environments, has There are several considerations that one must been shown to have a strong effect on commu- make when deciding whether network analysis nity structure (Gilbert et al. 2012). In spatial is appropriate for a given data set, including studies, comparisons between disparate loca- sample size, number of parameters measured, tions might cause a similar problem. To get and known relationships between those around these dominating factors, it is practical parameters. to separate the data set into multiple groups to be analyzed separately or to employ an analysis Sample Size procedure that factors out the dominant variable Larger sample sizes increase the power of statis- from other analyses. M tical tests (Pagano and Gauvreau 2000) thereby making accurate detection of associations more likely. With a small sample size, it may be diffi- Current State of Marine Microbial cult to detect associations in a way that is statis- Association Network Studies tically different from chance. Association patterns among the prokaryotic oper- Multiple Comparisons ational taxonomic units (OTUs), among eukary- In any analysis, comparing a large number of otic OTUs and between prokaryotic and bacterial variables leads to a high probability of obtaining OTUs were examined in both the San Pedro some false-positive results (Pagano and Ocean and Plymouth Marine lab time series. At Gauvreau 2000). Traditional corrections for mul- both sites, positive and negative time-lagged tiple comparisons such as Bonferroni, which associations were seen between microorganisms determine the probability of obtaining any false of both communities. positives tend to be prohibitively conservative in such analyses. To get around this, less conserva- SPOT tive corrections such as false discovery rate The San Pedro Ocean Time Series (SPOT), (Q value) are often used to estimate the fraction located off the coast of Southern California, of false-positive results in a network (Ruan examined bacterial and microscopic eukaryotic et al. 2006; Storey and Tibshirani 2003). As (protistan) communities, along with the abun- long as a small fraction of edges, e.g., 5 % or dance of some archaeal groups, using molecular 1 %, are due to random chance, it remains rea- methods (ARISA and TRFLP fingerprinting, sonable to draw conclusions about network pat- qPCR), from the deep chlorophyll maximum terns while not putting too much emphasis on any layer over a period of 3 years. The authors iden- individual correlation. tified that overall the communities showed M 312 Marine Bacterial, Archaeal, and Protistan Association Networks nonrandom patterns of association. Specifically, eukaryotes while bacteria correlated with bacte- they identified that bacteria are more likely to ria, while there were also cases in which particu- statistically associate with other bacteria and pro- lar taxa were shown to correlate with a number of tists with other protists, but there were many taxa from the other domain. examples of associations between the two domains. They identified at least one cluster of Nonmarine Microbial Systems strongly correlated eukaryotes that appeared to Outside of marine systems, network analysis has co-occur together. The authors hypothesized that been used to evaluate associations between such a pattern could be due to symbiosis, microbial taxa and between microbial genes in endoparasatism, or several genes within the various environments. Network analysis has been same organism. The authors examined overall employed to study microorganisms in lake sys- network topology and suggested that the network tems (Eiler et al. 2012; Kara et al. 2012), soil had a higher clustering coefficient and shorter (Barbera´n et al. 2011; Zhou et al. 2010), the path length than a less clustered network and human microbiome (Arumugam et al. 2011; suggested the network had “small world proper- Faust et al. 2012), and globally through meta- ties.” This meant that there were several particu- analysis across diverse sampling sites (Chaffron lar OTUs that were highly connected with many et al. 2010; Freilich et al. 2010). other OTUs, which they argue suggest these highly connected nodes may be keystone species (Steele et al. 2011). Publicly Available Tools

PML Network Analysis Calculation Programs: The Plymouth Marine Lab (PML) time series is Extended Local Similarity Analysis (eLSA): For located in the Western English Channel and networks of time series. examined microbes at the sea surface, along https://bitbucket.org/charade/elsa/ with many classically identified phytoplankton Co-Net: Provides several tools for identifying and zooplankton (Gilbert et al. 2012, 2009). linear associations. While this time series used high-throughput http://psbweb05.psb.ugent.be/conet/ sequencing, the network analysis component Maximal Information-Based Nonparametric examined the 300 most abundant microbes, Exploration (MINE): Identifies non-linear thereby returning a data set similar in size to the associations one investigated at SPOT (to make it computa- http://www.exploredata.net/ tionally manageable). There is greater seasonal Visualizing Association Networks variability in environmental conditions in the English Channel than in the San Pedro Channel, Cytoscape: and network patterns appear to be strongly http://www.cytoscape.org/ influenced by time of year. Overall network Network Topography structure was characterized by a few features. Topology Plugin for Cytoscape (Current version First, a large interconnected group of bacteria in in Cytoscape 2.8) which nodes were connected to most of the other Random Matrix Plugin (Note only works in nodes in the group. This group was statistically Cytoscape version 2.6) associated with winter months which coincides with high dissolved nitrate and nitrite concentra- tions. Second, there was a more loosely Summary connected group of eukaryotes and prokaryotes that appeared to be most abundant during the Association analysis is a tool that is beginning to spring. Generally, as seen at SPOT, it appeared identify patterns within marine microbiological that most eukaryotes correlated with other communities. There are a number of ways of Marine Invertebrate Animal Metagenomics: Porifera 313 M applying network analyses which promise to Kara EL, Hanson PC, Hu YH, et al. A decade of seasonal yield new insights into the factors that shape the dynamics and co-occurrences within freshwater bacterioplankton communities from eutrophic Lake structure of marine microbial communities. Mendota, WI, USA. ISME J. 2012;7:680–4. Pagano M, Gauvreau K. Principles of biostatistics. Acknowledgments The authors would like to thank the 2nd ed. Pacific Grove: Duxbury Press; 2000. Gordon and Betty Moore Foundation and the National Reshef DN, Reshef YA, Finucane HK, et al. Detecting Science Foundation for Support. novel associations in large data sets. Science. 2011;334:1518–24. Ruan Q, Dutta D, Schwalbach MS, et al. Local similarity analysis reveals unique associations among marine bacterioplankton species and environmental factors. Cross-References Bioinformatics. 2006;22:2532–8. Steele JA, Countway PD, Xia L, et al. Marine bacterial, ▶ Ocean Gyres, Metagenomics of archaeal and protistan association networks reveal ▶ ecological linkages. ISME J. 2011;5:1414–25. Ocean Metagenomics Storey JD, Tibshirani R. Statistical significance for genomewide studies. PNAS. 2003;100:9440–5. Xia LC, Steele J, Cram J, et al. Extended local similarity analysis (eLSA) of microbial community and other References time series data with replicates. BMC Syst Biol. 2011;5:S15. Agrawal R, Imielin´ski T, Swami A. Mining association Xia LC, Ai D, Cram J, et al. Efficient statistical signifi- rules between sets of items in large databases. New cance approximation for local association analysis of York: ACM SIGMOD Record; 1993. p. 207–16. high-throughput time series data. Bioinformatics. Arumugam M, Raes J, Pelletier E, et al. Enterotypes of the 2012;29:230–7. human gut microbiome. Nature. 2011;473:174–80. Zhou J, Deng Y, Luo F, et al. Functional molecular eco- Barbera´n A, Bates ST, Casamayor EO, Fierer N. Using logical networks. mBio. 2010;1:e00169–10. network analysis to explore co-occurrence patterns in soil microbial communities. ISME J. 2011;6:343–51. Borgelt C, Kruse R. COMPSTAT 2002 – Proceedings in M computational statistics: 15th Symposium held in Ber- lin, Germany, 2002. Physica-Verlag; 2002. p 395–400. Chaffron S, Rehrauer H, Pernthaler J, Von Mering C. A Marine Invertebrate Animal global network of coexisting microbes from environ- Metagenomics: Porifera mental and whole-genome sequence data. Genome Res. 2010;20:947–59. Jonathan Kennedy1, Stephen Jackson1, Deng Y, Jiang Y-H, Yang Y, et al. Molecular ecological 1 2 network analyses. BMC Bioinforma. 2012;13:113. John P. Morrissey , Fergal O’Gara and Eiler A, Heinrich F, Bertilsson S. Coherent dynamics and Alan D. W. Dobson1 association networks among lake bacterioplankton 1Marine Biotechnology Centre, Environmental taxa. ISME J. 2012;6:330–42. Research Institute, and School of Microbiology, Faust K, Raes J. Microbial interactions: from networks to models. Nat Rev Microbiol. 2012;10:538–50. University College Cork, Cork, Ireland Faust K, Sathirapongsasuti JF, Izard J, et al. Microbial 2BIOMERIT Research Centre and School of co-occurrence relationships in the human microbiome. Microbiology, University College Cork, Cork, PLoS Comput Biol. 2012;8:e1002606. Ireland Freilich S, Kreimer A, Meilijson I, et al. The large-scale organization of the bacterial network of ecological co-occurrence interactions. Nucl Acids Res. 2010;38:3857–68. Introduction Fuhrman J, Steele J. Community structure of marine bacterioplankton: patterns, networks, and relationships to function. Aquat Microb Ecol. 2008;53:69–81. The phylum Porifera is commonly known as Gilbert JA, Field D, Swift P, et al. The seasonal structure sponges, with the Latin name of the phylum of microbial communities in the Western English deriving from the large number of pores found Channel. Environ Microbiol. 2009;11:3132–9. on their surfaces. Sponges are the oldest meta- Gilbert JA, Steele JA, Caporaso JG, et al. Defining sea- sonal marine microbial community dynamics. ISME zoan animal phylum, with fossil records dating J. 2012;6:298–308. back to almost 600 Ma. The phylum contains M 314 Marine Invertebrate Animal Metagenomics: Porifera

Marine Invertebrate Animal Metagenomics: Porifera, Fig. 1 Sponge body plan and main cell types

around 6,000 species in a paraphyletic grouping that create the sponge’s water current and use consisting of three major sublineages, namely, microvilli to filter out particles from the water. the Calcarea (calcareous sponges), Bacteria within the seawater which are trans- Hexactinellida (glass sponges), and ferred into the mesohyl tissue are either ingested Demospongiae (demosponges), with the last by archaeocytes or survive and become group containing the majority of extant species. established as part of the sponge-specific Sponges are predominantly found in tropical and microbiota enclosed within the mesohyl matrix subtropical oceans as well as in the deep sea and and physically separated from the surrounding also in polar regions, with some sponges even seawater by the sponge pinacoderm. In this way being found in freshwater systems. Quite often marine sponges play host to significant microbial they are dominant members of particular benthic populations which include bacteria, archaea, and communities, and as a result sponges can exert single-celled eukaryotes (microalgae and fungi), important ecological influences on many benthic which play an important role on the biology of and pelagic processes. Sponges possess the host. a relatively simple body plan with a variety of This microbiota can be symbiotic or patho- overall body shapes which have adapted for max- genic, act as a food source, or be transiently imal efficiency of water flow through the sponge. associated with the sponge. In some instances, They are sessile filter feeders that remove bacte- up to 40–60 % of the animal’s total biomass can ria from the surrounding aqueous environment by comprise of endosymbiotic microorganisms, pumping up to 25 m3 of water per kg sponge per with densities of between 108 and 1010 bacteria day through their aquiferous system. Sponges per gram of sponge wet weight being reported. have an epidermal layer called the pinacoderm These bacteriosponges or the so-called high- which is composed of pinacocytes (Fig. 1). Sea- microbial-abundance (HMA) sponges such as water is drawn through tubular porocytes into an the Caribbean great barrel sponge Xestospongia area which is located between the outer and inner muta which contains in the region of 8 Â 109 cell layers in the so-called mesohyl layer of the bacteria per gram of sponge tissue, exceed the sponge, which is composed of a network of canal- number of bacteria typically found in seawater by like structures. The interior body of the sponge is at least two to four orders of magnitude. Not all lined by choanocytes which are flagellated cells sponges are classed as HMA with a number Marine Invertebrate Animal Metagenomics: Porifera 315 M containing relatively few bacteria, and are there- (FISH) studies were employed to identify the fore often termed as low-microbial-abundance numerous bacterial phyla that were closely asso- (LMA) sponges. Microbial communities within ciated with sponges. FISH involves the use of LMA sponges tend to be less diverse, as well as of rRNA-targeted oligonucleotide probes to specif- lower density. ically identify individual microbial cells. These Marine sponges are a very rich source of probes target 16S rRNA and are typically labeled natural products which are believed to have with fluorophores which allows visualization fol- evolved as a chemical defense mechanism lowing hybridization of the probe to the target against potential predators and to prevent 16S rRNA gene. Other culture-independent biofouling. Many of these have proven to be approaches which have proven useful in studying biologically active and pharmacologically valu- sponge microbial ecology involve the PCR able, with over 7,000 bioactive compounds hav- amplification of bacterial 16S rRNA genes ing to date been isolated from sponges. In fact directly from sponge metagenomic DNA and more novel bioactive metabolites are obtained subsequent analysis using denaturing gradient annually from sponges than from any other gel electrophoresis (DGGE) which is based on marine taxon. Many of these compounds display the electrophoretic separation of DNA based on anticancer, anti-infective, or other bioactivities, differences in their melting behaviour in either making them interesting lead compounds for a temperature- or denaturing agent-induced both medical and biotechnological applications. gradient. Many of these natural products are structurally Using these different approaches, >21 differ- similar and in some cases identical to bacterially ent bacterial phyla were reported to be present in derived compounds, and it is believed that many sponges, including Actinobacteria, Chloroflexi, of these products are produced by bacterial sym- Cyanobacteria, Nitrospira,andProteobacteria. bionts. Thus, there has been much interest in However, recently pyrosequencing of PCR M studying the microbiota associated with marine amplicon libraries from sponge metagenomic sponges, not only from a biotechnological per- sources has allowed for even deeper insights spective but also from a microbial ecology per- into environmental microbial community struc- spective, to gain insights into the exceptionally tures within sponges, thereby negating the diverse microbial communities present within requirement for a cloning step and providing the sponge ecosystem, with metagenomic- numbers of sequencing reads which are orders based approaches having proven particularly of magnitude greater than were previously pos- useful in facilitating this work on sponge- sible (Webster et al. 2010). These approaches microbe associations. haveresultedinanincreaseintheknownbacte- In common with terrestrial environments, rial phyla within sponges to >30 major phyla where more than 99 % of bacteria cannot cur- which have to date been found to be present in rently be cultured by conventional means, the close association with sponge species globally. vast majority of marine bacteria within marine As well as the aforementioned phyla, several environments have to date not yet been cultured, candidate phyla have been uncovered in sponges with as few as 0.001–0.1 % of microbes in sea- such as Poribacteria (Webster and Taylor 2012). water, for example, currently being culturable. Thus, culture-independent approaches have proven particularly useful in studying the micro- Natural Products and Bacterial bial ecology of marine sponges. Initial culture- Symbionts of Marine Sponges independent ecological investigations employed transmission electron microscopy (TEM) to As previously mentioned marine sponges are observe diverse bacterial cell types in sponge known to be a rich source of biologically active tissues (Vacelet and Donadey 1977). Subse- and pharmacologically useful natural products quently, fluorescence in situ hybridization (Mayer et al. 2010). The structures of many M 316 Marine Invertebrate Animal Metagenomics: Porifera sponge-derived natural products and in particular systematic investigation of 20 different the complex polyketides and modified peptides demosponge species, large numbers of highly structurally resemble bacterial compounds; and sponge-specific groups of PKS were present in thus, it is a widely held hypothesis that many of the sponge metagenomes. Many of these sponge- these products are produced by bacterial symbionts. specific PKS possessed an architecture Examples include jaspamide from the sponge suggesting that they were involved in the synthe- Jaspis spp. and the cyclodepsipeptide chondramide sis of methyl-branched fatty acids (Fieseler D isolated from the myxobacterium Chondromyces et al. 2007). crocatus and the myxobacterial metabolite Targeting PKS genes has proven successful in apicularen A which is almost identical to salicyli- the identification of novel bioactive metabolites halamide A from Haliclona sp. (Kennedy such as the identification of the putative et al. 2007). The study of the biosynthetic potential onnamide PKS gene cluster from the of sponge symbionts is gaining considerable recent metagenome of the marine sponge Theonella attention, given that it provides a potential novel swinhoei. Similarly PCR-based approaches source of marine-derived pharmacologically active targeting specific signature motifs within PKS compounds. With this in mind there has been much such as ketosynthase (KS) domains have been current interest in employing metagenomic-based successfully employed to clone the gene cluster approaches to exploit the biosynthetic potential of involved in mycalamide A biosynthesis from the marine sponges. sponge Mycale hentscheli (Piel 2011). In this respect, given that polyketides and With respect to NRPS, studies involving nonribosomal peptides are important classes of a whole gene amplification-based approach have bioactive bacterial secondary metabolites, much resulted in the identification of a novel NRPS of this effort has focused on isolating both poly- from the metagenomic of Aplysina aerophoba. ketide synthase (PKS) and nonribosomal peptide This NRPS which contains the unique domain synthetase (NRPS) gene clusters from sponge- architecture CATA (C, condensation; A, associated bacterial metagenomic libraries to adenylation; T, thiolation) shared some homol- identify symbionts of marine sponges with bio- ogy with genes involved in activity potential. In addition PKS enzymes are 2,3-dihydroxybenzoate synthesis in Bacillus known to be the source of natural products with subtilis and was located downstream of pharmacological activity such as the potential a putative efflux pump. In this study a novel antitumor agent psymberin isolated from PKS gene was also identified (Siegl and a symbiont of the sponge Psammocinia sp. aff. Hentschel 2010). NRPS have also been identified bulbosa. Thus, genes encoding PKS have been in fungi associated with marine sponges from the targeted in sponge metagenomes, and sponge South China Sea, with four NRPS genes being metagenomic libraries have been shown to con- identified from the 177 fungal isolates studied. tain numerous PKS genes. For example, seven A functional metagenomic-based approach PKS sequences have been reported from the has recently been successfully employed to sponge metagenome of Haliclona simulans. clone two genes with antibacterial activity from The analyses of the PKS sequences revealed the metagenome of the marine sponge a close affiliation with PKS from the members Cymbastela concentrica. These genes appeared of the Cyanobacteria, Myxobacteria, and to encode novel hydrolytic enzymes, with one of Dinoflagellata (Kennedy et al. 2008). Diverse these clones, ABg1, with activity against Staph- PKS gene clusters have also been reported in ylococcus aureus and Alteromonas sp. strain the metagenome of both Discodermia dissoluta CCSH174 displaying 31 % identity with a subtil- and Pseudoceratina clavata, and deep sequenc- isin from Bacillus amyloliquefaciens. The other ing of PCR products amplified from the sponge clone, ABg2, which displayed activity against the Cacospongia mycofijiensis revealed more than marine Bacillus strain Cc6 displayed some iden- 100 PKS homologs. In a study involving the tity to a beta-lactamase (Yung et al. 2011). Marine Invertebrate Animal Metagenomics: Porifera 317 M

Functional Metagenomics of Marine followed by characterization of the recombinant Sponges protein at the biochemical level. There are a number of examples of such an approach in Bacteria which are associated with marine various marine ecosystems, but not many in sponges are likely to have a number of environ- marine sponges. One example however involves mentally related constraints imposed upon them, the identification of a putative collagenase from due to the unique environment in which they find the metagenome of the marine sponge themselves. These constraints may include Cymbastela concentrica, with 49 % identity extremes in temperature with average seawater with the PrtC collagenase from Porphyromonas temperatures in the oceans of around 3 C, gingivalis. together with variations in hydrostatic pressures. The function-based screening approach In addition these bacteria will also have to com- involves directly screening the sponge pete under nutrient-replete conditions, as well as metagenomic library for detectable phenotypes. survive potential predation by grazers and infec- Metagenomic clones displaying the desired enzy- tions by viruses. Thus, to survive under these matic activity which is often determined as conditions, it is likely that they will have devel- a result of a colorimetric-based assay system oped quite unique cellular biochemistry and pos- can then be isolated. These clones can be subse- sess enzyme systems with atypical biochemical quently overexpressed in a relevant heterologous and physiological properties – such as increased host and then evaluated functionally at the bio- barophilicity and temperature adaptation, among chemical level. Functional-based approaches like others – therefore making them an interesting this have been successfully employed to clone source of novel marine-derived biocatalysts. novel genes encoding various enzymes from a Functional-based approaches have been variety of different marine sponge metagenomic employed to exploit the biotechnological poten- libraries. For example, a total of 58 clones which M tial of these marine sponge metagenomes. This displayed lipolytic activity on Luria-Bertani involves generating a marine sponge (LB) agar supplemented with 1 % tributyrin metagenomic DNA library by using a heterolo- have been isolated from a metagenomic library gous host expression system and the subsequent constructed from the marine sponge Haliclona screening of the library by employing either a - simulans. Heterologous expression of one of sequence-based or function-based approach. these clones, Lpc53E1, in E. coli and the subse- Sequence-based approaches involve targeting quent biochemical characterization of the recom- specific enzyme-coding genes by employing binant protein showed the enzyme to have the a PCR amplification-based approach. Primers are highest substrate specificity for long-chain fatty designed based on conserved sequences known to acyl esters. Optimal activity was observed with  be present in the targeted gene, and PCR amplifi- p-nitrophenyl palmitate (C16)at40 C, in the cation is then performed on the metagenomic presence of 5 M NaCl at pH 7; in addition the library. Following the cloning of the amplified recombinant enzyme displayed activity across PCR fragments, random clones can then be broad pH (3–12) and temperature (4–60 C) sequenced and subsequently analyzed. An exam- ranges and high levels of stability in the presence ple of such a successful PCR-based approach of various solvents at NaCl concentrations as involved the cloning of a novel laccase gene high as 5 M and at temperatures ranging from from a marine microbial metagenome by targeting 10 Cto80C. These biochemical characteristics the highly conserved copper-binding domain of expand the potential utility of this enzyme in laccases (Fang et al. 2011). various industrial applications. Other sequence-based approaches involve Another example is the cloning of a novel sequencing of metagenomic DNA and the subse- esterase from the metagenome of the marine quent identification of putative genes and expres- sponge Hyrtios erecta, which was isolated on sion in an appropriate heterologous host, LB agar supplemented with 0.5 % Tween-20. M 318 Marine Invertebrate Animal Metagenomics: Porifera

This novel esterase EstHE1 displayed the highest studied using both sequence-based and function- substrate specificity for short-chain fatty acyl based metagenomic approaches. This has led to esters such as p-nitrophenyl (pNP) acetate and the discovery of a number of genes encoding butylate with an optimum temperature for ester- novel laccases, collagenases, proteases, lipases, ase activity with pNP acetate as substrate of and esterases, among others. Given that marine 40 C. EstHE1 also displayed moderate thermo- sponges are a very rich source of biologically stability, retaining 58 % of its activity following active and pharmacologically valuable natural preincubation for 12 h at 40 C. Like the afore- products, many of which are produced by bacte- mentioned Lpc53E1, EstHE1 is also salt tolerant rial symbionts, then it is perhaps not surprising and maintained activity in high concentrations of that metagenomic-based approaches have also NaCl; and these thermal stability and salt toler- been employed to exploit this potential, with ance again, like the aforementioned Lpc53E1, genes encoding polyketide synthases and suggest potential utility as an industrial enzyme nonribosomal peptide synthases in particular (Okamura et al. 2010). being targeted in sponge metagenomes. Using Proteases are another industrially important these approaches putative onnamide PKS and group of enzymes, and while a number of novel mycalamide A biosynthetic gene clusters have proteases have been cloned from marine been identified. In addition genes with metagenomic libraries, there are surprisingly antibacterial activity have also been identified few reports to date of their isolation from sponge from sponge metagenomic libraries. This area is metagenomic DNA. There has been however one rapidly expanding, and with the ever increasing recent report of the isolation of two protease power of high-throughput DNA sequencing tech- clones from a metagenomic library from the nology coupled with the development of new sponge Haliclona simulans. sensitive and efficient high-throughput screening strategies, involving both sequence- and function-driven approaches, it is likely that Summary novel biotechnologically relevant enzymes and bioactive compounds will continue to be uncov- Sponges are the oldest metazoan animal phylum, ered from marine sponge metagenomes. containing around 6,000 species. They are mostly found in tropical and subtropical oceans as well as in the deep sea and also in polar regions and References have important ecological influences on many benthic and pelagic processes. Sponges remove Fang Z, Li T, et al. A bacterial laccase from marine bacteria from the surrounding seawater and play microbial metagenome exhibiting chloride tolerance and dye decolorization ability. Appl Microbiol host to significant numbers of endosymbiotic Biotechnol. 2011;89:1103–10. microorganisms, which play an important role Fieseler L, Hentschel U, et al. Widespread occurrence and in the biology of the host. Numerous culture- genomic context of unusually small polyketide synthase independent approaches including transmission genes in microbial consortia associated with marine sponges. Appl Environ Microbiol. 2007;73:2144–55. electron microscopy, fluorescence in situ hybrid- Kennedy J, Marchesi JR, Dobson ADW. Metagenomic ization, and denaturing gradient gel electropho- approaches to exploit the biotechnological potential resis have been employed to study the microbial of the microbial consortia of marine sponges. Appl ecology of marine sponges, resulting in the iden- Microbiol Biotechnol. 2007;75:11–20. > Kennedy J, Codling CE, et al. Diversity of microbes tification of 21 different bacterial phyla. More associated with the marine sponge, Haliclona recently pyrosequencing of PCR amplicon librar- simulans, isolated from Irish waters and identification ies from sponge metagenomic sources has of polyketide synthase genes from the sponge resulted in an increase in this number to >30 metagenome. Environ Microbiol. 2008;10:1888–902. Mayer AMS, Glaser KB, et al. The odyssey of marine major phyla. In addition the bacterial endosym- pharmaceuticals: a current pipeline perspective. bionts of marine sponges have been widely Trends Pharmacol Sci. 2010;31:255–65. Marine Sponge Craniella australiensis-Associated Bacterial Diversity 319 M

Okamura Y, Kimura T, Yokouchi H, Meneses-Osorio M, Introduction Katoh M, Matsunaga T, Takeyama H. Isolation and characterisation of a GDSL esterase from the metagenome of a marine sponge-associated bacteria. Marine sponges are benthic, sessile, and filter Mar Biotechnol. 2010;12:395–402. feeding, simplest form of multicellular ancient Piel J. Approaches to capturing and designing biologically metazoan group, having an outstanding impor- active small molecules produced by uncultured tance as a living fossil record dating back over microbes. Annu Rev Microbiol. 2011;65:431–53. Siegl A, Hentschel U. PKS and NRPS gene clusters from 600 Ma. In general, sponges are classified into microbial symbiont cells of marine sponges by whole three different groups, namely, Calcarea, genome amplification. Environ Microbiol Rep. 2010; Hexactinellida, and Demospongiae (Brusca and 2:507–13. Brusca 2002). The group Demospongiae is also Vacelet J, Donadey C. Electron microscope study of the association between sponges and bacteria. J Exp Mar called as demosponges. They encompass 95 % of Biol Ecol. 1977;30:301–14. the ca. 5,500 all known sponge species that have Webster NS, Taylor MW. Marine sponges and their so far been described. Marine sponges inhabit in microbial symbionts: love and other relationships. all the oceans in the world regardless of extreme Environ Microbiol. 2012;14:335–46. Webster NS, Taylor MW, et al. Deep sequencing reveals temperatures. They appear in a variety of colors, exceptional diversity and modes of transmission for shapes, and sizes. Sponges can be found hundreds bacterial sponge symbionts. Environ Microbiol. of meters under sea level but mostly are found 2010;12:2070–82. in 5–50 m deep. Sponges and their bacterial Yung PY, Burke C, et al. Novel antibacterial proteins from the microbial communities associated with the symbionts are one of the significant marine sponge Cymbastela concentrica and the green alga biota in the aspects of ecology, genetics, symbi- Ulva australis. Appl Environ Microbiol. 2011;77: osis, and pharmacology. This overview presents 1512–5. the diversity and functional potential of marine sponge-associated bacterial consortia, with special reference to Craniella australiensis. M Marine Sponge Craniella australiensis-Associated Bacterial Bacterial Diversity in Sponges Diversity Sponges are well known to harbor diverse micro- Ramasamy Anbuchezhian and Zhiyong Li bial communities (Hentschel et al. 2006; Taylor Marine Biotechnology Laboratory, State Key et al. 2007; Schmitt et al. 2012), with an amount Laboratory of Microbial Metabolism, School of that exceeding microbial population of seawater Life Sciences and Biotechnology, Shanghai Jiao by two to four orders of magnitude. The unique Tong University, Shanghai, China holobiont system in the sponges has complex arrangement to harbor diverse microbial commu- nities. Moreover, they become one of the impor- Synonyms tant microbial filters of the marine ecosystem. As the sponges become highly efficient filter feeders, Sponge-associated bacterial symbionts it is estimated that 1 kg of sponge can filter up to 24,000 L of seawater a day, which will include huge bacterioplankton (Vogel 1977). They feed Definition on bacteria, organic matters, and small organisms by drawing water inside the body through pores. Sponge: animals of the phylum Porifera. Some sponges that are adapted with thick Symbionts: living in symbiosis with another mesohyl contained abundant, dense, and morpho- organism or each other. logically diverse microbial communities Metagenomics: analysis of environmental (bacteriosponge), and those with a well- genomic DNA. developed aquiferous system and low-density M 320 Marine Sponge Craniella australiensis-Associated Bacterial Diversity mesohyl contained low bacterial cells and Natural products and their derivatives represent typically only single bacterial morphotypes majority of all drugs in clinical use. Marine (Hentschel et al. 2006). In bacteriosponges, bac- natural products have attracted the attention of terial densities may reach 108-1010 bacterial cells biologists and chemists because of their unique per g (wet weight) of sponge. In general, sponge- structure and biological potential. Many natural specific bacteria have been found mainly in the products isolated from sponge-associated bacte- bacteriosponges, and low bacterial abundance ria have already been proved to be significant sponges have the different bacterial communi- in industrial and biotechnological applications. ties. There are more than 30 sponge-associated The bioactive metabolites of sponge origin have bacterial phyla that have been reported so far been proven to be either from sponge, their sym- (Webster et al. 2010), mainly including bionts, or from communication between sponge Acidobacteria, Actinobacteria, Bacteroidetes, and symbionts (Faulkner et al. 1993). Many Chloroflexi, Cyanobacteria, Firmicutes, and investigations have proven that microorganisms Proteobacteria (Alpha-, Beta-, Delta-, and are the true producers of at least some of the Gamma-proteobacteria). biologically active metabolites which were orig- Investigations on the sponge-associated bacteria inally thought to be a product of host organisms have also explored novel bacterial groups, sponge- and which include sponges. Exploration of specific candidate phylum “Poribacteria” (Fieseler the nature of interaction between sponge and et al. 2004). Bacterial phylotypes Acidobacteria, bacterial associates is still becoming a challeng- Actinobacteria, and Chloroflexi have been identi- ing task. So, the documentation of sponge- fied most frequently from sponges by 16S rRNA associated bacterial consortia will play a major gene sequences. Some of the bacterial phyla have role as a scientific platform for understanding also been isolated by in vitro which includes the sponge-microorganism relationships, their Actinobacteria, Bacteroidetes, Cyanobacteria, relative functions, and exploration of their Firmicutes, Planctomycetes, Proteobacteria, and biotechnological potentials. Verrucomicrobia. But, the sponge-bacteria associ- ation is less well documented. The developments in microbial genomics and investigations by the Techniques for Sponge-Associated advanced sequencing techniques have found that Bacterial Diversity Investigation and bacteria residing in water column will not colo- Functional Potential Assessment nized well because of preadapted bacterial symbi- onts of the sponge mesohyl. It is well known that culture-dependent tech- niques are quite insufficient for exploiting the sponge-associated bacterial diversity, because Role and Importance of majority of them are not able to culture under Sponge-Associated Bacteria laboratory condition. So, culture-independent approaches are necessary to exploit true sponge- Sponge-associated bacterial symbionts will play associated bacterial diversity. The whole diver- a major role in the host chemical defense sity (including unculturables) and functions of (secondary metabolites) and many ecological sponge-associated bacteria can be explored by functions including photosynthesis, methane oxi- integrated approach of using metagenomics, dation, nitrification, nitrogen fixation, sulfate metatranscriptomics, and metaproteomics. This reduction, and dehalogenation along with host. can be performed by direct extraction of DNA, There is a great interest in marine sponges and RNA, and proteins. Metagenomics offers novel their bacterial symbionts because of their novel insights into the genetic and functional potential secondary metabolites. Natural products are of microbial communities. Metatranscriptomics a very important source of all drugs and it will is used to assess the expression gene profiles also play a key role in future drug discovery. of microbial communities. Metaproteomics is Marine Sponge Craniella australiensis-Associated Bacterial Diversity 321 M used to describe the expressed protein profiles of Bacterial Diversity Associated with microbial communities. The developments in Sponge C. australiensis sequencing technologies used in metagenomics, metatranscriptomics, and metaproteomics have Based on the representative sequences deposited made it possible to unravel complex bacterial in the GenBank, the whole bacterial diversity communities. Genomics-based studies in characterized by culture-dependent and culture- sponges described the bacterial diversity much independent studies from this sponge comprised higher than the results of culture-dependent four phyla, 26 genera, and 60 species. The total investigations. The advanced molecular tech- bacterial associates of sponge C. australiensis are niques have also made it possible the characteri- represented in Fig. 2. zation of the whole microbiota, their possible Different bacterial communities have been nutritional requirements, and physiological found to inhabit in the C. australiensis by total niches of many microorganisms from the available DNA sequence analysis (Li and Liu 2006). The data of known phylogenetic relatives. This may majority of bacterial sequences were related to also be helpful for the experimental manipulation Bacteroidetes (n ¼ 39; 43.9 %) and Gammapro- of culture conditions to provide the correct growth teobacteria (n ¼ 31; 34.0 %). Clones affiliated environment for targeted bacteria (Hentschel with Firmicutes (n ¼ 8; 8.8 %), Alphaproteo- et al. 2003). Many investigations have already bacteria (n ¼ 7; 7.7 %), and Betaproteobacteria succeeded in the isolation of biosynthetic genes/ (n ¼ 5; 5.5 %) have also been recorded. gene clusters from sponge unculturables, and it has Sequences related to Actinobacteria were only also led to isolation of symbiont-generated bioac- a minor component of the gene library. The bac- tive compounds (Li 2009). These different geno- terial diversity of this sponge recorded by DGGE mics techniques will also provide a platform to fingerprint approach has also harmony with the discover new molecules for therapeutics and bio- results of sponge total DNA sequence analysis M technological potentials of sponge-associated bac- (Li et al. 2006). The same trends of bacterial teria. The different techniques employed in the diversity have also been recorded in some characterization of sponge-associated bacterial sponges with the predominant component as community and their functional potential assess- Proteobacteria. The low diversity of Actinomy- ment are given in Fig. 1. cetes has also been recorded in different marine sponges. Some of the clones of C. australiensis were unculturable bacteria and a clone might be an Golf Ball Sponge C. australiensis unknown bacterium, which addressed a big chal- lenge in the recovery of sponge-associated bacte- Marine sponge C. australiensis (Porifera, Class ria. The culture-independent 16S rDNA-DGGE Demospongiae, Order Choristida, Family fingerprinting and phylogenetic analysis of com- Craniellidae) is widely distributed in Australia, munity structure associated with different sponges Western Indo-Pacific, South China Sea, etc. This (C. australiensis, Stelletta tenui, Halichondria sp., sponge is directly exposed to sunlight and is and Dysidea avara) have also shown that coated with a layer of sand grains through high bacterial diversity was associated with which megascleres protrude from the surface. C. australiensis, which comprised four phyla, Sponge C. australiensis is also characterized by i.e., Proteobacteria, Bacteroidetes, Firmicutes, a globular shape and commonly known as orange and Actinobacteria (Li et al. 2006). The predomi- or golf ball sponge. Craniella sp. sponges are nant phylum was Proteobacteria followed by comparatively less-studied genus for their Bacteroidetes, which have been documented microbiome. The unlocking of true bacterial bio- in sponges C. australiensis and D. avara.The diversity of marine sponge C. australiensis will different phylum Proteobacteria in four sponges be helpful in the documentation of their biodiver- and specific C. australiensis-associated phyla sity to avail their functional potentials. Actinobacteria and Firmicutes have reflected host M 322 Marine Sponge Craniella australiensis-Associated Bacterial Diversity

Marine Sponge Craniella australiensis-Associated Bacterial Diversity, Fig. 1 General scheme of the sponge- associated bacterial diversity and functional potential assessment approaches Marine Sponge Craniella australiensis-Associated Bacterial Diversity 323 M

M

Marine Sponge Craniella australiensis-Associated Bacterial Diversity, Fig. 2 Total diversity of marine sponge C. australiensis-associated bacteria particularity. This is the basis of host specificity of also been possible to in vitro culture, and they sponge-associated bacteria despite the similarity in included Alphaproteobacteria, Gammaproteo- predominant bacteria in these sponges which may bacteria, and Firmicutes. Meanwhile, some bac- result from the same seawater environment. teria that could not be detected by in vivo studies The diverse cultivable bacterial communities were also isolated by the mixed culture tech- have been isolated from the sponge niques, which included Alphaproteobacteria. C. australiensis which included Alphaproteo- Most of the sponge-associated bacterial commu- bacteria, Gammaproteobacteria, Firmicutes, and nities identified by molecular approaches are Actinobacteria. Alphaproteobacteria and still difficult for cultivation by in vitro culture, Gammaproteobacteria appear to form the major- especially the sponge-specific bacteria. So, ity of the C. australiensis-associated cultivable culturable sponge-associated bacterial communi- bacterial communities, and they contribute 78 % ties contributed only a fraction of total culturable of the total bacterial communities. The dominant microbial communities. Therefore, mixed cultiva- bacterial communities of C. australiensis tion techniques seemed to be a better option to recorded by in vivo approach using culture- recover diverse sponge-associated bacterial independent DGGE studies (Li et al. 2007) have communities. M 324 Marine Sponge Craniella australiensis-Associated Bacterial Diversity

Actinobacteria are filamentous gram-positive Chitinases are one of the important enzymes with prokaryotes and considered as an intermediate diverse applications. They play a significant role in group of bacteria and fungi. Actinobacteria from the decomposition of chitin and potentially in the sponges are one of the remarkable sources of utilization of chitin as a renewable source and also structurally novel bioactive metabolites. They very important in the fields of medicine, agricul- have also been widely recognized as one of the ture, biotechnology, waste management, and significant and sometimes sponge-specific bacte- industrial applications. Streptomyces sp. DA11 rial associates. Even though marine sponges have associated with sponge C. australiensis has been been recorded as a rich source of diverse proved as a potential source of chitinase (Han Actinobacteria, only a fraction have been isolated et al. 2009). The molecular weight of the Strepto- and exploited for their biotechnological poten- myces sp. DA11-derived chitinase was 34 kDa. tials. The majority of sponge-associated actino- Chitinase activity and cell growth were remark- mycetes are very difficult to isolate because of ably increased in the presence of optimal medium their low abundance and special nutrition require- conditions by 39.2-fold and 2.6-fold higher than ments. Based on the results of culture- that of the standard conditions (Han et al. 2008). independent techniques, it was found that the Chitinase also showed inhibition against Aspergil- sponge C. australiensis-associated actinobacteria lus niger (10.98 Æ 0.49 mm) and C. albicans were extremely low (Li and Liu 2006). A novel (10.48 Æ 0.45 mm), and it was also indicated that isolating strategy imitating the natural nutritional it has the potential to be an antifungal agent. state of actinobacteria within the sponge resulted Actinobacteria associated with the marine in the successful isolation of diverse sponge C. australiensis have also exhibited actinobacteria from the sponge C. australiensis. remarkable antimicrobial activity. Twenty out of The simulating natural conditions that were 23 actinobacterial strains have exhibited antimi- achieved by using only seawater and sponge crobial activity, and maximum inhibition was extracts yielded a better result in the successful recorded against P. fluorescens. Among them, fif- isolation. The phylogenetic analysis has also teen strains were active against multiple indicator showed the twenty-three actinobacterial strains strains with broad-spectrum antimicrobial activi- associated with C. australiensis, and they ties. Streptomyces sp. DA22 also exhibited signif- belonged to genera Actinomycetales and Strep- icant inhibitory activity against P. variotii, E. coli, tomyces (Li et al. 2006). P. fluorescens, C. albicans, B. subtilis and Staph- ylococcus aureus. A new indole alkaloid streptomycindole was also isolated from Strepto- Biotechnological Potential myces sp.DA22(Huangetal.2011). Overall, sponge C. australiensis-associated bacterial sym- Diketopiperazines (DKPs) are one of the smallest bionts with antimicrobial and chitinase activity cyclic peptides and represent an important class may contribute to chemical defense and the deg- of biologically active natural products. Four radation of chitin into nutrients such as diketopiperazines were isolated from sponge low-molecular-weight carbohydrates for sponge C. australiensis-associated Streptomyces or other sponge-associated organisms. And it sp. DA18 (Gao et al. 2010). These compounds could be a potential source for the production of have proved to play an important role in ecology chitinolytic enzymes and antimicrobials. as antifouling, antifungal, and antibacterial. Some of these isolated peptides have also shown moder- ate antimicrobial activity against Escherichia coli, Functional Genes Bacillus subtilis, Pseudomonas fluorescens,and Candida albicans. This also supported that Strep- Developments in the genetic screening of micro- tomyces sp. DA18 might provide antimicrobial bial biosynthetic genes lead to discovery of new defense for the host sponge C. australiensis. metabolites including novel polyketides, amino Marine Sponge Craniella australiensis-Associated Bacterial Diversity 325 M acid-derived compounds, and terpenes from in host defense as well as many ecological func- sponge-associated bacteria. A metagenomic tions. Unraveling the genetic diversity of bacteria library of C. australiensis has shown the presence associated with marine sponge is necessary to of 23 positive clones, and it has also recorded the assess their ecological and biotechnological presence of antimicrobial peptide genes potential. Sponge C. australiensis is one of the (Wu et al. 2006). Nonribosomal peptide synthe- hard sponge, and isolation of diverse bacteria tases (NRPSs) are large, multimodular enzymes from like these sponges is a challenging task. that are organized in modules containing specific The combination of genetic and traditional culti- domains that sequentially incorporate amino acid vation methods increases the culturability of building blocks into a growing peptide chain. C. australiensis-associated bacteria. This sponge NRPS gene clusters encode for a wide range of harbors diverse and host-specific bacterial nonribosomal peptides. An investigation communities, and the dominant group is performed on nonribosomal peptide synthetase Bacteroidetes and Actinobacteria is the minor (NRPS) adenylation (A) domain genes in component. The genomics-based approaches are 109 bacteria isolated from different marine significant in the screening of microbes for min- sponges, i.e., C. australiensis, S. tenuis, ing their whole biosynthetic potentials. The pres- H. rugosa, and D. avara, has shown the presence ence of PKS genes in C. australiensis-associated of NRPS genes in fifteen bacteria (Zhang bacteria and broad-spectrum antimicrobial spec- et al. 2009a). But, none of the sponge tra has revealed the chemical diversity of polyke- C. australiensis-associated bacteria have the tide metabolites. This characterized bacterial NRPS genes. Polyketide synthases (PKSs) are community will also serve as a key source for a family of multidomain enzymes or enzyme finding novel biosynthetic genes and natural complexes that produce polyketides, a large products. The different investigations of class of secondary metabolites. The screening C. australiensis evidently proved that only the M of 98 isolates from different sponges, i.e., integrated approaches of modern as well as tradi- C. australiensis, S. tenuis, H. rugosa, and tional techniques can play a vital role in the D. avara, has shown the presence of eighteen mining of taxonomical and functional diversity bacteria with KS (ketosynthase) genes (Zhang of sponge-associated bacteria. The mechanism et al. 2009b). Phylogenetic analysis has also behind the sponge bacterial host specificity is shown that 15 isolates belonged to the phylum still unknown. The information obtained from Firmicutes, among which fourteen isolates were different studies of the C. australiensis is provid- closely related to genus Bacillus and one to ing valuable insights into the comprehensive S. lentus. Two isolates were identified as taxonomy and functional diversity assessments Actinomycetes, and one as Alcaligenes sp. of sponge-associated bacteria. (Proteobacteria). Sponge C. australiensis- associated Streptomyces sp. DA23 and Actinomycetales bacterium DA20 have shown Cross-Reference the presence of PKS genes. The bacterial strains identified with PKS genes have also exhibited ▶ Marine Invertebrate Animal Metagenomics: broad-spectrum antimicrobial activities against Porifera fungi, gram-positive and gram-negative bacteria. ▶ Metagenomics of the Coral Holobiont ▶ Ocean Metagenomics

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Faulkner DJ, He HY, Unson MD, et al. New metabolites Wu J, Li Z, Zhang X. Construction of the metagenomic from marine sponges: are symbionts important? Gaz library of sponge Craniella australiensis and Chim Ital. 1993;123:301–7. antibacterial peptide gene preliminary screening. Fieseler L, Horn M, Wagner M, et al. Discovery of the Biotechnol Bull. 2006;3:95–103. novel candidate phylum “Poribacteria” in marine Zhang W, Li Z, Miao X, et al. The screening of antimi- sponges. Appl Environ Microbiol. 2004;70:3724–32. crobial bacteria with diverse novel nonribosomal pep- Gao Y, Yu L, Peng C, et al. Diketopiperazines from two tide synthetase (NRPS) genes from South China Sea strains of South China Sea sponge-associated micro- sponges. Mar Biotechnol. 2009a;11:346–55. organisms. Biochem Syst Ecol. 2010;38:931–4. Zhang W, Zhang F, Li Z, et al. Investigation of sponge- Han Y, Li Z, Miao X, et al. Statistical optimization of associated cultivable bacteria with polyketide synthase medium components to improve the chitinase activity genes and antimicrobial activity in the South China of Streptomyces sp. DA11 associated with the South Sea. J Appl Microbiol. 2009b;107:567–75. China Sea sponge Craniella australiensis. Process Biochem. 2008;43:1088–93. Han Y, Yang B, Zhang F, et al. Characterization of anti- fungal chitinase from marine Streptomyces sp. DA11 Marine Vertebrate Animal associated with South China Sea sponge Craniella australiensis. Mar Biotechnol. 2009;11:132–40. Metagenomics, Salmonidae Hentschel U, Fieseler L, Wehrl M, et al. Microbial diver- sity of marine sponges. Prog Mol Subcell Biol. Jaime Romero and Paola Navarrete 2003;37:59–88. Instituto de Nutricio´n y Tecnologı´a de los Hentschel U, Usher KM, Taylor MW. Marine sponges as microbial fermenters. FEMS Microbiol Ecol. Alimentos, Universidad de Chile, Santiago, Chile 2006;55:167–77. Huang X-L, Gao Y, Xue D-Q, et al. Streptomycindole, an indole alkaloid, from a marine Streptomyces Synonyms sp. DA22 associated with South China Sea sponge Craniella australiensis. Hel Chim Acta. 2011;94: 1838–42. Group of bacteria residing in the gut of salmo- Li Z. Advances in marine microbial symbionts in the nids; Salmonid gut microbiota China Sea and related pharmaceutical metabolites. Mar Drugs. 2009;7:113–29. Li Z, Liu Y. Marine sponge Craniella australiensis asso- ciated bacterial diversity revelation based on 16S Definition rDNA library and biologically active Actinomycetes screening, phylogenetic analysis. Lett Appl Microbiol. The salmon gut microbiota is the entire group of 2006;43:410–6. Li Z, He L, Wu J, Jiang Q. Bacterial community diversity bacteria that are present in the gastrointestinal associated with four marine sponges from the South tract of salmonids: rainbow trout China Sea based on 16S rDNA-DGGE fingerprinting. (Oncorhynchus mykiss), Atlantic salmon (Salmo J Exp Mar Biol Ecol. 2006;329:75–85. salar), and coho salmon (Oncorhynchus kisutch). Li Z, He L, Miao X. Cultivable bacterial community from South China Sea sponge as revealed by DGGE finger- printing and 16S rDNA phylogenetic analysis. Curr Microbiol. 2007;55:465–72. Introduction Schmitt S, Tsai P, Bell J, et al. Assessing the complex sponge microbiota: core, variable and species-specific bacterial communities in marine sponges. ISME The gastrointestinal microbiota of fish consists J. 2012;6:564–74. of a group of bacteria residing in the gut that Taylor MW, Radax R, Steger D, et al. Sponge-associated can reach 107–1011 bacteria/g of intestinal con- microorganisms: evolution, ecology, and biotechno- tent (Nayak 2010), with the highest values logical potential. Microbiol Mol Biol Rev. 2007;71: 295–347. observed in herbivorous tropical fish. The early Vogel S. Current-induced flow through living sponges in view (1970–1980s) about the existence of a stable nature. Proc Natl Acad Sci USA. 1977;74:2069–71. microbiota in the gastrointestinal tract of fish was Webster NS, Taylor MW, Behnam F, et al. Deep sequenc- controversial (Cahill 1990), principally due to the ing reveals exceptional diversity and modes of trans- mission for bacterial sponge symbionts. Environ constant contact of this system with the aquatic Microbiol. 2010;12:2070–82. environment. However, significant numbers of Marine Vertebrate Animal Metagenomics, Salmonidae 327 M studies have been performed during the past few production of rainbow trout was 0.58 million decades to characterize the microbiota in a wide tonnes. These salmonid species were considered range of fish species, and these topics have been to be dominant in diadromous fish production clarified. A stable microbiota can be established because Atlantic salmon represented 44 % and after the first feeding stages (Navarrete rainbow trout production was 17.4 %. Norway et al. 2010a; Hovda et al. 2012), and its major and Chile are the world’s leading aquaculture components can be derived from water and egg producers of salmonids, contributing 65 % of epibiota (Romero and Navarrete 2006). The fish world production. Other European countries gut appears to contain less microbial diversity added another 18.9 %, whereas Asia and North than the gut of homeothermic animals. Aerobes America contributed only 7.9 % and 7.4 %, and facultative anaerobes are predominant in the respectively. bacterial microbiota, whereas strict anaerobes are less frequent. The low concentration of short- chain fatty acids in the salmonid gut revealed Culture Analysis Versus Molecular that bacterial fermentation is not an important Techniques to Evaluate the Bacterial process (Holben et al. 2002). These observations Diversity of the Salmonid Gut can be explained by the carbohydrate-poor diet consumed, a short gastrointestinal tract The bacterial composition of the fish GI tract has (in comparison to herbivorous fish), and the low been studied previously using culture techniques ambient body temperature. (Cahill 1990; Hansen and Olafsen 1999); how- Currently, it is generally recognized that the ever, these methods are time consuming, and only gastrointestinal (GI) microbiota of animals serves part of the total bacterial community can be several functions, including nutrition, develop- recovered using traditional agar substrates. The ment, immunity, and xenobiotic metabolism. proportion of bacteria that can be cultivated M Recent studies performed in model vertebrates, (the ratio of the number of cultured bacteria to especially zebrafish, also provide insights into the the result of direct counts with microscopy) microbial-host molecular dialogs that impact sev- showed low cultivability rates ranging from 1 % eral functions of the host (Rawls et al. 2006). to 10 %, suggesting that the largest part of the These functions have been recently demonstrated microbiota failed to grow under the conditions in fish. An important study by Rawls et al. (2004) used for the isolation of salmon bacteria (Romero showed that the GI microbiota can regulate the and Navarrete 2006). However, the cultivability expression of 212 genes in the digestive tract of may increase to 50 % in rainbow trout (Navarrete zebrafish, some of them related to the stimulation et al. 2010a). of epithelial proliferation and the promotion of Alternative molecular methods based on PCR nutrient metabolism and innate immune amplification of DNA extracted from the samples response. An important aspect of these results have been shown effective for studying the GI was the specificity of the host response, which bacterial community of fish (Jensen et al. 2004; depends on the bacterial species that colonize the Romero and Navarrete 2006; Hovda et al. 2007; digestive tract (Rawls et al. 2004). Hence, it is Kim et al. 2007). The examination of bacterial relevant to know the composition of this communities has commonly been performed with microbiota in fish. Salmonids are important cul- a cloning approach (Holben et al. 2002), an anal- tured aquatic organisms. Atlantic salmon (Salmo ysis with temporal temperature gradient gel elec- salar) and rainbow trout (Oncorhynchus mykiss) trophoresis (TTGE), and denaturing gradient gel are the most common reared species, followed by electrophoresis (DGGE) of PCR-amplified DNA coho salmon (Oncorhynchus kisutch). According fragments. Such analyses produce a profile of all to the FAO World Aquaculture report for 2010, dominant bacterial species in environmental sam- the global production of Atlantic salmon reached ples or of a particular group, e.g., the genus level 1.5 million tonnes in 2008. During this year, the (Mangin et al. 2006). Because of its ubiquity and M 328 Marine Vertebrate Animal Metagenomics, Salmonidae the growing database, the gene encoding the 16S rRNA is usually used for the analysis. In addition, 2 1 3 these gene sequences contain highly conserved 10 Proteobacteria primer-binding sites and hypervariable regions Firmicutes that can provide species-specific signature 5 Actinobacteria Bacteroidetes sequences that are useful for bacterial identifica- Fusobacteria tion. However, the disadvantages of this method Tenericutes 6 include the heterogeneity of the different 16S 10 Deinococcus-Thermus rRNA genes and the poor discrimination between closely related bacteria. These disadvantages have led investigators to propose other genes, Marine Vertebrate Animal Metagenomics, such as the rpoB gene or cpn60, which have Salmonidae, Fig. 1 Bacterial phyla observed in gut microbiota of salmonids. The numbers correspond to the been used to analyze the bacterial diversity of number of reports describing a specific bacterial phylum rainbow trout (Navarrete et al. 2010a; Mansfield in the microbiota of salmonids, based on the review of et al. 2010). Recently, Roeselers et al. (2011) Nayak (2010) reported the first analysis of fish microbiota with a massive sequencing strategy in zebrafish. It is Pseudomonas can represent more than 60 % of expected that this technology significantly the community when ribosomal amplicons were improves our knowledge about the fish cloned and sequenced (Navarrete et al. 2009). microbiota and the factors that influence its The dominance of a particular bacterial group composition. has been observed in salmonid guts with similar culture-independent methods. Holben et al. (2002) reported that some genera were highly The Bacterial Composition of the Gut abundant in reared Atlantic salmon from two Microbiota of Salmonids Is Dominated different locations: in a Scottish hatchery, by a Few Genera Mycoplasma corresponded to 81 % of the clones retrieved, and in a Norwegian hatchery, Our current knowledge of the composition of the Acinetobacter represented 55 % of the clones microbiota of the fish gut is derived primarily retrieved. Although other genera were also present, from farmed fish. Among these fish, salmonids their abundance was closer to 2 %. Interestingly, in have received considerable attention. Figure 1, wild salmon (entirely carnivorous), Mycoplasma based on the recent review of Nayak (2010), represented 96 % of the clones analyzed. summarizes the most commonly reported bacte- Kim et al. (2007) later reported similar results. rial phyla in salmonids. Proteobacteria and In addition, they described the importance of Firmicutes are the most frequently reported Enterobacteriaceae in the trout microbiota. phyla in the salmonid gut microbiota, suggesting Recently, Mansfield et al. (2010)usedchaperonin that members of these bacterial classes are espe- (cpn60) instead of ribosomal RNA genes and cially well adapted to conditions in the fish intes- found that 80 % of the clones corresponded to tine. Recent reports have investigated the gut Carnobacterium, followed by Hafnia,which microbiota of salmonids with culture- represented approximately 10 % of the clones. independent methods. These studies, which Another study used a combination of 16S rRNA were conducted in Europe (Scotland, Denmark) gene and rpoB analysis to reveal that Lactococcus, and the Americas (Canada, Chile), reported that Citrobacter, Kluyvera, Obesumbacterium,and the composition of the gut microbiota can be Shewanella dominated the intestinal microbiota dominated by different bacterial groups. Huber (Navarrete et al. 2010a). Figure 2 shows a repre- et al. (2004) described Anaerofilum, sentation of the different bacterial taxa described Carnobacterium, and Clostridium as the most in the reports compiled in the review of important components of the gut microbiota. Nayak (2010). Marine Vertebrate Animal Metagenomics, Salmonidae 329 M

Marine Vertebrate Animal Metagenomics, compiled in the review of Nayak (2010). For example, Salmonidae, Fig. 2 Bacterial species observed in gut Acinetobacter and Pseudomonas were described in >5% microbiota of salmonids. The numbers represent the per- of the reports, whereas others corresponded to the bacte- centage of different bacterial taxa described in the reports rial taxa described in <1 % of the reports

The carnivorous diet of salmon may explain in is expected to influence interindividual variation in part the low number of taxa observed. Indeed, the intestinal microbiota. Studies in humans and a recent study indicated that diet influences the animals support the hypothesis that host-related bacterial diversity of the digestive tract. In that factors are involved in the determination of the report, a more comprehensive analysis of verte- gut microbiota. The possible involvement of host brate gut microbiota (although based primarily on genotype, particularly as it relates to the M mammalian data) indicates that bacterial diver- immunophenotype, has been frequently postulated sity increases from carnivory to omnivory to her- as a major influence on the composition and sta- bivory (Ley et al. 2008). This trend has recently bility of the microbiota, although this postulate has been observed in Antarctic fish, among which the been difficult to prove. The contribution of the host omnivorous Notothenia coriiceps exhibits greater to the composition of the microbiota was recently diversity than does the exclusively carnivorous determined by analyzing full siblings from four Chaenocephalus aceratus (Ward et al. 2009). different unrelated rainbow trout families, each This finding may indicate that increasing herbiv- derived from a single pair of breeders that had ory in fish leads to the diversification of the gut been previously identified and classified in microbiota, as found in mammals. a breeding program (Navarrete et al. 2012). The host influence on the gut microbiota was achieved in different non-related families of rainbow trout Host Influence that are fed controlled diets. The results showed that the main variations in microbiota composition Although these studies shed light on the composi- could be attributed to the hosts rather than to the tion of the gut microbiota, the available informa- diets. The compositions of the microbiota were tion does not fully clarify the factors involved in very different among families, although certain determining this composition. Exogenous and families may share some bacterial components. endogenous factors can affect the initial coloniza- In the families examined, a total of five phyla tion and nature of the microbial composition. were identified in the intestinal microbiota of the These factors include the developmental stage of trout: Proteobacteria, Firmicutes, Actinobacteria, the fish, the gut structure, the surrounding environ- Bacteroidetes,andFusobacteria. Certain phyla, ment (e.g., water temperature, salinity), and the such as Proteobacteria, Firmicutes,and rearing and farming conditions. The host genotype Actinobacteria, were detected in all families. M 330 Marine Vertebrate Animal Metagenomics, Salmonidae

Notably, these were the only phyla detected in molecular profiles, indicative of the stability of the certain families. In addition to the three phyla composition of the microbiota in both TVEO- mentioned, the other families showed one addi- treated and untreated fish. The stability of the tional phylum, Bacteroidetes or Fusobacteria. TTGE pattern over time was revealed by the sim- Furthermore, the results also showed that certain ilarity index (Dice, Cs), which exhibited average bacterial groups were significantly associated with values >65 % for both TVEO-treated and specific families, indicating that the host may untreated trout. Similarly, Hovda et al. (2012) influence microbiota composition. The principal used a molecular approach to examine the gut result obtained was that the response of the microbiota of farmed Atlantic salmon during an microbiota to diet depended on the host: the four annual cycle. These authors found that lactic acid trout families responded differently to diet. The bacteria (LAB) were the most highly dominant composition of the microbiota of certain families bacterial group and genera. Lactococcus, clustered together independently of diet, Weissella,andLactobacillus were observed in all suggesting that these families were less affected molecular profiles derived from the samples col- by diet than the remaining families. lected during the year-long study. These data sug- gest that the microbiota appear to be stable during the stages analyzed if the rearing conditions Effect of Diet on the Gut Microbiota of remain unchanged. Salmonids

A few previous studies address the issue of the Microbial Diversity in Wild Versus stability of the microbiota. Some of these studies Reared Salmon focus on the changes occurring over a short time scale (weeks, months), whereas others compare All studies performed to date in salmonids the composition of the microbiota over different included pond- or tank-raised fish. Only one seasons (years). A recent study evaluated the sta- study, using a cloning approach, analyzed the bility of the rainbow trout microbiota over a period bacterial diversity found in Scottish wild salmo- of weeks in the context of a diet assessment nids. This diversity was contrasted to the diver- (Navarrete et al. 2010b). The diet assessment sity found in Scottish- and Norwegian-raised sought to determine the effect of the dietary inclu- salmon (Holben et al. 2002). The first observation sion of Thymus vulgaris essential oil (TVEO) on was that the total bacterial density in the distal the microbiota composition. The fish fed the intestine of wild salmon was 2 log10 times lower experimental diet were compared with fish on than that of reared salmon. Genera belonging to a control diet without TVEO. The rainbow trout Mycoplasma were highly dominant in wild and used in the study were all reared under the same reared Scottish salmon (96 % and 81 % of the conditions, and the microbiota was followed over analyzed clones, respectively). In contrast, Nor- 5 weeks. The comparison of the composition of the wegian salmon showed a dominance of microbiota was performed with molecular profiles Acinetobacter. This genus represented 55 % of derived from samples collected at the same time. the clones. As discussed above, bacterial density This analysis showed high similarities (>71 %) and diversity were lower in wild salmon. It is between the TVEO-treated and untreated trout. probable that this difference resulted from the Thus, for these concentrations, TVEO induced no entirely carnivorous diet of these fish. changes in the gut microbiota profiles. If the molecular profiles within the same groups (treated or untreated) were compared throughout Summary the collection period, common bacterial compo- nents were primarily observed. These microbes, The literature shows that certain phyla or bacte- persistent throughout the trial, produced constant rial groups occur in the microbiota of certain Metabolic and Species Diversity Analysis for Metagenomics 331 M salmonids. Some evidence suggests that the com- Mangin I, Suau A, et al. Characterization of human intes- position of the microbiota may be influenced by tinal bifidobacteria using competitive PCR and PCR-TTGE. FEMS Microbiol Ecol. 2006;55:28–37. the genetics of the host to a certain extent, Mansfield GS, Desai AR, et al. Characterization of rain- irrespective of the diet. Certain components, par- bow trout (Oncorhynchus mykiss) intestinal ticularly Pseudomonas, Aeromonas, and other microbiota and inflammatory marker gene expression Proteobacteria, were commonly described in sal- in a recirculating aquaculture system. Aquaculture. 2010;307:95–104. monids. Lactic acid bacteria (LAB) have been Navarrete P, Espejo RT, et al. Molecular analysis of reported in many studies. This group has microbiota along the digestive tract of juvenile Atlan- received particular attention because it could tic salmon (Salmo salar L.). Microbiol Ecol. contribute common and stable bacterial compo- 2009;57:550–61. Navarrete P, Magne F, et al. Molecular analysis of intes- nents to the overall composition of the tinal microbiota of rainbow trout (Oncorhynchus microbiota. It is expected that massive sequenc- mykiss). FEMS Microbiol Ecol. 2010a;71:148–56. ing methods could furnish more comprehensive Navarrete P, Toledo MI, et al. Effect of Thymus vulgaris information about the stability of the composi- essential oil on intestinal bacterial microbiota of rain- bow trout Oncorhynchus mykiss (Walbaum) and bac- tion of the microbiota and the factors that influ- terial isolates. Aquacult Res. 2010b;41:e667–8. ence this composition. Further genomic and Navarrete P, Magne F, et al. PCR-TTGE analysis of 16S proteomic studies will help to elucidate rRNA form rainbow trout (Oncorhynchus mykiss) gut the importance of microbe-host interactions at microbiota reveals host-specific communities of active bacteria. PLoS ONE. 2012;7(2):e31335. doi:10.1371/ the mucosal interface. These studies will ulti- journal.pone.0031335. mately help to unravel the complexity of these Nayak SK. Role of gastrointestinal microbiota in fish. microbial ecosystems. Aquac Res. 2010;41:1553–73. Rawls JF, Samuel BS, et al. Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota. 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Kim DH, Brunt J, et al. Microbial diversity of the intesti- nal contents and mucus in rainbow trout Definition (Oncorhynchus mykiss). J Appl Microbiol. 2007;102: 1654–64. Ley R, Hamady M, et al. Evolution of mammals and their Metagenome is the entire collection of genetic gut microbes. Sci Express. 2008;320:1647–51. material of a microbial community. M 332 Metabolic and Species Diversity Analysis for Metagenomics

Species diversity of a community is defined as made feasible the metagenome shotgun sequenc- the number of species in the community, and in ing of environmental samples; however, most metagenomics, it is often estimated using the environmental communities are far too complex number of operational taxonomic units (OTUs). to be fully sequenced in this manner. Both Functional diversity of a microbial commu- approaches have their limitations – 16S rRNA nity refers to the variety of functions and biolog- sequencing may be biased because of unequal ical processes encoded by its metagenome. amplification of species’ 16S rRNA genes, whereas shotgun metagenomic sequencing may not be deep enough to detect the 16S rRNA genes Introduction of rare species in a complex community. Although some studies showed that these two Microbial communities are the major resources approaches give largely similar species profiles for genetic and metabolic diversity. for a few bacterial communities (Kalyuzhnaya Metagenomics, the direct analysis of DNA from et al. 2008), a study that systematically compared environmental samples, has been applied to stud- microbial community structures inferred from ies of microbial communities in various environ- 16S rRNA gene sequencing and shotgun ments, including soil, ocean water, and human metagenomics showed that these two approaches bodies, and has shown the impact of microbial may give significantly different community organisms on almost every aspect of life on Earth. structures even for the same microbial commu- Some microbial communities have rather simple nity (Shah et al. 2011). structures; for example, a biofilm from the acid Species diversity of a microbial community is mine drainage (AMD) was shown to contain often approximated as the number of operational merely several species (Tyson et al. 2004). Others taxonomic units (OTUs) that can be inferred from are more complex, including marine communi- sequencing data of 16S rRNA genes. OTU-based ties (on the order of 100–200 species per milliliter computational approaches cluster 16S rRNA of water (Curtis and Sloan 2004)), soil commu- gene sequences into OTUs at 97 % similarity nities (with an estimated species richness of about (3 % difference), which is generally recognized 4,000 species per gram of soil (Li et al. 2009)), as providing differentiation of bacterial organ- and human-associated microbial communities isms at the species level. Recent developments (which show great diversity within and across of OTU-based computational approaches focus individuals, as revealed recently through the on accurate and fast removal of chimeric human microbiome project (Peterson sequences that otherwise will cause inflated esti- et al. 2009)). From the functional aspect, micro- mation of species diversity (Caporaso et al. 2010) bial communities constitute a major asset in the and speedup of the clustering of sequences into search for new enzymes for various industrial OTUs (Ye 2011). processes, including the production of biofuels from plant biomass. Metabolic Diversity

Species Diversity Functional diversity of a microbial community refers to the variety of functions and biological 16S rRNA gene sequencing has been widely used processes encoded by the entire collection of for probing the species structure of a variety of genetic material of the microbial community environmental bacterial communities. 16S rRNA (i.e., the metagenome). Some scientists argue gene fragments can also be retrieved from shot- that examining functional diversity may be the gun metagenomic sequences and used for species most meaningful and practical way of assessing profiling. Advances in sequencing technologies biodiversity. Knowing the species diversity of Metabolic and Species Diversity Analysis for Metagenomics 333 M a microbial community is helpful for understand- structures and functional gene repertoires. ing its functional diversity, but functional analy- A study of gut microbial communities in three sis provides a more direct survey of the populations (Amazonas of Venezuela, rural functionality of microbial communities. It was Malawi, and US metropolitan areas) revealed shown that the human gut enterotypes are mostly functional maturation of the gut microbiome dur- driven by species composition, but abundant ing the first 3 years of life in all three populations molecular functions (e.g., two proteins associated (including age-associated changes in the genes with bacterial pilus assembly, FimA and PapC) involved in vitamin biosynthesis and metabo- are not necessarily provided by abundant species lism) and pronounced differences in bacterial (Arumugam et al. 2011). assemblages and functional gene repertoires Biological pathway reconstruction is essential between US residents and those in the other two for understanding the biological processes countries (Yatsunenko et al. 2012). Seasonal encoded by a metagenome. The common practice changes of bacterial communities – and changes of pathway reconstruction in metagenomics first in key genes among seasons and between day and identifies functions encoded by the metagenomic night (i.e., photosynthesis) – were also observed sequences and then reconstructs pathways from at a temperate costal site (Gilbert et al. 2010). the annotated functions, by mapping the func- Grzymski and colleagues (2012) reported intense tions to reference pathways, such as the KEGG inter-seasonal differences reflected through shifts pathways (http://www.genome.jp/kegg/pathway. in community composition and functional capac- html) and the SEED subsystems (http://www. ities encoded in winter and summer bacterio- nmpdr.org/FIG/subsys.cgi). Functional catego- plankton from the Antarctic Peninsula coastal ries used in metagenomic annotation include the surface waters, with significantly higher phyloge- KO families for the KEGG database and the FIG netic and functional diversity in winter. families for the SEED database. MG-RAST M (http://metagenomics.anl.gov/), one of the major metagenomic analysis servers, utilizes similarity Decreased Microbial Diversity and searches by BLAT for functional prediction. Com- Human Diseases monly, and straightforwardly, a complete biologi- cal pathway can be identified in a dataset if at least Studies have shown that reduced bacterial diver- one of the steps associated with the pathway is sities are associated with some human diseases. found. Ye and Doak (2009) discovered that this Investigation of the intestinal microbial diversity naı¨ve mapping approach may lead to an inflated allowed Manichanh and colleagues (2006)to estimate of biological pathways and thus overesti- detect a reduced complexity of the bacterial phy- mates the functional diversity of an environmental lum Firmicutes as a signature of the fecal sample from which the metagenomic sequences microbiota in patients with Crohn’s disease are derived. They also proposed a parsimony (CD). In another study, Qin et al. reported that approach, called MinPath, for biological pathway inflammatory bowel disease (IBD) patients reconstructions using protein family predictions, carry – in their gut microbial organisms – on which yields a more conservative, yet more faith- average 25 % fewer genes than the individuals ful, estimation of the biological pathways for not suffering from IBD (Qin et al. 2010). a query metagenomic dataset.

Unlocking the Promise of Metabolic Dynamics of Microbial Communities Diversity of Microbial Communities

Studies have shown that microbial communities Metagenomics represents a strategy for discover- are extremely dynamic with changing community ing – in nature – diverse enzymes for various M 334 Metabolic and Species Diversity Analysis for Metagenomics industrial processes, including the production of Caporaso JG, Kuczynski J, Stombaugh J, et al. QIIME biofuels from plant feedstocks. Plant biomass is allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7(5):335–6. the most abundant biopolymer on earth and has Curtis TP, Sloan WT. Prokaryotic diversity and its limits: long been recognized as a potential sustainable microbial community structure in nature and implica- source of mixed sugars for biofuel production tions for microbial ecology. Curr Opin Microbiol. (Li et al. 2009). The lack of enzymes that effi- 2004;7(3):221–6. Gilbert JA, Field D, Swift P, et al. The taxonomic and ciently deconstruct plant polysaccharides, how- functional diversity of microbes at a temperate coastal ever, represents a major bottleneck for industrial- site: a ‘multi-omic’ study of seasonal and diel temporal scale production of biofuels. Scientists have variation. PLoS ONE. 2010;5(11):e15545. started to look for biofuel-producing microbes Grzymski JJ, Riesenfeld CS, Williams TJ, et al. A metagenomic assessment of winter and summer in termite guts and cow rumens and mine for bacterioplankton from Antarctica Peninsula coastal genes encoding for glycosyl hydrolases for bio- surface waters. ISME J. 2012;6:1901. mass conversion in the terminate gut microbiome Hess M, Sczyrba A, Egan R, et al. Metagenomic discovery and cow rumen microbiome. Termites are noto- of biomass-degrading genes and genomes from cow rumen. Science. 2011;331(6016):463–7. rious for their voracious appetite for wood, yet Kalyuzhnaya MG, Lapidus A, Ivanova N, et al. High- they may provide a solution to a greener biofuel resolution metagenomics targets specific functional future. Warnecke and colleagues used types in complex microbial communities. Nat a metagenomic analysis of the bacterial commu- Biotechnol. 2008;26(9):1029–34. Li LL, McCorkle SR, Monchy S, et al. Bioprospecting nity resident in the hindgut paunch of a wood- metagenomes: glycosyl hydrolases for converting bio- feeding termite to show the presence of a large, mass. Biotechnol Biofuels. 2009;2:10. diverse set of bacterial genes for cellulose and Manichanh C, Rigottier-Gois L, Bonnaud E, xylan hydrolysis (Warnecke et al. 2007). Cow et al. Reduced diversity of faecal microbiota in Crohn’s disease revealed by a metagenomic approach. rumen microbes specialize in degradation of cel- Gut. 2006;55(2):205–11. lulosic plant material, but most members of this Peterson J, Garges S, Giovanni M, et al. The NIH human complex community resist cultivation. Hess microbiome project. Genome Res. 2009;19(12): et al. (2011) sequenced and analyzed 2317–23. Qin J, Li R, Raes J, et al. A human gut microbial gene 268 gigabases of metagenomic DNA from catalogue established by metagenomic sequencing. microbes adherent to plant fiber incubated in Nature. 2010;464(7285):59–65. cow rumen, and from these data, they identified Shah N, Tang H, Doak TG, et al. Comparing bacterial more than 27,000 putative carbohydrate-active communities inferred from 16S rRNA gene sequenc- ing and shotgun metagenomics. Pac Symp Biocomput. genes. 2011; 165–76. Tyson GW, Chapman J, Hugenholtz P, et al. Community structure and metabolism through reconstruction of Summary microbial genomes from the environment. Nature. 2004;428(6978):37–43. Warnecke F, Luginbuhl P, Ivanova N, et al. Metagenomic Metagenomic studies are revealing the tremen- and functional analysis of hindgut microbiota of a dous species diversity and metabolic diversity of wood-feeding higher termite. Nature. 2007; microbial communities. Applications of the 450(7169):560–5. Yatsunenko T, Rey FE, Manary MJ, et al. Human gut genetic and metabolic diversity of bacterial com- microbiome viewed across age and geography. Nature. munities have also surfaced. 2012;486(7402):222–7. Ye Y. Identification and quantification of abundant spe- cies from pyrosequences of 16S rRNA by consensus alignment. Proc IEEE Int Conf Bioinforma Biomed. References 2011;2010:153–7. Ye Y, Doak TG. A parsimony approach to biological Arumugam M, Raes J, Pelletier E, et al. Enterotypes pathway reconstruction/inference for genomes and of the human gut microbiome. Nature. 2011; metagenomes. PLoS Comput Biol. 2009;5(8): 473(7346):174–80. e1000465. Metagenomic Analyses in the Digestive Tract of the Leporidae 335 M

three regions also showing both cellulase and Metagenomic Analyses in the xylanase activity. Thus, the metagenomic profile Digestive Tract of the Leporidae of the microbial contents of their digestive tract is generally associated with organisms which have Neil R. McEwan evolved for an environment reflecting the host’s Institute of Biological, Environmental and Rural herbivorous lifestyle. Sciences, Penglais Campus, Aberystwyth Although fungi have been reported as being University, Aberystwyth, Wales, UK able to grow on the dung of both rabbits and hares (Webster et al. 1999) and also appear in fecal matter (e.g., Richardson 2005), fungi are not considered part of the microbial community The Family Leporidae and Comparisons of the rabbit’s digestive tract. Likewise, there are with Digestive Anatomy of Other no reports of ciliated protozoa being present in Herbivores the digestive tract. As such, although the func- tion of the microbial community of the rabbit’s The family Leporidae comprises around 60 spe- digestive tract is similar to that of the one in the cies of animals from 11 genera. Around half of rumen or equine cecum (i.e., to break down plant the species within this family belong to the genus material), a range of organisms involved are Lepus (the hares), while members of the different. Thus, with the exception of a few tran- remaining 10 genera are normally referred to as sient fungi and any parasites which might have rabbits. They are native to all parts of the world, infected the tract (e.g., Allan et al. 1999; with the exception of Antarctica and Oceania. Audebert et al. 2002), the metagenome of the However, they have been introduced into other digestive tract of these animals is regarded as parts of the world with considerable success (e.g., being restricted to the genetic material from M introduction to Australia), although in this exam- bacteria and archaea. ple this has caused problems for many of the At first it was generally thought that since endogenous mammalian population due to the microbes in the digestive tract of herbivores all rabbit’s ability to adapt and to exploit this new play a similar role (i.e., digestion of the plant environment. material ingested by the host animal), there Members of the Leporidae family are almost might be a similar group of organisms present exclusively herbivorous, lacking canine teeth, and so the metagenome of the digestive tract of although there have been reports of species one herbivorous species might act as a guide to exhibiting carnivorous behavior, e.g., Arctic the metagenome of other herbivores. However, hare (Lepus arcticus) having been reported to just as there are significant differences between eat frozen fish or taking meat used for bait from the metagenomes of foregut fermenters (i.e., a trap or feeding on the contents of eviscerated ruminants) and large hindgut fermenters (e.g., animals (Best and Henry 1994). However, as with horses), so too there are digestive differences other mammals, members of the Leporidae fam- employed by animals such as rabbits – most nota- ily do not have the enzymes necessary for diges- bly the evolution of cecotrophic practices and the tion of plant material and so any fiber-digesting presence of the sacculus rotundus and vermiform capacity is restricted to enzymes encoded by appendix, features anatomically absent in other genes in microbes within their digestive tract. species. However, even within the family Activity for digestion of plant material has been Leporidae, there are variations in the approach detected in four regions (stomach, small intestine, to cecotrophy, particularly on low-quality forage cecum, and colon) of the tract which were inves- (Kuijper et al. 2004), and by inference their tigated (Marounek and Vovk 1995), with all four microbial communities, meaning that there must regions showing pectinase activity and the last be differences between hares and rabbits in terms M 336 Metagenomic Analyses in the Digestive Tract of the Leporidae of their digestive metagenomes. Although the fibrolytic activity. Moreover, the bacteria of the microbial community produces many of the digestive tract are likely to play a role in the metabolites seen in the tracts of other species, digestive efficiency and gut health of the rabbit e.g., volatile fatty acids (VFAs) and ammonia, (Gouet and Fonty 1979), and particularly those in the relative abundance of some of the metabolites the cecum will have a role to play in reducing loss being produced differs. This is best illustrated by of farmed animals (Michelland et al. 2010). the fact that the acetate being produced in the As mentioned above, the microbial commu- cecum of the rabbit is about 30 % greater than nity of the rabbit gut is considered to lack eukary- the acetate in the rumen of the goat, but otic organisms, other than those which are there the butyrate produced in the rabbit cecum is transiently. This is one example of the difference only around a quarter of that in the goat’s seen in the microbial community of the rabbit gut cecum (Abecia et al. 2013). relative to that of the ruminant. Another is in the Primarily due to the rabbit being kept as a pet, or abundance of the methanogen community. a production animal in some countries, there is Although there are methanogens present in the more information regarding the microbial commu- cecal community of the rabbit, the relative abun- nity of the rabbit’s digestive tract than there is about dance of this community is a fraction of that in the the hare. However, there is no comprehensive rumen, having been estimated at having a relative report of the digestive metagenome of any animal abundance which is around 0.01 % of that seen in from the family Leporidae. Instead there are a few ruminants (e.g., Abecia et al. 2013). examples of functional genes isolated from Nevertheless, the comparison of bacteria from selected microbes from rabbit feces or the rabbit’s the digestive tracts of other herbivores identified cecum, 16S rRNA survey data or knowledge gained that some bacteria from the rumen were also from gene/genome sequencing of species described found in the cecum of the rabbit, including in the digestive tract of other herbivores. Likewise, Eubacterium cellulosolvens and species from although not explicitly described and sequenced, the genus Bacteroides (Boulharouf et al. 1991). knowledge of genes present has been derived from Moreover, there was enough evidence to mean either digestive or metabolic analyses. These areas that other species were also worthy of further of knowledge will provide the source of the current investigation: Fibrobacter intestinalis, review of the metagenome of the digestive tract of Fibrobacter succinogenes, Ruminococcus albus, these animals. and Ruminococcus flavefaciens (Bennegadi et al. 2003).

Microbes in the Digestive Tract of the Rabbit Sequences from Environmental Samples from the Digestive Tract of the Rabbit As mentioned above, the microbial community in the digestive tract of the rabbit is involved in To date there has been relatively little material breaking down plant material. A number of published on the metagenome of the rabbit’s reports exist which demonstrate that by doing digestive system. Two papers have been this, the bacteria produce VFAs and ammonia, published which examined the diversity of the substances which can be made use of by the host small ribosomal subunit (16S rRNA) sequences animal by absorption across the gut wall (e.g., detected following PCR (Abecia et al. 2005; Parker and McMillan 1976). Up to around 30 % Monteils et al. 2008). All other metagenomic of the energy obtained by the rabbit can come information is distributed as small numbers of from VFAs. Thus, the organisms of the digestive sequences in a range of papers, or as studies tract, and by inference their genetic composition, investigating changes in the composition of the are expected to be dominated by species which 16S rRNA genes by methods which do not deter- have evolved for a lifestyle which involves mine specific DNA sequences. Metagenomic Analyses in the Digestive Tract of the Leporidae 337 M

Early experimental work, based on culture- the sequences described in the previous work based techniques, suggested that the major of Abecia et al. (2005). organisms in the digestive tract of a rabbit were To date there has been no next-generation Eubacterium cellulosolvens and species from sequencing (NGS) work investigating the diges- the genus Bacteroides (Boulharouf et al. 1991). tive microbial community of any lagomorph However, the first attempts at molecular character- published in the peer-reviewed literature, ization, using oligonucleotide probes, identified although there has been at least one instance of four organisms previously describedintherumen NGS work being reported in poster format at an as being resident in the cecum of the rabbit: international meeting (Massip et al. 2012). Fibrobacter intestinalis, Fibrobacter succinogenes, Ruminococcus albus,andRuminococcus flavefaciens (Bennegadi et al. 2003). Sequences from Microbes Isolated from The first work which investigated the 16S the Digestive Tract of the Rabbit rRNA diversity of the rabbit cecum (Abecia et al. 2005) used 46 sequences to compare the One of the main ways to investigate microbial community of the rabbit’s cecum with a metagenome is to sequence DNA from individ- those of the principal sites of fermentation in the ual isolates within the niche as a means of pro- digestive tract of other species (e.g., hindgut of viding some form of scaffolding onto which horses, rumen of ruminants, etc.). This work con- subsequent environmental samples from the cluded that there was a cluster, comprising about niche may be assembled. In the case of the rabbit, half of the sequences which were genetically in comparison to the digestive tract of other her- distinct relative to those isolated from any other bivores such as ruminants, there have been rela- organisms previously described. Moreover, only tively few microbes isolated from the tract or one of the sequences was from an organism from fresh feces and then grown in vitro. M (using a 97 % identity threshold) which had pre- Although there are examples of such isolated viously been reported, although this organism bacteria having their metabolic characteristics had only been described in the digestive tract of investigated (e.g., Sirotek et al. 2001), very few the pig, and also in the rumen, but had never been of these have been studied at the molecular level, grown in culture. although sporadic examples of molecular studies These observations were corroborated on do exist. One such example was the recent isola- a larger scale by the second paper published on tion of Streptococcus thoraltensis from fecal this topic (Monteils et al. 2008) where 70 opera- samples from rabbits (Borø et al. 2010), an organ- tional taxonomic units (OTUs) were identified ism which had not previously been described in from 228 sequences. Of these OTUs were unable the rabbit. to be recognized as being represented within the NCBI database (cutoff threshold <97 % identity). Of those OTUs with 97 % or greater Factors Which Can Affect the Microbial identity to a sequence in the database, only one Community Structure representing a single sequence from the 228 was identified, the remainder being most similar to The use of denaturing gradient gel electrophore- environmental samples from fecal, cecal, or sis (DGGE) as a guide to population diversity ruminal sources. This sequence was shown to and species richness suggests that the populations have 99 % identity to a sequence from in the rabbit cecum have similar values to those Variovorax sp., with the particular sequence in seen in the rumen of the goat (e.g., Abecia the database having been identified in a soil sam- et al. 2013). ple. Interestingly one of the OTUs with an iden- Although DGGE and other hybridization-/ tity level of 98 %, and representing 13 of denaturation-based analytical methods, such as the 228 clones, was most similar to one of single-strand conformation polymorphism (SSCP), M 338 Metagenomic Analyses in the Digestive Tract of the Leporidae do not involve metagenomic analyses in the tract of rabbits can be extended to fecal samples strictest sense, these methods do act as from hares (e.g., Richardson 2005; Webster an indicator of changes in the population struc- et al. 1999), although these are again likely to ture of an environment in response to an envi- be transient organisms relative to the tract. ronmental stimulus. Thus, indirectly, they can Other work again dealt with the identification be used as an early indicator of the sort of organisms in the tract, rather than performing of factors which can be used to influence the genetical analysis on these organisms including microbial community, and by inference the parasitic infections (Dubinsky et al. 2010; metagenomic structure. DGGE has been used to Tizzani et al. 2011; Usai et al. 2012), E. coli investigate a number of different dietary factors, occurrence (Martinez et al. 2011), Mycobacte- including caprylic acid supplementation rium avium incidence (Salgado et al. 2011), (Skrivanova et al. 2010), soluble and insoluble Francisella tularensis occurrence (Sting fiber levels (Rodriguez-Romero et al. 2012, et al. 2013), and general digestive function 2013), antibiotics (Abecia et al. 2007a), medica- (Stott 2008). tion (Abecia et al. 2007b), and numbers of pups nursed by mothers (Abecia et al. 2007c). SSCP has been used to investigate postnatal develop- Conclusions ment of the cecal microbial community (Combes et al. 2011) and adaptation of the cecal commu- The metagenome of the microbial communities nity (Michelland et al. 2011). In addition to within Leporidae remains a poorly categorized the use of hybridization-/denaturation-based community. Although the digestive process of at analytical methods, other PCR-based techniques least some of the mammals within this group (i.e., have been used to investigate the effects of other farmed rabbits) is agriculturally important, little parameters on the digestive tract population, has been described in terms of microbial species e.g., enterobacterial repetitive intergenic con- present in the environment, and even less has sensus (ERIC)-PCR being used to look at the been performed in terms of the study of their effect of Entamoeba histolytica infection genetic material. Thus, the metagenome remains (He et al. 2012), or studies on Lawsonia poorly characterized, but in so doing presents intracellularis, Salmonella species, and Eimeria a tremendous opportunity for further investiga- species (Lim et al. 2012). tion in the digestive tract of another group of While none of these actually investigate the herbivore species. metagenome per se, these techniques have suc- cessfully allowed identification of factors which are able to influence the range of 16S rRNA genes References identified following PCR and by inference the factors which influence the microbial population Abecia L, Fondevila M, Balcells J, Edwards JE, Newbold and their associated metagenome. CJ, McEwan NR. Molecular profiling of bacterial spe- cies in the rabbit caecum. FEMS Microbiol Lett. 2005;244:111–5. Abecia L, Fondevila M, Balcells J, Edwards JE, Newbold Microbes in the Digestive Tract of Other CJ, McEwan NR. Effect of antibiotics on the bacterial Leporidae population of the rabbit caecum. FEMS Microbiol Lett. 2007a;272:144–53. Abecia L, Fondevila M, Balcells J, Lobley GE, McEwan As mentioned previously, there is very little NR. The effect of medicated diets and level of feeding known about the microbial community of the on caecal microbiota of lactating rabbit does. J Appl digestive tract of any other members of Microbiol. 2007b;103:787–93. the Leporidae and by inference about the Abecia L, Fondevila M, Balcells J, McEwan NR. The effect of lactating rabbit does on the development genetic composition of the associated region. of the caecal microbial community in the pups they The example cited above regarding fungi in the nurture. J Appl Microbiol. 2007c;103:557–64. Metagenomic Analyses in the Digestive Tract of the Leporidae 339 M

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Br F. tularensis subsp. holarctica in wild animals. Berl J Nutr. 1995;35:365–71. Munch Tierarztl Wochenschr. 2013;126:285–90. Martinez R, Garcia A, Blanco JE, Blanco J, Rey J, Alonso Stott P. Comparisons of digestive function between JM, Gomez L, Snchez S. Occurrence of verocytotoxin- the European hare (Lepus europaeus) and the producing Escherichia coli in the faeces of free- European rabbit (Oryctolagus cuniculus): mastication, ranging wild lagomorphs in southwest Spain. Eur gut passage and digestibility. Mamm Biol. 2008;73: J Wildl Res. 2011;57:187–9. 276–86. M 340 Metagenomic Study, Human Skin Microbiome Associated with Acne, Project

Tizzani P, Menzano A, Catalano S, Rossi L, Meneguz Description PG. First report of Obeliscoides cuniculi in European brown hare (Lepus europaeus). Parasitol Res. 2011;109:963–6. In this study, researchers investigated the skin Usai F, Rinnovati R, Trocchi V, Stancampiano L. Lepus microbiome associated with acne in three direc- corsicanus gastro-intestinal helminths: first report. tions. First, the composition and diversity of the Helminthologia. 2012;49:71–7. skin microbiome were characterized in a cohort Webster J, Whalley AJS, Thienhirun S, Richardson MJ. Wawelia argentea and W. microspore, two new of 49 acne patients and 52 individuals with species of xerophilous fungi on rabbit and hare dung in healthy skin. This demonstrated that P. acnes Britain. Mycol Res. 1999;103:1604–8. was the most dominant species in pilosebaceous units accounting for ~90 % of the microbiota (Fitz-Gibbon et al. 2013). Second, the strain Metagenomic Study, Human Skin diversity of P. acnes was characterized in these Microbiome Associated with Acne, subjects, and it was determined that certain Project strains of P. acnes were highly associated with the disease, while certain strains were enriched in healthy skin (Fitz-Gibbon et al. 2013). Third, Huiying Li more than 1,000 P. acnes strains from the skin Department of Molecular and Medical samples were isolated, and the genomes of Pharmacology, Crump Institute for Molecular 67 P. acnes isolates and three Propionibacterium Imaging, David Geffen School of Medicine, humerusii isolates were sequenced (Fitz-Gibbon UCLA, Los Angeles, CA, USA et al. 2013; Tomida et al. 2013). The large num- ber of Propionibacterium genomes enabled the Introduction researchers to identify the genetic differences in virulence properties of different strains, which This study is to characterize the skin microbiome may explain seemingly contradictory roles of in pilosebaceous units (hair follicles) to under- P. acnes in both health and disease (Fitz-Gibbon stand the role of the skin microbiota in health and et al. 2013; Tomida et al. 2013; Kasimatis et al. disease and its association with a common skin 2013). In addition, researchers are currently ana- disease, acne vulgaris. lyzing the genetic diversity and landscape of Acne vulgaris, commonly called acne, affects P. acnes bacteriophages isolated from these sam- 50 million Americans and many more individuals ples and the metatranscriptome of the skin globally. More than 80 % of the population suf- microbiota. Their goal is to understand the fers from acne at some point in their life. The molecular mechanism of the interactions between disease can be very painful and profoundly P. acnes and P. acnes bacteriophages and the affects patients’ self-esteem, especially in adoles- interactions between the microbes and the host cent population. Although its etiology still needs in disease pathogenesis. to be defined, the presence of Propionibacterium In conclusion, this study shows that the acnes in the pilosebaceous unit is a key factor. pilosebaceous unit, a specialized skin compart- However, P. acnes is a common skin commensal ment where acne arises, has a tractable and has not been considered pathogenic by nor- microbiome with a single dominant species, mal standards (Burkhart et al. 1999). Neverthe- P. acnes. This system offers a unique advantage less, antibiotic therapy targeting P. acnes has to allow in-depth analysis of a human been a mainstay treatment for more than microbiome at the strain level. The findings sug- 30 years (Leyden 2001), and the reduction in gest that the microbiome associated with acne numbers of P. acnes is a widely used parameter offers promise for understanding the relation- of therapeutic effectiveness of antibiotics ships between the microbiome and human health (Burkhart et al. 1999). and disease. Metagenomics of Deep Hypersaline Anoxic Basins 341 M

Cross-References the subducting margins of Southern Europe. This area contains one of the world’s highest abun- ▶ CRISPRs in the Human Microbiome dance of unique geological features called deep- ▶ Human Microbiome Project, Reference sea hypersaline anoxic lakes (DHALs). Since the Genomes, Rationale, Selection, Acquisition, discovery of the first Mediterranean DHAL Tyro Sequencing, and Annotation in 1983, six other salt-related structures have ▶ Propionibacteriaceae been unveiled in this area in the last decades: l’Atalante, Bannock, Discovery, Medee, Thetis, and Urania, and the existence of many other References DHALs has been speculated (Medriff Consor- tium 1995) (Fig. 1). Burkhart CG, Burkhart CN, Lehmann PF. Acne: a review of immunologic and microbiologic factors. Postgrad The hydrologic and geological settings of Med J. 1999;75:328–31. these fascinating builds likely share the same Fitz-Gibbon ST, Tomida S, Chiu B, Nguyen L, Du C, genesis, i.e., their formation is originated from Liu M, Elashoff D, Erfe MC, Loncaric A, Kim J, dissolution of Messinian evaporitic salt deposits Modlin RL, Miller JF, Sodergren E, Craft N, Weinstock GM, Li H. Propionibacterium acnes strain that underlie most of the Mediterranean basin. populations in the human skin microbiome associated The astonishing amount of evaporites (this sub- with acne. J Invest Dermatol. 2013;133:2152–2160. surface layer can be of up to 1,000–2,000 m Kasimatis G, Fitz-Gibbon ST, Tomida S, Wong M, Li thick) was formed when the Mediterranean Sea H. Analysis of complete genomes of Propioni- bacterium acnes reveals a novel plasmid and increased has been desiccated during the cyclic salinity pseudogenes in an acne associated strain. BioMed Res crisis of Messinian period – between 5,600,000 Int. 2013;2013:918320. and 5,300,000 years ago. As it generally Leyden JJ. The evolving role of Propionibacterium acnes accepted, the overpressured fluids coming from in acne. Semin Cutan Med Surg. 2001;20:139–43. the underlying sedimentary sequences might dis- Tomida S, Nguyen L, Chiu B, Liu J, Sodergren E, M Weinstock GM, Li H. Pan-genome and comparative solve the evaporites, thus creating the very dense genome analyses of Propionibacterium acnes reveal brines with salinities exceeding 300. These brine its genomic diversity in the healthy and diseased once expulsed on the surface of seabed are human skin microbiome. mBio. 2013;4(3):e00003–13. trapped in nearest and closed depressions. Owing to favorable bottom topography and slow current, they form very stable salinity-induced Metagenomics of Deep Hypersaline bottom water bodies (Cita` 2006). Through signif- Anoxic Basins icant difference in densities, all DHALs are sep- arated from the overlaying deep-seawater masses Michail M. Yakimov1, Violetta La Cono1, by a thin and very stable pycno-/chemo-/ Manuel Ferrer2, Peter N. Golyshin3 and redoxcline, which prevents both advective and Laura Giuliano1 convective exchange of oxygen and other chem- 1Department of Marine Molecular Microbiology, ical constituents across this boundary. As it was Institute for Coastal Marine Environment demonstrated previously (van der Wielen et al. IAMC-CNR, Messina, Italy 2005; Daffonchio et al. 2006; Yakimov et al. 2Institute of Catalysis, Spanish National 2007; Hallsworth et al. 2007; Borin et al. 2009), Research Council, CSIC, Madrid, Spain this thin interface (<2 m) acts as a hot spot of 3School of Biological Sciences, Bangor microbial activity. Restricted circulation across University, Bangor, Gwynedd, UK the seawater/brine interface has resulted in fast and complete biogenic depletion of oxygen in these brines and, as follows, the redox potential The Mediterranean Accretionary Ridge is formed decreased rapidly, usually within less than 1 m, along the rifted margins of Northern Africa and from values +200 to À100 mV in the upper M 342 Metagenomics of Deep Hypersaline Anoxic Basins

Metagenomics of Deep Hypersaline Anoxic Basins, Fig. 1 Location of the deep-sea anoxic hypersaline lakes (DHALs) in the Eastern Mediterranean Sea

interface. Thus, downwards the lower interface, indicated in Fig. 2. At this transition the salinity all compartments of DHALs represent a highly increases sharply from that of Mediterranean sea- reduced environment with the negative values water to almost ten times this value. All major often less than À400 mV. The Mediterranean ions, such as Na+,K+,Mg2+,ClÀ, and in less extent 2+ 2À DHAL brines are therefore the excellent objects Ca and SO4 , follow the rise of salinity conser- for the study of biogeochemical cycling of ele- vatively. The dramatic difference in density of the ments and of redox-related diagenetic reactions deep seawater and brines (from 1.03 to >1.27) at a distinct redox boundary that may supply serves as a trap for the organic matter which con- energy to various types of chemolitho- and sists of both organic detritus settled from above heterotrophic communities. and the organic matters produced there de novo (Yakimov et al. 2007). The total organic com- pounds (TOC), accumulated at the seawater/brine Environmental Settings Sustaining the interface evidently, support the various types of Microbial Life in DHALs aerobic and anaerobic organotrophy detected there. It seems likely therefore that intensive The behavior of major environmental parameters organic matter decomposition at the boundary is and occurrence of major donors/acceptors that the major mechanism defining a peculiar feature of determine the shaping of microbial communities all Mediterranean DHAL brines – the elevated inhabiting both redoxcline and brine of DHALs is concentration of ammonium (from >0.3 up to Metagenomics of Deep Hypersaline Anoxic Basins 343 M

Metagenomics of Deep Hypersaline Anoxic Basins, depth-increasing behavior of main parameters and occur- Fig. 2 Diagrammatic representation of environmental rence of major donors/acceptors couples settings sustaining the microbial life in DHALs: the

5,000 mM), which is more than two orders of low concentration of dissolved iron, usually less magnitude higher than in other marine anoxic than one hundredth of the average values of deep environments (Borin et al. 2009). Mediterranean seawater. Thus, the influence of The nitrate profiling detected within the inter- this metal on the DHAL ecosystem functioning face, i.e., sharp increase from 0.2 to 4–5 mM and mode of respiration is obviously not very within upper interface and then drop to 0.1 mM important, if ever occurred. in the lower interface, clearly indicated the strat- Mediterranean DHALs are the highly euxinic - M ified activities of (micro)aerophilic ammonium environments with HS concentrations varying and nitrite oxidizers in the upper part of interface from 2 to 20 mM (Daffonchio et al. 2006; Borin and denitrifiers underneath. Evidently, both et al. 2009). As predicted by thermodynamic anoxia and elevated salinity of the DHALs brines calculations, the DHAL interfaces have shaped the distribution of ammonium-consuming a typical vertical sequence of the dominant elec- À 2À energetic pathways and correspondingly limited tron acceptors (02 > NO3 ~ MnO2 > SO4 ), the activity of oxygen-requiring nitrification and indicating that oxygen, nitrate, manganese salinity-sensitive anaerobic ammonium oxida- oxides, and sulfate are used in succession as tion (anammox). electron acceptors with increasing depth and Downwards from seawater to the brine, the salinity. In fact, the maximum sulfate reduction concentration of dissolved manganese (Mn2+) rates (SRR, 10–12 mM dayÀ1) were detected in increases at the DHALs boundary almost conser- the interface of Urania brine lake just beneath the vatively from 3–20 nM to 5–7 mM. As it is shown layer, where the concentration of Mn2+ has in Fig. 2, there is a slight decline in the arising of stopped rising. Accordingly to the concentration manganese content, observed within the “sub- of nitrate and manganese oxides, the anaerobic oxic” zone, likely caused by the microbiologically energy-gaining processes, based on their reduc- mediated oxidation processes. As it was suggested tion, seem be quantitatively of minor importance, elsewhere (Daffonchio et al. 2006), dissolved compared with sulfate respiration. Moreover, it manganese is oxidized at the oxic-anoxic bound- seems that the high SRR, detected in the inter- ary and settles as “solidified” MnO2 into the anoxic face, is mainly responsible for the elevated con- brine, where it might subsequently undergo the centration of HS- in the brine of DHALs, because biological reduction by organotrophs leading to extremely low SRR values were obtained within high concentration of Mn2+ in the brine. Another this salt-saturated compartment (Daffonchio peculiar feature of Mediterranean DHALs is a very et al. 2006; Borin et al. 2009). By analogy with M 344 Metagenomics of Deep Hypersaline Anoxic Basins

Mn2+/Mn4+ behavior, the sulfide is rapidly electron donors under hypersaline conditions. reoxidized in the hypoxic part of DHAL These findings corroborate with generally redoxcline by both inorganic and biological pro- accepted statement that hydrogeno- and particu- cesses, leading to the formation of sulfate and, larly acetotrophic methanogeneses strongly likely, the intermediate sulfur species (ISS), such affected by salinity and failed to be operative 0 2À 2À as S ,S2O3 , and SO3 . The detailed picture of under hypersaline conditions (at salinities >200 ISS patterning in Mediterranean DHALs is yet to and >100, respectively) (Oren 2011). be clarified. In other anoxic marine ecosystems, As it was already mentioned above, DHALs i.e., beneath the Black Sea redoxcline, sulfide was and especially their interfaces represent a hot spot oxidized to ISS dominated for up to 80 % by of microbial diversity. Without any physical bar- thiosulfate. In the absence of other oxidizing rier other than density, the chemocline of DHALs agents, the ISS could be recycled in anoxic parts in the space of a few meters forced the evolution of DHAL to sulfate and sulfide by both hetero- of different microbial communities exposed to trophic and/or autotrophic S-disproportionating geochemical conditions that change drastically microorganisms. with depth. Notwithstanding harsh environmen- Inferring the description of environmental set- tal conditions, the Mediterranean brine lakes tings, one should mention that all Mediterranean inhabited by very peculiar microorganisms, DHALs contained the significant amount of belonging to all three kingdoms of life. Many of dissolved methane (up to 2.75 mM in the Lake them were found in Mediterranean DHALs for Urania) whose quantity rapidly declined to zero the first time and we correspondingly named in the presence of strong oxidizers within the them as MSBL (M editerranean SeaB rine L interface. Such elevated concentration of CH4 akes) candidate divisions (van der Wielen could be explained by high biological methane et al. 2005). Since the discovery in 2005 of the production rates (MPR) in some DHALs. Note- members of MSBL1 (deep-branching cluster in worthy, but in contrast to SRR and other meta- order Thermoplasmatales), now there are at least bolic activities almost inactivated in the brines, 11 other candidate MSBL divisions recognized the MPR exhibited the maximum activity (Fig. 3). À1 À1 (170 mmol CH4 l day ) more than 50 m beneath the redoxcline of the Lake Urania (Borin et al. 2009). Bearing in mind the fact that Metagenomic Study of Mediterranean sulfate reduction is more energetically favorable DHALs than the methanogenesis and the concentration of 2À SO4 is abundant in both redoxcline and in the The members of all these divisions, known just brine, it is not clear why MPR is so much higher by 16S rRNA gene sequences, have so far resisted than SRR in the saltiest parts of the DHALs. It to any cultivation attempts thus precluding the would be logical to assume that it might reflect elucidation of their metabolic preferences. In the presence in the brine of a series of substrates, other words, in case of DHAL ecosystems, we which can be uptaken by methanogens but not by are facing the existence of an unknown microbial sulfate reducers. Indeed, the incubations of the world whose genomic information is hidden from samples collected from the hypersaline deep-sea us up to now and we cannot go deeper into anal- ecosystems, similar to DHALs (mud volcanoes), ysis of functional genes simply because there have shown that the substrates, most important are no analogues known. On the other hand, the for methanogenesis in these environments, are environmental setting of DHALs can provide the the small methylated compounds such as methyl- only simplified speculation on major metabolic amines, dimethyl sulfide, and methanol – known pathways operating under such poly-extreme as noncompetitive substrates due to incapability conditions. So, to understand the functioning of of sulfate reducers to use them as a carbon source. DHAL ecosystem, one has to apply a cultivation-

Noteworthy, but H2 and acetate did not serve as independent approach created to handle with eaeoiso epHpraieAoi ais345 Basins Anoxic Hypersaline Deep of Metagenomics Marine Hydrothermal Vent Group

Archaea

Halobacteriaceae MSBL11

Deep Sea Euryarcheotic Group Methanobacteria

Marine Group I Marine Group II Methanomicrobia MSBL12 MSBL10 Marine Benthic Group E MSBL1 Cyanobacteria

Marine Benthic Group B Miscellaneous Crenarchaeotic Group OD1 Thermodesulfobacteria OP11 KB1 OP10 Actinobacteria

OP2 Chloroflexi Firmicutes NT-B4 MSBL5

Candidate division BRC1

Candidate division TM6 Nitrospina

MSBL9 OP8

Gamma Nitrospirae Beta Epsilon Delta DesulfobacteralesMSBL7 PlanctomycetalesOP9

Alpha MSBL3

SB-1 MSBL8MSBL2 MSBL4 Chlamydiae OP3 Deferribacteres Fusobacteria MSBL6

Planctomycetes Bacteria Lentisphaerae Fibrobacteres Spirochaetes

Verrucomicrobia 1.00 Bacteroidetes M

Metagenomics of Deep Hypersaline Anoxic Basins, Fig. 3 Overview on prokaryotic diversity of phylogenetic groups recovered from the Mediterranean DHALs M M 346 Metagenomics of Deep Hypersaline Anoxic Basins community genomic, i.e., metagenomic analysis interface community with overwhelming pre- of the environmental DNA. This rapidly growing dominance of Sulfurovum-like epsilonproteo- field is promoting our understanding on the func- bacterial species (Ferrer et al. 2012). The latter tions of microbial populations in marine environ- finding was also corroborated with the taxonomic ment and leads to unveiling the important binning of the interface metagenome where mechanisms of microbial metabolism on single- almost 62 % of the assembled contigs were attrib- cell and community levels, genetics, and evolu- uted to Sulfurovum-like species. These tion of deep-sea microbes. However, there are chemolithoautotrophic sulfur-oxidizing organ- very few examples of metagenomic studies isms were firstly isolated from deep-sea hydro- applied so far to both deep-seawater column and thermal vents (HTV). Recently it was shown that sediments and especially to deep-sea hypersaline they use reductive tricarboxylic acid cycle anoxic environments. Recently it was attempted (rTCA) for carbon fixation. The genome to gain insights into the genomics of microbial sequence data obtained from the Thetis interface communities inhabiting interface and brine of the metagenome strongly support this hypothesis. DHAL Thetis, a new bathyal thalassohaline for- Autotrophic carbon fixation genes for the rTCA mation, which belongs to the saltiest water bodies cycle were detected in the interface, namely, on Earth (La Cono et al. 2011; Ferrer et al. 2012; ATP-citrate lyase (two hits), malate dehydroge- Stock et al. 2012). The Lake Thetis is character- nase (one hit), fumarate hydratase (five hits), ized by extreme chemistry and the interface fumarate dehydrogenase (three hits), succinyl- between seawater and the anoxic salt-saturated CoA synthetase (six hits), 2-oxoglutarate synthase brine is a hot spot of microbial activity. As in (two hits), isocitrate dehydrogenase (eight hits), other DHABs, numerous redox combinations and aconitate dehydratase (two hits), with within this layer allow the occurrence of many 90 % of them affiliating to Epsilonproteobacteria. permutations of energy-generating reactions. As None of the genes indicative for two other autotro- other Mediterranean DHALs, the Lake Thetis is phic pathways that could be operative at a permanently euxinic formation. HS- and inter- (micro)oxic conditions (3-hydroxypropionate/4- mediate sulfur species, which diffuse to the sur- hydroxybutyrate and Calvin-Benson-Bassham face from the lake interior, are more reduced than cycles) were found. This finding strongly support ammonia and nitrite, and therefore their aerobic the statement that rTCA is a dominant carbon oxidation yields substantially more energy, thus fixation pathway within the Thetis interface. supporting the elevated biomass and diversity in Increase of the salinity in the brine drastically the interface. This was confirmed by analysis of affects the operation of autotrophic processes and

403 sequencing reads from the Thetis interface no CO2 fixation was detected at the salinities constituted partial 16S rRNA gene sequences above 200 (La Cono et al. 2011). A number of with lengths and quality sufficient to unambigu- indications for the reductive acetyl-CoA (Wood- ously taxonomically affiliate with source organ- Ljungdahl) pathway, namely, genes encoding isms. A surprisingly low number of these formylmethanofuran dehydrogenase (two hits) sequences (<1 %) were affiliated with and subunits of the CO dehydrogenase/acetyl- chemolithoautotrophic nitrifying members of CoA synthase (two hits), all associated with Marine Group 1 of Thaumarchaeota. Thus, the methanogenic Euryarchaeota, were found in the interface separates an extremely hypersaline Thetis brine. Besides this, additional three genes anoxic brine from overlaying oxygenated seawa- encoding subunits of CO dehydrogenase/acetyl- ter and likely acts as a barrier hampering vertical CoA synthase complexes of bacterial origin were migration of Thaumarchaeota typically dominat- found, indicating on eventual presence in the ing the bathypelagic microbial communities. brine of active acetogens. Both types of metabo- Similarly to classical clone libraries analysis lism are likely based on the disproportionation of (La Cono et al. 2011), Proteobacteria constituted methylated compounds (methylotrophy) with the bulk of assigned 16S rRNA genes in the production of CH4 and acetate and at less extent Metagenomics of Deep Hypersaline Anoxic Basins 347 M

H2 and CO2. Indeed, four Thetis brine contigs phylogenetically were distinct from Methanoha- harbor the genes related to four isoforms of lophilus but related to Thermoplasmatales, trimethylamine-corrinoid methyltransferase mmt a group of organisms where MSBL1 is phyloge- B. This enzyme catalyzes the transfer of methyl netically placed (Borin et al. 2009). group from TMA to a [Co(I) TMA-specific corrinoid protein], thus activating the methylotrophic type of reductive Wood- Summary Ljungdahl pathway. None of mmtB-related sequences other than of order Halanaerobiales Current endeavors to seek evidence of life on was found in Thetis brine metagenome. Produced other planets and the recent detection of hydrated hydrogen can be used by many extreme halo- salts on Mars generate an urgency to understand philes, including acetogenic fermenters of order microbial limits of survival in similar environ- Halanaerobiales and the Desulfohalobium-like ments on Earth. This can be achieved by the sulfate reducers, whose genomic signatures “omics” survey of the microbial communities were retrieved from the Thetis brine metagenome inhabiting the DHALs, the saltiest water bodies (Ferrer et al. 2012). As it was shown recently, the on our planet. There are very few reports describ- availability of hydrogen can support the presence ing microbial activity in seafloor hypersaline of hydrogenotrophic Desulfohalobium bacteria at brines and none of them delineates the eventual extremely high salinities. Possessing a high affin- trophic relation of metabolic processes. Based on ity to H2, these extremely halophilic sulfate the available data and the interpretations of the reducers are able to oxidize hydrogen and uptake metagenomic data obtained from the Lake Thetis acetate as carbon source (Oren 2011). The DHAL brine, we proposed the stratified trophic network brines are slightly acidic, thus favoring the free established in the interface and in salt-saturated energy change associated with proton-consuming brines of DHALs. Analysis of geochemical set- M hydrogenotrophic sulfate reduction. Noteworthy, tings and thermodynamic limits, coupled with the Halanaerobiales-related sequences were high salinity, let to anticipate the potential meta- never visualized by direct monitoring of bacterial bolic pathways, which may be operative under diversity in the brines of Mediterranean DHALs harsh conditions of DHALs (Fig. 4). (Borin et al. 2009; La Cono et al. 2011). Life in salt-saturated environments is energeti- According to available metagenomic data, the cally very expensive and only a limited number of presence in the brines of acetogenic organisms prokaryotes possessing certain modes of metabo- other than Halanaerobiales is evident and most lism can cope with the anoxic hypersaline condi- likely can be attributed to the members of KB1 tions. Along with halophilic methylotrophic division, dominating the brine. The presence of methanogens, only some of fermenters, sulfate- Acetohalobium-like contigs within KB1 16S reducing “incomplete oxidizers,” and rRNA-containing fosmid Medee_0001 homoacetogens can be metabolically active (JX454600) allowed us to propose metabolic above salinity 300 (La Cono et al. 2011;Oren sameness between these organisms. Similarly to 2011;Ferreretal.2012). Metagenomic data con- Halanaerobiales, Methanohalophilus-like organ- firmed the compositions of microbial assemblages isms were also never visualized in 16S rDNA and generally agreed with this hypothesis. The libraries of DHAL brine communities. The sequence reconstruction of chemolithotrophy, MSBL1 candidate division of Euryarchaeota which is highly active in the Thetis interface, dominating the salt-saturated anoxic environ- revealed that sulfur-oxidizing Sulfurovum-like ments was recently proposed to be epsilonbacteria are the key players in dark primary a methanogen (Borin et al. 2009). In concor- production of the interface. Thus, the activity these dance, analyzing the metagenome of Thetis chemolithoautotrophic organisms represents an brine, many gene clusters were found to harbor important source of de novo produced organic the methylotrophic methanogenic signatures that carbon both for deep-sea biota and the DHAL M 348 Metagenomics of Deep Hypersaline Anoxic Basins

Metagenomics of Deep Hypersaline Anoxic Basins, Fig. 4 Pivotal metabolic pathways operating in the interface and in salt-saturated brines as revealed by interpretations of the available DHALs metagenomic data. Abbreviations used: 3HP/ 4HB 3-hydroxypropionate/ 4-hydroxybutyrate autotrophic pathway; CBB Calvin-Benson-Bassham autotrophic cycle; rTCA reductive tricarboxylic acid autotrophic cycle; SR sulfate/sulfur reduction; MetMg methylotrophic methanogenesis; MetAc methylotrophic acetogenesis

interiors. Moving towards the bottom, the posses- Daffonchio D, Borin S, Brusa T, et al. Stratified prokary- sion of the methylotrophic modification of ancient ote network in the oxic-anoxic transition of a deep-sea halocline. Nature. 2006;440:203–7. r-acetyl-CoA (Wood-Ljungdahl) pathway seems Ferrer M, et al. Unveiling microbial life in the new deep- to be crucial for the life under salt-saturated con- sea hypersaline Lake Thetis. Part II: a metagenomic ditions of DHAL brines. The genomic signatures study. Environ Microbiol. 2012;14:268–81. of two forms of this pathway, respectively, attrib- Hallsworth JE, Yakimov MM, Golyshin PN, et al. Limits of life in MgCl2-containing environments: uted to the archaeal (methanogenic) and bacterial chaotropicity defines the window. Environ Microbiol. (acetogenic) variants, were found in the Thetis 2007;9:801–13. brine. Based on obtained data, we proposed that La Cono V, Bortoluzzi G, et al. Unveiling microbial life in these metabolic features belong to predominating new deep-sea hypersaline Lake Thetis. Part I: prokary- otes and environmental settings. Environ Microbiol. prokaryotic components of DHAL brine 2011;13:2250–68. microbiota, i.e., to the members of MSBL1 and MEDRIFF Consortium. Three brine lakes discovered in KB1 candidate divisions. the seafloor of the eastern Mediterranean. EOS Trans AGU. 1995;76:313. Oren A. Thermodynamic limits to microbial life at high salt concentrations. Environ Microbiol. 2011;13:1908–23. Stock A, Breiner H-W, Pachiadaki M, et al. Microbial References eukaryote life in the new hypersaline deep-sea basin Thetis. Extremophiles. 2012;16:21–34. Borin S, Brusetti L, Mapelli F, et al. Sulfur cycling and van der Wielen PW, Bolhuis HJJ, Borin S, et al. The methanogenesis primarily drive microbial coloniza- enigma of prokaryotic life in deep hypersaline anoxic tion of the highly sulfidic Urania deep hypersaline basins. Science. 2005;307:121–3. basin. Proc Natl Acad Sci U S A. 2009;106:9151–6. Yakimov MM, La Cono V, Denaro R, et al. Primary pro- Cita MB. Exhumation of Messinian evaporites in ducing prokaryotic communities of brine, interface the deep-sea and creation of deep anoxic brine-filled and seawater above the halocline of deep anoxic lake collapsed basins. Sediment Geol. 2006;188–189: L’Atalante, Eastern Mediterranean Sea. ISME J. 357–78. 2007;1:743–55. Metagenomics of Mangroves 349 M

literature are listed, in order to elucidate the Metagenomics of Mangroves underpinning processes of microbial communi- ties in this environment, additionally to foster our Fernando Dini Andreote understanding of these microbial assemblages in Department of Soil Science, University of Sao such particular ecosystem. Paulo, Sao Paulo, Brazil

Metagenomics of Mangroves: Introduction to Mangroves Pyrosequencing

Mangroves are boundary landform ecosystems The description of mangrove microbiology has present in tropical and subtropical regions, been recently made on the basis of located in the intersection between land and sea pyrosequencing, in a survey based on DNA (Holguin et al. 2001). This ecosystem acts as extracted directly from sediments of four dis- a buffer area being essential for maintenance of tinct mangrove areas along the coast of the Sao the sea level and for the protection of the coast Paulo State in Brazil (Andreote et al. 2012). (Duke et al. 2007). Environmental conditions Authors have based their assumptions in approx- particular to this biome are the salinity – which imately 217.5 Mb of information, retrieved is related to the influence of the sea – and the from approximately one million reads of envi- frequent fluctuation between aerobic and anaer- ronmentalDNA,withanaveragelengthof obic condition, caused by the fluctuation in the 236 base pairs. tidal regime (Holguin et al. 2001;Ferreira Initially, the datasets were used to depict the et al. 2010), which significantly causes an effect taxonomy of predominant microbes occurring in on redox potential that ranges from À200 to this niche, indicating that the particular combi- M +150 mV (Clark et al. 1998). Figure 1 illustrates nation of environmental conditions also deter- mangroves on its distinct aspects. mines the occurrence of particular microbial Mangrove sediments, although apparently assemblages in sediments of mangroves. By tax- harsh and inhospitable, harbor unique assem- onomically assigning the obtained data, using blages of microorganisms, which are able to 16S rRNA gene and also functional annotations, cope with the environmental conditions and a remarkable dominance of Proteobacteria- dynamics. Thus, such dynamics make this eco- related sequences was observed. Moreover, system a hotspot for biodiversity and also for the within this major phylum, the classes occurrence of different metabolic pathways and Gammaproteobacteria and Deltaproteobacteria nutrient transformations when compared to those were revealed to be the most abundant described for soil and water sources. Hence, (Andreote et al. 2012), distinguishing man- depicting mangrove microbiology is urgent in groves from other soils, where dominance is order to use this unique resource and also to observed for Alphaproteobacteria (Table 1). prevent mangrove devastations and degradation This observation is in accordance with previous due to contamination from different origins assessments of mangrove microbiology, where (Duke et al. 2007). the prevalence of such groups was evidenced in It is widely known that cultivation is very pristine areas, and also remained dominant when limiting in the assessment of the microbial exten- oil spill was simulated in macrocosm experi- sion in most of environments, the so-called great ments(DosSantosetal.2011). Summarizing, plate count anomaly. In this context, the use of these results suggest Gammaproteobacteria and culture-independent approaches to properly Deltaproteobacteria as important players in describe the microbial communities associated such environment. In this sense, much more with mangroves, on its distinct compartments, is focusedstudiesarerequiredtoshedlighton essential. Here some findings present in the untapped microbial functionality of such groups, M 350 Metagenomics of Mangroves

Metagenomics of Mangroves, Fig. 1 Illustrations of mangroves. The location between land and sea and detailed view of mangrove soils. Propagation of mangrove plants is also shown

Metagenomics of Mangroves, Table 1 Comparison major microbial groups inhabiting the rhizo- of major bacterial groups in common soils and mangroves. sphere of typical mangrove plant species. The values for common soil were obtained from Janssen (2006) and mangrove data was obtained by the The assessment was performed by an educated metagenomic approach (Andreote et al. 2012) measurement of rhizosphere community in Taxonomic group % in soils % in mangroves comparison to the groups occurring in bulk Proteobacteria 39.2 58.0 sediment samples. The obtained results highlight Alphaproteobacteria 18.8 12.0 the remarkable responsiveness of Acidobacteria, Betaproteobacteria 10.0 8.3 Actinobacteria, Verrucomicrobia, Burkholderiales, Gammaproteobacteria 8.1 17.3 Caulobacterales, and Rhizobiales to the plant- Deltaproteobacteria 2.3 20.4 released exudates, while other groups, as Acidobacteria 19.7 0.0 Chloroflexi, Firmicutes, and Desulfobacterales, Actinobacteria 12.7 4.4 were less abundant in the soil region affected by Verrucomicrobia 7.0 0.0 roots exudates. Planctomycetes 2.0 2.8 However, one can suggest that functioning is Bacteroidetes 5.0 0.7 more important than taxonomy for the mainte- Firmicutes 1.8 7.4 nance and functioning of microbial communi- ties, and selection can be exerted on the basis of functions that microbes can perform, instead profusely occurring in such environment its taxonomy. In order to attend to this issue, and possibly performing important roles in eco- Andreote et al. (2012) have also analyzed the system functioning and maintenance. Also metagenomic data based on the functions codi- assessing the mangrove microbiology, Gomes fied by the generated sequences. A robust anal- et al. (2010)hasfocusedondepictingthe ysis was performed, employing several Metagenomics of Mangroves 351 M databases and algorithms,whichresultedinthe Metagenomics of Mangroves: Fosmid description of the genetic basis for the occur- Libraries rence of several biogeochemical processes. Within the carbon cycle, it was possible to The beginning of metagenomics was based on observe the presence of complete sets of genes cloning high molecular weight DNA, used in required for the metabolism of methanol, while numerous studies to describe new coding genes genes involved in methane consumption were for enzymes and molecules, representing not present (possibly due to the low coverage a breakthrough in the biotechnology. Such of the used approach). Concerning the nitrogen approach has not been used to explore the man- cycle, genes related to biological nitrogen fixa- grove reservoir for the biotechnological explora- tion were found, and alternative metabolism of tion (Handelsman 2004). Considering the nitrate revealed to be present in mangroves, uniqueness of such environment, it is possible to being it consumed by the residing microbes by suggest that particular enzymes may occur in dissimilatory reduction of nitrate or denitrifica- such niche. An evidence of this characteristic is tion, while ammonium can also be consumed by the low rate of sequence affiliation observed for the anaerobic ammonium oxidation (anammox). the mangrove metagenomics when compared The overview of sulfur cycling indicated the with those generated from common soils and presence of genes involved in the sulfate reduc- water sources (Andreote et al. 2012). Based on tion, leading to the generation of H2S, which is a culture-dependent method, Dias et al. (2009) thegasresponsibleforthetypicalsmellof assessed the potential of bacterial isolates mangroves. obtained from mangrove sediments for the pro- By combining both approaches, these authors duction of several enzymes with industrial appli- suggested links between taxonomy and functions cations. The authors found several enzymatic of microbes in the transformations of nitrogen, activities, encompassing the production of amy- M carbon, and sulfur. The group identified, as lases, proteases, esterases, and lipases. These participant in the transformations of these three authors claim that due to the environmental con- elements in all mangrove areas, mostly the ditions in mangroves, these enzymes have its family Desulfobacteraceae, allocated within the optimal action in distinct conditions than those class Deltaproteobacteria, which also revealed to observed for other well-established and broadly be the dominant taxa in mangrove sediments. used enzymes. This suggested a possible link between function- Such tool has been recently used for other ality and abundance, what remains to be better purposes. An example of innovation in this covered, possibly on the basis of functional, as, approach is the replacement of screenings for for example, metatranscriptomics. enzymes codifying genes by the search of inserts Another approach supported by metagenomics containing genes related to specific functions as, is the comparison of the microbial patterns with for example, genes related to biogeochemical those found in other environments. Such analysis cycles (Suenaga 2012). In this sense, the use of indicated the allocation of mangroves between traditional metagenomics can add knowledge in samples from soils and water in a direct indication the establishment of links between taxonomy and that its location between land and sea also results function for the microbial community found in in a mixture of microbial assemblages within this mangrove sediments. For example, inserts niche. It can be that microbial cells delivered in containing similar genes to nifH can be targeted, mangroves are somehow related to those from and its sequencing and annotation can give clues nearby areas but also are subjected to specific about the type of organisms present in the sedi- selection pressures leading to evolution pro- ments, which are involved with biological nitro- cesses, which might result in mangrove specific gen fixation in this niche. This approach can also microbial groups. indicate the possible occurrence of specific M 352 Metagenomics of Mangroves genomic arrangements in such environment, oil contamination can exert influence on differ- where distinct genes can be combined within ent environmental parameters, where other a single genome in a mode not found in any forms of essential enzymes are necessary to other niche. By keeping the example aforemen- maintain the biogeochemical cycles function- tioned, the nifH-harboring insert can be used for ing. It indicates that, even after the oil degrada- comparison based on operon synteny, with other tion, the newly established community cannot nif operons found in N2 fixers living in other be converted into its natural stage and the con- environments. sidered recovered area run now on the basis of a distinct microbial community. It is not possi- ble to discuss about possible implications of this Modulation of Microbial Communities replacement, but this issue certainly composes in Mangroves the core of future studies of mangrove microbiology. Although metagenomics constitutes a robust Moreover, these studies based on fingerprint- assessment to microbial communities, the clear ing approaches were complemented with differentiation of these groups among areas is sequence-based analysis, used to describe the rarely described based on such approach. The major groups of microorganisms in this niche major limitation on this issue is the common and also to infer on groups harboring genes low coverage observed on such assessments. involved with specific activities in mangrove sed- The high diversity of microorganisms present in iments. An important remark, found in most of this niche make it difficult to cover its contents these surveys, is the occurrence of endemic properly, even using the most advanced resources organisms or genes in mangrove sediments to obtain environmental sequences. In this sense, (Taketani et al. 2010b; Dias et al. 2011, in other culture-independent approaches have been press). Authors describe the occurrence of spe- used to describe shifts caused in microbial com- cific clusters, composed by sequences retrieved munities residing in mangroves. from mangrove sediments, which have low sim- The compositions of bacterial and archaeal ilarities with sequences present in databases. It communities in mangroves were firstly deter- suggests that the prevalent conditions in man- minedbyLiangetal.(2007)andYan groves can lead to the specific formation of geno- et al. (2006). These authors have used the tradi- types not found in other environment. tional clone and sequencing approaches to infer about the most abundant groups of prokaryotes found. Concerning the shifts caused in such Open Issues in Mangrove Metagenomics communities, differences were firstly described based on fingerprinting of ribosomal genes, by The content of mangrove microbiology is far to using DGGE to demonstrate the distinct com- be completely understood, as it is for most of position of bacterial (Gomes et al. 2008), environmental microbial assemblages. Some fac- archaeal (Dias et al. 2011), fungal (Fasanella tors contribute to such initial stage of the knowl- et al. 2012), and cyanobacterial (Rigonato edge in this issue. et al. in press) in contaminated mangrove areas Firstly, the revolution in the power of sequenc- when compared to pristine conditions. Lately, ing has made the microbiologists very enthusias- function-related genes were used to infer about tic but also somehow frustrated, by seeing that the differential drivers of biogeochemical cycles in most recent approach used is already considered distinct mangrove areas, as observed for the old-fashion a few months later. It makes neces- genes nifH (Taketani et al. 2010a;Diasetal.in sary to keep on the wave with our mangrove press), dsrB and mcrA (Taketani et al. 2010b). samples, running after new results that confirmed The distinction between microbial groups in the observations made on the basis of the oiled from pristine mangroves indicates that presented approaches. Metagenomics of Mangroves 353 M

Secondly, the geographical distribution of the basis of microbial taxonomy or functioning. microbial groups found in mangroves. By one We have that, compared to other environments, side, the very particular environmental condition mainly the human body, the microbial communi- can give the idea that organisms present in such ties in mangroves are still in the infancy of their areas are similar in most of mangroves. However, description and comprehension. when considering the origin of these organisms, mainly delivered in mangroves from ocean waters during the tide regimes, it can be Summary suggested that distinct water sources can carry different microbes. In this case, the possible sim- The mangrove consists in a particular environ- ilarity between microbial assemblages in man- ment, and the initial depiction of its microbiology groves found in distinct areas could be the result has shown the occurrence of particular groups in of a convergent selection exerted by the particu- this niche, which can perform activities not found lar conditions on the introduced microbial groups in any other environment. It linked with its in mangroves. The fact is that a study on man- extinction process make urgent the better explo- grove biogeography is still needed to be ration of its natural resource for science and performed. biotechnology. Thirdly, another remarkable open point for discussion is that most of studies on mangrove microbiology were performed based on DNA analysis. As mangrove sediments are known to References accumulate organic matter, where its degradation Andreote FD, Jimenez DJ, Chaves D, Dias ACF, is delayed by the prevalent absence of oxygen, it Luvizotto DM, Dini-Andreote F, Fasanella CC, might be that most of recovered DNA from sed- Baena S, Lopez MV, Taketani RG, Melo IS. The M iment cores originated from dead or inactive microbiome of Brazilian mangrove sediments as cells. In this sense, the use of approaches more revealed by metagenomics. PLoS One. 2012;7(6): e38600. close to cellular activities is desired. For exam- Clark MW, McConchie D, Lewis DW, Saenger P. Redox ples, more functioning-based assessments of stratification and heavy metal partitioning in microbial communities in mangroves on the Avicennia-dominated mangrove sediments: bases of metatranscriptomics, metaproteomics, a geochemical model. Chem Geol. 1998;149:147–71. Dias ACF, Andreote FD, Dini-Andreote F, Lacava PT, or metabolomics approaches are still to be Sa’ ALB, et al. Diversity and biotechnological poten- presented. The use of such tools, and even an tial of culturable bacteria from Brazilian mangrove integrative use of them, will indubitably repre- sediment. W J Microbiol Biotechnol. 2009;25(7): sent a quantum leap forward in our understanding 1305–11. Dias ACF, Dini-Andreote F, Taketani RG, Tsai SM, of mangrove microbiology. Azevedo JL, et al. Archaeal communities in three Last but not least, several of the microbiology contrasting mangrove sediments. J Soils Sed. mysteries are also present in the mangrove con- 2011;11:1466–76. text. Unimaginable features of these organisms Dias ACF, Silva MCP, Cotta SR, Dini-Andreote F, Soares Jr. FL, Salles JF, Azevedo JL, van Elsas JD, Andreote are possible to be achieved if it is considered, for FD. Abundance and genetic diversity of nifH gene example, that speciation is driven by environ- sequences in anthropogenically affected Brazilian mental forces, which are distinct in mangroves mangrove sediments. Appl Environ Microbiol. 2012; when compared with other niches. Closely 78(22):7960–67. Dos Santos H, Cury JC, Lima do Carmo F, Lopes dos related to it is the presence of mobile elements, Santos A, Tiedje J, et al. Mangrove bacterial diversity which can induce a more diversification of micro- and the impact of oil contamination revealed by bial communities, transferring genomic informa- pyrosequencing: bacterial proxies for oil pollution. tion among its components (fragments of genes PLoS One. 2011;6(3):e16943. Duke NC, Meynecke J-O, Dittmann S, Ellison AM, responsible for key processes in mangroves), Anger A, et al. A world without Mangroves? Science. raising the question if there is selection made on 2007;317:41–2. M 354 Metagenomics of the Coral Holobiont

Fasanella CF, Dias ACF, Rigonato J, Fiore MF, Soares Jr FL, Melo IS, Pizzirani-Kleiner AA, van Elsas J, Metagenomics of the Coral Andreote FD. The selection exerted by oil contamina- tion on mangrove fungal communities. Water Air Soil Holobiont Pollut. 2012;223:4233–43. Ferreira TO, Otero XL, Souza-Junior VS, Vidal-Torrado- Mo´nica Medina1 and Shinichi Sunagawa2 P, Macias F, et al. Spatial patterns of soil attributes and 1Department of Biology, Pennsylvania State components in a mangrove system in Southeast Brazil (Sao Paulo). J Soils Sed. 2010;10(6):995–1006. University, University Park, PA, USA 2 Gomes NCM, Borges LR, Paranhos R, Pinto FN, Structural and Computational Biology, Mendonc¸a-Hagler LCS, et al. Exploring the diversity European Molecular Biology Laboratory, of bacterial communities in sediments of urban man- Heidelberg, Germany grove forests. FEMS Microbiol Ecol. 2008;66(1): 96–109. Gomes NCM, Cleary DFR, Pinto FN, Egas C, Almeida A, et al. Taking root: enduring effect of rhizosphere bac- Synonyms terial colonization in mangroves. PLoS One. 2010;5(11):e14065. Handelsman J. Metagenomics: application of genomics to Coral reef: scleractinian reef; Symbiodinium: uncultured microorganisms. Microbiol Mol Biol Rev. Zooxanthellae 2004;68(4):669–85. Holguin G, Vazquez P, Bashan Y. The role of sediment microorganisms in the productivity, conservation, and rehabilitation of mangrove ecosystems: an overview. Definitions Biol Fertil Soils. 2001;33:265–78. Janssen PH. Identifying the dominant soil bacterial taxa in Coral: Multicellular animal in the phylum libraries of 16S rRNA and 16S rRNA genes. Applied Cnidaria, host to a diverse microbial assemblage. and Environmental Microbiology. 2006;72:1719– 1728. Symbiodinium: The photosynthetic algal symbi- Janssen PH, Yates PS, Grinton BE, Taylor PM, Sait ont of corals and other animals. M. Improved culturability of soil bacteria and isolation Microbiome: The microbial assemblage and the in pure culture of novel members of the divisions corresponding genomes living in a particular Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. Appl Environ Microbiol. 2002;68: host. 2391–6. Coral holobiont: A coral host and its associated Liang JB, Chen YQ, Lan CY, Tam NFY, Zan QJ, Huang microbial community. LN. Recovery of novel bacterial diversity from man- Bleaching: Disruption of coral-Symbiodinium grove sediment. Mar Biol. 2007;150:739–47. Rigonato J, Kent A, Alvarenga DO, Andreote FD, Beirigo symbiosis. RM, Vidal-Torrado P, Fiore MF. Drivers of cyanobacterial diversity and community composition in mangrove soils in southeast Brazil. Environ Microbiol. 2013;15:1103–14. Introduction Suenaga H. Targeted metagenomics: a high-resolution metagenomics approach for specific gene clusters in Coral reefs are tropical and subtropical marine complex microbial communities. Environ Microbiol. oligothrophic environments. Despite the low 2012;14(1):13–22. Taketani RG, Franco NO, Rosado AS, van Elsas nutrient availability, they exhibit the largest bio- JD. Microbial community response to a simulated diversity estimates of any marine ecosystem. The hydrocarbon spill in mangrove sediments. estimated value of these ecosystems worldwide is J Microbiol. 2010a;48(1):7–15. of 375 billion dollars per year (Costanza Taketani RG, Yoshiura CA, Dias ACF, Andreote FD, Tsai SM. Diversity and identification of methanogenic et al. 1997) represented in the form of fisheries, archaea and sulphate-reducing bacteria in sediments tourism, and bioprospecting that benefit coastal from a pristine tropical mangrove. Antonie Van Leeu- communities. wenhoek J Microb. 2010b;97(4):401–11. Scleractinian corals are the key builders of Yan B, Hong K, Yu ZN. Archaeal communities in man- grove soil characterized by 16S rRNA gene clones. these magnificent structures. They live in an J Microbiol. 2006;44(5):566–71. obligate symbiosis with dinoflagellate algae Metagenomics of the Coral Holobiont 355 M

(Symbiodinium spp.), which restricts their distri- been applied to characterize bacterial and archaeal bution to shallow areas where light is available communities in corals (Rohwer et al. 2002; for photosynthesis. These algal symbionts, which Kellogg 2004). These efforts revealed unexpected provide their hosts with photosynthetic products, levels of microbial diversity as well as preliminary are diverse and relatively well characterized by evidence for host species specificity of the molecular systematics tools. A broad spectrum of microbiota (Rohwer et al. 2002). Next-generation other microbial species as well as viruses also sequencing approaches have followed up and coexists with this coral-algal partnership. revealed an even larger level of diversity in The term holobiont coined by Lynn Margulis the microbial community than predicted by the (Margulis 1981) refers to a multicellular host initial clone library sequencing-based approaches and its associated microbial community. This (Sunagawa et al. 2010). term has been incorporated in the recent coral Taken together, corals can be considered as symbiosis literature, initially to refer to the biological entities composed of the host organism coral-algal partnership as a holosymbiont and a complex, interacting network of microbial (Iglesias-Prieto and Trench 1997) to quickly associates. Metagenomic approaches will facili- become more inclusive and refer to the entire tate the study of their functional relationships, community (Rohwer et al. 2002). The holobiont e.g., cooperation; their interactions with biotic, concept is well established among coral biolo- e.g., pathogens, and abiotic factors, e.g., thermal gists,anditisnowexpandingtootherhost- stress; and their role in adaptive processes, e.g., microbial assemblages, as the study of microbial co-diversification. ecology in the context of a multicellular host permeates to macrobiology (Rosenberg 2011). The role of these additional complex microbial What Can Be Learned Now About and viral assemblages has received recent atten- Microbial Diversity in Corals? M tion, thanks to the rapid advances in DNA sequence-based technologies. Understanding the dynamics of these complex microbiomes and how they are shaping their host is of utmost importance today given the Microbial Diversity in Corals threats of climate change and coastal pollution. Time-series experiments in open water systems The long-lasting monospecific status of have shown clear correlational networks of Symbiodinium microadriaticum (Freudenthal microbial species; therefore, coral-associated 1962) gradually changed due to novel biochemi- microbiomes should reflect even more clear pat- cal and morphological evidence in the early terns of diurnal, seasonal, and annual oscillations. 1980s. But it was not until the 1990s that molec- Similar experiments with coral hosts will be ular genotyping tools revealed that Symbiodinium essential to link the physiological parameters represents a diverse genus whose member species with shifts in holobiont composition over predict- are now broadly grouped into eight different able seasonal expectations and delineate those clades (Coffroth and Santos 2005). While some from holobiont responses to coral bleaching, dis- coral species seem to associate with only one ease, and other environmental insults. particular clade of Symbiodinium, others, e.g., Biogeographic assessments of microbial diver- Orbicella faveolata, are known to establish asso- sity will address a gap in knowledge that is essential ciations with multiple Symbiodinium clades to resolve (i) how environmental factors drive the (Rowan and Knowlton 1995). composition of coral microbiomes – an ecological This degree of complexity only increased when time-scale from (ii) the deterministic potential of culture-independent efforts, initially based on host genetics on shaping its microbiome and vice clone library Sanger sequencing of the prokaryotic versa – an evolutionary time-scale. Phylogenetic small subunit ribosomal RNA gene (16S), have analysis of coral-associated microbial taxa is also M 356 Metagenomics of the Coral Holobiont relevant to understand the role of co-evolutionary coral susceptibility to develop diseases is corre- processes in the coral holobiont interaction net- lated with the extent of bleaching in corals work (Sunagawa et al. 2010). (Brandt and McManus 2009). Metagenomics has thus paved the road to a better understanding of the molecular mechanisms underlying the Coral Metagenomics response of the holobiont community to climate change perturbations as well as naturally occur- Metagenome analysis involves the sequencing of ring physiological variation. total genomic DNA extracted from environmen- Studies in other cnidarian hosts are also tal samples extending the effort to address micro- becoming available (Hewson et al. 2012), and bial diversity that was solely based on 16S model systems are emerging such as the sea ribosomal DNA sequencing surveys. This anemone Aiptasia pallida and the corals approach can provide information on the genetic Acropora digitifera, Acropora millepora, and content or in other words reveal the microbially- Orbicella faveolata to mention a few (Meyer encoded functional potential of the coral and Weis 2012). For example, research on dis- holobiont. Metagenomes from diverse coral sam- eased sea fans found a large number of new viral ples collected under different conditions show sequences that do not match any other organism a clear signature of coral-enriched-associated or viral environmental sample (Hewson functions in the microbial and viral communities et al. 2012), which suggests that there is (Dinsdale et al. 2008). In combination with other a widely underexplored virome not only in cni- macroecological methods such as species counts darian but also other marine multicellular hosts. and abiotic metadata, metagenomics is providing a more precise assessment of pristine vs. dis- turbed conditions in coral reef ecosystems What Else Can Be Learned from (Bruce et al. 2012). Metagenomic Approaches? Metagenome sequencing from stressed or dis- eased coral samples has shown the potential of Different combinations of coral-Symbiodinium the technique by illustrating how certain gene partnerships can affect important properties of functional categories are enriched in different the coral holobiont, including (i) adaptation to stress states. A stress response (i.e., nutrient different environmental conditions; (ii) growth, enrichment, thermal and pH changes) time-series carbon fixation, and photosynthate transfer rates; experiment illustrates how during a very short and (iii) susceptibility to thermal stress. Despite time span the microbial (Vega Thurber these apparent functional variations in different et al. 2009) and viral (Vega Thurber et al. 2008) host-Symbiodinium combinations, the effect of communities are shifting. Such high-throughput different Symbiodinium genotypes on global metagenomic data not only provide information coral gene expression has only started to be on changing community composition but also examined (DeSalvo et al. 2010). This, however, what are the most abundant gene functions poten- is an important step to elucidate the underlying tially being invoked by the microorganisms. molecular mechanisms that contribute to the phe- A shift from autotrophic to heterotrophic metab- notypic plasticity and environmental adaptation olism in coral microbial communities has been in corals. observed during a natural bleaching event Similarly, studying the interplay of prokary- (Littman et al. 2011), which was accompanied otic and viral members with their coral hosts can by and an increase in virulence genes and patho- now be tackled by metagenomics approaches. genic microorganisms. Similar results have been The correlation of environmental data with taxo- reported in the study of iron-enriched waters in nomic and functional signatures will help identify otherwise pristine reef environments (Wegley the main driving forces that determine the com- et al. 2012). It has also been documented that position and dynamics of the coral holobiont. Metagenomics of the Coral Holobiont 357 M

However, metagenome data have caveats in that diversity, benthic cover, and fish biomass data. PLoS not all the genomic information is fully recov- ONE. 2012;7(6):e36687. Coffroth MA, Santos SR. Genetic diversity of symbiotic ered. Rare gene functions, for instance, that may dinoflagellates in the genus Symbiodinium. Protist. be essential in getting a complete picture of what 2005;156(1):19–34. is the genetic repertoire of a species are not Costanza R, dArge R, de Groot R, Farber S, Grasso M, uncovered by the shallow nature of draft Hannon B, et al. The value of the world’s ecosystem services and natural capital. Nature. metagenome data. Metatranscriptome analysis 1997;387(6630):253–60. can provide further insight into what genes are DeSalvo MK, Sunagawa S, Fisher PL, Voolstra CR, being expressed and at what rate at different time Iglesias-Prieto R, Medina M. Coral host transcriptomic points or in different tissue samples. Library states are correlated with Symbiodinium genotypes. Mol Ecol. 2010;19(6):1174–86. preparation protocols for taxon-specific mRNA Dinsdale EA, Edwards RA, Hall D, Angly F, Breitbart M, libraries, e.g., fungal, algal, and prokaryotic, are Brulc JM, et al. Functional metagenomic profiling of now readily available and should therefore facil- nine biomes. Nature. 2008;452:629–33. itate such efforts. Examining holobiont metatran- Freudenthal HD. Symbiodinium Gen Nov and Symbiodinium microadriaticum sp nov, scriptomes in concert will add a new level of a Zooxanthella - taxonomy, life cycle, and morphol- understanding to this complex organismal ogy. J Protozool. 1962;9(1):45–52. network. Hewson I, Brown JM, Burge CA, Couch CS, LaBarre BA, In addition to these hypothesis-generating Mouchka MA, et al. Description of viral assemblages associated with the Gorgonia ventalina holobiont. “omics” approaches (genomics, metagenomics, Coral Reefs. 2012;31:487–91. metatranscriptomes, and metaproteomics), Iglesias-Prieto R, Trench RK. Photoadaptation, culturing-based techniques should complement photoacclimation and niche diversification in these studies and allow for hypothesis testing invertebrate-dinoflagellate symbioses. Proceedings of the 8th International Coral Reef Symposium. 1997. and refinement of models explaining the coral vol. 2, p. 1319–24. holobiont as one biological entity in the context Kellogg CA. Tropical Archaea: diversity associated with M of its surrounding environment. the surface microlayer of corals. Mar Ecol Prog Ser. 2004;273:81–8. 08 June 2004. Littman RA, Willis BL, Bourne DG. Metagenomic anal- ysis of the coral holobiont during a natural bleaching Summary event on the great barrier reef. Environ Microbiol Rep. 2011;3(6):651–60. Metagenomics is bringing broad insight into the Margulis L. Symbiosis in cell evolution: microbial com- munities in the Archean and Proterozoic Eons. San intricate interactions among coral holobiont Francisco: W. Freeman; 1981. members. Not only can we learn what microbial Meyer E, Weis VM. Study of Cnidarian-Algal Symbiosis and viral lineages coexist with a coral host but in the “Omics” age. Biol Bull. 2012;223(1):44–65. also what are the metabolic contributions and 1 Aug 2012. Rohwer F, Seguritan V, Azam F, Knowlton N. Diversity demands that these microbiome members impose and distribution of coral-associated bacteria. Mar Ecol on their host. We can use this information to Prog Ser. 2002;243:1–10. assess health state of coral reef ecosystems that Rosenberg, Eugene, Gophna, Uri (eds.), Beneficial micro- extends beyond macro-organismal scales. organisms in multicellular life forms, Springer Berlin Heidelberg. Rowan R, Knolwton N. Intraspecific diversity and ecolog- ical zonation in coral-algal symbiosis. Proc Natl Acad Sci U S A. 1995;92:2850–3. References Sunagawa S, Woodley CM, Medina MN. Threatened corals provide underexplored microbial habitats. Brandt ME, McManus JW. Disease incidence is related to PLoS ONE. 2010;5(3):e9554. bleaching extent in reef-building corals. Ecology. Vega Thurber RL, Barott KL, Hall D, Liu H, Rodriguez- 2009;90(10):2859–67. Mueller B, Desnues C, et al. Metagenomic analysis Bruce T, Meirelles PM, Garcia G, Paranhos R, Rezende indicates that stressors induce production of herpes- CE, de Moura RL, et al. Abrolhos bank reef health like viruses in the coral Porites compressa. Proc Natl evaluated by means of water quality, microbial Acad Sci U S A. 2008;105(47):18413–8. M 358 Methanogenic Archaea in the Human Microbiome

Vega Thurber R, Willner-Hall D, Rodriguez-Mueller B, and breath through blood circulation. However, Desnues C, Edwards RA, Angly F, et al. Metagenomic CH4 production varies significantly between analysis of stressed coral holobionts. Environ Microbiol. 2009;11(8):2148–63. individuals as not everyone will have detectable Wegley KL, Barott KL, Dinsdale E, Friedlander AM, CH4 (>1 ppm) in their breath. Methane produc- Nosrat B, Obura D, et al. Black reefs: iron-induced tion is dependent on fermentative production of phase shifts on coral reefs. ISME J. 2012;6(3):638–49. H2, which potentially explains differences in breath CH4 positive status prevalence across eth- nic groups. Methane breath tests are also widely used as a measurement of intestinal fermentation, Methanogenic Archaea in the for example, to determine lactose intolerance. Human Microbiome Substantial interindividual differences exist in colonic methanogenesis (Levitt et al. 2006). Franck Carbonero1 and H. Rex Gaskins2 A threshold value of 1 Â 108 methanogens per 1 Department of Food Science, University of gram of stool for CH4 to be detected in breath was Arkansas, Fayetteville, AR, USA defined. This was confirmed in a subsequent 2 Institute for Genomic Biology, University of study where breath CH4 excretors had an average Illinois at Urbana-Champaign, University of of 1 Â 109 CFU per g of methanogens in stool, Illinois Cancer Center, Urbana, IL, USA while nonexcretors had ~1 Â 104 CFU per gram or less (Miller and Wolin 1986). It was also shown that the structure of the cellulose- Synonyms degrading bacterial community in individuals differs according to their CH4 status. The rate Methanogens for carriage of methanogens was markedly greater in monozygotic adult twin pairs than in dizygotic twins, but carriage of methanogens Definition between mother and daughter was discordant (Hansen et al. 2011). Thus, host genotype and Methanogenic archaea are obligate anaerobic various environmental factors are among the archaea that produce methane as a metabolic potential determinants of persistent colonization by-product in anoxic conditions. Methanogens by methanogens. are usually coccoid (spherical) or bacilli (rod shaped). There are over 50 described species of methanogens, and they do not form Methanogenic Archaea and Interspecies a monophyletic group. There are three main Hydrogen Transfer metabolisms of methanogenesis: (1) acetate reduction, (2) methanol or methylamine reduc- Interspecies hydrogen transfer is a mutually ben- tion, and (3) H2 oxidation and CO2 reduction. In eficial, unidirectional process that plays a central the human gut, methanogenic archaea rely on role in the anaerobic fermentation of organic methanol and H2/CO2 metabolisms. matter in which one species degrades an organic substrate and releases reducing equivalents in form of hydrogen, which, in turn, is oxidized by Introduction the second species. Generally, the first organism profits from H2 removal by the syntrophic part- The human digestive tract contains variable ner. Colonic methanogenic archaea derive all amounts of gases, of which only H2 and CH4 are (or most) of their metabolic energy from produced by microorganisms. Methane is mainly methanogenesis by reducing CO2 or methanol to produced in the colon and is evacuated in flatus CH4 using H2 or formate as electron donors. Methanogenic Archaea in the Human Microbiome 359 M

Methanogenic archaea are crucial components of methanol as electron acceptor, but the genome the interspecies dihydrogen transfer, a syntrophic also harbors the genes for methanogenesis from relationship where groups of microorganisms con- methylamines; it is not known if this metabolism sume by-products of other microorganisms. While may be utilized in the colonic ecosystem. production of H2 by fermentative microbes is nec- essary for efficient fermentation, its accumulation would rapidly lead to a H2 partial pressure that Physiological Importance and would thermodynamically restrict further fermen- Links with Health tation. Thus, methanogenic archaea, among other hydrogenotrophic microbes, are responsible for H2 Methane has been linked to decreased colonic disposal (CH4 being an inert gas) (Carbonero et al. transit time in patients with irritable bowel syn- 2012). drome of the constipation-predominant type (IBS-C). A correlation was also observed

betweenhighbreathCH4 levels and the occur- Methanogenic Archaea Taxa Present rence of motility disorders. As demonstrated in in the Human Colon human and mammalian model systems, high levels of CH4 are correlated with decreased The two methanogenic species isolated from intestinal motility; however, it has not been con- the human colon, Methanobrevibacter smithii firmed that this increase in breath gases is asso- (Miller et al. 1982) and Methanosphaera ciated with increased abundance of colonic stadtmanae (Miller and Wolin 1985), have dif- methanogens. Global and deep microbial analy- ferent biochemical characteristics. M. smithii sis of stool samples frompatientswithIBSdem- converts CO2 and H2 to CH4, but M. stadtmanae onstrated the presence of methanogenic archaea uses H to reduce methanol to CH . To date, in a higher percentage of those with IBS-C than 2 4 M studies (using both culture-based and molecular- in healthy controls or in patients with diarrhea- based approaches) indicate that M. smithii is the predominant or alternating IBS. predominant methanogen in the human colon; A few, but consistent, reports indicate that M. stadtmanae has been isolated from the the prevalence of the methanogenic phenotype human intestinal tract at a lower abundance. is markedly lower in patients with Crohn’s Several different phylotypes closely related to disease or ulcerative colitis than in healthy M. smithii, M. stadtmanae, M. oralis,or individuals. However, these intriguing findings Methanosarcinales have been identified using have received limited attention, and it is not molecular fingerprinting studies targeting the known whether the potentially reduced preva-

16S ribosomal gene and the functional gene coen- lence of CH4 excretion in IBD is a cause or zyme M reductase (mcrA). The mcrA gene was consequence of, for example, reduced transit persistently detected in colonic biopsy samples time or pH. To date, only a single report, using from 25 healthy individuals with values ranging a molecular-based approach, has compared the from 3.0 Â 102 to 4.5 Â 109 copies per gram incidence and density of colonic methanogens (Nava et al. 2012). These data further confirm in healthy individuals versus patients with IBD. initial observations that breath CH4 concentration Targeting the mcrA gene, Scanlan et al. (2008) reflects the relative abundance and activity of reported that although the abundance of colonic methanogens, and not merely the pres- methanogens was reduced in both IBD groups ence or absence of this hydrogenotrophic group. relative to healthy controls, statistical signifi- A third colonic methanogenic archaea strain has cance was observed only for those with ulcera- recently been cultured to near purity and has been tive colitis. fully sequenced (Borrel et al. 2012). Candidatus In the 1970s and 1980s, numerous studies

Methanomethylophilus alvus was cultivated with reported a higher prevalence of methane CH4 M 360 Microbial Communities in a Shallow-Sea Hydrothermal System excretion among patients with colorectal cancer References (CRC) compared with healthy individuals and, in some cases, patients with other gastrointestinal Borrel G, Harris HMB, Tottey W, et al. Genome sequence disease. However, subsequent studies did not find of “Candidatus Methanomethylophilus alvus” Mx1201, a methanogenic archaeon from the human major differences in CH4 status between patients gut belonging to a seventh order of methanogens. with CRC and healthy individuals, and the use of J Bacteriol. 2012;194:6944–5. the breath test was apparently abandoned as Carbonero F, Benefiel AC, Gaskins HR. Contributions a possible CRC diagnostic tool. It was suggested of the microbial hydrogen economy to colonic homeostasis. Nat Rev Gastroenterol Hepatol. 2012; that observations of higher breath CH4 levels in 9:504–18. patients with CRC might have resulted from Hansen EE, et al. Pan-genome of the dominant human reduced transit time owing to at least partial gut-associated archaeon, Methanobrevibacter smithii, obstruction by tumor tissue. studied in twins. Proc Natl Acad Sci U S A. 2011;108: 4599–606. Obesity has been hypothesized to correlate Levitt MD, Furne JK, Kuskowski M, Ruddy J. Stability of with elevated levels of colonic CH4 and H2. human methanogenic flora over 35 years and a review This hypothesis is based on the assumption that of insights obtained from breath methane measure- increased methanogenesis would improve fer- ments. Clin Gastroenterol Hepatol. 2006;4:123–9. Miller TL, Wolin MJ. Methanosphaera stadtmaniae gen. mentation efficiency, resulting in increased pro- nov., sp. nov.: a species that forms methane by reduc- duction of short-chain fatty acids, which ing methanol with hydrogen. Arch Microbiol. potentially promotes adipogenesis by the host. 1985;141:116–22. An intriguing study detected markedly higher Miller TL, Wolin MJ. Methanogens in human and animal intestinal tracts. Syst Appl Microbiol. 1986;7: numbers of methanogenic archaea in obese indi- 223–9. viduals than in normal-weight individuals or Miller TL, Wolin MJ, de Macario EC, Macario AJ. Isola- patients after gastric bypass (Zhang et al. 2009). tion of Methanobrevibacter smithii from human feces. However, four reports demonstrate a reduced Appl Environ Microbiol. 1982;43:227–32. Nava GM, Carbonero F, Croix JA, Greenberg E, Gaskins number of CH4 excretors among obese individ- HR. Abundance and diversity of mucosa-associated uals compared with lean individuals, a lower hydrogenotrophic microbes in the healthy human level of M. smithii in obese individuals, and colon. ISME J. 2012;6:57–70. greater abundance of methanogens in those with Scanlan PD, Shanahan F, Marchesi JR. Human methanogen diversity and incidence in healthy and anorexia compared with obese and lean individ- diseased colonic groups using mcrA gene analysis. uals. Clearly, much additional work is needed to BMC Microbiol. 2008;8:79. Zhang HS, DiBaise JK, Zuccolo A, et al. Human gut determine the extent to which colonic H2 metab- olism might influence the development of microbiota in obesity and after gastric bypass. Proc Natl Acad Sci U S A. 2009;106:2365–70. obesity.

Summary Microbial Communities in a Methanogenic archaea are relatively rare compo- Shallow-Sea Hydrothermal System nents of the colonic microbiome, but their meta- bolic features are of significance for efficient Kai Tang fermentation and digestive health. State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, China

Cross-Reference Definition

▶ Sulfate-Reducing Bacteria in the Human Gut The cutoff between “shallow” and “deep” hydro- Microbiome thermal vent fields is at a depth of approximately Microbial Communities in a Shallow-Sea Hydrothermal System 361 M

200 m, based on faunal differences (Tarasov vent ecosystems (Fig. 1). However, in marine et al. 2005). Shallow-sea hydrothermal systems hydrothermal systems, the majority of micro- occur at depths of less than 200 m below sea bial surveys are in deep-sea systems, with very level. Metagenomic analysis provides useful little attention paid to their shallow-sea coun- insights into microbial composition and meta- terparts although they are much easier to access bolic potential encoded in genetic material and can often be explored via scuba diving. obtained from microbial communities in The microbial communities’ composition a shallow-sea hydrothermal system. has been investigated in the shallow-sea sys- tems (Fig. 1), including the sites at Kueishan Island, Taiwan, China (Zhang et al. 2012; Introduction Tang et al. 2013); Aeolian Islands (Vulcano Island and Panarea Island), Italy (Maugeri Approximately 50–60 shallow-sea hydrother- et al. 2009, 2010, 2013a, b;Huang2012; mal systems are currently known, occurring Manini et al. 2008); Ambitle Island (Meyer- active coastal or submarine volcanoes, with sys- Dombard et al. 2012); D. Joa˜o de Castro Bank, tems located along arcs, mid-ocean ridges, and Azores (Chellandi et al. 2012); Milos Island, in island arc-related environments and even in Greece (Brinkhoff et al. 1999;Sievert continental margins. The fluids of the examined et al. 1999, 2000a, b); Taketomi Island, Japan vent vary considerably in temperature, pH, and (Hirayama et al. 2007); and Eyjafjordur, Ice- chemical composition. Vent waters are charac- land (Marteinsson et al. 2001). With advances terized by wide temperature ranges in sequencing technologies, large-scale geno- (10–135 C). Main gas compositions observed mic surveys of microbial communities at shallow hydrothermal vents are usually dom- (metagenomics) have beenappliedtoreveala inated by carbon dioxide (CO ) with different new and enormous bacterial diversity and met- 2 M concentrationofhydrogensulfide(H2S), meth- abolic potential in shallow-sea hydrothermal ane (CH4), and hydrogen (H2). The presence of environments. Two of the better studied exam- gas phase and enrichment of oxygen compared ples are hydrothermal systems at Eolian Islands to deep-sea vents is as well a profound feature of (named Black Point and Hot Lake) (Huang shallow hydrothermal systems. Elemental sulfur 2012; Maugeri et al. 2013a) and Kueishan (S0) is naturally enriched in Kueishan Island Island (Fig. 2)(Zhangetal.2012;Tang shallow-sea hydrothermal fluids. An arsenic- et al. 2013). rich, shallow-sea hydrothermal system is located in Tutum Bay, Ambitle Island, Papua New Guinea. Geochemistry of shallow hydro- Microbial Diversity and Community thermal vents is strongly influenced not only by Structure the temperature and chemical composition of the hydrothermal fluids but also by the activity The most dominant sequences are Bacteria in of microorganisms. The extensive mixing of all the shallow-sea hydrothermal system thermal fluids with oxygenated seawater gener- metagenomes, contributing for more than 90 % ates microscale redox gradients within shallow- of the total sequences. The phototrophic mem- sea hydrothermal systems, thereby affecting bers of Cyanobacteria and Chlorobi are observed the phyletic composition and metabolic activi- in the shallow-sea hydrothermal systems. ties of microbial communities at these sites. Shallow-sea hydrothermal venting contains Moreover, the penetration of light might allow many relatively rare taxa. The percentages of for photosynthesis at shallow-sea hydrothermal archaeal sequences in shallow-sea systems systems. metagenomic datasets are significantly less than The biological data have been published for deep-sea hydrothermal vent, usually accounting approximately 22 shallow-water hydrothermal for less than 1 % of the total sequences. M 362 Microbial Communities in a Shallow-Sea Hydrothermal System

Microbial Communities in a Shallow-Sea Hydrother- communities. DGGE denaturing gradient gel electropho- mal System, Fig. 1 Shallow-water hydrothermal sys- resis, FISH fluorescence in situ hybridization, 16S rRNA tems around the world where biological data have been 16S rRNA gene clone libraries, RFLP restriction fragment published. Red circles indicate hydrothermal sites with length polymorphism analysis, 454 454 pyrosequencing, known data on microbial communities; gray circles indi- Illumina Illumina-based sequencing technology cate hydrothermal sites with no surveys on microbial

Microbial Communities in a Shallow-Sea Hydrothermal System, Fig. 2 Photographs of Kueishan Island (left) and venting (right) (Courtesy of Prof. Chen-Tung Arthur Chen (National Sun Yat-Sen University, Taiwan))

The phylogenetic groups retrieved from the Epsilonproteobacteria dominate the communi- shallow-sea hydrothermal metagenomes are ties from the white vent. A readily shift occurs represented in Table 1. in the predominant microbial community from a Ribosomal tag pyrosequencing based on 16S Gammaproteobacteria to Epsilonproteobacteria rRNA shows statistically significant differences across the redox gradients at the white vent sites. between the bacterial and archaeal communities Dominant Gammaproteobacteria are related to of the two Kueishan Island hydrothermal systems the sulfide-oxidizing genus Thiomicrospira, (yellow and white vents) with distinct geochem- amounting to 76–99 % of the total gammaproteo- ical parameters (Zhang et al. 2012). The bacterial tags in the DNA-based libraries. Gammaproteobacteria dominate the communi- Thiomicrospira is one of the most abundant ties from the yellow vent, while the culturable, sulfur oxidizers at shallow vents. Microbial Communities in a Shallow-Sea Hydrothermal System 363 M

Microbial Communities in a Shallow-Sea Hydrothermal System, Table 1 Taxonomic profiles of shallow-sea hydrothermal systems based on the metagenomic datasets of 16S rRNA Sample Site name Sampling location name Resource Bacterial phylotypes Archaeal phylotypes Black point The study site was Sediment Sediment Alphaproteobacteria Euryarchaeota located inside the area (Rhodovulum), (Methanosarcina, delimited by Dattilo, Gammaproteobacteria Halomicrobium, Bottaro, Lisca Nera (Thiohalospira, Halobiforma, islets, off the eastern Thiomicrospira), Halobacterium, coast of Panarea Island Actinobacteria, Natronomonas), (38380N, 15060E) Deltaproteobacteria, Crenarchaeota Bacteroidetes, (Staphylothermus, Acidobacteria, Thermocladium) Verrucomicrobia, Epsilonproteobacteria, Cyanobacteria, Deinococcus-Thermus, Planctomycetes, Firmicutes, Deferribacteres Fluid Fluid Chlorobi (Chlorobium), Euryarchaeota Betaproteobacteria, (Methanococcus), Alphaproteobacteria, Crenarchaeota Actinobacteria, Firmicutes, (Thermocladium) Gammaproteobacteria (Thiomicrospira), Epsilonproteobacteria (Sulfurimonas, Arcobacter, Sulfurospirillum), Bacteroidetes, Fusobacteria, M Deltaproteobacteria, Acidobacteria, Cyanobacteria Hot lake The study site was Hot Sediment Epsilonproteobacteria Crenarchaeota and located approximately lake I (Sulfurovum, Sulfurimonas, Euryarchaeota 2 km east of the main Arcobacter, (Thermococcaceae island of Panarea Sulfurospirillum, Thermoplasmatales, (38380N, 15060E) Campylobacter, Halobacteria) Nitratiruptor), Gammaproteobacteria (Thiomicrospira), Deltaproteobacteria (Desulfobacteraceae), Bacteroidetes, Chlorobi, Firmicutes, Aquificae, Cyanobacteria Hot Sediment Epsilonproteobacteria Crenarchaeota lake II (Sulfurovum, Nitratiruptor, (Desulfurococcaceae), Sulfurospirillum, Euryarchaeota Sulfurimonas, Wolinella, (Thermococcaceae, Arcobacter), Thermoplasmatales, Gammaproteobacteria, Halobacteria), Deltaproteobacteria, Korarcheota Firmicutes, Bacteroidetes, Chlorobi, Aquificae, Cyanobacteria (continued) M 364 Microbial Communities in a Shallow-Sea Hydrothermal System

Microbial Communities in a Shallow-Sea Hydrothermal System, Table 1 (continued) Sample Site name Sampling location name Resource Bacterial phylotypes Archaeal phylotypes Kueishan The study site was Yellow Fluid Epsilonproteobacteria Euryarchaeota (Marine Island located 1 km east of vent (Sulfurimonas, Nautilia, Group II, hydrothermal Kueishantao Island, near Arcobacter, Caminibacter, Thermococcus, Marine vent the southern end of the Lebetimonas, Group III), Okinawa Trough Thioreductor), Crenarchaeota (Marine (121570E, 24500N) Gammaproteobacteria Group I) (Thiomicrospira, Vibrio), Alphaproteobacteria (SAR11 clade, Rhodobacteraceae, Rhodospirillaceae), Cyanobacteria, Actinobacteria, Deltaproteobacteria, Bacteroidetes, Acidobacteria, Verrucomicrobia White Fluid Epsilonproteobacteria Euryarchaeota vent (Nautilia, Caminibacter, (Thermococcus, Lebetimonas, Thioreductor, Marine Group II), Campylobacter), Crenarchaeota (Marine Alphaproteobacteria Group I) (SAR11 clade, Rhodobacteraceae, Rhodospirillaceae), Gammaproteobacteria (Thiomicrospira, Hydrogenovibrio), Bacteroidetes, Cyanobacteria, Actinobacteria, Deltaproteobacteria, Acidobacteria, Verrucomicrobia aThe relatively rare taxa are not presented here and more taxa information are found in references (Maugeri et al. 2013a; Huang 2012; Zhang et al. 2012). Most of the significant genus are given in brackets

Members of the order Nautiliales (Nautilia, are also relatively abundant within the Caminibacter, and Lebetimonas) account for Alphaproteobacteria. Another significant group 77–90 % of the total Epsilonproteobacteria tags is Cyanobacteria at the shallow-sea hydrother- in the DNA-based libraries, and Epsilonproteo- mal system. Euryarchaeota is the dominant bacteria are typical members of hydrothermal group in both samples. Thermococcus and sites, both at deep- and shallow-sea vent loca- Marine Group II are most abundant in the tions, where they are numerically abundant bac- DNA-based libraries from the white and yellow teria and play a key role in catalyzing the elemental vents, respectively. Marine Group III also sulfur reduction and oxidation (Campbell accounts for a significant proportion of the et al. 2006). Alphaproteobacteria are, overall, DNA-based libraries from the yellow vent. dominated by the SAR11 clade. In addition, Marine Group I comprises the major fraction of the Rhodobacteraceae and Rhodospirillaceae the Crenarchaeota. Other bacterial and archaeal Microbial Communities in a Shallow-Sea Hydrothermal System 365 M taxa identified in the shallow-sea hydrothermal affiliated with Thiomicrospira-like organisms. systems are shown in Table 1. In addition, Sqr gene coding for sulfide:quinone At Black Point, the bacterial communities oxidoreductase involved in sulfide oxidation from sediment are dominated by sequences affil- sequences is detected in the vent and surface iated with members of Alphaproteobacteria water, indicating that sulfide oxidation might (Rhodovulum), Gammaproteobacteria (Thioha- be an important process in the shallow-sea lospira and Thiomicrospira), Deltaproteo- hydrothermal system. The bacterial community bacteria, and Epsilonproteobacteria, whereas in the surface water possesses genes encoding Betaproteobacteria, Alphaproteobacteria, for key enzymes of other pathways involved Gammaproteobacteria, and Epsilonproteo- in sulfur oxidation: genes encoding bacteria (Sulfurimonas, Arcobacter, and adenylylsulfate reductase, sulfide dehydroge- Sulfurospirillum) are the high abundant group in nase, sulfite oxidase, and thiosulfate sulfur trans- fluid (Maugeri et al. 2013a). Bacterial genera ferase. Genes encoding demethylase for affiliated with Actinobacteria and Bacteroidetes dimethylsulfoniopropionate (DMSP) degrada- are common to fluid and sediment samples, tion are found in the surface water dataset, and while Chlorobi (Chlorobium) are the high abun- the sequences are affiliated with those found in dant group only in fluid. The Euryarchaeota the Roseobacter and SAR11 clade within dominate archaeal communities. Euryarchaeotal Alphaproteobacteria. Genes encoding for poly- sequences are affiliated with the family sulfide reductase (Psr) are present in the vent Methanococcus and Methanosarcina (Maugeri metagenome, resulting the reduction of polysul- et al. 2013a). At Hot Lake, these bacterial gene fide derived from elemental sulfur to sulfide. Psr sequences are found to be mainly affiliated to gene sequences in the vent are annotated to Epsilonproteobacteria (Huang 2012). The species of Nautiliales. The genes coding for archaeal community is composed primarily of Ni–Fe hydrogenase are present in the vent M Euryarchaeota (Thermococcaceae, Thermo- metagenome, enabling bacteria to use H2 as an plasmatales, Halobacteria) (Huang 2012). energy source. Key genes for the reductive tricarboxylic acid (rTCA) cycle are found in the vent datasets, Novel Genes for Sulfur Metabolism and including genes coding for ATP-dependent cit- Carbon Fixation rate lyase, pyruvate:ferredoxin oxidoreductase, and 2-oxoglutarate:ferredoxin oxidoreductase. The functional metagenomic analyses reveal Genes encoding ribulose-1,5-bisphosphate car- that the metabolic profiles of the chemoautotro- boxylase (RuBisCO) and phosphoribulokinase phic members in the shallow-sea hydrothermal that mediate the Calvin–Bassham–Benson field are similar to those in deep-sea hydrother- (CBB) cycle are enriched in the surface water mal fields, with sulfur metabolism and carbon dataset. Thus, the chemoautotrophic microorgan- fixation being of particular importance. The isms in the vent and in the surface water might white vent and the surface water near Kueishan possess the rTCA cycle and the CBB cycle for Island differ significantly in microbial carbon carbon fixation in response to carbon dioxide fixation and sulfur metabolism (Tang highly enriched in the environment, which is et al. 2013)(Fig.3). Key genes for the Sox possibly fueled by geochemical energy with sul- pathway in the surface water metagenome are fur and hydrogen (Fig. 3). Similarly, more abundant than in the vent dataset, includ- sulfur-reducing Epsilonproteobacteria such as ing soxA, soxB, soxC, soxD, soxX, soxY, and Sulfurovum-like organisms are dominating sur- soxZ. The sox genes encode enzymes for the face sediments of Hot Lake where they gain oxidation of inorganic sulfur compounds to sul- energy from sulfur metabolism to fix CO2 by fate. Most of the sox gene sequences are the rTCAcycle (Huang 2012). Shallow-sea M 366 Microbial Communities in a Shallow-Sea Hydrothermal System

Microbial Communities in a Shallow-Sea Hydrothermal System, Fig. 3 A schematic diagram of metabolic profiling of shallow-sea hydrothermal systems near Kueishan Island inferred from metagenomic analysis

systems exist within the photic zone and photo- communities from the hydrothermal plumes synthetic organisms likely contributed to carbon are dominated by Euryarchaeota.Theyareall fixation as well using light as the alternative likely to contribute to local biogeochemical ele- energy source. No genes encoding the key ment cycles. enzymes in methanogenesis/methanotrophy (mcrA and pmoA) are found in a shallow-sea hydrothermal system near Kueishan Island. This Cross-References result suggests that these microbial processes are not dominant, although their fluids contained ▶ Ocean Metagenomics abundant CH4. References Summary Brinkhoff T, Sievert SM, Kuever J, Muyzer G. Distribution and diversity of sulfur-oxidizing The shallow-sea hydrothermal microbial com- Thiomicrospira spp. at a shallow-water hydrothermal munities are phylogenetically and metabolically vent in the Aegean Sea (Milos, Greece). Appl Environ diverse. Within individual communities, the Microbiol. 1999;65:3843–9. Campbell BJ, Engel AS, Porter ML, Takai K. The versa- distributions of organisms are affected by the tile epsilon-proteobacteria: key players in sulphidic environmental gradient created by the mixing of habitats. Nat Rev Microbiol. 2006;4:458–68. vent fluids with ambient seawater. The sulfur- Chellandi M, Raju R, Chinnarajan R, Ana C, Ricardo SS, reducing and sulfide-oxidizing chemolithoau- Ram MM. Bacterial diversity and their adaptations in the shallow water hydrothermal vent at D. Joa˜ode totrophs (such as Nautiliales-like organisms Castro Seamount (DJCS), Azores, Portugal. Cah Biol and Thiomicrospira-like organisms) account Mar. 2012;53:65–76. greatly for the primary biomass synthesis, and Hirayama H, Sunamura M, Takai K, Nunoura T, Noguchi T, that microbial sulfur metabolism fueled micro- Oida H, et al. Culture-dependent and -independent char- acterization of microbial communities associated with bial energy flow and element cycling in the a shallow submarine hydrothermal system occurring shallow hydrothermal systems. The taxes or within a coral reef off Taketomi Island, Japan. Appl genes associated with phototrophy and hetero- Environ Microbiol. 2007;73:7642–56. trophy are more prevalent in the shallow-sea Huang CI. Molecular ecology of free-living chemoauto- trophic microbial communities at a shallow-sea hydro- metagenomes than those typically found in thermal vent. Ph.D. dissertation, Universitaet Bremen; deep-sea hydrothermal systems. The archaeal 2012. Microbial Community in the Sputum of Patients with Pulmonary Tuberculosis 367 M

Manini E, Luna GM, Corinaldesi C, Zeppilli D, Bortoluzzi G, Caramanna G, et al. Prokaryote diversity Microbial Community in the Sputum and virus abundance in shallow hydrothermal vents of the Mediterranean Sea (Panarea Island) and the Pacific of Patients with Pulmonary Ocean (north Sulawesi-Indonesia). Microb Ecol. Tuberculosis 2008;55:626–39. Marteinsson VT, Hauksdo´ttir S, Hobel CFV, Zelin Cui Kristmannsdo´ttir H, Hreggvidsson GO, Kristja´nsson JK. Phylogenetic diversity analysis of subterranean Department of Laboratory Medicine, Shanghai hot springs in Iceland. Appl Environ Microbiol. First People’s Hospital, Medical College, 2001;67:4242–8. Shanghai Jiaotong University, Shanghai, China Maugeri TL, Lentini V, Gugliandolo C, Italiano F, Department of Medical Microbiology and Cousin S, Stackebrandt E. Bacterial and archaeal populations at two shallow hydrothermal vents off Parasitology, Shanghai Jiao Tong University Panarea Island (Eolian Islands, Italy). Extremophiles. School of Medicine, Shanghai, China 2009;13:199–212. Maugeri TL, Lentini V, Gugliandolo C, Cousin S, Stackebrandt E. Microbial diversity at a hot, shallow- sea hydrothermal vent in the southern Tyrrhenian Sea Synonyms (Italy). Geomicrobiol J. 2010;27:380–90. Maugeri TL, Gugliandolo C, Lentini V. Diversity of pro- Sputum microbiome in patients with pulmonary karyotes at a shallow submarine vent of Panarea Island tuberculosis (Italy) by high-throughput sequencing. Atti Accad Pelorit Pericol Cl Sci Fis Mat Nat. 2013a;91:A1. Maugeri TL, Lentini V, Spano A, Gugliandolo C. Abun- dance and diversity of picocyanobacteria in shallow Definitions hydrothermal vents of Panarea Island (Italy). Geomicrobiol J. 2013b;30:93–9. Meyer-Dombard DR, Price RE, Pichler T, Amend Pulmonary tuberculosis patients: the patients were clinically diagnosed with pulmonary tuber- JP. Prokaryotic populations in arsenic-rich shallow- M sea hydrothermal sediments of Ambitle Island, Papua culosis based on sputum smear, sputum culture, New Guinea. Geomicrobiol J. 2012;29:1–17. and computed tomography results. All patients Sievert SM, Brinkhoff T, Muyzer G, Ziebis W, Kuever J. Spatial heterogeneity of bacterial populations along an were free of HIV. None of the patients had environmental gradient at a shallow submarine hydro- taken antibiotics for at least 3 months before thermal vent near Milos Island, (Greece). Appl Envi- sampling. ron Microbiol. 1999;65:3834–42. Healthy participants: the participants were Sievert SM, Kuever J, Muyzer G. Identification of 16S ribosomal DNA defined bacterial populations at free of basic pulmonary diseases, severe lung a shallow submarine hydrothermal vent near Milos disease, severe oral disease, systemic disease, Island (Greece). Appl Environ Microbiol. 2000a;66: and other known diseases such as obesity or dia- 3102–9. betes, which could affect the microbial composi- Sievert SM, Ziebis W, Kuever J, Sah K. Relative abun- dance of Archaea and Bacteria along a thermal gradi- tion of the respiratory tract. Volunteers with ent of a shallow-water hydrothermal vent quantified by a history of smoking or drinking were also rRNA slot-blot hybridization. Microbiology. 2000b; excluded. The healthy participants had not taken 146:1287–93. any antibiotics for at least 3 months before sam- Tang K, Liu K, Jiao N, Zhang Y, Chen CT. Functional metagenomic investigations of microbial communities pling. The samples from healthy participants in a shallow-sea hydrothermal system. PLoS One. were a mixture of saliva and pharyngeal secre- 2013;8:e72958. tions collected by deep coughing in the early Tarasov VG, Gebruk AV, Mironov AN, Moskalev LI. morning before gargling. Deep-sea and shallow-water hydrothermal vent com- munities: two different phenomena? Chem Geol. Microbiota: the bacterial composition. 2005;224:5–39. Zhang Y, Zhao Z, Chen CT, Tang K, Su J, Jiao NZ. Sulfur metabolizing microbes dominate microbial communi- ties in andesite-hosted shallow-sea hydrothermal sys- tems. PLoS ONE. 2012;7:e44593. The essay mainly summarized cited from Cui et al. (2012). M 368 Microbial Community in the Sputum of Patients with Pulmonary Tuberculosis

Introduction The Human Sputum Microbiome in Pulmonary Tuberculosis Chronic pulmonary tuberculosis poses a global health re-emergency. It has been known In our work, we collected 31 sputum samples for many centuries and is mainly caused by the from pulmonary tuberculosis patients from bacillus Mycobacterium tuberculosis.Many Shanghai Pulmonary Hospital and 24 respiratory reports have revealed coinfection with different secretion samples from healthy participants in strains or species of Mycobacterium in pulmo- Shanghai, China, as controls, and investigated nary tuberculosis patients. Mixed infection the composition of the microbiota in the lower with Beijing and non-Beijing strains of respiratory tract of pulmonary tuberculosis M. tuberculosis (Huang et al. 2010) has been patients through the 16S rRNA V3 hyper- reported to mediate the increased reinfection variable regions amplified using bar-coded rate in regions with a high incidence of tubercu- primers and pyro-sequenced using Roche losis. Similarly, MAC (Mycobacterium avium 454 FLX. complex)andM. tuberculosis coexist in The similarities between the respiratory tract some patients with combined mycobacterial secretion microbiota of the healthy participants infections (Khan et al. 2010). The systems and sputum microbiota of the pulmonary tuber- biology concept of persistent infection is that culosis patients were estimated by calculating infectious diseases reflect an equilibrium UniFrac distances. The healthy participants between the host and the pathogen that is were clustered together, while the pulmonary established and maintained by a broad network tuberculosis patients were divided into several of interactions. These interactions occur across different sub-branches. scales that range from molecular to cellular, to A total of 24 phyla were detected in the pul- whole organism and population levels (Young monary tuberculosis samples, while 17 phyla et al. 2008). were detected in healthy participants. Actino- The development of nucleotide sequencing bacteria, Bacteroidetes, Proteobacteria, and has helped reveal the importance of microbiota Crenarchaeota were widely and abundantly dis- to human health (Blaser and Falkow 2009). tributed among nearly all of the samples. In light of the recent discovery of cystic Firmicutes (37.02 %), Bacteroidetes (29.01 %), fibrosis-associated lung microbiota, Delhaes and Proteobacteria (16.37 %), Crenarchaeota Monchy et al. discussed the microbial commu- (3.16 %), and Actinobacteria (2.89 %) were com- nity as a unique pathogenic entity (Delhaes mon in the healthy participants, while Firmicutes et al. 2012). Huang and Lynch emphasized that (41.62 %), Bacteroidetes (7.64 %), microbiota, as a collective entity, may contribute Proteobacteria (17.99 %), Actinobacteria to pathophysiologic processes associated with (21.20 %), and Crenarchaeota (7.5 %) were com- chronic airway disease (Huang and Lynch mon in the pulmonary tuberculosis patients. 2011). Robinson et al. also suggested the conser- Chlamydiae, Chloroflexi, Cyanobacteria/Chloro- vation or restoration of the normal community plast, Deinococcus-Thermus, Elusimicrobia, structure and function of host-associated Euryarchaeota, SR1, Spirochaetes, Synergistetes, microbiota should be included in the prevention and Tenericutes were found in both the healthy and treatment of human disease (Robinson participants and pulmonary tuberculosis patients, et al. 2010). Understanding the microbial compo- although they were rare in some samples. sition in the respiratory tract of pulmonary tuber- Aquificae, Caldiserica, Gemmatimonadetes, culosis patients may enhance our awareness of Lentisphaerae, Planctomycetes, Thermodesulfo- microbiota as a collective entity or even collec- bacteria, and Verrucomicrobia were unique to the tive pathogenic entity, and the role this entity pulmonary tuberculosis samples. Moreover, in plays in the onset and development of pulmonary healthy participants, Deinococcus-Thermus, tuberculosis. Bacteroidetes, and Fusobacteria accounted for Microbial Community in the Sputum of Patients with Pulmonary Tuberculosis 369 M

0.01 %, 29.01 %, and 8.06 %, respectively. How- Discussion and Conclusion ever, in pulmonary tuberculosis patients, Deinococcus-Thermus increased to 0.93 %, This study provides the first report on the micro- Bacteroidetes, and Fusobacteria decreased to bial composition of the lower respiratory tract of 7.64 % and 1.35 %, respectively. pulmonary tuberculosis patients. The results To better characterize the sputum microbiomes, revealed that the microbial composition of the the sequences were sorted to the genera level. lower respiratory tract in pulmonary tuberculosis A total of 614 genera were observed; 235 genera patients was more diverse (p < 0.05) than in were observed in healthy participants, and 564 gen- healthy participants. Charlson et al. reported era were found in pulmonary tuberculosis patients, that the microbial composition of saliva or phar- although more than half of these accounted for only ynx secretions can reflect the microbial commu- a small fraction of the total sequences. Streptococ- nities in the lower respiratory tract, and their cus, Granulicatella, Actinomyces, Prevotella,and results showed that there is a topographical con- Veillonella were predominant in the microbiota of tinuity of bacterial populations in the healthy both healthy participants and pulmonary tubercu- human respiratory tract (Charlson et al. 2011). losis patients. In contrast, Anoxybacillus, Klebsi- Therefore, we chose to use sputum and respira- ella, Acinetobacter, Pilibacter, Abiotrophia, tory secretions in this study. However, the best Paucisalibacillus,andRothia were more abundant samples to use would be lung lavage fluid, which in pulmonary tuberculosis patients than healthy perfectly reflects the lower microbial composi- participants. Neisseria, Porphyromonas, tion of the respiratory tract. However, obtaining TM7_genera_incertae_sedis, Parvimonas, Cam- lung lavage fluid is challenging, especially from pylobacter, Haemophilus,andFusobacterium healthy volunteers, because lung lavage is painful were less common in pulmonary tuberculosis and may even be harmful. This may raise some patients than healthy participants. Furthermore, ethical issues. In contrast, sputum and respiratory M Stenotrophomonas, Cupriavidus, Pseudomonas, secretions are easily obtained through noninva- Thermus, Sphingomonas, Brevundimonas, sive, patient-friendly collection methods. There- Brevibacillus, Methylobacterium, Diaphorobacter, fore, we chose to analyze sputum and respiratory Comamonas, Mobilicoccus,andFervidicoccus tract secretions in our study. were unique to and widespread among the pulmo- A previous study showed that less than 1 % of nary tuberculosis patients. commensal organisms are able to grow under lab- Many genera were unique to the sputum of oratory conditions (Staley and Konopka 1985); pulmonary tuberculosis patients. Phenyl- therefore, traditional cultivation-based strategies obacterium, Stenotrophomonas, Cupriavidus,and for analyzing the complexity and genetic diversity Pseudomonas were found in nearly half of the of microbial communities are strongly biased. tuberculosis patients we enrolled; furthermore, However, modern methods, based on bar-coded their total copies accounted for more than 1 % of primers and 454 pyro-sequencing, allow for the total sequences from the sputum of pulmonary a thorough profiling of the microbiota of each tuberculosis patients. Other genera such as enrolled person (Han et al. 2012; Zhou Sphingomonas, Mobilicoccus, Brevundimonas, et al. 2010). Published studies have also proved Brevibacillus,andDiaphorobacter were much that the 16S rRNA V3 region sequence is ideally more widely detected in pulmonary tuberculosis suited for distinguishing all bacterial species to the patients, even though they accounted for only genus level, except for closely related Enterobac- a small number of sequences. Several rare genera teriaceae (Chakravorty et al. 2007). were present in the sputum of pulmonary tubercu- The lower respiratory tract microbiome of pul- losis patients, such as Thermus, Pelomonas, monary tuberculosis patients was distinct from that Methylobacterium, Comamonas, Lactobacillus, of the healthy participants. The pulmonary tuber- Thermobacillus, Auritidibacter, Lapillicoccus, culosis patients formed a clear cluster that was and Devriesea. separate from the healthy participants based on M 370 Microbial Community in the Sputum of Patients with Pulmonary Tuberculosis their microbiota. The phyla Bacteroidetes and The host response to pathogens is characterized Fusobacteria were significantly underrepresented by rapid recognition combined with strong innate in pulmonary tuberculosis patients compared with (i.e., inflammatory) and adaptive immune healthy participants, while Actinobacteria was sig- responses, enabling microbial eradication often at nificantly overrepresented in pulmonary tubercu- the cost of significant tissue damage. Furthermore, losis patients. Moreover, bacteria from the phylum the host is constantly facing the challenge of dis- Deinococcus-Thermus were widely distributed in criminating between symbiotic and pathogenic bac- pulmonary tuberculosis patients (15/31), but rarely teria to organize an appropriately and adaptive found in healthy participants, and the phyla response (Sansonetti 2011). These responses lead Aquificae, Caldiserica, Gemmatimonadetes, to the extensive fibrosis associated with recurring Lentisphaerae, Planctomycetes, Thermodesulfo- infections, possibly leading to a decreased clear- bacteria, and Verrucomicrobia were unique to pul- ance of lymph and lymph-associated particles monary tuberculosis patients. The genera from the infected region (Ardies 2003). The lungs Klebsiella, Pseudomonas,andAcinetobacter were of individual patients typically contain diverse more common in pulmonary tuberculosis patients, lesions with varied overall structures that change and we postulated that these bacteria may aggra- over time (Young et al. 2008). Ultimately, a strong vate the syndrome of pulmonary tuberculosis in host response to the clearance of M. tuberculosis these patients. The genera Phenylobacterium, may produce local lesions in the lung. This may in Stenotrophomonas, Cupriavidus, Caulobacter, turn increase the possibility that foreign bacteria Pseudomonas, Thermus,andSphingomonas were will colonize or grow in the lower respiratory unique to and widely distributed in patients with tract. During the initial disease-causing invasion pulmonary tuberculosis. of the lung by M. tuberculosis, a strong host The respiratory tract microbiota of pulmonary immune response may kill or clear some normal tuberculosis patients who suffer from chronic bacteria in the lower respiratory tract of pulmonary infection might be important in the pathogenicity tuberculosis patients. This may be why the of this disease. The variety of bacterial genera, populations of many normal bacteria are decreased especially the presence of some abnormal genera or absent from the microbiota of the pulmonary in the sputum of pulmonary tuberculosis patients, tuberculosis patients. At the same time, a strong suggested that the pulmonary tuberculosis patient host strong immune response against the pathogen lung is an ecological niche that can support the may damage or produce lesions in the lung tissue, growth of a wide variety of bacteria, especially and consequently the microenvironment of the certain abnormal bacteria. These abnormal gen- lower respiratory tract may favor colonization or era are reportedly widespread in the environment, even host invasion by foreign microorganisms. and some have been reported to be associated These foreign bacteria may cooperate with with infectious diseases (Chuvochina M. tuberculosis to cause additional damage to the et al. 2011; Coenye et al. 2002; Fishman 2011; lung tissue. In this model, although M. tuberculosis Ryan and Adley 2010). Coenye et al. also plays a central role in the disease, the other bacteria reported the isolation of unusual bacteria from may assist in the destruction of the lung tissue, the respiratory secretions of cystic fibrosis especially in active tuberculosis. If M. tuberculosis patients (Coenye et al. 2002). However, there is eliminated promptly, however, lung function can are few reports on whether these organisms can be restored. Further investigation will be required to cause human disease. The lower respiratory tract determine whether pulmonary tuberculosis is the is an open system and can communicate freely cause of increased foreign bacterial colonization with the environment. We speculated that in pul- of the lower respiratory tract or vice versa (i.e., the monary tuberculosis patients, the lung microen- presence of foreign bacteria aggravates the symp- vironment may become more susceptible to toms of pulmonary tuberculosis). It is also possible colonization by some foreign microbes. that both occur simultaneously. Microbial Diversity and Novelty Along Salinity Gradients 371 M

Our study concluded that the microbial com- Huang YJ, Lynch SV. The emerging relationship between position of the respiratory tract of pulmonary the airway microbiota and chronic respiratory disease: clinical implications. Expert Rev Respir Med. tuberculosis patients is more complex than that 2011;5(6):809–21. of healthy participants and that many foreign Huang HY, Tsai YS, Lee JJ, Chiang MC, Chen YH, bacteria were found in the sputum of pulmonary Chiang CY, Lin NT, Tsai PJ. Mixed infection with tuberculosis patients. The roles of these foreign Beijing and non-Beijing strains and drug resistance pattern of Mycobacterium tuberculosis. J Clin bacteria in the onset or development of pulmo- Microbiol. 2010;48(12):4474–80. nary tuberculosis should be considered by Khan Z, Miller A, Bachan M, Donath J. Mycobacterium clinicians. Avium Complex (MAC) lung disease in two inner city community hospitals: recognition, prevalence, co-infection with Mycobacterium Tuberculosis (MTB) and Pulmonary Function (PF) improvements References after treatment. Open Respir Med J. 2010;4:76–81. Robinson CJ, Bohannan BJ, Young VB. From structure to function: the ecology of host-associated microbial Ardies CM. Inflammation as cause for scar cancers of the communities. Microbiol Mol Biol Rev. 2010;74(3): lung. Integr Cancer Ther. 2003;2(3):238–46. 453–76. Blaser MJ, Falkow S. What are the consequences of the Ryan MP, Adley CC. Sphingomonas paucimobilis: disappearing human microbiota? Nat Rev Microbiol. a persistent Gram-negative nosocomial infectious 2009;7(12):887–94. organism. J Hosp Infect. 2010;75(3):153–7. Chakravorty S, Helb D, Burday M, Connell N, Alland Sansonetti PJ. To be or not to be a pathogen: that is the D. A detailed analysis of 16S ribosomal RNA gene mucosally relevant question. Mucosal Immunol. segments for the diagnosis of pathogenic bacteria. 2011;4(1):8–14. J Microbiol Methods. 2007;69(2):330–9. Staley JT, Konopka A. Measurement of in situ activities Charlson ES, Bittinger K, Haas AR, Fitzgerald AS, of nonphotosynthetic microorganisms in aquatic Frank I, Yadav A, Bushman FD, Collman and terrestrial habitats. Annu Rev Microbiol. 1985; RG. Topographical continuity of bacterial populations 39:321–46. in the healthy human respiratory tract. Am J Respir Young D, Stark J, Kirschner D. Systems biology of per- Crit Care Med. 2011;184(8):957–63. sistent infection: tuberculosis as a case study. Nat Rev M Chuvochina MS, Marie D, Chevaillier S, Petit JR, Microbiol. 2008;6(7):520–8. Normand P, Alekhina IA, Bulat SA. Community var- Zhou Y, Lin P, Li Q, Han L, Zheng H, Wei Y, Cui Z, Ni Y, iability of bacteria in alpine snow (Mont Blanc) Guo X. Analysis of the microbiota of sputum samples containing Saharan dust deposition and their snow from patients with lower respiratory tract infections. colonisation potential. Microbes Environ. Acta Biochim Biophys Sin (Shanghai). 2010;42(10): 2011;26(3):237–47. 754–61. Coenye T, Goris J, Spilker T, Vandamme P, LiPuma JJ. Characterization of unusual bacteria isolated from respiratory secretions of cystic fibrosis patients and description of Inquilinus limosus gen. nov., sp. nov. J Clin Microbiol. 2002;40(6):2062–9. Cui Z, Zhou Y, Li H, Zhang Y, Zhang S, Tang S, Guo Microbial Diversity and Novelty X. Complex sputum microbial composition in patients Along Salinity Gradients with pulmonary tuberculosis. BMC Microbiol. 2012;12(1):276. Emilio Ortega Casamayor and Delhaes L, Monchy S, Frealle E, Hubans C, Salleron J, Leroy S, Prevotat A, Wallet F, Wallaert B, Dei-Cas E, Xavier Triado´-Margarit et al. The airway microbiota in cystic fibrosis: Biodiversity and Biogeodynamics Group, Center a complex fungal and bacterial community–implica- for Advanced Studies of Blanes-Spanish Council tions for therapeutic management. PLoS ONE. for Research, CEAB-CSIC, Girona, Spain 2012;7(4):e36313. Fishman JA. Infections in immunocompromised hosts and organ transplant recipients: essentials. Liver Transpl. 2011;17 Suppl 3:S34–7. Synonyms Han MK, Huang YJ, Lipuma JJ, Boushey HA, Boucher RC, Cookson WO, Curtis JL, Erb-Downward J, Lynch SV, Sethi S, et al. Significance of the microbiome in Genetic novelty; Hypersaline environments; obstructive lung disease. Thorax. 2012;67(5):456–63. Microbial biodiversity; Salt gradient M 372 Microbial Diversity and Novelty Along Salinity Gradients

Definition spring waters rich in sodium and chloride, man-made ponds that act as crystallizers where Microbial diversity is determined by the genetic salt precipitates have also been traditionally used diversity of ribosomal genes 16S for bacteria and for commercial salt exploitation. Paradoxically, archaea and 18S for protists. Microbial novelty hypersaline environments can also be found in indicates the uniqueness of the identity level of the sea ice, whereas the salinity of seawater and a given sequence based on BLAST search com- the exclusion of salt during freezing create exten- parison against the ribosomal gene sequences sive brine channels. Finally, deep-sea brine previously reported in GenBank (usually <97% anoxic basins have been also found in the Medi- identity). The gradient of saline concentrations terranean with in situ sulfate reduction, explored here ranged c. 20 times from 20 up to methanogenesis, and microbial heterotrophic 370 g/L, when NaCl precipitates. activities. Inland saline lakes are also numerous and widespread saline water bodies that occur on all Introduction continents (even in the Antarctica) with greater environmental variability than in coastal-marine Coastal salterns and inland salt lakes are widely areas. Salt composition has a larger repertory of distributed ecosystems around the world. In the concentrated cations such as calcium, magne- Mediterranean region, there has been a long tra- sium and lithium, and larger concentrations of dition to use solar saltern systems for salt produc- potassium and magnesium chlorides and sulfates tion in seawater evaporation ponds. Multipond (bitter lakes), sodium carbonate (alkali lakes), solar salterns are semi-artificial coastal systems and calcium and magnesium carbonates, with designed to harvest common salt (NaCl) from ionic rates and composition different from sea- seawater. Sequential precipitation of CaCO3 and water (athalassohalinity). Salt lakes are also het- CaSO4 occurs during the first stages of water erogeneous in their limnology and may contain evaporation and salt concentration. When seawa- water permanently, intermittently, or episodi- ter has been reduced to about one-tenth the orig- cally. Water levels may be constant or fluctuate inal volume, NaCl precipitates and can be widely, often in accord with salinity fluctuations collected in the crystallizer ponds. These ponds (Demergasso et al. 2008). Endorheic areas, in show very high population densities and very which precipitation never reaches the sea, harbor simple community structure. The extremely hal- most of the saline lakes in the world. In semiarid ophilic bacterium Salinibacter ruber inhabits regions the formation of saline lakes is more saltern crystallizer ponds worldwide, together widespread, adding high ecological and aesthetic with the square archaeon Haloquadratum values as naturally attractive landscapes and as walsbyi. Changes of one order of magnitude in attractors of emblematic organisms (e.g., flamin- salinity are easily found within a short walking gos). Important ecological changes occur through distance, from 37 g/L in seawater to 370 g/L in this gradient, with decreasing biodiversity as both the crystallizers, whereas changes in other envi- salinity and dominance of microbes increase. ronmental parameters such as temperature and Certainly, these are different and unique environ- pH are much less pronounced (Fig. 1). The ments not only from the microbial ecologists changes in prokaryote abundance along the salin- perspective despite the general biased perception ity gradient are also very pronounced and within that salt lakes are less valuable than other types of one order of magnitude from 106 cells/mL in inland water (Williams 1996). seawater to 107 cells/mL above 20 % salinities. Overall, high environmental dissimilarity is Important ecological changes occur through the present both at the global scale and within salinity gradient, with decreasing biodiversity as a short distance, and it is widely accepted that both salinity and dominance of microbes diversity decreases with increasing salinity pro- increase. In continental areas with hypersaline viding a working hypothesis about which Microbial Diversity and Novelty Along Salinity Gradients 373 M

Microbial Diversity and Salt lake Tebenquiche Novelty Along Salinity 35 Gradients, Salinity (%) Fig. 1 Environmental pH 30 heterogeneity in inland salt Temperature (°C) Lake Tebenquiche (Chile) and in coastal solar salterns 25 of Santa Pola (Spain). Changes of one order of 20 magnitude in salinity are easily found within a short walking distance, whereas 15 changes in other environmental parameters 10 such as temperature and pH are much less pronounced 5 (upper panel). Changes in prokaryotes abundance along the salinity gradient 0 are also within one order of 12 18 19 20 21 22 magnitude (lower panel) Sampling sites (within 1 km distance)

Sala Pola saltern ponds 40 1.4 × 107 Salinity (%) 35 1.2 × 107 30 1×107 25 M

20 8×106 Cells Salinity 15 6×106 10 4×106 5 Bacteria and Archaea/mL

0 2×106 12345678 Sampling sites (within 1 km distance) environments are high or low in diversity. Along Changes in Microbial Diversity Along the gradient several novel microorganisms of the Salinity Gradient environmental and biotechnological importance develop (Oren 2002), and these heterogeneous For many years hypersaline systems have proved habitats appear as a large unexplored reservoir to be an excellent source of new culturable micro- of unknown microbes (Triado´-Margarit and organisms forming colonies on agar plates using Casamayor 2013; Casamayor et al. 2013). common microbiological growth media. Studies Changes in microbial diversity, metabolic level, based on culturing showed a decrease in prokary- and genetic novelty are consistent along the gra- otic diversity recovered on plates as salt concen- dient. This background knowledge is essential to tration increased. At salinity up to 15 %, most guide hypothesis-driven metagenomic studies. isolates were those commonly found in seawater. M 374 Microbial Diversity and Novelty Along Salinity Gradients

Santa Pola Salterns

100 BACTERIA

80

60

ARCHAEA

40

EUKARYA

Percentage OTUs richness 20

PHYTOPLANKTON 0 0 5 10 15 20 25 30 35 40 Salinity gradient (%)

Santa Pola Salterns 8

7 BACTERIA ARCHAEA 6

5

4

3 Number of taxa 2

1

0 82232 Salinity (%)

Microbial Diversity and Novelty Along Salinity Gra- alpha-, beta-, gamma-, and epsilon-Proteobacteria, dients, Fig. 2 Changes in the microbial community Cyanobacteria, Actinobacteria, Bacteroidetes (non- richness along the salinity gradient in coastal solar salterns Salinibacter), Haloarchaea (non- Haloquadratum), of Santa Pola (Spain). OTUs detected as ssRNA gene Thermoplasmatales, Euryarchaota; 22 % salinity pond: bands in a denaturing gradient gel electrophoresis alpha- and gamma-Proteobacteria, Cyanobacteria, (DGGE) for bacteria, archaea, eukarya, and phytoplank- Bacteroidetes, Salinibacter, and Haloarchaea; and 32 % ton (algae and cyanobacteria) (upper panel). The taxa salinity pond: Cyanobacteria, Bacteroidetes, Salinibacter, found (bottom panel) are as follows: 8 % salinity pond: and Haloarchaea

Above 15 % salt, most strains were specialized et al. 2002). Interestingly, culture-independent halophilic. In the crystallizer, S. ruber and analyses have shown a similar threshold. Up to H. waslbyi had remained elusive to culturing for 15 % salinity, the richness of the different micro- many years, being Haloarcula and Halorubrum bial compartments in the genetic fingerprints the most frequently recovered genera but consid- (bacteria, archaea, microbial eukarya, and phyto- ered as weeds in these systems (Benlloch plankton) decreased consistently (Fig. 2)as Microbial Diversity and Novelty Along Salinity Gradients 375 M compared with the richness found in seawater uncultured microbes distantly related to cultured (Casamayor et al. 2002; Estrada et al. 2004). counterparts. Certainly, further culturing efforts Unexpectedly, at higher salinities no decrease in concentrated in these areas will provide a large the OTUs (operational taxonomic units, i.e., set of at least new bacterial and archaeal genera bands in a DGGE fingerprinting) richness was unknown to date. Microbial ecologists have observed. However, after cloning and sequencing shown where to search for it. The challenge now the number of taxa declined significantly (Fig. 2). for the microbiologists is to isolate this microbial Therefore, the 16S rDNA genetic diversity novelty in pure culture to analyze in detail their observed shifted from “macrodiverse” to specific adaptations to the fluctuating and stress- “microdiverse” microbial assemblages, and ful environmental conditions. salinity thresholds were detected along the gradi- ent (Benlloch et al. 2002; Casamayor et al. 2002). Regardless of their changes in abundance, both Changes in Microbial Productivity Along Bacteria and Archaea showed the same pattern: the Salinity Gradient as salinity increased, the number of different clusters decreased, and only one cluster became The picture usually observed along a salinity gra- dominant. This cluster, however, showed dient indicates heterotrophic prokaryotes being a considerable degree of microdiversity indicat- very abundant but growing at very low specific ing the coexistence of several closely related growth rates in ponds of the highest salt content. clones of microorganisms. Interestingly, the 16S In ponds of lower salt content, microbes are less rDNA approach expanded the range of known concentrated but grow at high specific rates diversity in the salterns with new clusters of hith- (Fig. 3). Bacteria are more abundant at lower erto uncultured microorganisms, but it is at inter- salinities, whereas archaea can reach between mediate salinities where the degree of novelty 80 % and 90 % of the total prokaryotic count in M was higher. There, uncultured counterparts of the high salinity ponds. The addition of Bacteroidetes, Alpha-, and Gammaproteo- taurocholate (a bile acid involved in the emulsi- bacteria distantly related to previously known fication of fats) has been used to lyse the archaea clones were the most abundant sequences recov- and prevent archaeal activity in experimental ered in coastal areas (Benlloch et al. 2002). In assays. These experiments indicated that most athalassaholine environments, Bacteroidetes and of the prokaryotic activity above 20 % salinity Gammaproteobacteria are the most frequently is carried out by Haloarchaea (Gasol et al. 2004). recovered in clone libraries with respect to all Protistan grazing has been considered one of the other bacterial groups (Demergasso the major factors regulating the variations in et al. 2004, 2008). Interestingly, the diversity of bacterioplankton abundance. Grazing pressure Bacteroidetes sequences is large and with in solar salterns decreases with increasing salinity a remarkable degree of novelty, forming specific up to a point where it is below detection. At the clusters with no cultured relatives in databases highest salinities, only viruses affect prokaryotes, and rather distantly related to any previously although the percentage of prokaryotic biomass known 16S rRNA sequence. Clones closely and production lost due to viruses is very small related to S. ruber can be also found in the saltiest (Guixa-Boixereu et al. 1996). Most likely, there- parts of athalassaholine lakes. fore, two different types of control of prokaryotic Overall, these ecosystems contain sequences abundance are involved: in the low-salinity that are candidates for new branches in the bac- ponds, high grazing pressure determines terial phylogenetic tree, novel clusters within a relatively low prokaryotic biomass with high well-characterized bacterial groups, and a large specific growth rates; in the high-salinity ponds, microdiversity within these novel clusters. Both low grazing pressure results in a relatively high intermediate salinities in solar salterns and inland prokaryote biomass with low specific growth salt lakes still contain a large repertory of rates. M 376 Microbial Diversity and Novelty Along Salinity Gradients

Santa Pola Salterns

100

ABUNDANCE 80 HIGH BIOMASS-LOW ACTIVITY

60

40

20 Percentage vs. maximal values LOW BIOMASS-HIGH ACTIVITY

PRODUCTIVITY 0 0 5 10 15 20 25 30 35 40 Salinity gradient (%)

Santa Pola Salterns

CONTROL NUTRIENTS ADDITION 200 DOM ADDITION

150

100

50 Percentage of productivity increment

0 4 8 22 37 Salinity (%)

Microbial Diversity and Novelty Along Salinity Gra- prokaryotic production values are found at salinities up to dients, Fig. 3 Functional changes in the microbial com- 10 % (upper panel). After bioassays experiments (bottom munity along the salinity gradient in coastal solar salterns panel) using control productivity as reference (100 % of Santa Pola (Spain). Maximal values of biomass are value), mostly organic carbon limitation was observed found at the highest salinities (>20 % salinity). Maximal all along the salinity gradient except in the crystallizers

On the other hand, bacterioplankton growth is glucose and acetate) (Gasol et al. 2004), it was expected to depend on the availability of organic examined the variability in nutrient limitation of and inorganic nutrients. Through a series of bacteria and archaea along the salinity gradient short-term enrichment bioassays (20 h incuba- (Fig. 3). In the first experiment, inorganic nutri- tion), adding inorganic nitrogen + phosphorous ents did not significantly increase prokaryotic (nutrient addition treatment) and dissolved productivity in any of the ponds above 10 %, organic carbon (DOM addition treatment, i.e., and only slightly in the ponds at lower salinities, Microbial Diversity and Novelty Along Salinity Gradients 377 M mainly at the 8 % pond. Thus, in general, micro- the ecology and microbiology of saline systems: bial assemblage productivity was not either inor- (i) “the cultured gap” for phylotypes >97 % ganic nitrogen or phosphorous limited along the identity with CEM and <97 % with CCM, gradient. Conversely, organic matter addition highlighting those microorganisms poorly stimulated prokaryotic productivity in all ponds represented in culture collections but detected along the gradient, except at 37 % salinity. These in environmental surveys that are good targets results also indicated decreased organic matter for culturing strategies and (ii) “the environmen- limitation with increasing salinity (Fig. 3). At tal gap” for phylotypes <97 % identity with the end, crystallizers appeared as very stable sys- CEM and >97 % with CCM, highlighting those tems, and neither organic matter limitation nor microorganisms represented in cultured collec- zooplankton predation pressure affected prokary- tions but previously not detected in environmen- ote development in the higher salinity ponds. In tal rRNA genes surveys, suggesting missing fact, high-salinity ponds only responded slightly niches or poor environmental relevance. Finally, to rain dilution (Gasol et al. 2004). All together, it can be also visualized those salinity ranges that microbial community structure and composition are poorly explored and need further research by consistently change along the gradient, but func- microbial ecologists and traditional microbiolo- tional aspects of the microbial food web also vary gists such as inland saline environments with at differing salinities. intermediate salt concentrations. In any case, novel sequences (i.e., <97 % identity) can be found all along the gradient, suggesting that Changes in Microbial Novelty Along the substantial genetic novelty remains still to be Salinity Gradient uncovered. Interestingly, each life domain shows Saline water bodies are abundant and heteroge- a different level of novelty, according to the M neous and at the global scale contain a mostly GenBank search criteria mentioned above. In unveiled genetic diversity and novelty of micro- general, protist and bacteria are closer to previ- organisms. Although these environments are an ously reported phylotypes both in culture and in excellent source of new culturable microorgan- the environment than Archaea (Fig. 4). There- isms, we are still far to understand how many fore, much more intensive efforts are needed for different microbial species exist adapted to col- this group of microorganisms in saline systems. onize these environments and which environ- Highly novel archaea of the enigmatic group mental conditions promote the highest and the deep-sea hydrothermal vent euryarchaeotic lowest genetic diversity for each life domain. In group 6 (DHVEG-6) and new phylotypes dis- fact, very few reports in the literature study the tantly related to well-known Halobacteriaceae three microscopic groups simultaneously are among the best candidates. Interestingly, the (Casamayor et al. 2013). The study of the highest novelty can be found for Archaea at genetic novelty can be carried out by BLAST the lowest saline concentrations, whereas for identity search of the ribosomal 16S and 18S bacteria and protists, no differences are observed RNA against GenBank sequences. The identity along the gradient (Fig. 4, Casamayor et al. of each single sequence can be then related both 2013). The best candidates to find high novelty to the closest environmental match (CEM) and are among uncultured members of the family to the closest cultured match (CCM) available in Cryomorphaceae (phylum Bacteroidetes) for GenBank. The highest novelty corresponds to bacteria and new chlorophyta substantially dif- microbial phylotypes matching <97 % identity ferent from any other green algae previously to both CEM and CCM; those phylotypes show- reported either in freshwater or in the sea. These ing >97 % identities in each case can be consid- new groups of microorganisms deserve to be ered of limited novelty. This exercise also shows brought into culture for detailed ecophysiological two important gaps in current investigations of studies. M 378 Microbial Diversity and Novelty Along Salinity Gradients

100 BACTERIA ARCHAEA EUKARYA 80

60

40 % of “new species”

20

0 2-5% 5-10% 10-15% >15% salinity gradient (%)

Microbial Diversity and Novelty Along Salinity Gra- Oct 2012). The highest values of highly novel sequences dients, Fig. 4 Changes in genetic novelty along the were found for archaea at salinities 2–10 %. Eukaryotes salinity gradient in microbial communities from inland showed the highest novelty at salinities 10–15 %. Bacteria saline lakes, Monegros desert (Spain). Percentage of was the domain with the lowest percentage of phylotypes “new species”: phylotypes with < 97 % identity to previ- not previously reported ously reported sequences in databases (GenBank search,

Summary along the gradient. This background knowl- edge is essential to guide hypothesis-driven Coastal salterns and inland salt lakes are metagenomic studies. widely distributed ecosystems around the world. These systems are characterized by a high environmental variability both at the Cross-References global scale and within a short distance. It is widely accepted that diversity decreases with ▶ Brine Pools, Metagenomics of increasing salinity providing a working ▶ Metagenomics of Deep Hypersaline Anoxic hypothesis about which environments are high Basins or low in diversity. Along the gradient several ▶ Salt Lakes, Metagenomics of novel microorganisms of environmental and biotechnological importance develop, and these heterogeneous habitats appear as a large References unexplored reservoir of unknown microbes. Microbial community structure and composi- Benlloch S, Lo´pez-Lo´pez A, Casamayor EO, tion consistently change along the gradient, et al. Prokaryotic genetic diversity throughout the but functional aspects of the microbial food salinity gradient of a coastal solar saltern. Environ web also vary at differing salinities. Changes Microbiol. 2002;4:349–60. Casamayor EO, Massana R, Benlloch S, et al. Changes in in microbial diversity, metabolic level, and archaeal, bacterial and eukaryal assemblages along a genetic novelty are consistent and predictable salinity gradient by comparison of genetic Microbial Dysbiosis and Esophageal Diseases 379 M

fingerprinting methods in a multi-pond solar saltern. Definition Environ Microbiol. 2002;4:338–48. Casamayor EO, Triado´-Margarit X, Castan˜eda C. Micro- bial biodiversity in saline shallow lakes of the Monegros Dysbiosis: Trillions of bacteria inhabit the human Desert, Spain. FEMS Microbiol Ecol. 2013;85:503–18. body. They form ecological communities on every Demergasso C, Casamayor EO, Galleguillos P, external (skin) and internal (mucosal) surface of et al. Distribution of prokaryotic genetic diversity in our body. Some of bacteria are beneficial to us, athalassohaline lakes from the Atacama Desert, North- ern Chile. FEMS Microbiol Ecol. 2004;48:57–69. some are neutral, and others are harmful. Normally Demergasso C, Escudero L, Casamayor EO, et al. Novelty they maintain a balanced community structure and and spatio-temporal heterogeneity in the bacterial a symbiotic relationship with the host. Dysbiosis is diversity of hypersaline Lake Tebenquiche (Salar de defined as a state of altered microbial community Atacama). Extremophiles. 2008;12:491–504. Estrada M, Hendriksen P, Gasol JM, et al. Diversity of that disrupts the symbiotic relationship and causes planktonic photoautotrophic microorganisms along or contributes to disease/dysfunction. a salinity gradient as depicted by microscopy, flow cytometry, pigment analysis and DNA-based methods. FEMS Microbiol Ecol. 2004;49:281–93. Gasol JM, Casamayor EO, Join I, et al. Control of hetero- Introduction trophic prokaryotic abundance and growth rate in hypersaline planktonic environments. Aquat Microb The human body plays host to a diverse and Ecol. 2004;34:193–206. abundant population of microbes. All surfaces Guixa-Boixereu N, Caldero´n-Paz JI, Heldal M, et al. Viral lysis and bacterivory as prokaryotic loss factors of the body exposed to the environment are pop- along a salinity gradient. Aquat Microb Ecol. 1996; ulated by microbes. The term “microbiome” 11:215–27. refers to the collection of all members in Oren A. Halophilic microorganisms and their environ- a complex microbial community (Lederberg and ments. Dordrecht: Kluwer, Scientific Publishers; 2002. Triado´-Margarit X, Casamayor EO. High genetic diversity McCray 2001). The host relationship with the microbiome can be commensal, symbiotic, and high novelty in planktonic protists inhabiting M inland and coastal high salinity water bodies. FEMS dysbiotic, or pathogenic. Bacterial mutualists Microbiol Ecol. 2013;85:27–36. within the gastrointestinal tract are beneficial to Williams WD. The largest, highest and lowest lakes of the world: saline lakes. Verh Internat Verein Limnol. the host as they assist in the synthesis of vitamins, 1996;26:61–79. promote development of the gut immune system, and provide competitive barriers to pathogen invasion. This complex microbial population influences an estimated 10 % of all metabolites Microbial Dysbiosis and Esophageal in our body (Wikoff et al. 2009). In return, the Diseases host provides bacteria with safe housing and food during lean times. In order for this symbiotic Liying Yang1, Carlos Wolfgang Nossa2 and relationship to be sustained, the immune system Zhiheng Pei3 has to balance permissive, tolerogenic responses 1Department of Medicine, New York University to food antigens and commensal microbes with School of Medicine, New York, NY, USA potentially damaging, inflammatory responses to 2Gene by Gene Ltd., Houston, TX, USA ward off pathogens. This delicate balance is 3Departments of Pathology and Medicine, New maintained by the constant interplay between York University School of Medicine, New York, the microbiome, the host gastrointestinal barrier, NY, USA and the host mucosal immune system – which is a prerequisite for normal gut homeostasis. Dysbiosis, often represented by alteration of the Synonyms microbial community, may lead to innate immune (inflammation) and adaptive immune Alteration of the microbiome in esophageal (infectious pathology) responses and/or disease. disease While pathogenic bacteria have long been M 380 Microbial Dysbiosis and Esophageal Diseases studied for their specific disease-causing traits, the principle of dysbiosis as a causative agent of chronic disease has recently been recognized as an important field of study. The esophagus, as with other luminal organs of the digestive system, represents a suitable environment for bacteria to inhabit. Besides res- ident bacteria, extraneous bacteria can be intro- duced into the esophagus by swallowing or by reflux from the stomach. However, compared with the well-studied colonic and oral microbiomes, characterization of the esophageal microbiome has received much less attention. Previous culture-based studies suggested that the esophagus is either sterile or contains only few transient bacteria (Gagliardi et al. 1998); but later studies using cultivation-independent PCR have consistently identified bacteria associated with mucosal surfaces in tissue biopsies (Narikiyo et al. 2004; Pei et al. 2004, 2005; Yang et al. 2009). Furthermore, the bacteria are visible by microscopy on the mucosal surfaces of the distal esophagus (Fig. 1; Pei et al. 2004). Microbial Dysbiosis and Esophageal Diseases, Fig. 1 Visualization of bacterial in the distal esophagus by Gram stain in normal Alteration of the Esophageal Microbiome in Esophagitis and Barrett’s Esophagus no “smoking gun” risk factors correlated to gas- troesophageal reflux disease (GERD), it was The increase in esophageal disease in recent proposed that the esophageal microbiome may decades has generated much interest in esopha- play a role in disease onset. Part of the rationale geal research. Esophageal adenocarcinoma for this proposal is that gastroesophageal reflux (EAC), the malignant transformation at the end impairs the mucosal barrier and exposes the of a spectrum of diseases related to gastroesoph- squamous epithelium and lamina propria to ageal reflux, is now the most rapidly increasing (1) the microbes swallowed from the oral cavity, cancer in the Western world. Barrett’s esophagus colonized in the esophagus, and regurgitated (BE) is defined as the metaplastic columnar epi- from the stomach; (2) the acidic gastric contents; thelium that replaces squamous mucosa and pre- and (3) the bile from the duodenum. Reflux disposes to cancer development (Spechler esophagitis and Barrett’s esophagus represent et al. 2011). For unclear reasons, the incidence phenotypes of inflammation of the esophageal of esophageal adenocarcinoma in the USA has mucosa induced by long-term gastric acid and risen approximately 600 % since the 1970s bile reflux into the esophagus, potentially alter- (Devesa et al. 1998; Haggitt 1992; Shaheen and ing the diverse microbial ecosystem and induc- Ransohoff 2002), and since its development is ing chronic inflammation. Much of the interest not universal among patients with Barrett’s to further study the microbiome comes from its esophagus, it is important to understand and to confirmed link to esophageal disease. gauge the factors that influence risk of progres- A recent study of the human distal esophagus sion to dysplasia and cancer. Because there are microbiome (Yang et al. 2009) linked Microbial Dysbiosis and Esophageal Diseases 381 M

Microbial Dysbiosis and Esophageal Diseases, Fig. 2 Typing of esophageal microbiome. Detection of Microbial Dysbiosis and Esophageal Diseases, natural microbiome groups by unsupervised cluster anal- Fig. 3 Taxonomic definition of microbiome types. Clas- ysis. The dendrogram was constructed using the average sification of microbiome by the relative abundance of linkage algorithm and cosine measure of the genetic dis- Streptococcus. An outlier (solid circle) was excluded tance calculated from samples of the microbiome. Sam- using a box plot in which the upper whisker length is ples are represented by colored rectangles (green for 1.5*IQR. The 95 % normal reference range (NRR) normal, red for esophagitis, and black for Barrett’s (mean Æ 1.96 S.D.) was calculated by the relative abun- esophagus) dance of Streptococcus after excluding the outlier. The dotted line (50.3 %) is the upper limit of the 95 % normal inflammation and BE to changes in the esopha- reference range (NRR), which separates the 34 samples into normal (inside the NRR) and abnormal taxonomic geal microbiome. The study used 16S rRNA gene types (outside the NRR) surveys to characterize the bacterial communities in biopsy samples taken from the distal esopha- gus of 34 individuals with either normal mucosa closely associated with the normal esophagus (n ¼ 12), esophagitis (n ¼ 12), or Barrett’s (11/12, 91.7 %), whereas the type II microbiome M esophagus (intestinal metaplasia) (n ¼ 10). Two is mainly associated with an abnormal esopha- hundred 16S rRNA genes were cloned and gus (13/22, 59.1 %) ( p ¼ 0.0173, among group sequenced from each sample. comparison), including both esophagitis (7/12, Overall, the 6,800 sequences represented 58.3 %, OR ¼ 15.4) and Barrett’s esophagus 9 phyla, 70 genera, and 166 species. Firmicutes (6/10, 60.0 %, OR ¼ 16.5). is the only phylum consistently detected in all The alteration of the microbiome from type I to 34 samples, whereas 8 other phyla, the type II in distal esophagus, thus, is associated with Bacteroidetes, Proteobacteria, Actinobacteria, host phenotypes and its disease progression. Strep- Fusobacteria, TM7, Spirochaetes, Cyanobacteria, tococcus is the most dominant genus in the esoph- and unclassified bacteria, were less common. ageal microbiome, and its relative abundance The samples from healthy subjects were dom- differs between the two types of microbiome and inated by Streptococcus species. On average, decreases in disease states. Overall, the 20 type 76 % of the sequences from healthy esophageal I samples had a mean of 78.8 % Streptococcus mucosa were categorized to belong to streptococ- (range, 60.5–97.0 %), whereas the 14 type II sam- cal species, while the abundance of some other ples had a mean of 30.0 % (range, 8.0–46.5 %) genera was low, but significantly increased in ( p < 1 Â 10À10). The mean of relative abun- esophagitis and Barrett’s esophagus. dance of Streptococcus in the normal esophagus With an unsupervised approach, samples of group (75.9 %) was significantly higher than that the microbiome form two distinct clusters or in the esophagitis (50.5 %) and Barrett’s esopha- two microbiome types, type I and II, based on gus (54.1 %) groups (Fig. 3). combined genetic distance between samples In the disease-associated type II microbiome, (Fig. 2). Although neither of the two types of the decrease in the relative abundance of Strep- microbiome exclusively correlated with the tococcus is compensated by an increase in the phenotypes, the type I microbiome is more relative abundance of 24 other genera. M 382 Microbial Dysbiosis and Esophageal Diseases

a Aerobes b 100 P = 1.0x10–10 100 Gram-negative P = 8.0x10–10 Anaerobes –5 P = 1.2x10 Gram-positive P = 9.0x10–10 50 Microaerophils 50 P = 1.1x10–4 Undetermined P = 8.7x10–3 Abundance (%) Abundance Undetermined (%) Abundance P = 2.0x10–2

0 0 Type I Type II Type I Type II

Microbial Dysbiosis and Esophageal Diseases, microbiome types according to culture conditions (Panel Fig. 4 Taxonomic characterization of microbiome by a) and staining properties (Panel b) population of main bacterial groups. Comparisons of

Microbial Dysbiosis and Esophageal Diseases, (b) Shannon-Wiener evenness index. (c) Richness by Fig. 5 Difference between the two types of microbiome observed and estimated SLOTUs (Chao 1984). Mean Æ in biologic diversity. (a) Shannon-Wiener diversity index. 1.96 SD is indicated by horizontal lines

Specifically, the most prominent increase (Shannon-Wiener evenness index mean 0.78 involves Veillonella, Prevotella, Haemophilus, vs. 0.51, p ¼ 4.2 Â 10À8) than the type Neisseria, Rothia, Granulicatella, Campylobac- Imicrobiome(Fig.5). The type II microbiome ter, Porphyromonas, Fusobacterium, and Actino- appears to be the strongest (OR > 15) among all myces, many of which are Gram-negative known environmental factors that are associated anaerobes or microaerophiles and are putative with the pathological changes related to gastro- pathogens for periodontal disease. Anaerobic esophageal reflux, such as hiatus hernia with an (type I, 11.0 % vs. type II, 38.2 %, OR 4.2 for RE and 3.9 for BE and tobacco use with À p ¼ 1.2 Â 10 5) and microaerophilic bacteria an OR 2.6 for RE (Yang et al. 2009). (5.4 % vs. 23.0 %, p ¼ 1.1 Â 10À4) are more These findings have opened a new understand- abundant in the type II microbiome than in the ing of the recent surge in the incidence/prevalence type I microbiome (Fig. 4a). Gram-negative bac- of GERD, Barrett’s esophagus, and esophageal teria comprise 53.4 % of type II microbiome but adenocarcinoma and suggest the possible role of only 14.9 % of type I microbiome dysbiosis in their pathogenesis. The diverse type II À (p ¼ 8.0 Â 10 10)(Fig.4b). Overall, the type II community composed of a larger proportion of microbiome is significantly more diverse Gram-negative bacteria might interact with the (Shannon-Wiener diversity index mean of 2.69 innate immune system of the epithelial cells in vs. 1.51, p ¼ 1.3 Â 10À7) and more even a different way from the type I microbiome, by Microbial Dysbiosis and Esophageal Diseases 383 M releasing a larger spectrum of microbial compo- several intriguing observations. It speculates on nents, such as lipopolysaccharide (LPS) of Gram- the role of the type II microbiome in the diseases negative bacteria that may stimulate pattern recep- of reflux esophagitis, Barrett’s esophagus, and tors (e.g., Toll-like receptors) (Suerbaum 2009; esophageal adenocarcinoma (Yang et al. 2012). Yang et al. 2012). Furthermore, the type II The type II microbiome with stepwise increase in microbiome that contains significant numbers of Gram-negative bacteria in the esophagitis, potential pathogens, such as Campylobacter, Barrett’s esophagus, and probably in esophageal Veillonella, Prevotella, Haemophilus, Neisseria, adenocarcinoma could contribute to carcinogen- Porphyromonas, Fusobacterium,andActinomy- esis by induction of chronic inflammation. The ces, and a significantly higher percentage of type II microbiome could be used as a novel Gram-negative bacteria might play a role with biomarker for risk assessment in clinical manage- relevance in the maintenance of inflammation. ment. Antibiotic/probiotic treatment could Further in vitro testing models, currently under- reverse the type II microbiome back to the type way, are needed to verify the potential causative I microbiome and decrease the detrimental role of the microbiome in GERD via the mecha- effects of Gram-negative bacteria in those dis- nisms mentioned above. eases development. Alternatively, the type II microbiome might be To understand the range of human genetic and secondary to changes caused by gastric reflux. The physiological diversity, it is important to under- type I microbiome could represent a direct exten- stand the factors that influence the distribution sion of the normal oral microbiome via saliva, and evolution of the microbiome, which is to while the type II microbiome could represent understand and expand on the connection these regurgitated bacteria in gastric juice or organisms have with their human hosts. This a microbiome modified by gastric acid by knowledge can be used to better treat and diag- selecting against acid-sensitive bacteria in the nose disease and perhaps find ways to prevent M esophagus. However, at this stage, it is unclear some diseases by elucidating the link(s) between whether the presence of type II microbiome microbiota and the disease. (or the absence of type I bacteria) plays a causal role in the pathogenesis of esophageal inflamma- tion or Barrett’s esophagus. These hypotheses will Cross-References have to be addressed by future studies, which should be conducted with a prospective design ▶ Foregut Microbiome, Development of and involve a finer characterization of the Esophageal Adenocarcinoma, Project microbiomes (Suerbaum 2009). The microbiome ▶ Microbiome, Foregut alteration from type I to type II might prove to be an important step in the pathogenesis of esopha- geal tumorigenesis and represent a biologically more plausible microbial component in GERD- References BE-EAC progression. Consequently, it is essential Chao A. Nonparametric estimation of the number of to assess the type II microbiome and/or numbers of classes in a population. Scand J Statist. 1984;11: its potential pathogens as either a sole or a panel of 265–270. biomarkers in order to decipher its relevance in Devesa SS, Blot WJ, Fraumeni Jr JF. Changing patterns in GERD-BE-EAC progression. the incidence of esophageal and gastric carcinoma in the United States. Cancer. 1998;83(10):2049–53. Gagliardi D, Makihara S, Corsi PR, Viana Ade T, Wiczer MV, Nakakubo S, et al. Microbial flora of the normal Summary esophagus. Dis Esophagus. 1998;11(4):248–50. Haggitt RC. Adenocarcinoma in Barrett’s esophagus: a new epidemic? Hum Pathol. 1992;23(5):475–6. The 16S rRNA gene survey of the literature on Lederberg J, McCray AT. ‘Ome Sweet’Omics – a the esophageal microbiome has uncovered genealogical treasury of words. Scientist. 2001;15:8. M 384 Microbial Genomes That Drive Earth’s Biogeochemical Cycles

Narikiyo M, Tanabe C, Yamada Y, Igaki H, Tachimori Y, Definition Kato H, et al. Frequent and preferential infection of Treponema denticola, Streptococcus mitis, and Strep- tococcus anginosus in esophageal cancers. Cancer Sci. Biogeochemical cycle: the combined move- 2004;95(7):569–74. ment, by biology and geology, of chemical Pei Z, Bini EJ, Yang L, Zhou M, Francois F, Blaser elements between Earth’s biosphere and its abi- MJ. Bacterial biota in the human distal esophagus. otic reservoirs. Especially important are those Proc Natl Acad Sci U S A. 2004;101(12):4250–5. Pei Z, Yang L, Peek Jr RM, Levine SM, Pride DT, involving the main building blocks of life: car- Blaser MJ. Bacterial biota in reflux esophagitis and bon, hydrogen, oxygen, nitrogen, phosphorus, Barrett’s esophagus. World J Gastroenterol. 2005; and sulfur. 11(46):7277–83. Shaheen N, Ransohoff DF. Gastroesophageal reflux, Barrett esophagus, and esophageal cancer: clinical applications. JAMA. 2002;287(15):1982–6. Introduction Spechler SJ, Sharma P, Souza RF, Inadomi JM, Shaheen NJ. American Gastroenterological Association medi- A network of biotic and abiotic mechanisms cal position statement on the management of Barrett’s esophagus. Gastroenterology. 2011;140(3):1084–91. maintains global elemental cycling, also called Suerbaum S. Microbiome analysis in the esophagus. Gas- biogeochemistry. An important step in under- troenterology. 2009;137(2):419–21. standing global elemental cycling is determining Wikoff WR, Anfora AT, Liu J, Schultz PG, Lesley SA, the role of organisms and their communities in Peters EC, et al. Metabolomics analysis reveals large effects of gut microflora on mammalian blood metab- these processes. At the interaction of the living olites. Proc Natl Acad Sci U S A. 2009;106(10): and nonliving world, life is powered by coupling 3698–703. its endergonic reactions to favorable electron- Yang L, Lu X, Nossa CW, Francois F, Peek RM, Pei transfer reactions, changing the environment in Z. Inflammation and intestinal metaplasia of the distal esophagus are associated with alterations in the the process. microbiome. Gastroenterology. 2009;137(2):588–97. The present network of Earth’s electron Yang L, Francois F, Pei Z. Molecular pathways: patho- donors, acceptors, and electron-transfer pathways genesis and clinical implications of microbiome alter- uses mainly solar energy to support life and to ation in esophagitis and Barrett esophagus. Clin Cancer Res. 2012;18:2138–44. continue cycling of its most basic building blocks, H, C, N, O, and S as well as important metals like Fe and Mn. The system has adjusted to major perturbations, such as the Great Oxida- Microbial Genomes That Drive tion Event (GOE) and has survived each of Earth’s Biogeochemical Cycles Earth’s mass extinctions. As all living organisms exchange gas with their environment (Vernadsky Paul Falkowski1 and Benjamin I. Jelen2 1926), this network of redox reactions (Fig. 1) 1Departments of Geological Sciences and exerts great control over Earth’s inventory of Marine and Coastal Sciences, Institute of Marine gases, in turn playing a great role in phenomena and Coastal Sciences, Rutgers University, like global climate change. New Brunswick, NJ, USA The blueprints for the biological components 2Environmental Biophysics and Molecular of biogeochemistry, i.e., the genes passed on Ecology Laboratory, Institute of Marine and through evolution, are stored by Earth’s diverse Coastal Sciences, Rutgers University, microbial life (Falkowski et al. 2008). New Brunswick, NJ, USA Completely ubiquitous, this core set of genes responsible for the biological side of elemental cycling on our planet is represented by less than Synonym 1,000 orthologous groups in online databases. Metagenomics, sampling the entire genetic Metagenomics for the analysis of biogeochemi- makeup of a given community, is a high- cal pathways throughput method to collect these genes, Microbial Genomes That Drive Earth’s Biogeochemical Cycles 385 M

O2-mediated Mn-mediated Fe-mediated S-mediated N-mediated

H2- or C-mediated

2+ NH + 2+ − H [CH O] Mn 4 Fe HS CH4 2 2

O2 Respiration Mn4+ − NO3 N2 fixation N2 Fe3+ 2− SO4

CO2

H2O +CO2 Photosynthesis

Microbial Genomes That Drive Earth’s Biogeochem- information stored in the planet’s microbial metagenomes ical Cycles, Fig. 1 Earth’s interconnected network of (Falkowski et al. 2008). Pathway groups I–VI correspond biogeochemical reactions, depicted here, is driven by with those in Fig. 3 M including those from the unculturable majority of of importance can be isolated and cultured, so not microbes. Allowing unprecedented insight into all have been individually sequenced, but for the metabolic potential of today’s microbial com- those fully sequenced organisms, lifestyles are munities, the deluge of data presented by revealed. Each genome indicates heterotrophy metagenomics also promises further advances in or autotrophy, whether an organism feeds on understanding the evolution, current regulation, fixed carbon from the environment or is able to and possible futures of biogeochemistry on Earth. fix its own from CO2. If the organism is capable of fermentation, the substrates it is capable of metabolizing can be detected. If the organism Environmental Genomics uses an electron transport chain, the redox pairs it is capable of using for energy transduction are In recent years, environmental genomics has clar- uncovered. The ability to photosynthesize or fix ified the interaction between microbes and their nitrogen can quickly be highlighted, information environment. Inclusive of environmental critical to understanding an organism’s place in metagenomics, this discipline strives to predict biogeochemical cycling. organismal response to environmental change. Using databases of known genes with annotated function, the sequenced genome of a given Genome and Pathway Databases microbe can now quickly infer biochemical capacity by homology of its computationally A service such as the Kyoto Encyclopedia of derived open reading frames (ORF), to known Genes and Genomes (KEGG) or JGI’s Integrated genes (Brown 2002). Of course, not all microbes Microbial Genomes (IMG) is able to link M 386 Microbial Genomes That Drive Earth’s Biogeochemical Cycles

Microbial Genomes That Drive Earth’s Biogeochem- expanded to their KO definitions, representing the com- ical Cycles, Fig. 2 Screenshot of KEGG pathway map plete catalog of genes needed for denitrification, a small, for nitrogen metabolism. EC#s highlighted in pink are conserved set (Kanehisa et al. 2012)

a genome (or metagenome) in its database to a list biogeochemistry reveals a surprisingly small of genes, which is subsequently linked together to set, significantly less than 1,000 orthologous show pathway and biogeochemical capacity. In groups. KEGG, these genes are represented by KO IDs, or orthologous groups of genes, which are in turn organized into pathway modules (Kanehisa Environmental Metagenomics et al. 2012). KEGG pathway maps are organized in a hierarchical fashion to organize pathway For a system-level understanding of biogeochem- modules into computationally friendly and bio- istry, looking at individual genomes of each logically meaningful networks. After condensing known organism in a sample is not nearly as mass amounts of genetic information into KEGG revealing as sequencing the entire community. metabolic pathway maps, an example (Fig. 2) Community sequencing, or metagenomics, shows that the total number of gene families allows for examination of an environmental sam- carrying out denitrification, a major energy trans- ple’s entire biochemical capacity rather than indi- duction in biogeochemical cycling, is a highly vidual pieces of the community’s metabolic limited set. The theme encountered here with puzzle. denitrification is repeated in the remaining bio- Metagenomics, striving to understand biology geochemical pathways; they are controlled by at the aggregate level, is often used in analysis of a small core set of genes. Adding the KO groups communities so complex that they can only be that are directly involved in Earth’s known sampled, never completely characterized Microbial Genomes That Drive Earth’s Biogeochemical Cycles 387 M

(National Research Council 2007). Many ORFs Pathway Interaction in a typical metagenome are annotated with unknown or hypothetical function, genes either Characterizing a sample with an interconnected homologous to a gene of unknown function or set of pathways is desirable, as the pathways those without a known homolog. A typical new involved do not stand alone, but have significant genome contains 20–30 % genes of unknown interaction with one another. Biogeochemical function. This lack of complete information, pathways have strong effects on their immediate though representing a significant area for environment, which can in turn affect expression improvement in genomics in general, is not as of genes and ultimately the environment’s path- big of a problem when studying biogeochemistry, way capacity. The waste product from one path- where relevant pathways are represented by way can be the substrate for another, as is the case a core set of well-known genes. Though new with CO2 from aerobic respiration feeding into electron-transfer pathways based on favorable photoautotrophic pathways. In the nitrogen cycle, redox couples have been predicted, they have several species of organisms are often required yet to be found in nature. Novel metabolism for complete denitrification. One strain will typ- will be discovered, but only so many options are ically be capable of reducing nitrate to nitrite, available to the microbial community another from nitrite to N2O, and a final player (McCollom and Shock 1997). capable of converting N2O to free N2 gas If a target organism represents a significant (Madigan and Brock 2009). Pathways can also percentage of the community, its genome can be be “leaky,” letting substrates out into the envi- reconstructed from the community metagenome ronment, freeing them to interact with the net- because a good percentage of the reads produced work where they may preclude the need for from sequencing will have ends that align, another pathway (Morris et al. 2012). With cell enabling construction of contigs, scaffolds, and, death occurring, all components are potentially M in some cases, an entire genome (Iverson leaked. Rather than ideal end products linking et al. 2012); this same concept works in favor of pathways, as in KEGG pathway maps, interme- using metagenomics to study biogeochemical diate products also have this potential, contribut- capacity. Just as the most representative organ- ing to the system’s complexity. For these reasons, ism in a sample will give a strong signal, so will the approach to understanding biogeochemical the most representative genes. As discussed pre- cycling can be greatly simplified by involving viously, with the relatively small set of highly metagenomic analysis at the system level. conserved genes responsible for elemental Metagenomics has provided a new way to cycling, it stands to reason that this set will measure the genetic potential of a community. appear repeatedly and in high frequency, making For measuring the more immediate genetic the signal for biogeochemical capacity of expression of a sample, metatranscriptomics, or a sample clearly detectable by metagenomics. the sequencing of a community’s active mRNA Metagenomics is a method of sampling the transcripts, reveals the current biochemical state genes in a given sample, far from producing of the sample (Moran 2009). Change to a system a comprehensive library of every gene present. may result in different metatranscriptomes, giv- Less common genes in a sample are difficult to ing important clues as to the response of the find, but manipulating an environment, letting the system to any kind of environmental perturba- microbial population adjust, or resequencing the tion. Modeling future states of planetary biogeo- community can enrich for pathways of interest. chemistry will be greatly aided by comparing Nonconventional parts of the metagenome have metatranscriptomic and other “omics” data been accessed with methods such as fractionating (proteomics, metabolomics) from various envi- metagenomic DNA by cell-density gradients or ronments under different stresses, directly under- DNA fragment size distribution (Delmont standing how changes to one part of the system et al. 2011). can affect the network as a whole. M 388 Microbial Genomes That Drive Earth’s Biogeochemical Cycles

VI: Oxygenic Photosynthesis Photosystem I V: Methanogenesis

Cytochrome b6t complex and II, I: Aerobic sugar Reductive Acetyt CoA metabolism II: Sulfate Coenzyme F420 Reduction Pathway NAD(P)H Quinone Oxidoreductase APS Reductase Sulfite Reductase Sulfate Reductase

TCA Cycle Glycolysis, Pyruvate

Oxidation

Cytochrome c

Intermediate Cytochromes, Iron sulfurQuinones Proteins,

IV: Hydrogen Oxidation III: Denitrification

Hydrogenase Nitrate Reductase Nitrite Reductase Nitric Oxide Reductase Nitrous Oxide Reductase

Microbial Genomes That Drive Earth’s Biogeochem- photosynthesis.” Pathway group III: “Denitrification” ical Cycles, Fig. 3 Venn diagram depicting the uses unique reductases to reduce nitrates and other oxi- interconnected and promiscuous nature of biogeochemi- dized nitrogen compounds but still requires the common cal pathway groups I–VI from Fig. 1, based on shared components of the electron transport chain as well as components. Pathway groups I to VI (large font) are glycolysis and the TCA cycle as a source of reductant. shown to have redox components of electron transport Pathway Group IV: “Hydrogen oxidation” uses an elec- chains as well as auxiliary pathways in common (small tron transport chain but is not dependent on glycolysis or font). Pathway group I: “Aerobic sugar metabolism” is the TCA cycle for producing reductant. Pathway Group V: shown to have components like an NAD(P)H reductases, “Methanogenesis” does not use an electron transport chain as well as membrane-localized cytochromes and qui- and is the only pathway utilizing coenzyme F420. Size of nones, in common with pathway group VI: “Oxygenic circles is irrelevant (Kim et al. 2013)

Evolutionary History environmental metadata have provided examples of geochemical constraints on metabolism In addition to better understanding the current (Inskeep et al. 2010). and future state of elemental cycling on Earth, Evolution during the Archaean period during metagenomic methods can also help unravel the which the first metabolic pathways were being history of how genes and biogeochemical cycles invented was highly permissive of HGT (David have coevolved. For example, analysis of path- and Alm 2011). The genes responsible for today’s ways occurring in supposed early-earth ana- pathways of anabolism and catabolism are widely logues such as anaerobic niches, deep-sea vents, distributed through both bacteria and archaea, or hot springs gives insight into life’s early suggesting horizontal gene flow from a common metabolism. Contrasting the metabolic catalogs gene pool before vertical modes of transmission from different environments and incorporating became more important. With each microbe on Microbial Genomes That Drive Earth’s Biogeochemical Cycles 389 M the planet, a temporary “guardian” of its own The great diversity of the microbes temporar- metabolism, the entire variety of metabolism is ily guarding the blueprints for life’s reactions is conserved by a diverse set of organisms, render- largely responsible for the system’s resilience to ing the robust nature of biogeochemical cycles on environmental change. The rare biosphere pre- Earth more comprehensible. With its assets serves pathways not relevant to a current ecolog- spread, the majority of metabolism does not rely ical context, giving the system great flexibility on any single taxon for its conservation. In addi- should the context change. Though a tool only tion to this, distribution of genomic information able to sample this diversity, metagenomics is through the planet’s diverse set of microbes nonetheless powerful when analyzing how the allows for important information, not relevant to biogeochemical network operates, adjusts, and the current ecosystem state, to still be preserved is regulated. Studying the current, past, and pos- in low copy number by the “rare biosphere” sible futures of global elemental cycling is (Sogin et al. 2006). This ancient rare biosphere greatly aided by this new technique. is highly innovative, with highly divergent mem- bers. Depending on the environmental factors at a given time, these genes may have played impor- Cross-References tant parts in the evolution of planetary processes. Pathways, representing large and highly con- ▶ Brine Pools, Metagenomics of served modules of evolution, are now passed ▶ Forest Soil Metagenomics primarily through vertical descent, but because ▶ Ocean Gyres, Metagenomics of many pathways use common components, it is ▶ Ocean Metagenomics possible that horizontal transfer of part of ▶ Rhizosphere Metagenomics a pathway can give new capability to an organism ▶ Rivers, Metagenomics of genetically prepared for it. In the past, this has ▶ Salt Lakes, Metagenomics of M allowed the pathways to evolve in modules, more ▶ Seafloor, Metagenomics of freely than if built from completely unique parts ▶ Soil Metagenomics (Fig. 3). References Summary Brown TA. Genomes, 2nd ed. Oxford: Wiley-Liss; 2002. Chapter 7, Understanding a genome sequence. http:// Earth’s biogeochemical cycles are built upon www.ncbi.nlm.nih.gov/books/NBK21136. Accessed a relatively small set of highly conserved genes, 15 May 2013. widely distributed throughout life. This set, David LA, Alm EJ. Rapid evolutionary innovation during an archaean genetic expansion. Nature. 2011; enabling elemental cycling on Earth, is 469(7328):93–6. represented by well under 1,000 orthologous Delmont TO, Robe P, Cecillon S, Clark IM, groups of genes assembled into less than 150 path- Constancias F, Simonet P, et al. Accessing the soil ways in the KEGG database. Small gene sets metagenome for studies of microbial diversity. Appl Environ Microbiol. 2011;77(4):1315–24. representing common pathways appear in large Falkowski PG, Fenchel T, Delong EF. The microbial numbers, even if from a variety of organisms, engines that drive Earth’s biogeochemical cycles. rendering biogeochemistry highly amenable to Science. 2008;320:1034–39. metagenomic analysis. Whereas genomics has Inskeep WP, Rusch DB, Jay ZJ, Herrgard MJ, Kozubal MA, Richardson TH, et al. Metagenomes from high- uncovered lifestyles of culturable and fully temperature chemotrophic systems reveal geochemi- sequenced organisms, metagenomics has revealed cal controls on microbial community structure and a massive unculturable world of microbes, com- function. PLoS ONE. 2010;5(3):e9773. prehensible by homology to known genes. The Iverson V, Morris RM, Frazar CD, Berthiaume CT, Morales RL, Armbrust EV. Untangling genomes from biogeochemical capacity of an environmental metagenomes: revealing an uncultured class of marine sample can be inferred from this data. euryarchaeota. Science. 2012;335(6068):587–90. M 390 Microbial Infection, Leukemia and Lymphoma Associated with

Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe factors such as state of disease, cytotoxicity M. KEGG for integration and interpretation of large- induced by immunosuppressive medications, scale molecular datasets. Nucleic Acids Res. 2012;40: D109–14. intensity and depth of neutropenia, defects in Kim JD, Senn S, Harel A, Jelen BI, Falkowski host humoral and cellular defense mechanisms, P. Discovering the electronic circuit diagram of life: or immune system impairment can make a patient structural relationships among transition metal binding more susceptible to infection. The infectious sites in oxidoreductases. Phil Trans R Soc B 2013, June (in press)20120257 http://dx.doi.org/10.1098/ agents are present as cofactors and at times as rstb.2012.0257. causative agents. Infections and the associated Madigan MT, Brock TD. Brock biology of microorganisms. disease are many times endemic to particular San Francisco: Pearson/Benjamin Cummings; 2009. geographical area(s). It is estimated that almost McCollom TM, Shock EL. Geochemical constraints on chemolithoautotrophic metabolism by microorgan- 80 % of acute leukemia patients and 70 % of isms in seafloor hydrothermal systems. Geochim lymphoma patients develop infection during Cosmochim Ac. 1997;61(20):4375–91. their disease course (Rolston and Bodey 2010). Moran MA. Metatranscriptomics: eavesdropping on Multiple episodes of infections in one patient are complex microbial communities. Microbe. 2009; 4(7):329–35. not uncommon. Morris JJ, Lenski RE, Zinser ER. The black queen hypoth- esis: evolution of dependencies through adaptive gene loss. MBio. 2012;3(2):1–6. Risk Factors for Infectivity by Sogin ML, Morrison HG, Huber JA, Mark Welch D, Huse SM, Neal PR, et al. Microbial diversity in the deep sea Microorganisms in Leukemia and and the underexplored “rare biosphere”. Proc Natl Lymphoma Acad Sci U S A. 2006;103(32):12115–20. National Research Council. The new science of Infections are generally caused by organisms that metagenomics: revealing the secrets of our microbial planet. Washington, DC: The National Academies colonize the patient, although they can also be Press; 2007. acquired once the patient is in the hospital. Vernadsky VI. ([1926] 2007). Geochemistry and the bio- Polymicrobial bacterial infections occur more sphere. Translated by Olga Barash (Synergetic Press, often in patients with acute leukemia, because Santa Fe, 2007). of intense immunosuppressive therapy and prolonged neutropenia or when the absolute neu- trophil count is less than or equal to 500/mm3 Microbial Infection, Leukemia and (Rolston and Bodey 2010). Approximately 23 % Lymphoma Associated with of bacterial infections associated with hemato- logic malignancies are polymicrobial, and these Preeti Zanwar often result in higher mortality than infection by Sealy Center on Aging, University of Texas a single species (Rolston and Bodey 2010). The Medical Branch, Galveston, TX, USA degree and intensity of neutropenia is a critical factor that can substantially affect the develop- ment of infections in leukemia patients. An abso- Introduction lute neutrophil count less than 1,000/mm3 increases the risk of infections, yet neutropenic Microbial infections by bacteria, viruses, fungi, patients seldom develop signs and symptoms of and opportunistic pathogens are a common infection because of their inability to mount an occurrence in leukemia and lymphoma and inflammatory response (Freifeld and Kaul 2008). account for a large share of infections. They Fever, therefore, is often the only early sign of cause significant morbidity and mortality to infection (Rolston and De 2004). Defects in neu- their host. Each malignant disease harbors trophil function can also exist in spite of adequate a unique set of infections, and the frequency of number of circulating neutrophils. Neutrophil infection depends on the underlying neoplasm defects include impairments in the mobilization and state of the disease. Several predisposing function of neutrophils, which limits their ability Microbial Infection, Leukemia and Lymphoma Associated with 391 M to migrate to the site of inflammation and the et al. 2007). Defects in lymphocyte function and inability to phagocytose and kill organisms neutropenia are present in hairy cell leukemia, (Rolston and De 2004; Rolston and Bodey 2010). and infections with gram-negative bacilli, gram- positive cocci, and mycobacteria (including nontuberculous) have been reported. Hairy cell Common Microbial Infections in leukemia, however, is not associated with EBV Leukemia/Lymphoma infection (Hasserjian 2011). Exogenous viruses that transmit horizontally, Infections by bacteria, viruses, fungi, and other such as oncogenic retrovirus and lentivirus, have opportunistic pathogens in leukemia are summa- been strongly associated with lymphoid malig- rized in Table 1. According to M. D. Anderson nancies. Human T-cell leukemia/lymphoma virus Cancer Center data (MDACC), gram-positive type 1 (HTLV-1) was the first human oncogenic bacteria cause nearly 50–55 % of recognized retrovirus isolated in 1980 by Gallo’s group. It infections in neutropenic patients (Rolston and belongs to the family of exogenous retroviruses Bodey 2010), including 65–75 % of bloodstream and is the known etiologic agent for adult T-cell infections. In acute leukemia, gram-positive leukemia (ATL). It is the only human retrovirus cocci, gram-negative bacilli, Candida, Aspergil- that is conclusively accepted as the causative lus, Fusarium, and Trichosporon are common agent of a human leukemia or lymphoma and occur in combination with neutropenia. (Mahieux and Gessain 2005). ATLL is Acute lymphoblastic leukemia (ALL) is a rare a malignant lymphoproliferation of CD4+ acti- clonal proliferation of cancerous lymphoblasts vated T cells with at least one clonally integrated in adults. No direct evidence of a viral role in HTLV-1 provirus (McAdam and Sharpe 2009). ALL causation is present, however an indirect ATLL is present in all endemic areas associated route of viral involvement has been suggested with HTLV-1 infection, namely, Japan, Carib- M (Hoelzer and Go¨kbuget 2005). bean, intertropical Africa, Central and South In chronic lymphocytic leukemia (CLL), America, and some restricted areas of the Middle humoral immune dysfunction and reduction in East and Melanesia. Its occurrence is sporadic in opsonizing antibodies render the defense process the United States (Stricker and Kumar 2009). of bacterial pathogen ingestion and destruction In high endemic areas of viral infection such as by phagocyte defective. Therefore, CLL patients Jamaica in the Caribbean, prevention of early are especially at risk of infections by encapsu- HTLV-1 infection from seropositive mothers by lated organisms such as Streptococcus not breast-feeding their babies can significantly pneumoniae, Haemophilus influenzae, and reduce HTLV-1-associated non-Hodgkin’s T-cell Neisseria meningitidis (Montserrat 2005; Rolston lymphomas (Mahieux and Gessain 2005). Associ- and Bodey 2010). Richter syndrome (RS) is also ations of HTLV-1 and HTLV-2 with other lym- known as the development of high-grade phoid neoplasms remain inconclusive at this time. non-Hodgkin’s lymphoma (NHL) or small lym- phocytic leukemia in those with CLL. At M. D. Anderson Cancer Center (MDACC), samples of Specific Infectious Agents Associated RS and fludarabine-refractory CLL were positive with Lymphomas for Epstein-Barr virus (EBV) by polymerase chain reaction (Tsimberidou et al. 2007). Increasing numbers of infectious, mostly viral These results are suggestive that EBV, a linked, associations have now been found in lym- B-lymphotropic human herpes virus, may play phomas. Usually the infection, exists in persistent a role in RS and in the progression of CLL. The form and requires additional cofactors for the precise role of EBV in Richter’s transformation is malignancy to develop (Kinlen 2004). Microbial not known, and the causal link between EBV and infections and the specific lymphoma types are RS has not been established (Tsimberidou summarized in Table 2. Mechanisms of M 392 Microbial Infection, Leukemia and Lymphoma Associated with

Microbial Infection, Leukemia and Lymphoma Associated with, Table 1 Microbial infections associated with leukemia Defect in host defense mechanism/disease Leukemia type Microbial infection(s) setting Acute lymphoblastic Common bacteria: gram-positive cocci Prolonged neutropenia, intense leukemia (ALL) (Streptococcus, Streptococcus pneumoniae, chemotherapy, humoral immune Streptococcus mitis, Staphylococcus, dysfunction Corynebacterium sp.), gram-negative pathogens (Escherichia coli, Klebsiella, Pseudomonas aeruginosa) Bacillus sp., Clostridium difficile, Clostridium septicum, Listeria monocytogenes, Chlamydia New gram-positive and gram-negative pathogens: Stenotrophomonas (Xanthomonas) maltophilia, Bacillus cereus, Stomatococcus mucilaginosus, Corynebacterium jeikeium, Rhodococcus sp., Leuconostoc sp., Burkholderia cepacia, Bartonella sp. Virus: herpes simplex virus (HSV), varicella zoster virus (VZV), Cytomegalovirus (CMV), parvovirus B19, human herpes virus 6, respiratory syncytial virus, influenza virus, parainfluenza Fungi: Candida, Aspergillus, Fusarium, Mucor, Fusarium, Trichophyton, C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, A. flavus, A. fumigatus Yeast: non-Candida species, Trichosporon, Malassezia furfur, Blastoschizomyces capitatus, Rhodotorula rubra, Saccharomyces cerevisiae, Clavispora lusitaniae, Cryptococcus sp. Protozoa: Pneumocystis carinii P. carinii pneumonia (PCP) common due to ubiquitous prophylaxis Burkitt’s leukemia Epstein-Barr virus (EBV) Endemic, Africa Chronic lymphocytic Encapsulated bacteria: Streptococcus Hypogammaglobulinemia leukemia (CLL) pneumoniae, Neisseria meningitidis, Staphylococcus, Haemophilus influenzae Common complication: herpes zoster Opportunistic organisms: Legionella Posttreatment with new pneumophila, Pneumocystis carinii, Listeria immunosuppressive agents such as monocytogenes, CMV fludarabine and prednisone Fungi: Candida, Aspergillus Virus: EBV Richter syndrome (high-grade NHL/small lymphocytic leukemia) Acute myelogenous Common bacteria: coagulase-negative leukemia (AML) staphylococci, Staphylococcus aureus (including MRSA*), alpha-hemolytic (viridians) streptococci, Enterococcus sp. (including VRE**), Enterobacteriaceae (Escherichia coli, Klebsiella sp., Proteus sp., Serratia sp., Citrobacter sp., Enterobacter sp.), Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter sp. Common fungi: Candida sp., Aspergillus sp. Common viruses: HSV, VZV, CMV, community respiratory viruses (continued) Microbial Infection, Leukemia and Lymphoma Associated with 393 M

Microbial Infection, Leukemia and Lymphoma Associated with, Table 1 (continued) Defect in host defense mechanism/disease Leukemia type Microbial infection(s) setting Chronic myelogenous Common bacteria: encapsulated S. pneumoniae, P. aeruginosa frequently fatal in advanced leukemia (CML S. aureus, gram-negative bacteria, P. aeruginosa, disease and prolonged myelosuppression H. influenzae, Legionella sp., Salmonella sp. Fungi: Candida and Aspergillus Viruses: HSV, Advanced stage disease, prolonged VZV neutropenia Opportunistic pathogens: mycobacteria, Listeria Patients treated with fludarabine monocytogenes, Nocardia sp., Pneumocystis carinii, Candida, Aspergillus, VZV Hairy cell leukemia Common bacteria: P. aeruginosa, other enteric Neutropenia, impaired lymphocyte (HCL) gram-negative bacilli, gram-positive function (Staphylococcus) Nontuberculous mycobacteria: M. kansasii, M. avium-intracellulare, M. gordonae, M. scrofulaceum, M. chelonae, M. fortuitum, M. malmoense Unique fungi: Histoplasma capsulatum, Coccidioides immitis, Cryptococcus neoformans, Blastomyces dermatitidis, Sporothrix schenckii, Pseudallescheria boydii T-cell prolymphocytic HTLV-1 Tax sequence in DNA samples of some leukemia (T-cell PPL) Japanese patients, single case of EBV-related T-PLL exists NK-cell large granular Aggressive NK-cell LGLL EBV lymphocytic leukemia associated (NK-cell LGLL) Adult T-cell leukemia Etiologic agent: human T-lymphotropic Endemic (Japan, Caribbean, intertropical M (ATL) virus 1 (HTLV-1) Opportunistic microbes: Africa, Central and South America, Pneumocystis carinii, Cryptococcus meningitis, restricted areas of Middle East and herpes zoster, Strongyloides stercoralis (SS) Melanesia) MRSA* – methicillin-resistant Staphylococcus aureus VRE** – vancomycin-resistant Enterococcus

infections in non-Hodgkin’s lymphoma (NHL) the corresponding higher risk for NHL subtypes. can be categorized in three broad groups: Group three includes hepatitis C virus linked to (1) direct transformation of lymphocytes, NHL and Helicobacter pylori linked to mucosa- (2) depletion of CD4+ lymphocytes, and associated lymphoid tissue (MALT) NHL. Advent (3) chronic immune stimulation. Group one com- of highly active antiretroviral therapy (HAART) prises of EBV as the etiologic cofactor in the has mitigated the risk of NHL in those with causation of endemic Burkitt lymphoma in HIV in the recent years (Crump 2011). Africa, NHL in immunocompromised hosts, Human immunodeficiency virus, a lentivirus, extranodal natural killer/T-cell NHL, human her- does not directly cause any human lymphoid pes virus 8 (HHV-8) linked to primary effusion malignancy. In HIV-infected patients, the lymphoma, and HTLV-1 linked to ATLL common B-cell lymphomas are a result of the (Hoelzer and Go¨kbuget 2005; Engels 2007; reactivation of latent EBV virus (Mahieux and Fowler and McLaughlin 2011). Group two includes Gessain 2005). Similarly, reactivation of human human immunodeficiency virus (HIV) linked to herpes virus 8 (HHV-8) or Kaposi’s sarcoma- acquired immunodeficiency syndrome (AIDS) and associated herpesvirus (KSHV) is implicated in M 394 Microbial Infection, Leukemia and Lymphoma Associated with

Microbial Infection, Leukemia and Lymphoma Associated with, Table 2 Microbial infections associated with lymphoma Microbe Microbe specification Predominant infectious agents Lymphoma type Virus Lymphotropic Epstein-Barr virus (EBV) EBV coinfection; endemic Burkitt lymphoma viruses (Africa); concomitant infection with malaria Natural killer/T-cell nasal lymphoma AIDS-related non-Hodgkin’s lymphoma (ARS) [most CNS] Hodgkin’s lymphoma (in the setting of HIV infection) Hodgkin’s lymphoma (infectious mononucleosis-associated lymphoma) Posttransplant lymphoproliferative disorder (PTLD) Human T-cell lymphotropic Adult T-cell lymphoma virus I (HTLV-I) Human herpes virus 8 (HHV-8)/Kaposi’s Primary effusion lymphoma sarcoma-associated herpes virus (KSHV) Plasmablastic lymphoma Lymphoid neoplasia HHV-8 HIV-associated body cavity lymphoma Diffuse large B-cell lymphoma (DLBCL) Polyomavirus Simian virus 40 AIDS-associated non-Hodgkin’s lymphoma Herpes simplex virus (HSV) Lymphoma RNA virus Hepatitis C virus (HCV) Splenic marginal zone lymphoma Indolent B-cell lymphomas Lymphoplasmacytic lymphoma Bacteria Gram- Chlamydia psittaci Ocular adrenal MALToma (NHL) negative Helicobacter pylori Gastric MALToma (indolent B-cell lymphoma bacteria of mucosa-associated lymphoid tissue) Borrelia burgdorferi Primary cutaneous B-cell lymphoma/marginal zone B-cell lymphoma (MZL) Borrelia afzelii Cutaneous MALT NHL Campylobacter jejuni Mediterranean lymphoma (a heavy-chain disease)/small intestine MALT lymphoma Salmonella sp. Lymphoma Gram-positive Mycobacterium sp. Hodgkin’s disease (impairment of the cellular bacteria component of the host defense mechanisms) Protozoa Parasitic Toxoplasma gondii Lymphoma (60 %) protozoa

the development of primary effusion lymphomas HIV-associated central nervous system lympho- in those with AIDS (Mahieux and Gessain 2005). mas and nearly with all cases of posttransplant It is estimated that Kaposi’s sarcoma, an uncom- lymphoma (Fowler and McLaughlin 2011). mon cancer of the lymphoid organ, is 10,000 EBV plays a fundamental role in at least some times more likely in HIV-infected than non-HIV- Hodgkin’s lymphomas. Increased incidence of infected individuals. Hodgkin’s disease and Hodgkin’s lymphoma has been recognized with NHL, the other two tumors linked with AIDS, EBV infection (Crump 2011). EBV-positive are associated with EBV (MacMohan 2008). Hodgkin’s lymphomas have better outcomes than EBV is also correlated with 100 % of EBV-negative Hodgkin’s lymphoma, and the Microbial Infection, Leukemia and Lymphoma Associated with 395 M underlying etiologies of these two lymphomas leukemia surpass the immune surveillance system are different. Infectious mononucleosis occurs and are undetected. Therefore, broad range antibi- approximately three years post-EBV infection otic therapy is suggested for these patients. There and is only seen in EBV-positive Hodgkin’s is an alarming trend towards multidrug resistance lymphoma. Hodgkin’s lymphoma is not an among newer gram-negative isolates (Rolston AIDS-defining malignancy in the HIV-positive KVI and De 2004). Viral associations in lym- population; however, its incidence is higher phoma patients are becoming increasingly more fivefold in those with HIV infection as compared common, and at least one virus is an established to the general population (Crump 2011). Subtypes causative agent for leukemia/lymphoma. of Hodgkin’s lymphoma in the setting of HIV infection occur more often in advanced stage disease, often involve extranodal sites, are Cross-References often of mixed cellularity and of predominant lymphocyte histology, and often more strongly ▶ Cancer, Definition associated with EBV infection (Crump 2011). ▶ Corynebacteriaceae The associations of gram-positive bacteria ▶ Fungus in the Human Microbiome, Definition with various MALTomas, gram-negative bacte- and Examples ria with Hodgkin’s disease, and parasitic proto- zoa with lymphoma are summarized in Table 2. The association of hepatitis C virus in lymphoplasmacytic lymphoma is contentious References (Viswanatha et al. 2011). More definite studies Butel JS. Simian virus 40, human infections, and cancer: are needed to associate HTLV-1 with cutaneous emerging concepts and causality considerations. In: T-cell lymphomas such as mycosis fungoides Khalili K et al., editors. Viral oncology. New Jersey: M (MF) and Se´zary syndrome (Mahieux and Wiley; 2010. p. 165–89. Gessain 2005). No serologic and molecular evi- Crump M. Hodgkin lymphoma. In: Hussain SI, Ghulam MJ, editors. Advances in malignant hematology. 1st ed. West dence decisively suggests association of Sussex: Wiley-Blackwell; 2011. p. 296–314. lymphoproliferative diseases with HTLV-2 Engels EA. Infectious agents as causes of non-Hodgkin (Mahieux and Gessain 2005). Sequences of lymphoma. Cancer Epidemiol Biomarkers Prev. SV40’s T oncoprotein have been reported in 2007;16:401–4. Fowler N, McLaughlin P. Non-Hodgkin lymphoma. In: high proportions of NHL and AIDS-related lym- Hussain SI et al., editors. Advances in malignant phomas; however, heterogeneity, inconsistencies hematology. 1st ed. West Sussex: Wiley-Blackwell; in findings, and a dearth of mechanistic insights 2011. p. 274–95. among studies regarding SV40 functionality in Freifeld AG, Kaul DR. Infection in the patient with can- cer. In: Abeloff MD et al., editors. Abeloff’s clinical lymphoid cells make the role of SV40 in NHL oncology. 4th ed. Philadelphia: Churchill Livingstone/ premature (Jarret 2006; Vilchez and Butel 2007; Elsevier; 2008. p. 1–21. Butel JS 2010). Hasserjian R. Hairy cell leukemia. In: Jaffe ES et al., editors. Hematopathology. Philadelphia: Saunders/ Elsevier; 2011. p. 247–55. Hoelzer D, Go¨kbuget. Acute lymphoblastic leukemia in Summary and Findings adults. In: Degos L et al., editors. Textbook of malig- nant hematology. 2nd ed. Boca Raton: Taylor & A large number of bacteria, viruses, and fungi are Francis; 2005. p. 501–20. Jarret RF. Viruses and lymphoma/leukemia. J Pathol. associated with infections in those with leukemia 2006;208:176–86. and lymphoma. In leukemic patients, bacterial Kinlen L. Infections and immune factors in cancer: the infections arise during the early phase of neutro- role of epidemiology. Oncogene. 2004;23:6341–8. penia, while fungal infections are more predomi- MacMohan B. Accomplishments in cancer epidemiology. In: Adami HC et al., editors. Textbook of cancer epi- nant during the state of persistent neutropenia. demiology. 2nd ed. New York: Oxford University Many pathogenic infections in patients with Press; 2008. p. 3–33. M 396 Microbiome, Bladder

Mahieux R, Gessain A. Lymphoproliferations associated Definition with human T-cell leukemia/lymphoma virus type 1 or 2 infection. In: Degos L et al., editors. Textbook of malignant hematology. 2nd ed. Boca Raton: Taylor & A microbiome is the totality of microbes, their Francis; 2005. p. 187–206. genetic elements (genomes), and environmental McAdam AJ and Sharpe AH. Infectious diseases. In: interactions in a particular environment, in this Robbins and Cotran pathologic basis of disease, pro- instance, the human urinary tract. fessional edition. 8th ed. Philadelphia: Saunders Elsevier; 2009. Chapter 8. Montserrat E. Chronic lymphoid leukemia’s. In: Degos L et al., editors. Textbook of malignant hematology. Introduction 2nd ed. Boca Raton: Taylor & Francis; 2005. p. 521–42. Rolston KVI, Bodey GP. Infection in patients with cancer. In: Waun KH et al., editors. Holland-Frei cancer med- External body sites (epithelial-lined cavities icine. 8th ed. Shelton: People’s Medical Publishing that are contiguous with the environment) House-USA; 2010. Chapter 137. include the skin, mouth, ear, gastrointestinal Rolston KVI, De I. Infection in patients with acute mye- tract, respiratory tract, vagina, and the urinary logenous leukemia. In: Greene JN, editor. Infections in cancer patients. New York: Marcel-Dekker; 2004. tract. These sites are subject to colonization by p. 47–64. environmental organisms. Most of these micro- Stricker TP, Kumar V. Neoplasia – microbial carcinogen- organisms are of low virulence and have little esis. In: McAdam AJ, Sharpe AH, editors. Robbins and pathogenic potential or may even be of some Cotran pathologic basis of disease, professional edi- tion. 8th ed. Philadelphia: Saunders Elsevier; 2009. benefit in preventing colonization by pathogenic Chapter 7. bacteria. Others may be virulent with high path- Tsimberidou AM, Keating MJ, Wierda WG. Richter’s ogenic potential wherein colonization pro- transformation in chronic lymphocytic leukemia. gresses to symptomatic infection at the body Curr Hematol Malig Rep. 2007;2:265–71. Vilchez RA, Butel JS. Polyomavirus SV40 and AIDS- site of entry. The distal portion of the urethra related systemic non-Hodgkin lymphoma. In: is generally colonized with a variety of bacteria. Meyers G, editor. Cancer treatment and research. The remainder of the urinary tract, which New York: Springer; 2007. p. 215–40. includes the bladder, ureters and kidneys, is Viswanatha DS, Montgomery KD, Foucar K. Mature B-cell neoplasms: chronic lymphocytic leukemia- historically thought of as essentially sterile small lymphocytic lymphoma, B-cell prolymphocytic based upon lack of cultivatable bacteria in leukemia, and lymphoplasmacytic lymphoma. In: urine samples. Urinary tract colonization with Jaffe ES et al., editors. Hematopathology. Philadel- virulent bacteria may result in symptomatic uri- phia: Saunders/Elsevier; 2011. p. 221–46. nary tract infection (UTI) (Hooton and Stamm 1997). Alternately, bacterial colonization of the bladder may occur in the absence of signs and Microbiome, Bladder symptoms of UTI. The so-called asymptomatic bacteriuria (ABU) occurs spontaneously in cer- A Metagenomic Approach to Understanding the tain patient groups, such as those who rely upon Human Urinary Tract Microbiome catheterization for bladder drainage, and is thought to be beneficial in some instances Richard Hull (Nicolle 2012). These concepts are based upon Department of Molecular Virology and analysis of bacteria cultured from urine sam- Microbiology, Baylor College of Medicine, ples. Results of recent metagenomic studies Houston, TX, USA suggest that the urinary tract in most individuals may be colonized with uncultivated bacteria without associated signs or symptoms of urinary Synonyms tract infection. These findings challenge the “sterile bladder” paradigm and suggest that 16S rRNA gene sequencing; Uncultured urinary ABU may be far more prevalent than previously bacteria; Urinary microbiome thought. Microbiome, Bladder 397 M

Urethra Bladder

Several studies have focused on the microbiome Other studies were more focused upon UTI. of the male urethra. Although these studies were Imirzalioglu et al. (2008) used a 16S rDNA designed primarily to identify bacteria associated approach to survey for uncultured or unculturable with sexually transmitted infections, they provide bacteria that might be associated with symptom- some insight into the multiple bacterial genera atic UTI; 1449 serial urine samples were col- found in voided urine and in the distal urethra of lected over a 2-month period at a university asymptomatic males. Riemersma et al. (2003) hospital. Some urine samples were associated studied the microbial population in voided urine with symptomatic UTI, whereas others were col- of healthy males as part of their control group. lected for other reasons. The primary goal of this First-pass urine samples were collected from study was to illustrate the value of the male volunteers who presented with no signs of metagenomic approach for detection of fastidious urethritis. Total 16S rDNA was PCR amplified bacteria that might be associated with UTI symp- from urine samples, the products cloned, and toms. Routine bacteriological culture and PCR restriction fragment length polymorphism analy- amplification of 16S rDNA was performed on sis used to identify unique clones. Selected clones all urine samples. The authors reported that were subjected to DNA sequencing. The authors 165/1449 urine samples were positive by culture reported significant inter- and intrapersonal vari- and/or PCR and that 37/165 (34 female, 3 male) ability of the urine flora. One RFLP type occurred samples were positive for PCR only. DNA in nine of ten of the control urine samples but in sequence analysis of PCR products for the none from their urethritis symptomatic group. No 34 female subjects revealed bacteria associated other correlation between organism and disease with vaginal flora. Flaws in the methods used in state was seen. The authors noted that 7 of this report may limit interpretation of results. The M 18 clones were matched to microbial species method of urine collection was not reported but that had never been cultured in vitro or had not may have included samples of voided urine been classified as a species. which, for females, was likely contaminated Dong et al. (2011) collected urine samples and with vaginal flora. The study also did not distin- urethral swab samples from 22 male subjects who guish between asymptomatic colonization and had no evidence of urethritis, also as part of a study symptomatic UTI for most samples. Finally, an of the urethra microbiome associated with sexually unspecified number of patients had received anti- transmitted infection. Genomic DNA was biotic treatment prior to sample collection, which extracted and subjected to 16S rDNA amplifica- the authors note may explain the high frequency tion and subsequent sequencing. As in an earlier of culture and PCR-negative samples (89 %). study from the same group (Nelson et al. 2010), the Siddiqui et al. (2011) used a metagenomic majority of sequences corresponded to a few abun- approach to investigate the bacterial diversity in dant genera. All of the abundant genera were also urine from healthy females. Urine was collected reported present in the vaginal microbial commu- via the clean-catch method from eight healthy nities of healthy individuals (Kim et al. 2009; female subjects. PCR amplification of 16S rDNA http://www.hmpdacc-resources.org/). As the was performed on all urine samples followed by authors note, it is premature to conclude that any DNA sequencing. PCR amplicons were detected of the strains represent part of the normal flora of for DNA preparations from all six subjects. DNA the male urinary tract. However, it is tempting to sequence analysis revealed that the bacterial com- speculate that bacterial genera such as Lactobacil- position in the urine specimens was polymicrobial lus that are thought to suppress uropathogenic and that there was considerable variation between bacteria in the vagina may also colonize the male urine samples. The bacterial genera detected were urethra and serve a similar prophylactic role nearly identical with those typically associated (Stapleton et al. 2011). with the vaginal microbiome. M 398 Microbiome, Bladder

A recent study by Wolfe et al. (2012) more bladder pain, urgency, and frequency. However, directly addressed the hypothesis that ABU may attempts to identify an infectious etiology by be more prevalent than previously thought. Urine standard culture methods have not yielded defin- was obtained from 12 control subjects and 11 cul- itive results. Several 16S rDNA-based surveys of ture negative subjects who reported symptoms of the bladder microbiome were conducted to show pelvic distress. The pelvic distress concept a possible bacterial etiology for IC (Domingue encompasses a wide variety of interrelated clini- et al. 1995; Haarala et al. 1996; Heritz et al. 1997; cal conditions that include urinary incontinence, Keay et al. 1998). Bladder tissue samples were fecal incontinence, pelvic organ prolapse, collected by cystoscopy in each study, and urine voiding dysfunction, and defecatory dysfunction was collected by SP aspirate in one study. Overall that can adversely impact the lives of women the studies were limited in scope with regard to (Barber et al. 2005). The urine collection elucidation of the bladder microbiome. Results methods were carefully controlled to reduce the were variable, in one instance finding no bacteria likelihood of result bias due to introduction of and in others finding vaginal or enteric organ- bacteria into the bladder urine from other sites. isms. Based upon the results of these studies, Experimental samples were collected by trans- the general conclusion was that no single micro- urethral catheterization (TUC) and suprapubic organism appears to be associated with IC. (SP) aspiration. Control experiments included sampling of clean-catch, midstream voided urine samples for comparison and also sampling Discussion of skin organisms at the site of transcutaneous puncture that was associated with SP aspirate The metagenomic approach for description of the collection. Samples for each site were examined urinary tract microbiome has proven of value for by light microscopy, by standard microbiological identifying both cultured and uncultivated culture cultivation methods, and by metagenomic microbes in the urinary tract samples. However, methods. a review of the composition and potential signif- Voided samples contained both culturable and icance of the urinary tract microbiome in health nonculturable bacteria. The bacterial genera and disease may be premature. The application of found in voided urine samples were similar to 16S rDNA survey methods to the urinary tract the vaginal flora collected from the same patient. microbiota is still in the earliest stage, and inter- The authors concluded that voided urine samples pretation is still subject to limitations that have were contaminated with vaginal bacteria and may been thoughtfully presented in recent reports. not be representative of bacteria present in the The method of sample collection and patient bladder. In contrast, none of the SP or TUC urine demographic are two important considerations. samples contained culturable bacteria. However, Many of the studies have used voided urine or 21/23 (91 %) contained non-cultivatable bacteria urethral swabs as a source of clinical material. To as evidenced by production of 16S rDNA PCR paraphrase Dr. Foxman (2010), the urethra is product. The bacterial genera found in TUC and a portal for the both exit of urine and the entry SP samples, upon deep 16S rDNA sequence of microbes from the environment. Bacteria live analysis, were generally representative of those around the urethral opening in both men and typically associated with vaginal microbial com- women and routinely colonize urine in the ure- munities, but were not necessarily identical with thra. The majority of these bacteria are washed vaginal flora of the same patient. No significant out during micturition, but a few likely remain to differences were noted between colonization of be detected by the sensitive molecular methods control and symptomatic subjects. currently in use. While the clean-catch and swab Interstitial cystitis (IC) is a chronic disease collection methods are appropriate for that is characterized by symptoms that are similar metagenomic analysis of the distal urethra as with those associated with UTI, including was the purpose in the Riemersma, Nelson, and Microbiome, Bladder 399 M

Dong studies, results obtained from voided urine microbiome of the urethra or bladder, including samples may not be representative of bacteria in uncultivated bacteria, serve a similar probiotic the bladder. In addition the Nelson study illus- service. trates that patient behavioral characteristics, such as sexual activity, may significantly affect uri- Is There a Future for a Metagenomic nary tract microbiome results. Approach for Prevention of Urinary Tract Infection? Do Uncultivated Bacteria Have a Role in Efficacy of the metagenomic approach for iden- Protecting the Healthy Bladder from tification of disease-causing organisms as causa- Symptomatic Infection? tive agents in symptomatic UTI has been The normal, healthy state of the bladder, whether demonstrated in specific instances. For example, it is populated by low-virulence organisms or is it has been effective in identifying the slow- a sterile environment, is yet to be determined. growing microorganism Actinobaculum schaalii Sequence-based studies have revealed that the as an opportunistic uropathogen that affects pri- bladder and urethra may be populated by a far marily elderly patients who have underlying uro- more diverse microbiome than previously logical dispositions (Tschudin-Sutter 2011). thought. Additional studies will be needed to However, a large majority of acute urinary tract distinguish between the contrasting conditions infections can be attributed to one or more known wherein (1) microbial colonization with uropathogenic genera; they respond to antimicro- low-virulence organisms represents the normal bial therapy based upon the susceptibility of well- steady state of the urinary tract and (2) microbial known uropathogenic bacteria (Foxman 2010). In colonization results from transient events that are these instances, the metagenomic approach may of little medical significance. The studies to date be only marginally cost-effective. Metagenomic primarily represent temporal snapshots of the approaches are well suited for the study of the M urinary tract microbiome. They revealed substan- interaction of resident flora with pathogenic tial variability at both the genus and species level organisms. Studies described here suggest that in the composition of the urethra and bladder uncultivated bacteria may be more abundant in microbiomes. In most instances, the urinary the urinary tract than previously thought. tract microbiome appears to be comprised of Whereas culture methods find only one organism a subset of the bacterial genera that populate the in 95 % of acute uncomplicated UTI, vagina. The composition varies among different metagenomic methods reveal multiple species people and within the same person at different in many instances. Do these organisms commu- times. nicate with uropathogenic bacteria? Do they There is ample evidence that stable bladder influence urovirulence gene expression? Even at colonization by low-virulence organisms may this early stage, metagenomic discovery has be beneficial in at least some patient groups. opened new avenues for investigation into mech- Hansson et al. (1989) reported that elimination anisms of urinary tract disease. of bacteria from the urine of patients who presented with ABU resulted in increased risk of subsequent acute kidney infection. This Summary implied that ABU offered some protection against subsequent UTI. More recent clinical In this review, the potential for application of studies revealed that deliberate colonization of metagenomic approaches for understanding the the bladder with low-virulence bacteria resulted human urinary tract microbiome was addressed. in significant reduction in the incidence of symp- Although few studies specifically address the tomatic UTI (Darouiche et al. 2011; Sunde´n microbial composition of the urinary tract, results et al. 2010). Future metagenome-based studies of these studies challenge the existing paradigm may find that organisms that constitute the that the bladder is essentially a sterile M 400 Microbiome, Eye environment. The microbiomes of the urethra and Nicolle LE. Urinary catheter associated infections. Infect bladder are diverse and may have a role in urinary Dis Clin N Am. 2012;26:13–27. Riemersma WA, van der Schee CJ, van der Meijden WI, tract health. et al. Microbial population diversity in the urethras of healthy males and males suffering from nonchlamydial, nongonococcal urethritis. J Clin Cross-References Microbiol. 2003;41:1977–86. Siddiqui H, Nederbragt AJ, Lagesen K, et al. Assessing diversity of the female urine microbiota by high ▶ Microbiome, Vagina throughput sequencing of 16S rDNA amplicons. BMC Microbiol. 2011;11:244. Stapleton AE, Au-Yeung M, Hooten TM, et al. Randomized, placebo-controlled phase 2 trial of a References Lactobacillus crispatus probiotic given intravaginally for prevention of recurrent urinary tract infection. Clin Barber MD, Walters MD, Bump RC. Short forms of two Infect Dis. 2011;52:1212–7. condition-specific quality-of-life questionnaires for Sunde´nF,Ha˚kansson L, Ljunggren, et al. Escherichia coli women with pelvic floor disorders (PFDI-20 and 83972 bacteriuria protects against recurrent lower PFIQ-7). Am J Obstet Gynecol. 2005;193:103–13. urinary tract infections in patients with incomplete Darouiche RO, Green BG, Donovan WH, bladder emptying. J Urol. 2010;184:179–85. et al. Multicenter randomized controlled trial of bac- Tschudin-Sutter S, Frei R, Weisser M, et al. terial interference for prevention of urinary tract infec- Actinobaculum schaalii – invasive pathogen or inno- tion in patients with neurogenic bladder. Urology. cent bystander? A retrospective observational study. 2011;78:341–6. BMC Infect Dis. 2011;11:289. Domingue GJ, Ghoniem GM, Bost KL, et al. Dormant Wolfe AJ, Toh E, Shibata N, et al. Evidence of microbes in interstitial cystitis. J Urol. uncultivated bacteria in the adult female bladder. 1995;153:1321–6. J Clin Microbiol. 2012;50:1376–83. Dong Q, Nelson DE, Toh E, et al. The microbial commu- nities in male first catch urine are highly similar to those in paired urethral swab specimens. PLoS ONE. 2011;6:e19709. Microbiome, Eye Foxman B. The epidemiology of urinary tract infection. Nat Rev Urol. 2010;7:653–60. 1 2 Haarala M, Jalava J, Laato M, et al. Absence of bacterial Diane S. Hutchinson , Stephen C. Pflugfelder DNA in the bladder of patients with interstitial cystitis. and Joseph F. Petrosino3 J Urol. 1996;156:1843–5. 1Interdepartmental Program in Translational Hansson S, Jodal U, Lincoln K, et al. Untreated asymp- Biology and Molecular Medicine, Baylor tomatic bacteriuria in girls: II – effect of phenoxymethylpenicillin and erythromycin given for College of Medicine, Houston, TX, USA intercurrent infections. BMJ. 1989;298:856–9. 2Department of Ophthalmology, Baylor College Heritz DM, Lacroix JM, Batra SD, et al. Detection of of Medicine, Houston, TX, USA eubacteria in interstitial cystitis by 16S rDNA ampli- 3Department of Molecular Virology and fication. J Urol. 1997;158:2291–5. Hooton TM, Stamm WE. Diagnosis and treatment of Microbiology, Alkek Center for Metagenomics uncomplicated urinary tract infection. Infect Dis Clin and Microbiome Research, Baylor College of N Am. 1997;11:551–81. Medicine, Houston, TX, USA Imirzalioglu C, Hain T, Chakraborty T, et al. Hidden path- ogens uncovered: metagenomic analysis of urinary tract infections. Andrologia. 2008;40:66–71. Keay S, Zhang CO, Baldwin BR, et al. Polymerase chain Synonyms reaction amplification of bacterial 16S rRNA genes in interstitial cystitis and control patient bladder biopsies. Ocular microbiome; Ocular microbiota J Urol. 1998;159:280–3. Kim TK, Thomas SM, Ho M, et al. Heterogeneity of vaginal microbial communities within individuals. J Clin Microbiol. 2009;47:1181–9. Introduction Nelson DE, Van Der Pol B, Dong Q, et al. Characteristic male urine microbiomes associate with asymptomatic sexually transmitted infection. PLoS ONE. 2010;5: Similar to other mucosal sites, the ocular e14116. surface is colonized by microbial communities. Microbiome, Eye 401 M

For over a century, clinicians have employed culturing methods to identify bacteria responsi- ble for infections of the ocular surface. How- ever, these cultures frequently revealed the commensal bacteria colonizing the eye. The bacterial populations of the eye resemble those of the skin and upper respiratory tract, with Staphylococcus species being the most com- monly identified microorganisms (McClellan 1997; Brinser and Burd 2001; Miller and Iovieno 2009). The microbiome of the eye has been observed to change with age and is highly dependent on climate and locale, although seasonal changes are not observed (Brinser and Burd 2001). A better understanding of the normal bacteria colonizing the ocular surface will provide clinicians with resources to improve diagnoses of ocular infections and The tear film covers the surface of the corneal diseases. and conjunctival epithelium and is composed of three layers: aqueous (produced by the lacrimal gland), oil (produced by the meibomian glands), The Ocular Surface and mucus (produced by the goblet cells of the conjunctiva). Immunomodulatory molecules are The eye is generally considered an immune present in the tear film including immunoglobulins M privileged site; however, this status is limited to (predominantly IgA) and complement. Impor- the enclosed portions of the eye. Sites of the tantly, the tear film contains the antibacterial pro- ocular surface are continually exposed to the teins lysozyme, lactoferrin, and lipocalin. environment (including microbes); therefore, we Lysozyme acts to destroy the outer cell wall of will only consider the ocular surface in the con- Gram-positive bacteria. The iron-sequestering text of the microbiome. activities of lactoferrin and lipocalin slow the The ocular surface consists of the cornea growth of bacteria. The tear film plays an impor- and conjunctiva. Both the corneal and conjunc- tant role in the protection of the ocular surface by tival epithelia are composed of several layers providing lubrication to the eye and helping to of nonkeratinized squamous epithelium. The remove foreign particles and organisms. structure of the corneal epithelium is highly The eyelids provide an external physical bar- organized, while the conjunctiva is comprised rier to the eye and are lined by the conjunctiva. of less organized epithelial cells interspersed The blinking action of the eyelids is responsible with goblet cells and lymphoid tissue (Miller for spreading the tear film across the ocular sur- 1979). The organized structure of the cornea face (Miller 1979). This motion also removes aids in the focusing of images by the lens, and foreign particles and microbes. damage to the cornea, through trauma or infec- tion, can greatly reduce vision. The conjunctiva protects the eye from environmental insults, Microbes Colonizing the Ocular Surface including recognition of nonself by the muco- sal immune system (McClellan 1997). The Culture-dependent studies have identified a goblets cells are integral in the production of variety of species of aerobic and anaerobic mucins that populate the mucus layer of the bacteria on the ocular surface. The most common tear film. genus identified is Staphylococcus, including M 402 Microbiome, Eye both S. epidermidis and S. aureus. Other common immune system. Like other mucosal surfaces, the aerobic bacteria include Corynebacterium spe- ocular epithelial cells express pattern recognition cies, Haemophilus influenzae, and Pseudomonas receptors (PRR), including Toll-like receptors aeruginosa. The anaerobic bacteria most com- (TLR) and NOD-like receptors that detect micro- monly isolated are Propionibacterium species organisms (Miller and Iovieno 2009). However, (McClellan 1997; Brinser and Burd 2001; Miller microbes present in the ocular microbiome fail to and Iovieno 2009). Although these bacteria are elicit an innate immune response under normal considered normal residents of the ocular surface, conditions. Although all TLR and NOD-1 are conditions that alter the ocular surface, such as expressed by corneal and conjunctival epithelial use of contact lenses, antibiotics, and surgery, cells, cellular localization may play an important allow bacteria to invade the epithelial surface role in whether activation of the immune system causing infections and other diseases (Miller occurs. Invasion of the epithelial barrier by and Iovieno 2009). Fungal populations that colo- microbes presents a mechanism for infection by nize the eye are less well characterized, but some commensals and abnormal activation of the fungi commonly present are Alternaria, immune system leading to ocular surface disease. Cladosporium, Aspergillus, and Candida species (Brinser and Burd 2001). Recent studies of the ocular surface Sampling Techniques microbiome in healthy human subjects revealed the presence of bacteria that had previously been The most common tools for sampling the ocular undetected, such as the genera Erwinia and surface are swabs and impression cytology (IC). Rhodococcus (Graham et al. 2007). In a study Both methods have been applied to collect samples with five healthy human subjects, a “core” for metagenomic studies. One 16S rRNA gene microbiota of the human conjunctiva was pro- survey determined that samples collected by posed to contain 12 genera, including Pseudomo- swabs produced positive PCR results more often nas, Propionibacterium, Bradyrhizobium, than those collected by IC (Graham et al. 2007). Corynebacterium, Acinetobacter, Brevundimonas, Sampling methods remain an important consider- Staphylococcus, Aquabacterium, Sphingomonas, ation for studies of the ocular microbiome, and Streptococcus, Streptophyta,andMethyl- further investigation is recommended to determine obacterium (Dong et al. 2011). These studies the appropriate method for new studies. indicate that the diversity of the ocular surface exceeds what has previously been characterized and suggest that further characterization of the Summary ocular microbiome is required to determine the ocular microbiome of “healthy” individuals. Loca- The ocular surface plays an important role in tion and the age of human subjects will be impor- providing a barrier function to the environment tant considerations for future studies. through the cornea, conjunctiva, tear film, and eyelids. The physical action of blinking and the immune reactions of the tear film and conjunctiva Interactions of the Microbiome with the maintain the barrier of the ocular surface. Ocular Surface Immune System Although culture-dependent techniques have been employed for decades to characterize the The interactions of the ocular surface epithelial ocular microbiome, metagenomic studies are in and immune cells with microbes are thought to their infancy. Culture-dependent studies demon- resemble those seen in the intestine. Microbes strated that the microbiome of the eye resembles colonizing the ocular surface interact with epithe- that of the upper respiratory tract and the skin with lial and immune cells to act as a barrier to patho- Staphylococcus, Streptococcus,andPropioni- gens, aid in wound repair, and maintain the bacterium species being commonly detected. Microbiome, Foregut 403 M

16S rRNA gene analysis confirmed the presence of these genera and revealed additional genera in the ocular microbiome. The ocular microbiome plays a role similar to that of the gut microbiome especially in maintenance of the immune system.

Cross-References

▶ Immunity, Innate: Definition and Examples

References

Brinser J, Burd E. Principles of diagnostic ocular micro- biology. In: Tabbara K, Hyndiuk R, editors. Infections of the eye. Boston: Little, Brown and Company; 2001. p. 69–84. Dong Q, Brulc J, Iovieno A, et al. Diversity of bacteria at healthy human conjunctiva. Invest Ophthalmol Vis Sci. 2011;52:5408–13. Graham J, Moore J, Jiru X, et al. Ocular pathogen or commensal: a PCR-based study of surface bacterial Microbiome, Foregut, Fig. 1 The human foregut flora in normal and dry eyes. Invest Ophthalmol Vis Sci. 2007;48:5616–23. McClellan K. Mucosal defense of the outer eye. Surv duodenum leading to the bile duct (Fig. 1). The Ophthalmol. 1997;42:233–46. M Miller D. Structure and Function of the Eye. In: Rubin- accessory organs regarded as being part of the stein, M, editor. Ophthalmology: The Essentials. New functional unit of the foregut include the liver, York: Houghton Mifflin Professional Publishers; gallbladder, and pancreas. The study of the fore- 1979. p. 1–25. Miller D, Iovieno A. The role of microbial flora on the gut microbiome (with perhaps the exception of ocular surface. Curr Opin Allergy Clin Immunol. the oral cavity) has been greatly overshadowed 2009;9:466–70. by the study of the microbiome represented in feces. This has been due to past assumptions regarding the sterility and/or lack of diversity Microbiome, Foregut within the foregut, as well as the difficult and invasive nature of collecting foregut samples, Carlos Wolfgang Nossa1, Liying Yang2 and which involves endoscopic probing. Zhiheng Pei3 Research attempting to characterize the fore- 1Gene by Gene Ltd., Houston, TX, USA gut microbiome has increased dramatically as the 2Department of Medicine, New York University use of culture-independent microbiome charac- School of Medicine, New York, NY, USA terizations has been facilitated by next- 3Departments of Pathology and Medicine, generation sequencing technologies. While there New York University School of Medicine, was already significant research involving the New York, NY, USA oral microbiome, studies involving the microbiome of the rest of the foregut were sparse; most involved few samples and were very shal- Introduction low characterizations. Recent publications have contributed much more in-depth characterization The foregut is comprised of the mouth, esopha- of the foregut microbiome, although there are still gus, and first two proximal portions of the relatively few groups focused on the esophageal M 404 Microbiome, Foregut and none on the duodenal microbiota. There have rRNA genetic marker, which was necessary due also been groups interested in the microbiome of to sequencing capability restraints. the foregut as a whole, as interest has been grow- ing about the nature of the human gut microbiome that cannot totally be extrapolated Oral Microbiome from stool specimens. The foregut microbiome shares some common The best studied portion of the foregut microbiome characteristics with the much better understood by far is the oral cavity. The oral microbiome has colonic microbiome, and the gut microbiome as a been shown to have an impact on oral health and whole. There are also several distinct properties of diseases such as gingivitis, periodontal disease, the foregut microbiome, and its individual sub- dental caries, halitosis, canker sores, and oral can- units, that differentiate it from the mid- and hind- cer (Belda-Ferre et al. 2012). It has also been gut. Mouth to colon analyses of the gut shown to serve as an indicator of disease state in microbiome have been performed, although with other locations in the body such as cardiac and few samples (Stearns et al. 2011). These have liver disease. The oral microbiome is represented provided invaluable glimpses into the continuity not only by the commensal species of the mouth and progression of the gut microbiota through the but also transient species that colonize down- GI tract. As the microbiome is characterized from stream in the GI tract, using the oral cavity as the the mouth to the anus, it can be seen that there are first point of entry. certain phyla of bacteria common throughout the Among all GI sites, the oral microbiome GI tract, but not in constant ratios throughout the exhibits the most variability in overall composi- gut, and some species dominantly colonize specific tion from subject to subject with over niches quite successfully. 600 observed prokaryotic species identified in There are also proven associations between one study (Dewhirst et al. 2010). While the core the foregut microbiome and several chronic dis- microbiome may be relatively similar between eases, some of which are systemic or not local- subjects (Table 1), the lesser represented micro- ized to the foregut itself. Whether these bial components may differ greatly. This is associations are causative or symptomatic still a consequence of the mouth being open to the remains to be seen in most cases, although environment and thus susceptible to entry by research on these microbiome disease associa- a great variety of microbes, some of which do tions is still in the discovery phase in most not colonize, but may be present transiently at the cases, while in some a direct correlation has time of testing. Several research groups have been hypothesized. defined a putative core microbiome for the oral It should also be noted that the vast majority of cavity. The consensus seems to be one of a core research into the foregut microbiome focuses on microbiome almost entirely represented by members of the Kingdom Bacteria. Therefore, 5 common phyla with the remainder showing technically speaking it is only the “bacteriome” a great degree of variation from subject to sub- that has been well characterized. This is due to ject. This variation may be due to transient envi- the use of the 16S rRNA gene marker using ronmental exposure, diet, and oral hygiene primers specifically designed for conserved bac- habits. However, since current testing methods teria sequences for phylogenetic analysis. The provide only a “snapshot” of the microbial com- composition of the archaeome, the mycobiome, munity at the time of sampling, transient bacteria the protistome, and the virome still needs further versus truly endogenous bacteria can only be attention, although this will probably be achieved determined either by comprehensive temporal as more research groups are harnessing the ever- sampling or by comparing human oral increasing power of high-throughput sequencing microbiome results with results from environ- and performing more metagenomic sequencing mental microbiome studies (such as air, food, efforts rather than focusing only on the 16S drink, etc.) (Dewhirst et al. 2010). Microbiome, Foregut 405 M

Microbiome, Foregut, Table 1 Summary of published oral microbiome results. Compilation of data and metadata from six comprehensive surveys of gastric oral microbiota. All surveys were culture-independent 16S rRNA gene based. Data are shown only for normal and control subjects. n/a denotes data not available from publication (Ahn data from Ahn et al. 2011) Aas Zaura Dewhirst Ahn Belda-Ferre Study et al. (2005) et al. (2009) et al. (2010) et al. (2011) Stearns et al. (2011) et al. (2012) Sample site Various Saliva Plaque, Oral wash Sub-/supragingival Supragingival subgingival plaque, tongue pockets # Sequences 2,589 298,261 34,753 79,000 11,511,138 4,254 (avg/subject) (518) (99,427) (55) (3,950) (2,877,785) (170) # Phylotypes 141 >500 >600 n/a n/a 186 observed Phylum/avg% Core phyla relative abundance relative abundance Firmicutes n/a 36 % 42 % 52 % 29 % n/a (40 %) Bacteroidetes n/a 11 % 13 % 16 % 19 % n/a (15 %) Proteobacteria n/a 22 % 20 % 20 % 21 % n/a (21 %) Actinobacteria n/a 25 % 11 % 7 % 4 % n/a (11 %) Fusobacteria n/a 4 % 4 % 5 % 22 % n/a (9 %) M

The five phyla making up the core oral complement of metabolic genes to colonize and microbiome include the Firmicutes, thrive in the various oral niches (Nobbs Bacteroidetes, Actinobacteria, Proteobacteria, et al. 2009). and Fusobacteria (Table 1). The representative Members of the microbial population are not genera of interest from each phylum include the distributed equally throughout the oral cavity following: Firmicutes (Streptococcus, Veillonella, (Aas et al. 2005). This is due to the variable Granulicatella), Proteobacteria (Neisseria, physical terrain of the mouth. Unlike the rest of Haemophilus), Actinobacteria (Corynebacterium, the GI tract, which is relatively uniform with Rothia, Actinomycetes), Bacteroidetes a lining of epithelial cells and mucosa, the (Prevotella, Capnocytophaga, Porphyromonas), mouth consists not only of epithelial cell sur- and Fusobacteria (Fusobacterium). So, it is faces but includes teeth. The soft surfaces of evident that while there is a good amount of the oral cavity represent distinct terrains includ- bacterial diversity within the mouth, most of the ing invaginations of the tongue, areas surround- microbial population is dominated by a few genera ing the salivary glands, and the subgingival from the five core phyla. pockets. The hard surface of the teeth also rep- The most abundant bacterial genera in the resents a different surface for bacteria to colo- healthy oral microbiome are streptococci nize. Unlike the epithelial surfaces, there is no (Dewhirst et al. 2010). One potential reason for shedding and sloughing off of surface layer, so this is that streptococcal species possess genes longer-term colonization can persist on tooth enabling them to successfully adhere to dental surfaces in the form of biofilms. Other surfaces surfaces, thus making them good primary colo- that can promote long-term colonization in the nizers. Additionally, they possess the proper mouth include the space between the teeth. Both M 406 Microbiome, Foregut of these hard to reach areas can sometimes evade which includes not only these species, but also normal oral hygiene techniques and topical anti- S. mutans, acts synergistically in tooth coloniza- biotic treatment. tion and caries formation (Belda-Ferre et al. Because of the variable terrain of the mouth, 2012). Aside from the disease-carrying species different results can be obtained from different of dental caries, other disease-carrying species sampling sites (Zaura et al. 2009). Lower species have been identified in healthy mouths, diversity is seen in cheek samples, while the suggesting that the balance between commensals highest diversity is observed in dental samples and pathogens is tenuous and can possibly be obtained from approximal surfaces (Zaura shifted by factors such as age, diet, tobacco and et al. 2009). Furthermore, using principal com- alcohol use, poor hygiene, and the presence of ponent analysis, microbial sequences from sur- dental prosthetics. faces with shedding epithelial cells clustered The fungal microbiome is also of interest, as together, while sequences from solid teeth sur- several fungal species present problematic oppor- faces clustered together. The formation of long- tunistic infections, for example, the association of lived biofilms on dental surfaces most likely Candida infections with immunodeficiency. The explains the increased diversity in dental sur- oral mycobiome was characterized in healthy faces. The approximal surfaces most likely individuals, and it was observed that there were exhibit higher diversity because the region is 85 fungal genera detected within 20 subjects protected from regular tooth brushing. Because (Ghannoum et al. 2010). However, more than of these factors, the site(s) of oral sampling must half of the genera were present in a single subject be considered when interpreting data from oral and none were present in all subjects, making it samples. difficult to establish a core oral mycobiome. The The most complex microenvironment within most frequently detected species were Candida the entire human body may be within dental (75 % of sequences obtained), Cladosporium plaques (Jenkinson 2011). These complex com- (65 %), Aureobasidium (50 %), Saccharo- munities represent populations of bacteria that mycetales (50 %), Aspergillus (35 %), Fusarium may be interdependent for structural support (30 %), and Cryptococcus (20 %). While several (production of the biofilm glycoprotein matrix), of these genera are known pathogens, they are metabolites (some bacteria feed exclusively on usually harmless except in opportunities, such as the metabolites and waste products of other bio- immunocompromise or antibacterial treatment, film members), and protection against host where they can flourish. While in-depth analysis defenses (such as protection against host antimi- of the fungal microbiome throughout the GI tract crobials within the matrix). is lacking, it would be interesting to see which Another unique niche presented within the species also colonize downstream GI sites. oral cavity is dental caries, which, like dental biofilms, may form a distinct microbiome within a microbiome. In fact, the microbiota found in Esophageal Microbiome these disease sites are more diverse than their healthy counterparts (Jenkinson 2011). While The rise of esophageal disease, such as esopha- streptococcal species are the most abundant in geal adenocarcinoma, in the past few decades has the mouth, they are also believed to be the main led to a rise in the interest level in esophageal causative agent of dental caries – particularly the microbiome research. The dearth of esophageal species S. mutans. A recent metagenomic analy- microbiome research was mostly attributable to sis of dental caries revealed an almost complete the notion that the esophagus harbored very little absence of S. mutans and a diverse community bacteria, either by biomass or diversity. Addition- that includes genera such as Veillonella, Coryne- ally, extraction of esophageal samples is difficult bacterium, and Leptotrichia. Nevertheless, it is and invasive, unlike oral swabbing or stool thought that a complex biofilm community, sampling. Microbiome, Foregut 407 M

The first comprehensive analysis of the esoph- due to the hostile, acidic nature of the gastric ageal microbiome revealed a bacterial diversity lumen. However, with the discovery of much higher than previously reported or expected Helicobacter pylori colonization, it was shown (Pei et al. 2004). It is estimated that the esopha- that certain specialized microbes could inhabit geal microbiome is comprised of approximately the gastric niche. Until recently though, it was 200 bacterial species (Yang et al. 2009). still assumed that although some bacteria could Although higher than previously assumed, this survive in the stomach, that overall species diver- makes it much less diverse than the oral or sity would be low. The reality is while the overall colonic microbiome, and the biomass is also biomass may be relatively low, the species diver- much lower. sity within the gastric mucosa is surprisingly While not enough subjects have been sampled robust. Although in subjects with active to truly determine a core esophageal microbiome, H. pylori colonization, most species are based on two previous studies, a speculative core outcompeted and Helicobacter is the predomi- microbiome of the esophagus is said to be com- nant bacteria. prised of five phyla: Firmicutes (83 % of The first comprehensive gastric microbiome sequences obtained from healthy esophagus sam- analysis showed that there were over 100 bacterial ples), Bacteroidetes (7 %), Actinobacteria (2 %), species residing in the normal stomach (Bik Proteobacteria (8 %), and Fusobacteria (1 %) et al. 2006). Most of the subjects (19 out of 23) (Yang et al. 2009). This is similar to the oral had H. pylori present, even though some had not microbiome. The dominant genus within the tested positive for H. pylori. In those subjects esophagus was found to be Streptococcus,in who had tested positive for H. pylori, it was the a relative abundance of 76 %. Other high- predominant species (72 % relative abundance), abundance genera of note were Prevotella and while in those tested negative for H. pylori, its Veillonella. relative abundance was only 11 %. The core M Because of the lack of a known common caus- phyla of the gastric microbiome identified in ative risk factor for gastroesophageal reflux dis- this study are Proteobacteria (representing ease (GERD), Pei et al . hypothesized that the H. pylori) (50 % of sequences obtained), esophageal microbiome could have been an Firmicutes (30 %), Bacteroidetes (10 %), overlooked contributor to esophageal disease, Actinobacteria (9 %), and Fusobacteria (5 %). such as reflux esophagitis, Barrett’s esophagus, Other later studies showed similar results among and esophageal adenocarcinoma. It was shown the core phyla (Table 2). While H. pylori was that most subjects with esophageal disease har- found to be the most abundant phylotype in the bored an esophageal microbiome distinct from study (42 % of all sequences obtained), the next the “normal” microbiome type. The difference most dominant genera were Streptococcus (6 %) between the two microbiomes is that in the and Prevotella (8 %) – which are abundant in type II microbiome, there is a shift from Gram- upstream oral and esophageal microbiomes. positive species to Gram-negative species, A follow-up study to Bik’s work by Li et al . mainly due to a loss in abundance of Streptococ- aiming to view the gastric microbiome away cus species. This could provide an attractive tar- from the context of H. pylori infection reported get for therapeutics aimed at preventing or very similar results. The total number of observed ameliorating esophageal disease via the use of phylotypes were similar (133 vs. 128), and the probiotics, prebiotics, or antibiotics. predicted number of species present was about 200 (Li et al. 2009). It was also observed that there was no significant difference in the Gastric Microbiome microbiome of the body versus the antrum of the stomach, which is important information for Much like the esophageal microbiome, the gas- future sampling considerations. The core phyla tric microbiome was long considered to be barren were similar in the absence of H. pylori; the M 408 Microbiome, Foregut

Microbiome, Foregut, Table 2 Summary of published gastric microbiome results. Compilation of data and metadata from four comprehensive surveys of gastric microbiota. All surveys were culture-independent 16S rRNA gene based. Data are shown only for normal and control subjects. Excluded subjects include those with H. pylori infection (Andersson et al. 2008), non-H. pylori, and nonsteroidal anti-inflammatory drug use (Li et al. 2009) Study Bik et al. (2006) Andersson et al. (2008) Li et al. (2009) Stearns et al. (2011) # Subjects 23 3 5 4 Sample site Corpus (9), antrum (14) Corpus Body, antrum Body, antrum # Sequences 1,833 9,958 612 2,612,339 (avg/subject) (80) (3,319) (122) (870,780) # Phylotypes observed 128 262 133 n/a # Phylotypes predicted 193 375 200 n/a Phylum Core phyla % relative abundance (avg% relative abundance) Firmicutes 25 % 30 % 22 % 53 % (33 %) Bacteroidetes 11 % 11 % 28 % 9 % (15 %) Proteobacteria 52 % 11 % 37 % 33 % (33 %) Actinobacteria 9 % 47 % 8 % >1% (14 %) Fusobacteria 3% 4% 4% 4% (3 %)

abundance of Proteobacteria was reduced (from The five core phyla previously identified were 52 % to 37 % relative abundance) with the abun- still present, but with the addition of Chlamydiae dance Bacteroidetes increased (from 10 % to and Cyanobacteria phyla. 28 %). This shows that in the absence of the Each of the core phyla within the stomach is dominating H. pylori, the core stomach represented by only one or two genera, which microbiome is more representative of those may explain the decrease of species diversity upstream in the mouth and esophagus. down the GI tract from the mouth to the stomach. Although it was shown that H. pylori is not the What may be occurring is the selection of more only species comprising the gastric microbiome, specialized genera for the more demanding as previously assumed, it is still the most abun- niches found within the GI tract. As bacteria dant and most relevant species. H. pylori has been borne-materials (i.e., food, saliva) pass through shown to be a causative factor in peptic ulcers and the alimentary canal, those that cannot survive gastric adenocarcinoma – the first example of the environments of the esophagus and stomach, a bacterial infection linked to oncogenesis. which are less nutrient rich and harsher, perish, Because of its widespread colonization and infec- while those that possess specialized traits or have tion rate, even in asymptomatic individuals, it evolved specializations to survive and thrive remains the main species of interest when remain and colonize, outcompeting less well- discussing the gastric microbiome. equipped organisms. The highest reported estimate of bacterial diversity within the stomach is 375 phylotypes. This was derived from higher coverage and Duodenal Microbiome deeper sequencing facilitated by next-generation sequencing (Andersson et al. 2008). The The duodenal microbiome is the least studied, observed 276 phylotypes represented 13 phyla. and therefore the least characterized portion of Microbiome, Foregut 409 M the foregut microbiome. No studies exist that have discovery. Helicobacter species have also been attempted a high-coverage sequencing of a large found in the gall bladder and have been impli- population of duodenal samples. A deep sequenc- cated in the creation of gallstones (Lee, Lee ing effort of the duodenum of four subjects identi- et al. 2010). H. pylori was found in different fied many of the same phyla reported upstream in types of gallstones along with Escherichia coli, the foregut (Stearns et al. 2011). The most pre- Pseudomonas, Citrobacter, and Klebsiella. dominant were Bacteroidetes, Firmicutes, Whether these bacterial species were causative Proteobacteria, Fusobacteria, Actinobacteria, agents of the gallstones is unclear; they may have TM7, SR1, and also, interestingly, the significant just been trapped during its formation. It has been presence of an unclassified Cyanobacteria in one hypothesized that biofilm formation, excessive sample. Homogeneity between the four samples mucin production by epithelial cells in reaction was low, so it is hard to establish from this data to bacteria, or bacterial metabolites changing bile a “core” duodenal microbiome. juice formation may be contributing factors to the Although it is not surprising that the same production of gallstones. Salmonella species, phyla are present, it was of note that there was, specifically Salmonella enterica subspecies on average, a higher proportion of Acidobacteria enterica serovar Typhi (S. typhi) has also been than the other sites, and much like the stomach, associated with the hepatobiliary system. It is the duodenum was dominated by only a few gen- suggested that S. typhi colonizes either the liver era. This is most likely due to selection for those or gall bladder, particularly in non-symptomatic species equipped to survive the harsh environ- carriers (Nath et al. 2010). ment presented by sodium bicarbonate and bile Whether the above examples are representa- salts. Overall, due to lack of comprehensive tive of the microbiomes of normal individuals or published studies up to this point, it is difficult rather rare pathogen-carrying examples remains to define a true “core” microbiome for the duo- to be determined in the absence of microbiome M denum or an estimate of species richness and profiling efforts of control subjects. Whether the diversity. hepatobiliary system can be sampled in control subjects is questionable, since this would be inva- sive; however, if possible it could be rather Hepatobiliary and Pancreatic informative. Microbiomes There are no studies of microbiome in the pancreas, but bacteria can cause pancreatitis The hepatobiliary system and pancreas are con- when the pancreatic duct is obstructed. The sidered accessory organs in the human foregut. source of bacteria could reside in the pancreatic They are connected to the intestinal tract of the duct or be translocated from the duodenum. The foregut via the duodenum. These organs were finding of pancreatic stellate cells expressing once considered sterile for the most part, but Toll-like receptors 2, 3, 4, and 5 and the associ- connection via ducts links them to the GI tract, ated molecules CD14 and MD2 suggest the pan- which is connected to the environment, and thus creas is not a sterile organ but may host they are susceptible to exposure to microbes. indigenous bacteria. It would be interesting to While no core microbiome has been established see what type of bacteria might survive the alka- for these organs, bacteria have been known to line environment rich with digestive enzymes colonize, but mostly in terms of bacterial infec- (Masamune et al. 2008). tion, and not much can be said for the commensal microbiota in healthy individuals. Some exam- ples of known colonization include Helicobacter Summary species within the liver and gall bladder. Helicobacter hepaticus was first isolated from Foregut microbiome studies have revealed that the liver tissue and named for its niche of the four distinct sites of the foregut (the mouth, M 410 Microbiome, Foregut esophagus, stomach, and small intestine) all share of sequences), another is dominated by similar common core microbiomes. Although all Firmicutes (52 %), another has an unusually five core phyla are represented within the differ- high proportion of the SR1 phylum (17 %), and ent niches of the foregut, they are represented in yet another sample is populated with a significant different proportions. This is likely due to the amount of Cyanobacteria (15 %). So it is clear varying conditions within each portion of the from this small sample set that it would be impos- foregut. While the less harsh environments in sible to establish a core microbiome that would the mouth and esophagus are dominated by phy- be expected to appear in other samples, unless lum Firmicutes (particularly genus Streptococ- they all happened to fall within the similar rela- cus), the harsher environment of the stomach tive abundance profiles – in which case a small can be dominated by the acid-resistant subset could still be informative. Helicobacter pylori. Although a core Another potential pitfall when comparing microbiome for the duodenum is not well results from different studies is the sampling defined, the limited data available show that methods used. We have already seen that sam- there can be a great variety of dominant phyla pling different areas within the oral cavity results within this degradative environment. Whether in markedly different microbial profiles. The this will hold true once more samples are ana- sampling method could also affect the results lyzed remains to be seen. particularly when choosing collection methods. Current research has also documented the sur- For GI samples, many groups relied on tissue prising diversity of bacteria within the foregut, biopsies. However, biopsies only recover revealing that not only the mouth is populated by a small portion of epithelial tissue, which may hundreds of species, but so are the previously not yield enough bacterial biomass to success- underestimated regions of the esophagus, stom- fully extract enough bacterial for sequencing ach, and duodenum. The decrease of species efforts, or even for PCR amplification of 16S diversity from the mouth to the duodenum most rDNA. Tissue brushings are able to yield likely represents an environmental selection for a larger surface of bacteria containing epithelial species capable of withstanding the harsh interior cells and provide a more accurate microbial of the digestive tract. Those species that can profile. thrive within the gastric lumen and duodenum Sequencing considerations can also affect must possess an array of genes that allow them microbiome surveys. It has been reported that to resist degradation and digestion, as will be different variable regions of the 16S rRNA discovered once more metagenomic research is gene, as well as size of the amplicon being done in these areas. sequenced, can affect the classification accuracy It should be noted that comparing the results of the sequences obtained (Nossa et al. 2010). from different studies, as has been done here, is Therefore, different research groups utilizing dif- not ideal. Differences in factors in experimental ferent regions of the 16S rRNA gene for sequenc- design and data analysis could all contribute to ing may come up with different results, making minute or significant differences in results comparisons between studies difficult and further obtained, as can be seen in some of the compar- complicating the definition of a core microbiome. isons in Tables 1 and 2. Sample size differences Other considerations that varied between stud- can lead to extreme variations, as one aberrant ies compared in this review include sequence sample (out of 3 or 4) can skew the relative depth (more sequences per sample would give abundance percentage for the whole sample set. more accurate results, better determine expected For example, the survey of the duodenum phylotypes, and reveal rarer phylotypes), presented here (Stearns et al. 2011) samples sequencing platform used, homo- or heterogene- four subjects, all of which have extremely differ- ity of subject demographics, and dates of ana- ent phyla relative abundance percentages. One lyses (more recent research has had the sample is mostly Proteobacteria (82 % percent advantage of better microbial 16S rDNA Microbiome, Stomach 411 M databases, higher throughput and longer length Nath G, Singh YK, et al. Does Salmonella typhi primarily sequencing technologies, and computational reside in the liver of chronic typhoid carriers? J Infect Dev Ctries. 2010;4(4):259–61. tools). Nobbs AH, Lamont RJ, et al. Streptococcus adherence and As more, and deeper, microbial surveys are colonization. Microbiol Mol Biol Rev. 2009;73(3): performed and published, we will be closer to 407–50. being able to define a true core microbiome for Nossa CW, Oberdorf WE, et al. Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut the foregut. This accomplishment will be impor- microbiome. World J Gastroenterol. 2010;16(33): tant as more diseases are being associated with 4135–44. dysbiosis, such as esophagitis, gastritis, and oral Pei Z, Bini EJ, et al. Bacterial biota in the human distal cancer. Establishment of a core microbiome will esophagus. Proc Natl Acad Sci U S A. 2004;101(12): 4250–5. allow for diagnosis of microbiome-related dis- Stearns JC, Lynch MD, et al. Bacterial biogeography of ease and perhaps therapy geared towards the human digestive tract. Sci Rep. 2011;1:170. reestablishment of the normal core microbiome. Yang L, Lu X, et al. Inflammation and intestinal metapla- This work was supported by grants from the sia of the distal esophagus are associated with alter- ations in the microbiome. Gastroenterology. 2009; National Cancer Institute, the National Institute 137(2):588–97. for Allergy and Infectious Diseases, and the Zaura E, Keijser BJ, et al. Defining the healthy “core National Institute of Dental and Craniofacial microbiome” of oral microbial communities. BMC Research (UH3CA140233, R01AI063477, Microbiol. 2009;9:259. R01CA159036, and U19DE018385).

References Microbiome, Stomach Aas JA, Paster BJ, et al. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol. 2005; Erik C. von Rosenvinge M 43(11):5721–32. Department of Medicine, University of Maryland Ahn J, Yang L, et al. Oral microbiome profiles: 16S rRNA School of Medicine, Baltimore, MD, USA pyrosequencing and microarray assay comparison. PLoS ONE. 2011;6(7):e22788. Andersson AF, Lindberg M, et al. Comparative analysis of human gut microbiota by barcoded pyrosequencing. Synonyms PLoS ONE. 2008;3(7):e2836. Belda-Ferre P, Alcaraz LD, et al. The oral metagenome in health and disease. ISME J. 2012;6(1):46–56. Gastric microbiome; Gastric microbiota; Stom- Bik EM, Eckburg PB, et al. Molecular analysis of the ach microbiota bacterial microbiota in the human stomach. Proc Natl Acad Sci U S A. 2006;103(3):732–7. Dewhirst FE, Chen T, et al. The human oral microbiome. J Bacteriol. 2010;192(19):5002–17. Definition Ghannoum MA, Jurevic RJ, et al. Characterization of the oral fungal microbiome (mycobiome) in healthy indi- The community of microorganisms living within viduals. PLoS Pathog. 2010;6(1):e1000713. the human stomach. Jenkinson HF. Beyond the oral microbiome. Environ Microbiol. 2011;13(12):3077–87. Lee JW, Lee DH, et al. Identification of Helicobacter pylori in gallstone, bile, and other hepatobiliary tissues Introduction of patients with cholecystitis. Gut Liver. 2010;4(1):60–7. Li XX, Wong GL, et al. Bacterial microbiota profiling in Historically, the human stomach was believed gastritis without Helicobacter pylori infection or sterile due to the harsh environment created by non-steroidal anti-inflammatory drug use. PLoS the presence of gastric acid and digestive enzymes. ONE. 2009;4(11):e7985. The 1983 report of resident gastric bacteria, Masamune A, Kikuta K, et al. Pancreatic stellate cells express toll-like receptors. J Gastroenterol. 2008; later named Helicobacter pylori, dramatically 43(5):352–62. altered this belief (Warren and Marshall 1983). M 412 Microbiome, Stomach

Initially, H. pylori was thought unique; however, separated based on the presence or absence of traditional culture methods also suggested the gastritis and analyzed by temperature gradient presence of additional gastric microbial inhabi- gel electrophoresis (TTGE) and 16S rDNA tants (Adamsson et al. 1999). Dramatic advance- sequence analysis. Five histologically normal ments in the field of genomics have led to samples were analyzed and found dominated by non-culture-based methods for detecting micro- the genera Pseudomonas, Staphylococcus, and bial populations, principally through 16S rRNA Enterococcus. Helicobacter species were also gene sequencing. Application of these tech- present in these histologically normal specimens, niques has once again transformed our under- but at significantly lower amounts when com- standing of microbial life within the human pared to gastritis specimens. A more recent stomach. It is now clear that a complex commu- Swedish study used mucosal biopsies taken nity of organisms is present – the human stom- from six healthy older individuals and assessed ach microbiome. This entry will summarize the stomach microbiome using barcoded current knowledge of the human stomach pyrosequencing (Andersson et al. 2008). Three microbiome in health and disease and efforts to of the samples were H. pylori negative and their determine the resident bacterial and fungal analysis revealed a diverse microbial community members of the stomach microbiome. including 262 phylotypes representing 13 phyla, many of which were also found in the throats of different patients participating in the same study, The Stomach Microbiome in Health suggesting that some sequences may represent transient ingested bacteria. The majority of the The healthy stomach serves as a reservoir for phylotypes found in the stomach but not in the ingested food and as a barrier to ingested patho- throat belonged to the Proteobacteria. gens (Martinsen et al. 2005). Organisms that are A recent study of gastric mucosal biopsies and resident, ingested with food, swallowed from the gastric juice samples obtained from 12 healthy mouth or esophagus, and refluxed from the small persons has provided further insights into the intestine are all possible members of this com- stomach microbiome in health (Delgado et al. munity. As H. pylori is present in the majority of 2013). All samples were subjected to routine human stomachs, it can be considered a “normal” culture and four mucosal biopsy samples member of the stomach microbiome present in underwent 16S rRNA gene amplicon health; however, as H. pylori is associated with pyrosequencing. Using conventional culture upper gastrointestinal disease and was classified techniques, the total number of cultivable micro- as a carcinogen in 1994 by the International organisms ranged from 102 to 104 cfu/g or ml, Agency for Research on Cancer of the World and most isolates belonged to four genera: Health Organization, it will primarily be Propionibacterium, Lactobacillus, Streptococ- discussed in subsequent sections on the stomach cus, and Staphylococcus. Pyrosequencing of the microbiome in disease. four mucosal samples yielded 15,622 high- The exact composition of the healthy stomach quality 16S rDNA sequence reads, which analy- microbiome is unknown; most reports on the sis grouped into 59 families and 69 genera. In stomach microbiome have characterized patients these samples, the most abundant phyla were undergoing clinically indicated upper endoscopy Firmicutes, Proteobacteria, and Actinobacteria, (Dicksved et al. 2009; Maldonado-Contreras and the most abundant operational taxonomic et al. 2011; von Rosenvinge et al. 2013) or have units (OTUs) belonged to Streptococcus, provided little clinical information other than Propionibacterium, and Lactobacillus. H. pylori status (Bik et al. 2006). An early geno- One additional study may shed light on the mic study of the stomach microbiome used sam- microbiota of the normal stomach ples obtained from asymptomatic volunteers in (Li et al. 2009). This study, performed in Hong Sweden (Monstein et al. 2000). Samples were Kong, included patients undergoing endoscopy, Microbiome, Stomach 413 M but subsequently grouped samples based on conventional testing, and (3) samples that tested whether there was gastritis present on endoscopy positive for H. pylori by at least two conventional or if the stomach was endoscopically normal. methods (i.e., culture, rapid urease test, serum 16S rRNA gene clone libraries were constructed IgG, and histopathology). H. pylori sequences and sequenced. In the endoscopically normal dominated in the group that tested positive for group, the major phyla present included H. pylori by conventional means, comprising an Proteobacteria (37 %), Bacteroidetes (28 %), average of 72 % of clones. When present in those Firmicutes (22 %), Actinobacteria (8 %), and negative for H. pylori by conventional testing, Fusobacteria (4 %), which together represented H. pylori reads comprised 11 % of clones. All 99 % of all clones present. Comparison of biop- samples without H. pylori reads were considered sies from the normal-appearing gastric antrum H. pylori negative by conventional methods. and body showed little difference, only the fam- Analysis of the phylum distribution of ily Prevotellaceae and the genus Prevotella non-H. pylori phylotypes in the three groups of showed a significant increase in the antrum rel- samples demonstrated no gross differences in ative to the gastric body. While endoscopically taxonomic patterns. The dominance of normal, presumably these patients underwent H. pylori, when present, found in this study was endoscopy for a clinically indicated reason; also identified, but even more pronounced, in therefore, results of this particular study may subsequent study that utilized barcoded not accurately reflect the stomach microbiome pyrosequencing to analyze mucosal biopsy sam- in health. ples obtained from three H. pylori-positive indi- viduals – finding that H. pylori comprised 93–97 % of sequence reads (Andersson The Stomach Microbiome During et al. 2008). These findings in studies of mucosal Disease biopsy samples are divergent from that reported M in a study of gastric fluid (von Rosenvinge Helicobacter pylori Infection et al. 2013) where H. pylori, when present, H. pylori is a gram-negative bacterium of the accounted for <0.4 % of sequence reads, and phylum Proteobacteria, and H. pylori gastric are consistent with a body of literature showing infection is present in at least half of the human that H. pylori colonizes the mucous layer and population, primarily in developing countries. adheres to epithelial cells (Amieva and El-Omar H. pylori infection is most commonly asymptom- 2008). atic; however, it is strongly associated with the A more recent study evaluated the structure of presence of gastritis, gastric and duodenal ulcers, the human stomach microbiome in relation to gastric cancer, and gastric mucosa-associated H. pylori status through use of DNA microarrays lymphoid tissue (MALT) lymphoma (McColl (Maldonado-Contreras et al. 2011). Gastric 2010). mucosal biopsies were obtained from patients The first in-depth molecular characterization undergoing upper endoscopy: ten adult Amerin- of the bacterial microbiome of the human stom- dians in Venezuela and two recent adult immi- ach (Bik et al. 2006) used mucosal biopsy sam- grants to the United States. Eight subjects were ples and the majority of 16S rRNA gene H. pylori positive and four (all Amerindians) sequences identified (1,833 total) were assigned were H. pylori negative by glmM-PCR testing. to the Proteobacteria, Firmicutes, Actinobacteria, A total of 44 bacterial phyla were detected with Bacteroidetes, and Fusobacteria phyla. Samples a strong dominance of four phyla: Proteobacteria, were separated into three groups based on Firmicutes, Actinobacteria, and Bacteroidetes. H. pylori status: (1) samples without H. pylori Comparison of H. pylori-positive and sequences and negative for H. pylori by conven- H. pylori-negative subjects revealed similarity tional testing, (2) samples with H. pylori in the representation of these four major phyla, sequences and negative for H. pylori by and H. pylori status did not appear to affect phyla M 414 Microbiome, Stomach richness. Helicobacteriaceae taxa were detected 16S rRNA gene cloning and sequencing, stomach in all samples, although to a much lesser degree biopsies from noncancerous areas of gastric can- in those that tested negative for H. pylori. cer patients were analyzed. One hundred two Multidimensional clustering analysis showed phylotypes were found and these clustered into that about 28 % of the total variance in the stom- five bacterial phyla: Firmicutes, Bacteroidetes, ach microbiome of the 12 subjects was explained Actinobacteria, Proteobacteria, and Fusobacteria. by H. pylori status. Bacterial communities in The Firmicutes were most highly represented H. pylori-negative patients had greater abun- with the majority corresponding to Streptococ- dances of Actinobacteria and Firmicutes, while cus, Lactobacillus, and different Clostridiales. H. pylori-positive patients had greater abun- The phylum Bacteroidetes was the second most dances of non-H. pylori Proteobacteria and commonly represented and was primarily com- Actinobacteria. These results suggest that posed of various species of Prevotella. When H. pylori status alters the structure of the stomach compared with samples from five dyspeptic con- microbiome. trol patients, there were no significant differences in diversity indices. However, when samples Immunosuppression were clustered according to their terminal restric- The stomach environment is subject to active tion fragment patterns, four of the five healthy control by host innate and adaptive immune controls clustered together, suggesting these sam- responses (Vorabjova et al. 2008). Information ples’ bacterial communities are more similar to about the role of immunosuppression on the each other than to samples obtained from cancer stomach microbiome is limited; however, inter- patients. Further studies will be needed before estingly, a recent study found that gastric fluid conclusions can be drawn. samples could be divided into two groups based on microbiota species richness and composition, which coincided best with differences in the Investigations of the Resident Stomach immune status of the host (von Rosenvinge Microbiome et al. 2013). Bacterial richness was reduced on average 42 % in samples obtained from immu- Using 16S rRNA gene sequencing techniques, nocompromised individuals (transplant recipi- one cannot distinguish between resident, meta- ents and HIV/AIDS patients) when compared bolically active organisms and dead organisms to non-immunocompromised patient samples. that are passing through the stomach after being At the phylum level, significant changes in the ingested with food or swallowed from the mouth bacterial composition included increased abun- or esophagus. It is established that H. pylori lives dances of Firmicutes and reduced abundances of and thrives in the gastric mucus layer, and Bacteroidetes; at the genus level, markedly H. pylori can be cultured from gastric biopsy increased Lactobacillus spp. and reduced specimens using conventional techniques Fusobacterium spp. and Prevotella spp. were (Warren and Marshall 1983). Conventional cul- found. There was considerable overlap between ture of gastric biopsy samples washed under immunosuppression, antibiotic use, and proton harsh conditions, followed by 16S rRNA gene pump inhibitor therapy, so further studies are sequencing, has identified Streptococcus species needed before conclusions can be drawn. that are both living and adherent to the mucosa (Lietal.2009). The identification of a transcrip- Gastric Cancer tionally active microbiome in stomach fluid was A single study has investigated the stomach recently reported (von Rosenvinge et al. 2013). microbiota in patients with gastric cancer In this study, 16S rRNA transcripts were reverse (Dicksved et al. 2009). Using the molecular pro- transcribed into cDNA, PCR amplified, and the filing approach of terminal restriction length amplicons sequenced. Results were compared to polymorphism (T-RFLP), in combination with 16S rRNA gene amplicon sequencing data Microbiome, Stomach 415 M created from the same samples using identical Summary primers and sequencing conditions. Phyloge- netic distance calculations using weighted The human stomach harbors a complex commu- UniFrac analysis showed significant differences nity of microorganisms, including bacteria and between the 16S rRNA transcript and gene fungi. H. pylori, when present, is a dominant data. Shifts in the relative distribution of the member of the mucosal but not the stomach major taxonomic phyla were apparent and fluid microbiome, and H. pylori status influences included a decrease in Actinobacteria and the structure of the microbiome. Differences Firmicutes and an increase in Bacteroidetes identified through comparisons of 16S rRNA and Proteobacteria in the transcript data when gene and 16S rRNA gene transcript sequencing compared to the gene data. Members of the class suggest that a subset of the bacteria present in the Actinobacteria, including the oral commensals stomach are metabolically active and, therefore, Rothia dentocariosa and Actinomyces more likely able to survive the harsh environment odontolyticus, had a lower relative abundance of the stomach. There appear to be differences in 16S rRNA transcripts compared with genes, between the stomach microbiome during health while the relative abundance of H. pylori was on and disease (e.g., H. pylori infection or gastric average 19.9 times higher in the 16S rRNA tran- cancer); however, further work is needed to con- scripts than the genes. These findings support firm these findings and determine their impor- the utility of this approach for characterization tance to human health. of the metabolically active microbiome in stomach fluid. References

Adamsson I, Nord CE, Lundquist P, Sjӧstedt, Edlund The Stomach Fungal Microbiome C. Comparative effects of omeprazole, amoxicillin plus metronidazole versus omeprazole, clarithromycin M A stomach fungal microbiome was recently dis- plus metronidazole on the oral, gastric and intestinal microflora in Helicobacter pylori-infected patients. covered in gastric fluid samples obtained from J Antimicrob Chemother. 1999;44:629–40. patients undergoing upper endoscopy (von Amieva MR, El-Omar EM. Host-bacterial interactions in Rosenvinge et al. 2013). In this study, internal Helicobacter pylori infection. Gastroenterology. transcribed spacers of the rRNA gene cluster 2008;134:306–23. Andersson AF, Lindberg M, Jakobsson H, B€ackhed F, were amplified in nine gastric fluid samples. Nyre´n P, Engstrand L. Comparative analysis of Amplicon pyrosequencing revealed between human gut microbiota by barcoded pyrosequencing. 19 and 81 genus-level OTUs (operational taxo- PLoS ONE. 2008;3:e2836. nomic units) per sample. On average 77.5 % of Bik EM, Eckburg PB, Gill SR, et al. Molecular analysis of the bacterial microbiota in the human stomach. Proc the ITS reads could not be taxonomically Natl Acad Sci U S A. 2006;103:732–7. assigned, likely due to incomplete representation Delgado S, Cabrera-Rubio R, Mira A, Sua´rez A, Mayo of these fungi in available ITS sequence refer- B. Microbiological survey of the human gastric eco- ence collections. Candida and Phialemonium system using culturing and pyrosequencing methods. Microb Ecol. 2013;65:763–72. were the only two genera found in all samples. Dicksved J, Lindberg M, Rosenquist M, Enroth H, Jansson The known pathogens C. albicans, C. tropicalis, JK, Engstrand L. Molecular characterization of the and C. parapsilosis were identified based on phy- stomach microbiota in patients with gastric cancer logenetic tree predictions. A correlation between and in controls. J Med Microbiol. 2009;58:509–16. Li XX, Wong GLH, To KF, et al. Bacterial microbiota fungal microbiota richness or composition and profiling in gastritis without Helicobacter pylori infec- host immune status was not identified; however, tion or non-steroidal anti-inflammatory drug use. PLoS a single HIV/AIDS patient treated with trimeth- ONE. 2009;4:e7985. oprim/sulfamethoxazole and azithromycin for Maldonado-Contreras A, Goldfarb KC, Godoy-Vitorino F, et al. Structure of the human gastric bacterial commu- infection prophylaxes exhibited markedly nity in relation to Helicobacter pylori status. ISME reduced fungal microbiota richness. J. 2011;5:574–9. M 416 Microbiome, Vagina

Martinsen TC, Bergh K, Waldum HL. Gastric juice: However, relatively little is known about the func- a barrier against infectious diseases. Basic Clin tion of these communities or how their constituent Pharmacol Toxicol. 2005;96:94–102. McColl KEL. Helicobacter pylori infection. N Engl members interact with each other and the host to J Med. 2010;362:1597–604. form a dynamic ecosystem that responds to envi- Monstein HJ, Tiveljung A, Kraft CH, Borch K, Jonasson ronmental disturbances. Major efforts are cur- J. Profiling of bacterial flora in gastric biopsies from rently underway to better understand the role of patients with Helicobacter pylori-associated gastritis and histologically normal control individuals by tem- these communities in health and disease. perature gradient gel electrophoresis and 16S rDNA sequence analysis. J Med Microbiol. 2000;49:817–22. von Rosenvinge EC, Song Y, White JR, Maddox C, Common Wisdom and General Blanchard T, Fricke WF. Immune status, antibiotic medication and pH are associated with changes in the Characteristics of the Vaginal stomach fluid microbiota. ISME J. 2013;7:1354–66. Microbiota Vorabjova T, Watanabe T, Chiba T. Helicobacter pylori immunology and vaccines. Helicobacter. 2008;13 Anatomy and Environmental Characteristics Suppl 1:18–22. Warren JR, Marshall BJ. Unidentified curved bacilli on of the Vagina gastric epithelium in active chronic gastritis. Lancet. The vagina is a fibromuscular organ situated 1983;1:1273–5. between the rectum, urethra, and bladder (Fig. 1). It acts as the gateway from the outer genitalia (vulva) to the opening of the uterus (cervix). When standing upright, the vagina of Microbiome, Vagina a reproductive-age woman exists in a relaxed state and is similar in shape to a convex curve Jian Shen1,2, Roxana Hickey3 and with a near right angle from the upper to lower Larry J. Forney3 portions. The anterior and posterior walls are 1Department of Gynaecology and Obstetrics, generally slack and remain in contact with each Ruijin Hospital Affiliated to Shanghai Jiaotong other, whereas the lateral walls of the vagina are University School of Medicine, Shanghai, China fairly rigid. As such, the vagina is characterized 2Institute for Bioinformatics and Computational as a potential, not actual, open space. The size Biology, University of Idaho, Moscow, ID, USA and shape of vaginas vary among reproductive- 3Institute for Bioinformatics and Computational age women, but on average the length of the Biology, Department of Biological Sciences, anterior vaginal wall is 6–8 cm and the posterior University of Idaho, Moscow, ID, USA wall up to 14 cm, including the cervix. The inte- rior surface area ranges widely from 65.7 to 107.1 cm2 (average 87.5 cm2), but these are The human vagina is home to a milieu of almost certainly underestimates, as they do not microbes that are thought to play a critical role include the area of vaginal rugal folds (Ferguson in maintaining health and protecting their host and Rohan 2011). from infectious disease. These microbes, collec- The vaginal mucosa is lined by nonkeratinized, tively known as the vaginal microbiome, exist in stratified squamous epithelial cells connected by a finely balanced mutualistic association with gap junction nexuses that create an open channel their hosts and play a central role in the dynamic system allowing for traversal of molecules and interplay between health and risk to disease. The electrolytes between adjacent cells (Ferguson exact mechanisms mediating these interactions and Rohan 2011). Due to a limited blood supply, are not currently well understood. Recent studies the epithelium must depend on the underlying have begun to shed light on how vaginal micro- submucosal tissues for diffusion of glucose, bial communities vary among and within indi- oxygen, and various essential nutrients. Under viduals at various stages of a woman’s lifespan. the resulting relatively anoxic conditions, the Microbiome, Vagina 417 M

Microbiome, Vagina, Fig. 1 Female genital tract anatomy. (a) Represents a side view of the gross anatomy of the female genital tract with respect to other organs. (b) Represents tissue types of the upper vagina and cervix. (c) Display of cells composing the vaginal and ectocervical tissue and display of cells composing the endocervical tissue (Reprinted from Ferguson and Rohan 2011)

M estrogen-dependent vaginal epithelial cells Lactobacilli and Their Protective Role in (primarily the intermediate layer) convert glyco- Reproductive Health gen to glucose, which is then metabolized to pyru- Lactobacilli have been considered the keystone vate and subsequently lactate, that diffuses from species of the normal postpubertal vaginal the cells and accumulates in the extracellular microbiota since 1892 when renowned German milieu. Lactic acid bacteria (LAB) that predomi- gynecologist Albert Do¨derlein first cultured the nate in the vaginas of reproductive-age women organisms from vaginal secretions obtained from also metabolize extracellular glycogen into lactic healthy, pregnant women. These long, slender acid by anaerobic glycolysis and also release the rods, originally termed “Do¨derlein’s bacillus,” acid into the vaginal lumen. Epithelial cells pro- were renamed Lactobacillus acidophilus in duce only the L-lactate isomer, while many bacte- 1928 in light of their ability to produce lactic ria are capable of producing both D-andL-lactate. acid and reduce the vaginal pH. In the 1980s Moreover, many organisms produce organic acids L. acidophilus was determined to be a group of such as formic acid, acetic acid, propionic acid, closely related, obligately homofermentative succinic acid, and others through fermentative species collectively known as the Lactobacillus metabolism and thereby contribute to the acidifi- acidophilus complex. Since species in this com- cation of the vaginal environment. The environ- plex are difficult to differentiate phenotypically mental pH of the vagina, traditionally considered or biochemically, they were differentiated on the to be around 4.0–4.5 in healthy women, is presum- basis of DNA homology and include all species ably determined by the organic acid contributions that have been reportedly found in the human from both epithelial cells, resident lactic acid bac- vagina to date (Danielsson et al. 2011). teria (LAB), and other strictly anaerobic bacteria While there is compelling evidence indicating (Linhares et al. 2010). that lactic acid and the resulting low pH are M 418 Microbiome, Vagina important in precluding the colonization of At the onset of puberty, adrenal and gonadal nonindigenous organisms in the vagina maturation causes an increase in estrogen produc- (Redondo-Lopez et al. 1990), lactic acid may tion, which once again causes thickening of the also play a direct role in protecting the host by vaginal epithelium and production of glycogen. acting as an antimicrobial compound. In addition, These environmental conditions selectively favor previous in vitro studies on the colonization of the proliferation of glycogen-fermenting LAB vaginal epithelial cell monolayers with bacteria and the concomitant acidification of the vaginal such as Lactobacillus crispatus, Prevotella bivia, environment (pH ~4.5), which is sustained and Atopobium vaginae have demonstrated these throughout the reproductive years. The vaginal bacteria may regulate epithelial innate immunity bacterial communities of adolescent girls in a species-specific manner (Fichorova (13–18 years) are comparable to those found in et al. 2011). In addition to lactic acid, vaginal adults, but less is known whether this is also the lactobacilli are also capable of producing other case for premenarcheal or perimenarcheal girls antimicrobial compounds such as antibiotics, (Farage and Maibach 2006). target-specific bacteriocins, and hydrogen perox- In reproductive-age women a low vaginal pH ide. However, since oxygen levels in the vagina creates an environment thought to restrict or are typically low, it is unlikely that hydrogen preclude the growth of many pathogenic organ- peroxide is produced and accumulates to levels isms. However, recent findings demonstrate that that are bactericidal (O’Hanlon et al. 2011). the average vaginal pH of healthy women in some ethnic groups is closer to 5.0 or higher, Changes in the Vaginal Microbiota over calling into question what constitutes a “normal” a Woman’s Lifespan vaginal pH (Ravel et al. 2011). Furthermore, Vaginal microbial communities undergo signifi- vaginal pH and bacterial species composition cant structural changes at various stages in do not always remain stable (Gajer et al. 2012). a woman’s lifespan that are directly linked to Fluctuations in vaginal communities may reflect the level of estrogen in the body (Farage and personalized responses to various kinds of Maibach 2006). Initial colonization occurs at chronic and acute disturbances caused by birth when the infant is first exposed to the human behaviors such as the use of antibiotics, mother’s vaginal microbiota via passage through hormonal contraceptives, sexual activity, vagi- the birth canal or by the skin bacteria of persons nal lubricants, and douching, in addition to handling infants delivered via Cesarean section. numerous other host intrinsic factors such as This initial colonization event is believed to innate and adaptive immunity. establish the gut, skin, and vaginal microbiota, During menopause, a decrease in estrogen allowing them to differentiate into habitat- levels and the cessation of menstruation are specific communities in the weeks and months accompanied by atrophy of the vaginal epithe- following birth (Koenig et al. 2011). During the lium and reduced cervicovaginal secretions first 2–4 weeks of life, maternal estrogen medi- (Farage and Maibach 2006). In many women ates thickening of the vaginal epithelium and the this is accompanied by a shift in the vaginal production of glycogen that is fermented by microbiota from populations of lactic acid- indigenous bacteria resulting in a lowering of producing bacteria to an assortment of species the vaginal pH (~5.0). This effect is transitory, that include strictly anaerobic bacteria compara- however, as waning maternal estrogen, subse- ble to those found during childhood or bacterial quent thinning of the vaginal mucosa, and vaginosis. Likewise, vaginal pH typically rises to a concomitant increase in vaginal pH occur. Dur- near-neutral levels (6.5–7.0) in women who do not ing childhood, the vagina is colonized by diverse use hormone-replacement therapy (HRT), whereas assemblages of aerobic, strictly anaerobic, and women who do use HRT typically maintain enteric species of bacteria, and the pH is nearly a vaginal pH comparable to that of reproductive- neutral (Farage and Maibach 2006). age women (4.5–5.0) (Danielsson et al. 2011). Microbiome, Vagina 419 M

The dynamic nature of this ecosystem underscores and among women. There are a limited number of the importance of resolving its microbial constitu- different kinds of vaginal microbial communities ents at different stages of human development in asymptomatic, apparently healthy women. and the important role of estrogen on the vaginal Ravel et al. (2011) reported the vaginal bacterial environment. communities of White, Black, Hispanic, and Asian women in North America clustered into five groups, four of which were dominated by Bacterial Composition of the Vaginal Lactobacillus iners, L. crispatus, L. gasseri,or Microbiome L. jensenii. The fifth cluster included communi- ties that had lower proportions of lactic acid Cultivation-Dependent and Cultivation- bacteria and higher proportions of strict and fac- Independent Studies ultative anaerobes. The latter community type Until recently our understanding of the composi- accounted for about 25 % of women, a notable tion and ecology of human-associated microbial finding considering the prevailing view that high communities was derived from studies that relied numbers of Lactobacillus are necessary for on cultivation-dependent methods . While these a healthy vaginal tract. These microbiota include pioneering studies provided a fundamental under- members of the genera Atopobium, Corynebacte- standing of microbes in vaginal health, they were rium, Anaerococcus, Peptoniphilus, Prevotella, limited due to inherent biases of the methods used Gardnerella, Sneathia, Eggerthella, Mobiluncus, that precluded the cultivation of many microbial and Finegoldia, among others. The distribution species. More recently, cultivation-independent of community types also varies significantly methods reliant on DNA sequencing of 16S among women from the ethnic backgrounds sam- rRNA genes have provided a means to study pled. Vaginal bacterial communities dominated fine-scale variation in host-associated microbial by Lactobacillus spp. were found in 80.2 % and M communities within and among individuals and 89.7 % of Asian and White women, respectively, exploration of ecological relationships between but just 59.6 % and 61.9 % of Black and Hispanic bacterial species and with the host. Typically, par- women, respectively. On the other hand, occur- tial 16S rRNA gene sequences are amplified using rence of communities with low proportions or no primers that anneal to highly conserved sequences detectable Lactobacillus species was more often in the gene, and the resulting amplicons are seen in Hispanic (38.1 %) and Black (40.4 %) sequenced. Phylogenetic analyses of the sequences women compared with Asian (19.8 %) and White allow for classification of phylotypes and determi- (10.3 %) women (Fig. 2). Furthermore, high bac- nation of the numerically dominant taxa in terial species diversity exists in all vaginal com- a community. Other methods that rely on other munities, even those in which the phylotype conserved genes (cpn60, rpoC, uvrB,orrecA) abundance distribution was highly skewed have also been developed but are not as widely toward one or very few numerically dominant used. Thus, major advances in DNA sequencing phylotypes. technologies over the last decade have fundamen- tally changed the way we assess microbial com- Community Dynamics and Stability munity structure and composition, and this has To date, most vaginal microbial studies facilitated more expansive and intensive studies have employed cross-sectional designs, wherein of vaginal community structure and dynamics. samples are obtained from any number of individuals at a single point in time or with Community Types multiple sampling points separated by relatively The vaginal microbial community is established long intervals (weeks or months). While these in puberty and may reside in women until meno- studies have yielded valuable information pause. In healthy, asymptomatic, reproductive- in terms of species composition, they do not age women, its composition differs both within allow for any assessment of community M 420 Microbiome, Vagina

Microbiome, Vagina, Fig. 2 Representation of vaginal and L. jensenii, respectively, while community group IV bacterial community groups within four ethnic groups of contains a diverse assemblage of facultative and strictly women. The number of women from each ethnic group is anaerobic bacteria. Percent values are the percentages of in parentheses. The roman numerals indicate the five women in each ethnic group whose vaginal bacterial com- common vaginal bacterial community groups described munity clustered with a particular community group by Ravel et al. (2011). Community groups I, II, III, and (Reproduced from data in Ravel et al. 2011) V are predominated by L. crispatus, L. gasseri, L. iners, stability, therefore painting an incomplete The study by Gajer et al. highlights the poten- picture of vaginal microbial ecology. tial of prospective longitudinal studies to eluci- Daily fluctuations in the composition of the date the cause and etiology of vaginal diseases vaginal microbiota have been previously and conditions. Prospective longitudinal studies documented primarily by microscopy. More in which samples and detailed behavioral meta- recently, a longitudinal study assessed the tem- data are frequently collected would provide poral dynamics of 32 healthy, asymptomatic a way to observe events that occurred prior to women sampled twice weekly over a 16-week the onset of disease or infection and follow the period. The vaginal communities of nearly all sequence of events that occur during treatment the women assessed exhibited some degree of and recovery. For example, it has been widely variability, with some changing markedly over recognized that a variety of risk factors are asso- a short time and others remaining relatively con- ciated with a high prevalence of vaginal infective stant. Usually these shifts involved changes in the disease, including sexual behaviors (frequent relative proportions of species present, but in intercourse, multiple male sexual partners in the some cases, a distinct and persistent turnover in last 12 months, lesbian or bisexual intercourse, species composition occurred, marking the pres- lower age of first intercourse, oral-anal sex, and ence of an apparent alternative equilibrium state. use of vaginal lubrication or sex toys) and Several factors may have contributed to these nonsexual behaviors (cigarette smoking, vaginal differing levels of community stability. For douching). Additionally, the selection of instance, menses was identified as having the a contraceptive method may also significantly largest effect on changes in community compo- impact susceptibility to certain diseases, for sition, while periods of the menstrual cycle example, consistently using condoms may be an marked by high levels of estrogen (late follicular effective and protective strategy for women to phase) or estrogen and progesterone (luteal prevent bacterial vaginosis (BV) (Smart 2004) phase) had tended to stabilize composition and reduces the likelihood of its relapse. Host- (Gajer et al. 2012). Other factors that may influ- related risk factors should also be taken into ence community dynamics include the frequency account, such as antibiotic use, uncontrolled dia- and type of feminine hygiene products used, betes, conditions with high reproductive hormone aging, sexual practices and frequency, and other levels (gravity, luteal phase of menstrual cycle), host factors such as genetics, stress, and diet. genetic predisposition, as well as chronic stress. Microbiome, Vagina 421 M

Host-Microbe Mutualism of nonspecific antimicrobial molecules including mannose-binding lectin, complement compo- Nutrients Provided by Host nents, defensins, secretory leukocyte protease Bacteria in the vagina are dependent on their inhibitors, heat shock proteins, and nitric host for nutrients. A major source of nutrients oxide (Witkin et al. 2007). Additionally, cyclical is almost certainly vaginal fluid, which origi- hormonal changes influence innate immunity nates from serum transudate as well as by upregulation during post-menses and salpingeal and mucous membranes. Other com- downregulation in the luteal phase. ponents of the vaginal fluid may include cervical The release of cytokines by the innate mucus, endometrial and oviductal fluids, immune system leads to the activation of the sloughed vaginal epithelial cells, macrophages, antigen-specific acquired immune system medi- lymphocytes, Langerhans cells, and other com- ated by T and B lymphocytes. Upon activation, pounds or molecules associated with the innate these lymphocytes initiate antibody-mediated microbiota. This mixture may also include com- responses to specific microbial antigens. ponents such as carbohydrates (such as glycogen Whereas activation of innate immunity occurs derived from sloughed vaginal epithelial cells), immediately upon recognition of a pathogen, amino acids, proteins, ions, and lipids (Ferguson several days are required to mount a response and Rohan 2011). Vaginal fluid therefore consti- from the acquired immune system. Antibodies tutes an essential source of nutrients for resident transude from the systemic circulation into bacterial populations. By modulating the kind the vagina and, in turn, result in microbial killing and amounts of resources in vaginal secretions, by a complement-dependent mechanism or the host metabolically influences the composi- opsonization by recognizing and binding to spe- tion and dynamics of microbial communities. cific antigens on microorganisms. Both IgG and IgA classes of antibodies can be locally pro- M Immune Regulation duced. The antibody-producing B lymphocytes In addition to protection conferred by the com- are present principally in the endocervix but also mensal vaginal microbiota against infectious in the vagina. The elaboration of antibodies in agents, invasion and infection by pathogens are the female lower genital tract provides a rapid prevented by components of the local innate and mechanism for combating pathogenic microor- acquired immune systems. The vaginal innate ganisms (Witkin et al. 2007). immune system, which is composed of membrane-associated components such as Toll- like receptors (TLRs), soluble factors, and phago- Vaginal Maladies cytic cells, provides the first line of defense against bacterial pathogens by recognizing Bacterial Vaginosis pathogen-associated molecular patterns Bacterial vaginosis (BV) is the most frequently (PAMPs) on microbial invaders. At least cited cause of vaginal discharge and malodor and 11 TLRs have been identified in human vaginal the most common vaginal condition of epithelial cells and are considered major determi- reproductive-age women, affecting millions of nants in the recognition of microbial pathogens women each year. The prevalence of BV among and subsequent activation of the innate response. women varies widely and depends on the subject This recognition process triggers a sequence population. In nonpregnant women, BV is asso- of events leading to the production of ciated with serious adverse sequelae including pro-inflammatory cytokines. This cytokine infertility, endometritis, and pelvic inflammatory release subsequently stimulates phagocytic and disease as well as an increased risk of acquiring natural killer cells to attack and quell HIV, Neisseria gonorrhoeae, and other sexually the invading pathogen. Human vaginal epithelial transmitted diseases. During pregnancy, BV is cells are also capable of producing a suite associated with several adverse outcomes M 422 Microbiome, Vagina including preterm delivery of low birth weight (Amsel et al. 1983), wherein three of the follow- infants, spontaneous abortion, premature rupture ing four symptoms must be evident: (a) a homog- of membranes, preterm birth, amniotic fluid enous, white, noninflammatory discharge that infections, postpartum endometritis, and endo- smoothly coats the vaginal walls; (b) the pres- metritis following Caesarian section. In most ence of clue cells (squamous epithelial cells cov- women, the symptoms of BV are resolved on ered with adherent bacteria) on microscopic their own without intervention. In other cases, examination; (c) a vaginal fluid pH over 4.5; symptoms and signs of infection ensue and anti- and (d) a fishy odor of vaginal discharge before biotic therapy is required. The standard treatment or after addition of 10 % KOH (potassium for bacterial vaginosis is metronidazole in hydroxide). The Nugent Gram stain score a dosage of 500 mg taken orally twice daily for (Nugent et al. 1991), which is used in clinical 7–10 days. Equivalent effective alternatives research settings, is scored on a scale that reflects include metronidazole in a single 2 g oral dose, the proportions of (a) Gram-positive rods oral clindamycin, and intravaginal clindamycin (Lactobacillus morphotypes), (b) Gram-variable cream or ovules. Recurrent bacterial vaginosis is rods and cocci (G. vaginalis, Prevotella, common and requires longer treatment Porphyromonas, and Peptostreptococci mor- (10–14 days) with any of the recommended or photypes), and (c) curved Gram-variable rods alternative therapies (Centers for Disease Control (Mobiluncus spp. morphotypes). In a formal and Prevention 2007). sense, an obvious potential problem with the Despite decades of research, attempts to find Nugent score premise is that high numbers of a single causative agent for BV have failed. Lactobacillus spp. define “health,” and this Indeed, BV is characterized and perhaps caused imposes a bias against normal vaginal microbial by disruption of the vaginal ecosystem, which is communities that lack appreciable numbers of reflected in alterations to the composition and lactobacilli, yet maintain a low pH. The Amsel structure of vaginal microbial communities. Typi- test, on the other hand, may lack sensitivity due cally the numbers of lactic acid-producing bacteria to the subjectivity of a clinician’s interpretation. are decreased and the diversity and numbers These measures can lead to opposing conclusions of strictly anaerobic bacteria are increased, includ- and have led many to suspect the accuracy of ing species of Gardnerella vaginalis, Atopobium these tests. To date, neither of the two diagnostic vaginae, Leptotrichia amnionii, Sneathia methods nor community composition and struc- sanguinegens, Porphyromonas asaccharolytica, ture can fully explain symptomatic BV, which and novel members of the order Clostridiales appears to be a multifactorial clinical syndrome referred to as BV-associated bacteria (BVAB) with complex and still unknown etiologies. (Fredricks et al. 2005). It is unclear if these bacte- The causes and cures of BV will continue to be rial species are causally related to the symptoms of enigmatic until it is recognized that while BV or whether the association is contingent on the “normal and healthy” can be equated with high criteria used to diagnose BV. These organisms numbers of lactobacilli, the converse statement might not be “infectious agents” in the strict that “unhealthy” is equated with low numbers of sense, but when present in high numbers they or no lactobacilli is not necessarily true. may elicit some or all of the symptoms classically associated with BV. Yeast Infections The confusion about BV partly stems Vulvovaginal candidiasis (VVC), more com- from the fuzzy definition of BV (Centers for monly known as vaginal yeast infection, is the Disease Control and Prevention 2007) and the second most common vaginal disorder, being diagnostic criteria commonly used that remain diagnosedinupto40%ofwomenwithvaginal mired in controversy. In clinical settings, BV symptoms seeking primary care (Anderson is commonly diagnosed by an Amsel test et al. 2004). An estimated 75 % of all women Microbiome, Vagina 423 M will experience at least one episode of VVC (150 mg oral fluconazole). This treatment during their reproductive-age years; approxi- regimen has been shown to be effective in over mately half of these women experience recur- 90 % of patients with RVVC (Achkar and Fries rence. VVC is diagnosed by signs and symptoms 2010). of inflammation in the presence of Candida spp. and in the absence of other infectious etiol- Intrauterine Infection and Preterm Birth ogy. Recurrent VVC (RVVC) is defined as hav- Preterm birth is the leading cause of neonatal ing four or more episodes of VVC per year. In mortality worldwide and it continues to increase addition to the characteristic symptoms of vul- yearly. Intrauterine infection may account for var pruritus and burning, women with VVC fre- 25–40 % of preterm births as a conservative esti- quently experience soreness and irritation mate. Intrauterine infection begins in the decidua, leading to dyspareunia and dysuria. Vulvar and extends to the space between amnion and cho- vaginal erythema, edema, fissures, and a thick, rion, and finally reaches the amniotic cavity and curdy vaginal discharge are commonly found fetus. Although bacteria are found in the mem- upon physical examination. Approximately branes of up to 70 % of pregnant women, the half of patients who have positive microscopy presence of bacteria in chorioamnion alone is results for yeast cells and hyphal elements have not always sufficient to cause an inflammatory what is typically considered a normal vaginal pH response that leads to preterm birth. Two condi- in the range of 4.0–4.5. tions are essential for intrauterine infections to VVC is caused by the yeast Candida,most cause preterm birth. First, the infectious organ- commonly Candida albicans, which colonizes isms must enter the amniotic cavity and be rec- the genitourinary tracts of 20–30 % of asymp- ognized as foreign by the host immune system. tomatic, nonpregnant women at any given point Second, the bacterial numbers must breach some in time and in up to 70 % of women over a year- threshold to trigger an intra-amniotic inflamma- M long period without immune suppression, tory response, which in turn induces preterm damaged mucosa, or other signs of disease. labor (Zhou et al. 2010). Therefore, Candida is often considered an Through cultivation-independent methods, opportunistic pathogen. Several attributes of Mycoplasma, Ureaplasma, Streptococcus, Lacto- C. albicans may contribute to virulence, includ- bacillus, Prevotella, Delftia, Neisseria, ing hyphal formation, adhesion, phenotypic Fusobacterium, Sneathia, Leptotrichia, Shigella, switching, extracellular hydrolytic enzyme pro- Bacteroides, Bergeyella, and Peptostreptococcus duction, and biofilm formation. Moreover, host- were found in amniotic fluids of women with related factors such as antibiotic use, preterm labor and intact membranes. Most are uncontrolled diabetes, conditions with high related to those found in the human vagina. reproductive hormone levels, and genetic pre- Thus, there is a potential connection between dispositions have been significantly associated the bacterial species in amniotic fluid with those with VVC and RVVC. Standard antibiotic treat- in the vagina, with the latter being a potential ment of VVC involves single-dose or short- source of infecting organisms (Goldenberg course therapy for which both topical and oral et al. 2008). Coincidentally, bacterial vaginosis, treatments are available. Although relapse of present in up to 15–20 % of pregnant women, has VVC following treatment is common, been identified as a risk factor for spontaneous C. albicans rarely exhibit resistance to azoles preterm delivery, premature rupture of mem- during VVC. RVVC requires topical therapy branes, and postpartum endometritis. Neverthe- administered intravaginally on a daily basis for less, it is recommended that only patients at high at least 1 week or multiple doses of oral flucon- risk for preterm delivery, specifically those with azole (150 mg every 72 h for three doses), a previous history of a spontaneous preterm birth, followed by long-term weekly treatment should receive antibiotic treatment to prevent M 424 Microbiome, Vagina preterm birth if they are found to have bacterial References vaginosis (Zhou et al. 2010). Achkar JM, Fries BC. Candida infections of the genitouri- nary tract. Clin Microbiol Rev. 2010;23(2):253–73. Consideration of Differences Between Amsel R, Totten PA, Spiegel CA, Chen K, Eschenbach D, Women: Toward Personalized Holmes KK. Nonspecific vaginitis: diagnostic criteria and microbial and epidemiologic associations. Am Medicine? J Med. 1983;74(1):14–22. Anderson MR, Cohrssen A, Klink K. Evaluation of vagi- The species that make up human microbial com- nal complaints. 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Redondo-Lopez V, Cook RL, Sobel JD. Emerging role of of animal behaviors with relevance to pain, lactobacilli in the control and maintenance of the vag- cognition, depression, and anxiety. This has pro- inal bacterial microflora. Rev Infect Dis. 1990;12(5): 856–72. found implications across preventative, diagnos- Smart S. Social and sexual risk factors for bacterial vag- tic, and therapeutic domains. It is clear that inosis. Sex Transm Infect. 2004;80(1):58–62. continued efforts to understand and favorably Witkin SS, Linhares IM, Giraldo P. Bacterial flora of the manipulate the gut microbiome have the potential female genital tract: function and immune regulation. Best Pract Res Clin Obstet Gynaecol. 2007;21(3): to be a transformative venture and will likely 347–54. accrue multiple health benefits. Zhou X, Brotman RM, Gajer P, Abdo Z, Schuette€ U, Ma S, Ravel J, Forney LJ. Recent advances in understanding the microbiology of the female reproductive tract and the causes of premature birth. Infect Dis Obstet Synonyms Gynecol. 2010;2010:737425. Brain–gut–enteric microbiota axis; Brain–gut– microbiome axis

Microbiome–Gut–Brain Axis Definition Gerard Clarke1, Ted Dinan2 and John Cryan3 1Alimentary Pharmabiotic Centre, University A bidirectional communication network between College Cork, Cork, Ireland the brain and the gastrointestinal tract that 2Department of Psychiatry, University College includes the central nervous system, the sympa- Cork, Cork, Ireland thetic and parasympathetic arms of the auto- 3Department of Anatomy and Neuroscience, nomic nervous system, the neuroendocrine and University College Cork, Cork, Ireland neuroimmune systems, the enteric nervous sys- M tem, and the collection of microorganisms and their genomes in the gut habitat. Technological advances have facilitated a huge increase in our understanding of the composition and diversity of the gut microbiome, and efforts Introduction to define the influence of this virtual organ on human physiology are a burgeoning area of One of the most important scientific pursuits in research. Important concepts have emerged recent times has been the concerted effort to including that of a core microbiome, distinct understand the role of the gut microbiome in enterotypes, and age-related shifts in composi- health and disease (Sekirov et al. 2010). Compel- tion, which are being linked to adverse health ling evidence from diverse disciplines has illumi- outcomes. In tandem, a new research narrative nated a role for the gut microbiota in virtually all in the field of neuroscience positions the gut aspects of physiology and in disease states rang- microbiome as a key player in communication ing from inflammation (Hooper et al. 2012)to along the gut–brain axis. This has heralded the obesity (Tremaroli and B€ackhed 2012). This revised concept of the microbiome–gut–brain positioning center stage has been facilitated by axis. Important research has highlighted alter- large-scale metagenomic projects such as the ations in the composition and stability of the Human Microbiome Project (https:// gut microbiome in clinical populations linked to commonfund.nih.gov/hmp/), which aims to char- CNS-related disorders such as irritable bowel acterize the composition, diversity, and temporal syndrome (IBS), obesity, and autism spectrum stability of the gut microbiome. Aligned with disorders. A variety of preclinical strategies these initiatives is an emerging narrative, which have underlined the importance of the gastroin- posits a crucial role for the intestinal microbiota testinal microbial community for the expression in gut–brain communication, brain function, and M 426 Microbiome–Gut–Brain Axis

Microbiome–Gut–Brain Axis, Fig. 1 The microbiome– influence gut physiology leading to inappropriate gut–brain axis. There is a bidirectional communication microbiome–gut–brain axis signaling and associated con- network between the brain and the gastrointestinal tract. sequences for CNS functions and disease states. Gut path- A stable gut microbiota is a key component of this axis and ogens, for example, can induce such a dysbiosis. Stress at is essential for normal gut physiology. This stability con- the level of the CNS can also impact gut function and lead tributes to appropriate signaling along the microbiome– to perturbations of the microbiota. Probiotics may produce gut–brain axis and to the healthy status of the individual. their effects in part through a metagenomic shift leading to Conversely, dysbiosis of the gut microbiota can adversely the restoration of a healthy microbial composition even behavior (Rhee et al. 2009; Cryan and Dinan spectrum disorders (Mulle et al. 2013). Also 2012). This has seen the gut microbiome recog- highlighted are a variety of preclinical strategies nized as a crucial node in the gut–brain axis with that have confirmed the importance of the gut its increasingly appreciated importance signified microbiome to the expression of animal behav- by a revision of the concept to that of the iors with relevance to pain, cognition, depression, “microbiome–gut–brain axis” (see Fig. 1). and anxiety (see Fig. 2). The implications of these Outlined here are the critical components and findings are profound and bestride preventative, functional relevance of this axis and a sketch of diagnostic, and therapeutic domains. Indeed, the the picture that has arisen from research showing National Institute of Mental Health (NIMH) alterations in the composition and stability of the states that understanding how differences in gut microbiome in clinical populations linked to microbiota profiles influence the development of CNS-related disorders such as IBS (see also brain and behavior will be one of the great fron- “▶ Intestinal Microbiota, Alterations in Irritable tiers of clinical neuroscience in the next decade Bowel Syndrome” in this encyclopedia), obesity (http://www.nimh.nih.gov/about/director/2012/ (Tremaroli and B€ackhed 2012), and autism the-top-ten-research-advances-of-2012.shtml). Microbiome–Gut–Brain Axis 427 M

Microbiome–Gut–Brain Axis, Fig. 2 Investigating the applied to gauge the effects of pathogenic bacteria at the role of the microbiome in health and disease. The impact level of the CNS. The administration of probiotic strains to of the gut microbiome on brain and behavior can be adult animals or humans can similarly be used to appraise assessed using a variety of approaches. Germ-free (GF) the effects of these beneficial bacterial on the studies can be used to assess how the complete absence of microbiome–gut–brain axis. The ability of antibiotic a microbiota during development affects CNS function. drugs to transiently alter microbiota composition can GF animals can also be employed to study the “humani- also be exploited. The combined information gleaned M zation” of the gut microbiota by transplanting fecal from these studies to date implicates the microbiome in microbiota from specific clinical populations or indeed a variety of complex behaviors and functions including from animal models of disease. Infection studies can be depression, anxiety, cognition, and pain

The Gut–Brain Axis conversely, visceral messages from the GIT can influence brain function (Cryan and O’Mahony The realization that the microorganisms resident 2011). in our gastrointestinal tract impact on brain func- If this axis malfunctions and homeostasis is tion was preceded by the emphasis placed on disrupted, pathophysiological consequences can a more established construct in the field of ensue and alterations in gut–brain interactions are neurogastroenterology known as the gut–brain associated with gut inflammation, chronic axis (Mayer 2011). The general scaffolding of abdominal pain syndromes, and eating disorders. this axis includes the central nervous system Moreover, gut–brain axis dysfunction is associ- (CNS), the neuroendocrine and neuroimmune ated with agitation of the stress response and systems, the sympathetic and parasympathetic behavior, and there is a high comorbidity arms of the autonomic nervous system (ANS), between stress-related psychiatric symptoms and the enteric nervous system (ENS). These such as anxiety and gastrointestinal disorders, components interact to form a complex reflex including IBS and inflammatory bowel disease network that allows for a top-down and bottom- (IBD) (Scott et al. 2013). Studies of this ilk now up flow of information. It is essentially through include the gut microbiota, which can be this bidirectional communication network that regarded as a metabolic organ in its own right signals from the brain can influence the functions (O’Hara and Shanahan 2006). Crucially, there is of the gastrointestinal tract (GIT) and, a growing appreciation that this is an integral M 428 Microbiome–Gut–Brain Axis component of the axis that can influence CNS highlighted that the gut microbiota is essential for function and can in turn be influenced by the normal GIT motility and in the maintenance of CNS through the latter’s effects on the gastroin- barrier function (Mayer 2011). Germfree studies testinal tract (see Fig. 1) (Rhee et al. 2009). also drew attention to the essential role of the Stress and the associated activation of the microbiota in the development of the hypothalamus–pituitary–adrenal (HPA) axis can gut-associated lymphoid tissue (GALT) in influence the composition of the gut microbiota, sculpting the immunological repertoire of the and the functional consequences of this influence GIT and specifically the mucosal immune sys- are currently under investigation (Dinan and tem. Indeed, the absence of a gut microbiota Cryan 2012). For example, early-life stressors compromises appropriate immune responses to such as maternal separation, which induces pathogenic threats (Grenham et al. 2011). long-term increases in HPA axis activity, can The microbiota is also pivotal for normal have both short- and long-term effects on the digestion and host metabolism. There are two composition of the gut microbiota (O’Mahony main mechanisms by which it can maximize et al. 2011). Chronic stress in adulthood can nutrient availability, either by the release of cal- also modulate gut bacterial populations in ories from otherwise unavailable oligosaccha- a manner that correlates with alterations in rides or by modulating absorption (Nicholson pro-inflammatory cytokines. An additional con- et al. 2012). A significant energy source for sequence of chronic stress is a disruption of the humans is the bacterial metabolism of dietary intestinal barrier, and this “leaky barrier” leads to fiber to short-chain fatty acids (SCFAs) increased circulating levels of immunomodula- (Tremaroli and B€ackhed 2012). Finally, the pre- tory bacterial cell wall components such as lipo- vention of colonization by pathogens by the res- polysaccharide (LPS) (Scott et al. 2013). Of note ident microbiota is largely accomplished by is that these effects can be reversed by probiotics competing for nutrients and receptors and by and that some clinical studies show increased production of antimicrobial compounds (O’Hara bacterial translocation in disorders such as and Shanahan 2006). depression. Consequently, targeting the gut– brain axis is viewed as an attractive option for the development of novel treatments for a wide Microbiota Composition variety of disorders including obesity and stress- related psychiatric and gastrointestinal disorders Although there is a well-developed understand- (Cryan and Dinan 2012; Scott et al. 2013). ing of the reciprocal communication between the ENS and the CNS, the role of the gut microbiota is less well delineated. This knowledge gap, Functional Relevance of the Microbiota which is largely due to a poor understanding of our gastrointestinal inhabitants and their func- At a more general level, it is clear from early tional capability, is being bridged by the studies in germ-free (GF) animals (see below) metagenomic revolution (Weinstock 2012). One that the microbiota has an extensive repertoire outcome of such studies is the consideration that of structural, protective, and metabolic functions humans are more microbe than man: the (O’Hara and Shanahan 2006). The consequences microbes in our gastrointestinal tract (1013–1014 of growing up germ-free from a morphological microorganisms) outnumber the human cells in perspective include a greatly enlarged cecum, our bodies by a factor of 10 and contains reduced intestinal surface area, increased entero- 150 times as many genes as our genome. This chromaffin cell area, smaller Peyer’s patches, and microbial community counts among its constitu- smaller villous thickness in these animals com- ents greater than 1,000 species and more than pared to conventional controls. Unsurprisingly, 7,000 strains (Scott et al. 2013). Although domi- in the light of such alterations, these studies also nated by bacteria (mainly strict anaerobes), Microbiome–Gut–Brain Axis 429 M viruses, protozoa, archaea, and fungi are also pres- primed, it has been suggested that they group ent. Nevertheless, the microbiota is largely marked into just three distinct types – enterotypes – by two bacterial phyla, the Bacteroidetes and defined by their bacterial composition. These Firmicutes, with Proteobacteria, Actinobacteria, enterotypes are dominated by high levels of Fusobacteria, and Verrucomicrobia present at a particular microbial genus from a palette of a lower abundance (Grenham et al. 2011). three: Bacteroides, Prevotella,orRuminococcus, The historical view of these commensal bac- respectively (Ursell et al. 2012). Current thinking teria as “benign” components too troublesome to makes it clear that a microbiota with a diverse but study, due to the majority not being amenable to balanced compositional motif confers health ben- culture, has largely been supplanted in the efits and that a perturbation of this balance con- culture-independent and metagenomic age by fers disease susceptibility (see Fig. 1) (Sekirov a broader appreciation of intestinal community et al. 2010). One of the factors that influences this composition and diversity (Sekirov et al. 2010). balance is diet, while other influences, including Furthermore, a previously limited but rapidly infection, disease, and antibiotics, may tran- advancing suite of techniques (metabolomics, siently alter the stability of the gut microbiota metaproteomics) is beginning to open up the and impact adversely on host well-being secretory and metabolic capability of the (Grenham et al. 2011). Interestingly, the core microbiome in addition to its phylotype compo- microbiota in the elderly has been reported to be sition. It is also now evident that the microbial different from that of younger adults, and its diversity within the gut has a developmental composition is directly correlated with health timeline and a longitudinal variation outcomes (O’Toole 2012, see also (Adlerberth and Wold 2009). Because microbial “▶ ELDERMET (Ireland)” in this encyclopedia). penetration of the amniotic cavity is considered Arising from this improved appreciation of the extremely rare, the developing fetus is judged influence of gut bacteria on health is a growing M essentially sterile. The initial microbiota then is body of literature, both clinical and preclinical, simple and has a high interindividual variability that focuses on the impact of enteric microbiota with maternal microbial signatures, and delivery on brain and behavior (Collins et al. 2012). mode is a key determinant of community compo- sition (Grenham et al. 2011). This is an imprint that can last for months, and it can influence The Gut Microbiome in CNS-Related health outcomes (Adlerberth and Wold 2009). Conditions Prior to the acquisition of the stable adult microbiome after 1–3 years of life, there are shifts The links between microbiome dysbiosis and in composition that likely reflect key early-life a variety of CNS-related conditions have come events such as weaning or infections (Cryan and under intense scrutiny of late. Although obesity Dinan 2012). The functional capability of the has both central and peripheral components, the microbiome parallels infant host requirements, fact that the microbiota plays a key role in host and infants who are breastfed exhibit an increased digestion and metabolism (see above) suggests abundance of Bifidobacterium species that are that obesity could indeed have a microbial basis equipped to utilize human milk oligosaccharides. (Nicholson et al. 2012; Tremaroli and B€ackhed As the infant progresses toward adulthood, each 2012). However, clinical studies to date have not dietary epoch is distinguished by changes in the defined whether the altered microbiota precedes microbiota and the enrichment of genes special- the development of obesity or represents the ized toward microbial digestion of that diet upshot of dietary factors and alterations in host (Lepage et al. 2013). physiology. Data from the preclinical literature Although gastrointestinal bacterial communi- does argue in favor of such a linkage, and it is ties have a composition that varies greatly interesting to note that olanzapine, an atypical between individuals and may be genetically antipsychotic that causes weight gain, has been M 430 Microbiome–Gut–Brain Axis shown to alter the composition of the gut defecatory patterns, bloating, and the absence of microbiota in rats. However, recent studies have reliable biological markers. The composition also confirmed that high-fat diets influence of the microbiota, both qualitatively and in microbiome composition (Cryan and Dinan terms of temporal instability, has been reported 2012). Besides such caveats, obesity is best con- to be altered in IBS (Grenham et al. 2011). ceptualized as multifactorial with hypothalamic Involvement of the microbiome in the generation dysfunction also likely to be involved of IBS symptoms can be inferred by the develop- (Schellekens et al. 2012), and it is as yet unclear ment of the condition following an episode of whether CNS control of food intake is subject to bacteriologically confirmed gastroenteritis in modulation by the microbiome (Cryan and Dinan a significant proportion of individuals (known as 2012). postinfectious IBS) and also by the reports of The term autism spectrum disorders (ASD) a chronic low-grade inflammation in IBS subjects describes neurodevelopmental disorders charac- (Scott et al. 2013). Indirectly, the finding that terized by deficits in social interaction and com- specific probiotic strains can relieve particular munication accompanied by the presence of symptoms of IBS supports this concept (Clarke limited, repetitive stereotyped interests and et al. 2012a). This seems particularly relevant for behaviors. Gastrointestinal symptoms and distur- the pain component of the disorder, which is bances are commonly reported in children with a symptom likely due to visceral hypersensitivity. ASD, and it has been suggested that there might Visceral pain perception is complex and involves be a role for an abnormal composition of the gut both central and peripheral mechanisms that can microbiome in this disorder (Mulle et al. 2013). potentially be affected by alterations in the intes- To date, studies probing for compositional per- tinal microbiota (Cryan and Dinan 2012). How- turbations of the microbiome in ASD have ever, not all studies have reported disturbances in yielded conflicting results, and interpretation is the microbiota composition in IBS, and it remains complicated by the fact that individuals suffering unclear, where alterations have been reported, from ASD frequently use antibiotics and have whether they are primary or secondary in nature. diets which often diverge from those of healthy populations (Grenham et al. 2011). On the other hand, lending credence to this concept is a study Strategies to Investigate showing that vancomycin (a minimally absorbed Microbiome–Brain–Gut Axis antibiotic that targets gram-positive anaerobes in Perturbation the gut following oral administration) can tran- siently improve symptoms in regressive onset Although the microbiome–gut–brain axis is autism. Fecal concentrations of SCFAs a relatively new concept to the neuroscience com- (potentially neuroactive microbial metabolites) munity, the transformative potential of this have also been reported to be altered in research has seen a burgeoning interest and the ASD. Of further note, the administration of one identification of many promising avenues of of these SCFAs (propionic acid) to animals via inquiry. Information about communication along the intracerebroventricular route results in some this axis has been charted using a variety of alter- autistic-like behaviors, suggesting a potential native but converging means (see Fig. 2). GF mice mechanism through which an altered microbiome canbeusedtoassesshowthecompleteabsenceof might induce symptoms of ASD (Cryan and a microbiota during development affects CNS Dinan 2012). Clearly, this is an area that warrants function (Cryan and Dinan 2012). As indicated further investigation. earlier, this is a strategy that has already contrib- IBS, a functional gastrointestinal disorder, can uted greatly to our appreciation of the role of the be regarded as a prototypical stress-related brain– microbiota in general health and well-being. Tak- gut axis disorder (Kennedy et al. 2012). It is ing advantage of the principle of a sterile uterine characterized by abdominal pain, altered environment, it is possible to prevent the normal Microbiome–Gut–Brain Axis 431 M postnatal colonization of the gastrointestinal tract of the gut microbiome on brain and behavior. The by combining a surgical delivery mode with range of complex behaviors that fall under the a germfree rearing environment in gnotobiotic remit of the gastrointestinal microbiome is now units. This allows for direct comparison with con- known to include pain, anxiety, depression, and ventionally colonized counterparts (Grenham aspects of cognitive function (Cryan and Dinan et al. 2011). Although the clinical translation of 2012; Foster and McVey Neufeld 2013). Studies this tactic is limited and GF mice do not model any in GF animals, for example, from independent clinical conditions per se, these studies are critical research groups and using a variety of assessment in establishing the aspects of brain and behavior methods, have reproducibly shown a reduced that depend on an intact microbiome (Cryan and anxiety phenotype (Collins et al. 2012). It also Dinan 2012). GF mice also permit the study of how appears that the exploratory behavioral profiles of one particular entity (e.g., a probiotic) impacts on specific mouse strains are transmissible as indi- the microbiome–gut–brain axis in isolation and cated by microbiota transplant studies in previ- permit manipulations aimed at reintroducing ously GF animals. These findings are bolstered by a more complete gut microbiome later in life. the data from infection studies (e.g., Trichuris Moreover, GF mice studies can be employed to muris and Citrobacter rodentium) that demon- study the “humanization” of the gut microbiota by strate that the disruption of the gut microbiota in transplanting fecal microbiota from specific clini- this manner can increase anxiety-like behaviors cal populations or indeed from animal models of (Cryan and O’Mahony 2011). Antimicrobials can disease (Collins et al. 2012; Cryan and Dinan also increase exploratory behavior in mice. 2012). Moreover, probiotic treatment can either prevent The administration of probiotic strains to adult such behavioral alterations or reverse them animals or humans can be used to appraise the (Foster and McVey Neufeld 2013). Indeed, cer- effects of these beneficial bacteria on the host, tain probiotics or combinations of probiotics can M and it is increasingly apparent that any effects on reduce anxiety-like behaviors even in situations the gut–brain axis that are observed are most without any previous dysbiosis of the microbiota likely strain and species specific (Clarke (Cryan and Dinan 2012). et al. 2012a). Infection studies have also been Studies in GF animals have also repeatedly applied to gauge the effects of pathogenic bacte- highlighted that the microbial content of the ria at the level of the CNS, and many, though not gastrointestinal tract is essential for the develop- all, of the observations arising from these studies ment of an appropriate stress response later in are mediated through the activation of the life and also that there is a critical window in immune system. Finally, the ability of antibiotic early life during which colonization must occur drugs to transiently alter microbiota composition to ensure normal development of the HPA axis in a translationally relevant fashion is also (Dinan and Cryan 2012). Certain probiotic a powerful and clinically relevant instrument in strains can also abrogate infection-induced the study of the microbiome–gut–brain axis increases in serum corticosterone levels in mice (Cryan and O’Mahony 2011). One should how- (Cryan and Dinan 2012)aswellasreduceserum ever bear in mind when investigating the effects cortisol concentrations in humans (Clarke of these antimicrobial agents that many are also et al. 2012a). At the cognitive level, GF mice systemically toxic (Cryan and Dinan 2012). display deficits in simple nonspatial and working memory tasks. Cognitive impairments are also apparentinGFmicesubjectedtoaninfectious Impact of the Microbiome on Brain and agent. Interestingly, a similar intervention in Behavior conventionally colonized animals required an acute stressor to unmask the infection-induced A number of important recent advances arising cognitive dysfunction. This effect was prevented from these strategies have unmasked the impact by a pretreatment regimen with a combination of M 432 Microbiome–Gut–Brain Axis probiotics (Cryan and Dinan 2012). Although of growing up germ-free are only evident in male there are calls to expand the repertoire of behav- animals (Cryan and Dinan 2012). ioral cognitive assays employed, it should be There are molecular alterations not restricted noted that maintaining animals in a GF state and to GF studies, and both bacterial infection and conducting complex behavioral studies present antimicrobial regimens can alter BDNF levels in the researcher with significant logistical hurdles. the hippocampus and amygdala (Collins Similarly, there is a need for GF studies to et al. 2012). Additionally, bacterial infections interrogate in greater detail the impact of the gut can increase the plasma kynurenine to tryptophan microbiome on behavioral patterns relevant to ratio (indicative of alterations in tryptophan depression (Scott et al. 2013). The best evidence metabolism), alter markers of neuronal activation comes from probiotic studies where the (cFOS expression), and increase plasma levels of depression-related behavior observed post- the pro-inflammatory cytokines tumor necrosis myocardial infarction in rats was overturned by factor a and interferon g (Cryan and Dinan a cocktail of probiotics, while ingestion of Lac- 2012). Treatment with the anti-inflammatory tobacillus rhamnosus JB1 reduced depression- agents etanercept and budesonide normalizes like behaviors in mice (Cryan and Dinan 2012). many of the observed infection-induced Further evidence of positive effects of probiotics alterations, with the exception of the infection- on behavior arises from studies that demonstrate induced changes in hippocampal BDNF expres- that the probiotic agent Bifidobacterium infantis sion (Foster and McVey Neufeld 2013). Probiotic has antidepressant-like effects in an early-life treatment in some instances can restore hippo- stress model (Clarke et al. 2012a). However, at campal BDNF mRNA levels but without modu- the neuronal level, GF animals have repeatedly lating plasma cytokine or kynurenine levels. been shown to have molecular alterations of par- Probiotics can also alter the mRNA expression ticular relevance to depression. This includes of specific GABA receptor subtypes, which have decreased hippocampal levels of brain-derived been associated with anxiety- and depression-like neurotrophic factor (BDNF), a key neurotrophin behaviors, in several brain regions. Interestingly, involved in neuronal growth and survival, and vagotomy prevented the anxiolytic and antide- decreased expression of the NMDA receptor pressant effects of the probiotic, as well as the subunit 2a (NR2a) in the cortex and hippocampus effects on central GABA receptor mRNA levels. compared to conventionally colonized controls. It seems likely then that parasympathetic inner- GF animals also have increased concentrations of vation is necessary for certain probiotics to influ- serotonin (5-HT) and its main metabolite ence microbiome–gut–brain communication 5-hydroxyindoleacetic acid (5-HIAA) in the (Cryan and Dinan 2012). Normalization of hippocampus, compared with conventionally peripheral pro-inflammatory cytokine and trypto- colonized control animals. Tryptophan, the phan concentrations, both of which have been precursor of serotonin, is also increased in the implicated in depression, has been achieved fol- plasma of GF animals, suggesting a humoral lowing probiotic treatment in the maternal sepa- route through serotonergic transmission in the ration model of depression (Scott et al. 2013). CNS can be influenced by the microbiota Finally, recent exciting studies have shown that (Clarke et al. 2013). Interestingly, although brain fatty acid concentrations of arachidonic peripheral tryptophan availability can be normal- acid and DHA can be manipulated by probiotic ized by the colonization of the GF animals agents. These fatty acids are known to play postweaning, the changes in 5-HT concentrations important roles in neurodevelopmental processes in the hippocampus in adulthood that are induced such as neurogenesis, neurotransmission, and by an absent microbiota in early life appear not to protection against oxidative stress and their con- be reversible. Of note is that there are sex differ- centrations in the brain influence anxiety, depres- ences in these effects and many of the neuro- sion, and learning and memory (Cryan and Dinan chemical, but not endocrine or immune, effects 2012). A similar antimicrobial regimen induced Microbiome–Gut–Brain Axis 433 M an increase in exploratory behavior and altered assess the impact on the brain of widespread BDNF levels in hippocampus and amygdala in antibiotics use in humans. mice (Collins et al. 2012). One of the most consistent findings from antibiotic-induced dysbiosis of the microbiome Potential Mechanisms by Which in preclinical studies is the increased visceral Microbiota Affect CNS Function hypersensitivity in response to colorectal disten- sion, an effect that can be reversed in some There are multiple pathways involved in the bidi- instances by subsequent administration of rectional communication between gut microbiota a probiotic (e.g., Lactobacillus paracasei) and the brain (Rhee et al. 2009). They include (Cryan and O’Mahony 2011). These effects are endocrine (cortisol), immune (cytokines), and not due to any off-target, systemic effects of these neural (vagus, enteric nervous system) pathways. medications as they failed to affect behavior in As indicated above, the brain can recruit these germfree conditions or affect gut inflammation mechanisms to influence the composition of the per se (Cryan and Dinan 2012). Interestingly, gut microbiota under conditions of stress. Con- neither vagotomy nor sympathectomy affected versely, this gut–brain axis scaffolding also per- the ability of the antimicrobials to induce their mits the microbiota to exert a marked influence effects on behavior (Collins et al. 2012). This on brain function, and there are a number of suggests that other, as yet unidentified mecha- potential mechanisms through which this might nisms (but see also below) are involved in occur (Mayer 2011). gut–brain communication in this model of dysbiosis-induced behavioral change. Altering Microbial Composition Taken together, these studies certainly con- The administration of putative probiotic bacteria firm the various aspects of brain function and or infectious agents can alter the composition of M behavior that fall under the remit of the the gut microbiota in multiple ways: increase microbiome and also highlight the utility of competition for dietary ingredients such as such strategies in parsing the role of microbiota growth substrates, produce fermentation products in gut–brain function, specifically brain function with inhibitory properties, produce growth sub- and physiology. However, caution clearly needs strates (e.g., polysaccharides or vitamins) benefi- to be exercised, for example, when generalizing cial for specific bacteria, produce bacteriocins probiotic effects from one bacterial strain to that target specific bacteria, increase competition another (Clarke et al. 2012a). Further efforts for binding sites, improve gut barrier function, also need to be directed toward identifying the alter intestinal properties for colonization and mechanism by which each probiotic strain persistence by reducing inflammation, and stim- induces its own particular effects. Moreover, ulate innate immune responses. All of these fac- clinical validation is urgently required to trans- tors can have a pronounced impact on gut–brain late these important and encouraging findings signaling (O’Hara and Shanahan 2006; Grenham into humans. In this vein, it is of interest to note et al. 2011). the recent report that a probiotic mixture can substantially alter brain activity in brain regions Immune Activation relevant to emotion. Probiotic administration has As indicated above, the microbiota is essential for also shown beneficial effects for anxiety- and normal immune function, and certain probiotic depression-like behaviors, albeit in healthy vol- agents can have direct effects on the immune unteers (Cryan and Dinan 2012). Probiotics can system (Hooper et al. 2012). Furthermore, the also manipulate the expression of opioid and innate and adaptive immune systems combine to cannabinoid receptors in the gut epithelium ensure homeostasis at the luminal surface of the (Clarke et al. 2012a). More work remains to be intestinal host–microbial interface, which is crit- done and one important implication is the need to ical for maintaining health and well-being M 434 Microbiome–Gut–Brain Axis

(O’Hara and Shanahan 2006). The immune sys- Microbial Metabolites tem is an integral component of the gut–brain The gut bacteria are key to a variety of metabolic axis and may be key to transducing the effects reactions, many of which are essential for host of bacteria to the CNS. The effects of the gut health (e.g., by production of bile acids, choline, microbiota and probiotics on the innate immune and SCFAs; see above) (Tremaroli and B€ackhed system can modulate circulating levels of pro- 2012). SCFAs, such as n-butyrate, acetate, and and anti-inflammatory cytokines that directly propionate, are known to be neuroactive affect brain function (Cryan and Dinan 2012). (Forsythe et al. 2010). Some bacteria and yeasts can also generate neurotransmitters and Vagus Nerve neuromodulators. For example, Lactobacillus The vagus nerve (cranial nerve X) is the major and Bifidobacterium produce GABA; nerve of the parasympathetic division of the auto- Escherichia, Bacillus, and Saccharomyces pro- nomic nervous system, another critical compo- duce noradrenalin; Candida, Streptococcus, nent of the gut–brain axis. This nerve has both Escherichia, and Enterococcus produce seroto- efferent and afferent projections and polices nin; Bacillus produce dopamine; Lactobacillus a number of organ functions, including bronchial produce acetylcholine (Lyte 2011). constriction, heart rate, and gut motility (Rhee et al. 2009; Mayer 2011). The activation of the Bacterial Coat Sugars vagus nerve can be markedly anti-inflammatory The outer exocellular polysaccharide coating of (e.g., protecting against microbial-induced sep- probiotic bacteria shields the bacteria from acid sis), an effect mediated by the nicotinic acetyl- and bile in the gut and protects the bacteria from choline receptor. As indicated above, vagotomy the host immune response (O’Hara and Shanahan studies have shown that many of the effects of the 2006). This coating has been shown to be respon- microbiota or potential probiotics on brain func- sible for many of their health-promoting effects tion are dependent on vagal activation (Cryan and (Cryan and Dinan 2012). This may imply that, in Dinan 2012). The precise mechanisms through some instances, nonviable bacterial components which vagal afferents become activated by gut might be used as microbial-based therapeutic microbiota are currently unclear. alternatives to probiotics. The cell wall compo- nents of microorganisms in the intestinal lumen Tryptophan Metabolism or attached to epithelial cells are thought to Tryptophan is an essential amino acid and encourage the release of transmitter molecules a precursor to many neuroactive agents, includ- from epithelial cells that in turn influence neural ing the neurotransmitter 5-HT. An increasing signaling or act directly on primary afferent number of studies implicate dysregulation of the axons (Rhee et al. 2009; Mayer 2011). often-overlooked but physiologically dominant kynurenine arm of tryptophan metabolic pathway (Ruddick et al. 2006). Alterations in this pathway Implications and Future Directions have been reported in many stress-related psychi- atric and gastrointestinal disorders (Scott The metagenomic revolution has resulted in et al. 2013). This initial rate-limiting step in the a greatly enhanced understanding of the compo- kynurenine metabolic cascade is catalyzed either sition and diversity of the gut microbiome and by the immunoresponsive indoleamine-2,3- opened up new avenues of investigation with dioxygenase or by the largely hepatic-based associated preventative, therapeutic, and diag- and glucocorticoid-stimulated tryptophan nostic opportunities. It is now apparent that the 2,3-dioxygenase (Ruddick et al. 2006). There is human gut is home to a core microbiome with some evidence that certain probiotic strains (e.g., a functional repertoire that is essential for health B. infantis) can alter concentrations of and which varies considerably at the extremes of kynurenine (Clarke et al. 2012a). life (Adlerberth and Wold 2009;Sekirov Microbiome–Gut–Brain Axis 435 M et al. 2010;O’Toole2012;Urselletal.2012). established (Cryan and Dinan 2012;Scott Future studies need to clarify how this core et al. 2013). The restoration of a normal gut microbiome can have its composition altered to microbiome after dysbiosis does appear to be elicit beneficial effects. The identification of clinically beneficial (Lemon et al. 2012), and enterotypes means it is likely that multiple stable there may be therapeutic openings early in life states are possible for the human microbiome, during where intervention might bestow health but the functional consequences for general benefits. Moreover, narrow- rather than broad- health and well-being or in terms of brain func- spectrum antibiotics might offer a more nuanced tion of these alternative compositions remain approach to the elimination of harmful commu- to be fully defined. Further integration of nity members. The use of microbial-based ther- these insights from metagenomic studies with apeutics to beneficially influence the CNS is now the preclinical strategies outlined above will a possibility although this is likely still some address some of these queries. GF animals, for way off until the interesting candidates that example, when colonized in a targeted manner have emerged are more rigorously evaluated. with either single strains or representative These options include the probiotic strains microbial compositions, can provide some that have already shown promise in stress- invaluable information. related brain–gut axis disorders (Clarke To date, we only have correlative studies et al. 2012a)aswellasthesomewhatmore linking the gut microbiota to disease states. Pro- controversial fecal transplantation strategies for spective studies are now warranted to establish the treatment-resistant Clostridium difficile causality and are essential to channel the diag- cases (Lemon et al. 2012). Therapeutic mining nostic potential of an altered gut microbiome. of the gut microbiome also represents a viable Preclinical data implicates the gut microbiome optionandisalreadyinprogress(O’Haraand as a key regulator of CNS function and strongly Shanahan 2006). M suggests studies in human populations should be expanded to include psychiatric cohorts. These studies will be the need to adequately powered Summary taking into account the inherent variation of the gut microbiome. Close attention should also be Tremendous advances in the last decade now paid to the long-term consequences of gut leave researchers poised to beneficially exploit microbiome dysbiosis (e.g., antibiotics) or the gut microbiome for health gains, diagnostic altered development patterns (e.g., caesarean innovations, and novel therapeutics. The section vs. vaginal delivery, breastfeeding metagenomic revolution has made it clear that vs. formula fed) early in life. This may have the microbiome is both amenable to quantitation particular relevance at key brain centers. The and is malleable. In tandem, a new field of neu- contributing factors to detrimental health out- roscientific investigation has prospered and comesinoldagearealsonowverymuchon brought an enlightened understanding of the the agenda and are topical in the light of an microbiome–gut–brain axis. This has broad and aging worldwide population (http://www.who. exciting implications in the areas of pain, cogni- int/topics/ageing/en/). Dietary inputs to gut tion, depression, and anxiety to name a few, and microbiome reshaping and the deleterious con- as the comprehension of the human relationship sequences of reduced microbial diversity are with its microbial self increases, so too will the particularly noteworthy (O’Toole 2012). amassed health benefits. In terms of therapeutic opportunities, the The Alimentary Pharmabiotic Centre is a microbiome can be regarded as a potentially research center funded by Science Foundation druggable target once the conditions which pro- Ireland (SFI), through the Irish Government’s mote the growth of desirable species at the National Development Plan (NDP). The authors expense of less sought-after members are fully and their work were supported by SFI (grant nos. M 436 Microbiome–Gut–Brain Axis

02/CE/B124 and 07/CE/B1368). GC was in Dinan TG, Cryan JF. Regulation of the stress response by receipt of a research grant from the American the gut microbiota: implications for psychoneuroendo- crinology. Psychoneuroendocrinology. 2012;37(9): Neurogastroenterology and Motility Society 1369–78. (ANMS). TD, also as part of the NDP, Forsythe P, Sudo N, Dinan T, Taylor VH, Bienenstock J. is supported by way of a Department of Agricul- Mood and gut feelings. Brain Behav Immun. ture Food and Marine and Health Research 2010;24(1):9–16. Foster JA, McVey Neufeld KA. Gut-brain axis: how the Board FHRI award to the ELDERMET project. microbiome influences anxiety and depression. Trends GC,JFC,andTDarealsofundedbytheIrish Neurosci. 2013;36(5):305–12. Health Research Board (HRB) Health Research Grenham S, Clarke G, Cryan JF, Dinan TG. Brain-gut- Awards (grant no HRA_POR/2011/23). The microbe communication in health and disease. Front Physiol. 2011;2:94. authors would like to thank Dr. Marcela Julio Hooper LV, Littman DR, Macpherson AJ. Interactions for the assistance in preparing the images used between the microbiota and the immune system. Sci- in this review (http://www.facebook.com/ ence. 2012;336(6086):1268–73. imagenesciencia) and Dr. Sue Grenham for Kennedy PJ, Clarke G, Quigley EM, Groeger JA, Dinan TG, Cryan JF. Gut memories: towards a cognitive helpful comments and discussions. neurobiology of irritable bowel syndrome. Neurosci Biobehav Rev. 2012;36(1):310–40. Lemon KP, Armitage GC, Relman DA, Fischbach MA. Cross-References Microbiota-targeted therapies: an ecological perspec- tive. Sci Transl Med. 2012;4(137):137rv135. Lepage P, Leclerc MC, Joossens M, Mondot S, Blottiere ▶ Animal Diseases, Applications of HM, Raes J, Ehrlich D, Dore J. A metagenomic insight Metagenomics into our gut’s microbiome. Gut. 2013;62(1):146–58. ▶ ELDERMET (Ireland) Lyte M. Probiotics function mechanistically as delivery ▶ vehicles for neuroactive compounds: microbial endo- Immunity, Innate: Definition and Examples crinology in the design and use of probiotics. ▶ Intestinal Microbiota and Aging Bioessays. 2011;33(8):574–81. ▶ Intestinal Microbiota, Alterations in Irritable Mayer EA. Gut feelings: the emerging biology of Bowel Syndrome gut-brain communication. Nat Rev Neurosci. ▶ 2011;12(8):453–66. Microbiome, Foregut Mulle JG, Sharp WG, Cubells JF. The gut microbiome: a new frontier in autism research. Curr Psychiatry Rep. 2013;15(2):337. References Nicholson JK, Holmes E, Kinross J, Burcelin R, Gibson G, Jia W, Pettersson S. Host-gut microbiota metabolic Adlerberth I, Wold AE. Establishment of the gut interactions. Science. 2012;336(6086):1262–7. microbiota in Western infants. Acta Paediatr. O’Hara AM, Shanahan F. The gut flora as a forgotten 2009;98(2):229–38. organ. EMBO Rep. 2006;7(7):688–93. Clarke G, Cryan JF, Dinan TG, Quigley EM. Review O’Mahony SM, Hyland NP, Dinan TG, Cryan article: probiotics for the treatment of irritable bowel JF. Maternal separation as a model of brain-gut syndrome–focus on lactic acid bacteria. Aliment axis dysfunction. Psychopharmacology (Berl). 2011; Pharmacol Ther. 2012a;35(4):403–13. 214(1):71–88. Clarke G, Grenham S, Scully P, Fitzgerald P, Moloney RD, O’Toole PW. Changes in the intestinal microbiota from Shanahan F, Dinan TG, Cryan JF. The microbiome-gut- adulthood through to old age. Clin Microbiol Infect. brain axis during early life regulates the hippocampal 2012;18 Suppl 4:44–6. serotonergic system in a sex-dependent manner. Mol Rhee SH, Pothoulakis C, Mayer EA. Principles and clinical Psychiatry. 2013;18(6):666–73. implications of the brain-gut-enteric microbiota axis. Collins SM, Surette M, Bercik P. The interplay between Nat Rev Gastroenterol Hepatol. 2009;6(5):306–14. the intestinal microbiota and the brain. Nat Rev Ruddick JP, Evans AK, Nutt DJ, Lightman SL, Rook GA, Microbiol. 2012;10(11):735–42. Lowry CA. Tryptophan metabolism in the central ner- Cryan JF, Dinan TG. Mind-altering microorganisms: the vous system: medical implications. Expert Rev Mol impact of the gut microbiota on brain and behaviour. Med. 2006;8(20):1–27. Nat Rev Neurosci. 2012;13(10):701–12. Schellekens H, Finger BC, Dinan TG, Cryan JF. Ghrelin Cryan JF, O’Mahony SM. The microbiome-gut-brain signalling and obesity: at the interface of stress, mood axis: from bowel to behavior. Neurogastroenterol and food reward. Pharmacol Ther. 2012;135(3): Motil. 2011;23(3):187–92. 316–26. Mobile Metagenome 437 M

Scott LV, Clarke G, Dinan TG. The brain-gut axis: a Introduction target for treating stress-related disorders. In: Halaris A, Leonard BE, editors. Inflammation in psychiatry. Basel: Switzerland; 2013. p. 28. The adult human gastrointestinal tract (GIT) is Sekirov I, Russell SL, Antunes LC, Finlay BB. Gut home to a dense population of microorganisms, microbiota in health and disease. Physiol Rev. reaching an estimated 1013–1014 individual pro- 2010;90(3):859–904. karyotic cells in the distal colon and dominated Tremaroli V, B€ackhed F. Functional interactions between the gut microbiota and host metabolism. Nature. by bacteria from ~150 to 800 species (Ley 2012;489(7415):242–9. et al. 2006; Qin et al. 2010). Collectively this Ursell LK, Metcalf JL, Parfrey LW, Knight R. Defining community of microbes is referred to as the gut the human microbiome. Nutr Rev. 2012;70 Suppl 1: microbiome and undertakes a wealth of functions S38–44. Weinstock GM. Genomic approaches to studying the beneficial to the human host. human microbiota. Nature. 2012;489(7415):250–6. However, just as humans play host to this diverse population of microbes, members of the gut microbial community also play host to their own array of “hitchhikers” in the form of mobile Mobile Metagenome genetic elements (MGEs). MGEs may be defined as discrete genetic units capable of moving between Brian V. Jones distinct molecules of DNA and/or host cells and Center for Biomedical and Health Science for this reason are often dubbed “mobile DNA.” Research, School of Pharmacy and Biomolecular Collectively, the total pool of MGEs associated Sciences, University of Brighton, Brighton, East with the gut microbiota is referred to as its mobile Sussex, UK metagenome (Jones and Marchesi 2007;Jones et al. 2010; Jones 2010; Ogilvie et al. 2012a), and there is growing interest in understanding how this M Synonyms flexible gene pool is involved in the development of the gut microbiota and its effects on human health. Gut microbiota; Lateral gene transfer; Mobile MGEs encode a wide range of functions microbiome; Phage advantageous to host bacteria, including those with a direct bearing on human health and sur- vival in the human gut (reviewed in Jones 2010; Definitions Ogilvie et al. 2012a). These encompass traits such as antibiotic resistance and virulence fac- Metagenome: The collective genomes of all tors, as well as genes conferring tolerance to members of a bacterial community. environmental factors, those facilitating nutrient Mobile metagenome: The total pool of acquisition, and mechanisms underpinning inter- mobile genetic elements associated with a bacte- actions of host and microbe. MGEs also facilitate rial community. the spread of these genes within a bacterial com- Mobile genetic element (MGE): A discrete munity and frequently acquire “new” genetic genetic unit capable of mediating its own transfer material from their bacterial hosts, which may between distinct DNA molecules, and/or between then be disseminated to other species as MGEs distinct host cells of the same or different species. move from cell to cell. This process is termed Plasmids, transposons, insertion sequences, horizontal gene transfer (HGT) and allows recip- conjugative transposons, integrons, and bacterio- ient cells to rapidly acquire new traits and genes phage are all examples of MGE. outside the normal process of inheritance (the Horizontal gene transfer: Transfer and acqui- vertical transmission of genes from “parent” to sition of genetic material between distinct cells or “offspring”). This facilitates the formation of species, outside of and in addition to the normal new functional pathways and dissemination of process of inheritance (vertical gene transfer). adaptive traits among members of a bacterial M 438 Mobile Metagenome community, and HGT is considered a key process which act as vehicles for these elements. Once in bacterial evolution and adaptation (Ochman transported into a new host cell, these MGEs can et al. 2000; Smalla et al. 2000). subsequently initiate their own transfer to other This naturally leads to questions regarding the DNA molecules in the cell, such as the chromo- activities encoded by the gut mobile metagenome, some or resident plasmids. In contrast, and how attributes of this flexible gene pool relate mobilizable plasmids lack the ability to initiate to the development of the gut microbiota, and the conjugal transfer independently, but can utilize impact of these microbes on human health (Jones the transfer machinery setup by conjugative plas- and Marchesi 2007; Jones et al. 2010;Jones2010; mids and CTn to move from cell to cell, and also Ogilvie et al. 2012a). Overall, we remain relatively act as vehicles for transposons and integrons. ignorant of the vast majority of MGEs associated Therefore, in terms of gene flow and HGT, self- with this community, but the adaptation and appli- transmissible elements are of particular impor- cation of new strategies to study the mobile tance and have been the focus of many studies. metagenome (including metagenomic approaches) Figure 1 illustrates the potential interactions of are now beginning to change this. Recent studies different MGEs within a host cell and how they employing such techniques have now started to may be transferred to new host cells. provide intriguing insights into the nature of the Far less is known regarding MGE diversity mobile metagenome, MGEs associated with the and abundance, and there are currently few stud- human gut microbiome, and the role of this flexible ies that have attempted to survey MGEs associ- gene pool in the development of the gut microbial ated with the human gut microbiome. The most community and its impact on human health. extensively studied fraction of the gut mobile metagenome are bacteriophage. Initial surveys of the gut-associated phage community estimated Characteristics, Diversity, and that there are around two to five times more viral Abundance of MGEs in the Gut Mobile genotypes than bacterial species in the human gut Metagenome microbiota (Breitbart et al. 2003). However, more recent surveys of phage populations in the human Plasmids, transposons, integrons, and bacterio- gut microbiome suggest the ratio of phage types phage are all examples of MGEs found in the to bacterial species may approach 1:1, with tem- gut mobile metagenome, and Table 1 summarizes perate phage dominating (Reyes et al. 2010). the main characteristics and general properties of There are currently no detailed surveys of other these MGEs. These elements can be further MGE types such as plasmids, transposons, and divided into two broad groups based on their integrons in the gut mobile metagenome, and ability to move between host cells: their abundance or diversity in the gut microbial 1. Self-transmissible MGEs are capable of medi- community remains unknown. Despite this, the ating their own autonomous transfer between diversity of these types of MGEs in the gut mobile host cells. This group includes conjugative metagenome is predicted to be high, and it is plasmids, bacteriophage, and conjugative likely that MGEs account for a significant frac- transposons (CTn). tion of the novel and uncharacterized “biological 2. Nonself-transmissible MGEs include transpo- dark matter” associated with the gut microbiome. sons (Tn), insertion sequences (IS), integrons, and mobilizable plasmids. These elements are unable to move between host cells indepen- Overview of Functions Encoded by the dently. However, Tn, IS, and integrons can Human Gut Mobile Metagenome mediate their own transfer between distinct molecules of DNA within a cell. Antibiotic Resistance Tn, integrons, and IS are spread between host The human gut is generally regarded to be a reser- cells when carried by self-transmissible MGEs, voir for antibiotic resistance genes (ABRGs). Mobile Metagenome 439 M

Mobile Metagenome, Table 1 Types and general properties of MGE present in the gut mobile metagenome Group MGE Characteristics Role in HGT Self-transmissible Conjugative • Circular DNA molecule encoding all • Often encode beneficial accessory plasmid genes necessary for autonomous functions transfer between host cells • Can act as vehicles for other MGE, • Non-integrative and replicates particularly transposons and integrons independently of host chromosome as • Interaction between IS/Tn and extra-chromosomal element plasmids can lead to acquisition of • Generally mosaic genetic structure “new” genes and accessory functions form host chromosome • Many plasmids capable of transfer to a broad range of bacterial host species • Facilitate cell to cell transfer of mobilisable plasmids Conjugative • Integrative element capable of • Integrative nature facilitates stability transposon autonomous excision from the bacterial of new genetic material in new host (CTn) chromosome species • Encodes all genes necessary for • Can act as vehicles for other MGE, autonomous transfer between host cells, particularly transposons and integrons and subsequent integration into host • Interaction between IS/Tn and chromosome plasmids can lead to acquisition of • No independent replication, and “new” genes and accessory functions copied along with host chromosome form host chromosome during normal cell growth • Facilitate cell to cell transfer of mobilisable plasmids Bacteriophage • Viruses that specifically infect • Bacteriophage may facilitate HGT by bacteria, and typically exhibit narrow either: host ranges limited to specific species or 1. Specialized transduction. Associated strains with lysogenic cycle. Aberrant excision M • Replication takes one of two broad of phage genome results in flanking forms (although details vary by phage regions of host chromosome also being host combination): excised and packaged into new capsids. 1. Lytic cycle. Phage replicate This then transferred to new host during autonomously, and independently of phage infection. Transfer is limited to chromosome, usually as a circular genes adjacent to phage insertion site in intermediate. Produces new viral chromosome particles that typically lyse host cells on 2. Generalized transduction. Accidental exit packaging of non-phage DNA into 2. Lysogenic cycle. Involves site specific capsids, which can subsequently integration of phage genomes into the host transfer this genetic material to new host chromosome and subsequent stable cells. Some reports also indicate that maintenance as a prophage element. plasmids may be transferred this way in During this time prophage are replicated some specialized examples, although during normal cell growth and inherited this is not generally known to be by daughter cells. Reversion to lytic cycle a widespread occurrence can subsequently occur, and often in 3. Act as vehicles for non-self- response to physiological status of host transmissible MGE cell Non-self- Mobilisable • As for conjugative plasmids, but • Utilize conjugation machinery set up transmissible plasmid lacking genes necessary to permit by conjugative plasmids or transposons autonomous conjugal transfer between • “Mobilized” to new host cells by host cells conjugative elements when these initiate conjugal transfer Insertion • Small integrative elements capable of • IS give rise to composite transposons sequence (IS) autonomous transfer between distinct (see below) DNA molecules • Two IS elements in close proximity • Consist of genes required for can result in recognition of repeat transposition (transposes) flanked by sequences in both IS by transposases (continued) M 440 Mobile Metagenome

Mobile Metagenome, Table 1 (continued) Group MGE Characteristics Role in HGT short inverted or direct repeat sequences • Results in excision of both IS and any which act as transposase recognition intervening chromosomal sequences as sites one unit • Do not encode accessory functions • May be transferred to new host cells • Replicated along with host DNA when inserted in self-transmissible molecule MGE Transposon • Integrative elements that may insert • Can readily transfer between different (Tn) randomly or in a site-specific fashion DNA molecules in host cells into host chromosome or other DNA • IS elements may excise and relocate molecule independently of Tn as a whole, which • Consist of two IS elements with can give rise to new Tn encoding traits intervening DNA region encoding from host genome accessory functions. This often derived • May be transferred to new host cells from genome of original host bacteria when inserted in self-transmissible • Capable of independent transfer MGE between distinct DNA molecules in the • Integrative nature facilitates stability same cell, but not independent transfer of new genetic material in new host between distinct cells species • Replicated along with host DNA molecule Integrons • Consist of an integrated, non-mobile • Gene cassettes may transfer between segment that controls integration, integrons located on the host excision and expression of transferable chromosome and other DNA molecules gene cassettes in a site-specific fashion in the same cell, including MGE such as • Gene cassettes consist solely of one or plasmids and phage more promotorless open reading frames, • Acquisition of gene cassettes by self- and an integrase recognition site. transmissible or mobilisable MGE Known to encode diverse accessory facilitates their transfer to new host cells functions • Integrative nature provides stability for • Multiple gene cassettes may be newly acquired gene cassettes, and integrated in one site to form linear expression facilitated by integron arrays termed integrons promoter • Replicated along with host DNA molecule • When excised gene cassettes form circular intermediates, but are not capable of independent replication or transfer between cells

This general view of the gut microbiome as an chloramphenicol, and trimethoprim, have been important repository of ABRGs has been strength- isolated from the human gut microbiota (Licht ened by recent function-driven metagenomic stud- and Wilcks 2005; Ogilvie et al. 2012a;Salyers ies, which highlighted not only the high level of et al. 2004). Such plasmids are also frequently potential novel resistance mechanisms in the gut mobile, and autonomous transfer between mem- microbiome but also their frequent association bers of the mammalian gut community, as well as with genes and sequences related to MGEs between commensal organisms and species con- (Sommer et al. 2009). sidered to be transient colonizers, has been dem- Plasmids encoding resistance to a wide range of onstrated both in vitro and in vivo (Licht and antibiotics, including b-lactams, nitroimidazoles, Wilcks 2005; Ogilvie et al. 2012a;Salyers sulfonamides, aminoglycosides, tetracyclines, et al. 2004). Similarly, the dissemination of Mobile Metagenome 441 M

M

Mobile Metagenome, Fig. 1 Overview of interaction including other MGEs, and facilitate the movement of between MGE within host cells and transfer to new host genetic material between the different DNA molecules cells. Self-transmissible MGEs constitute the main vehi- in a cell. Nonself-transmissible MGEs are transported to cles for cell-cell spread of MGE and associated HGT. new host cells when carried or mobilized by self- Nonself-transmissible MGEs may move independently transmissible elements. See Table 1 for details on charac- between distinct molecules of DNA in the host cell, teristics of different MGEs and their role in HGT erythromycin and tetracycline resistance genes MGEs also appear to facilitate maintenance of between Bacteroides species, which are important ABRGs in a bacterial community, with many and numerically prevalent members of the gut MGEs apparently stable in long-term hosts even microbiome, has been largely attributed to hori- in the absence of direct selective pressure for the zontal transfer of the relevant genes by conjugative ABRGs they encode. This is exemplified by transposons (Salyers et al. 2004). A high level of the presence of MGEs encoding resistance to ABRG transfer was also recently observed tetracycline in gut isolates of Bacteroides between the gut microbiome and bacteria associ- sp. collected prior to the widespread clinical use ated with food or farm animals, highlighting the of these drugs (Salyers et al. 2004). Such stability potential for acquisition of these genes from die- may be the result of selection for other traits tary sources (Smilie et al. 2011). encoded by the MGEs, the presence of active M 442 Mobile Metagenome stabilization mechanisms such as toxin-antitoxin In particular, mechanisms that contribute to addiction modules, or, in the case of integrative colonization of and persistence within the elements, incorporation into the host chromosome. human GIT, as well as host-microbe signaling and immune modulation, may be put to use by Virulence both beneficial species and pathogens, albeit for Many pathogenic species associated with the different purposes and with very different human gut have been found to harbor MGEs impacts on host health. This raises important that encode virulence determinants or provide questions as to what extent, if any, MGEs resident evidence for a role of MGEs and HGT in the in the gut mobile metagenome are a reservoir of development of a pathogenic lifestyle (reviewed potential virulence attributes that may be in Ogilvie et al. 2012a). Plasmids have been well accessed by emerging pathogens and/or tran- studied in this regard and provide prime examples siently colonizing species. Recent studies of of MGE-encoded virulence attributes. The pro- gene exchange in the human microbiome as duction of toxins and hemolysins, cell invasion a whole suggest virulence genes are certainly and survival within macrophages, attachment to transferred between related species occupying host tissues, immune system evasion, and type III a particular niche and may contribute to the emer- secretion systems and associated effectors have gence of new pathogenic strains (Smilie all been found encoded by plasmids harbored in et al. 2011). These observations already indicate gut-associated pathogens. Bacteriophage have that analysis of the mobile metagenome from this also been frequently implicated in the horizontal perspective will yield clinically valuable insights transfer of virulence factors and the development into microbial pathogenesis (Smilie et al. 2011). of pathogenic lifestyles in host species. An excellent example of both plasmids and Adaptation, Survival, and Persistence in the bacteriophage contributing to pathogenesis is Gut Environment E. coli O157:H7, in which several of the major Characterization of plasmids and other MGEs virulence factors have been acquired by HGT from cultivatable species found in the human (Croxen and Finlay 2010). These include the GIT has revealed that these often encode a wide Shiga toxin which has been acquired from the range of functions relevant to survival in this closely related Shigella sp. (also highly virulent environment and interaction with the human gut pathogens) through transduction by bacterio- host (Jones and Marchesi 2007; Jones 2010; phage capable of infecting both species. Plasmids Ogilvie et al. 2012a; Claesson et al. 2006). encoding hemolysins, toxins, and factors mediat- Among these are genes involved in the utilization ing attachment to host cells are also common in of carbohydrates and other nutrients, bacteriocin isolates of E. coli O157:H7 (Croxen and production, adhesion to host cells, and resistance Finlay 2010). to bile acids. In a number of instances, plasmids The presence of MGEs encoding virulence fac- encoding multiple functions that increase fitness tors also has important implications for the poten- in the GIT have been isolated and characterized tial development of deleterious host-microbe (reviewed in Ogilvie et al. 2012a). interactions in the gut microbiome and for the A particularly interesting example of MGE emergence of new pathogenic species. Although encoding such functions is the large the presence of such traits in the gut microbiomes megaplasmids (~120–490 kb) that appear to be of healthy individuals may at first seem at odds widespread in Lactobacillus (Claesson with the view of this community as generally ben- et al. 2006). One of the best characterized exam- eficial and benign, many members retain the abil- ples comes from studies of the probiotic organism ity to act as opportunistic pathogens, and a wide and gut commensal L. salivarius UCC118, which variety of mechanisms for host-microbe interac- harbors three plasmids, including a megaplasmid tion are of utility to both commensals and patho- encoding genes related to bile tolerance, redox gens alike (Ogilvie et al. 2012a). balance, cell wall biosynthesis, and bacteriocin Mobile Metagenome 443 M production (Claesson et al. 2006). Collectively, Wilcks 2005; Jones and Marchesi 2007). Despite these accessory functions are proposed to endow evidence for a role in attachment to abiotic sur- L. salivarius UCC118 with a greater degree of faces, a general role for conjugative plasmids in genetic and biochemical flexibility, ultimately gut colonization has yet to be proven, although enhancing its fitness and ability to survive in the plasmid-mediated mechanisms of attachment to gut environment. In particular, the proposed flex- host tissues have been described (reviewed in ibility offered by the cache of accessory functions Ogilvie et al. 2012a). encoded by the UCC118 megaplasmid should permit this organism to adapt to transient ecolog- ical niches that arise in this community; tolerate An Arbitrary Assemblage or fluctuations in diet, community structure, and a Community-Specific Mobile Gene properties of the gut environment; as well as Pool? overcome intrinsic barriers to colonization of the GIT (Claesson et al. 2006). Activities of MGEs are generally studied in terms The “probiotic effect” of this organism has of their impact on fitness of the bacterial host. also been attributed to megaplasmid-encoded However, bacteria belonging to communities functions: the ability of UCC118 to protect mice associated with higher host organisms, such as against infection by the foodborne pathogen the gut microbiota, are also subject to selection Listeria monocytogenes is associated with the from the level of the host. The human gut megaplasmid-encoded bacteriocin system microbiota is squeezed between selective pres- (Claesson et al. 2006; Corr et al. 2007). This sures imposed by the human host, due to the demonstrates the potential for MGE-encoded effects of activities of these microbes on host functions to have a positive influence on human fitness (top down), and those generated by com- health, while the commonality of megaplasmids petition for resources between individual species M among numerous isolates of lactobacilli tested in this community (bottom up) (Ley et al. 2006). illustrates the potential for such traits to be mobi- This has resulted in a microbial community with lized within a community (Claesson et al. 2006). many emergent properties that directly benefit In conjunction with the potential pharmaceutical the host, and the gut microbiome we see today applications of bacteriocins as alternatives to is generally accepted to be the outcome a long antibiotics, and the use of probiotics to manipu- coevolutionary relationship between human and late the gut microbiome for therapeutic purposes, microbe, resulting in a microbial community the UCC118 megaplasmid illustrates that func- performing functions integral to our health and tions of significant biotechnological potential are well-being. encoded by the gut mobile metagenome. It has been proposed that MGEs comprising In the case of attachment to host tissues, it has the gut mobile metagenome also reflect the been proposed that this may be a feature of coevolution of host and microbe in the human conjugative plasmids and transposons in general GIT and effectively constitute an additional res- that facilitates gut colonization (Licht and Wilcks ervoir of human-associated microbial genetic 2005). Pili and associated conjugation machin- information (Jones 2010). If so, then functions ery, assembled by these MGEs in order to transfer encoded by the mobile metagenome will be between cells, may also act as adhesins and shaped by key stresses, host-microbe, and directly mediate attachment of host bacteria to microbe-microbe interactions, which are impor- human cells and tissues. In this scenario, species tant for survival and persistence in the human gut. harboring conjugative elements may be better Table 2 summarizes evidence for coevolution of able to colonize the GIT which not only would gut-associated MGEs with the human host and serve to enhance the fitness of the host organism the role of the gut mobile metagenome in com- but may also serve to enrich and stabilize such munity function, development, and host-microbe plasmids within the gut microbiome (Licht and interaction. M 444 Mobile Metagenome

Mobile Metagenome, Table 2 Evidence for coevolution of gut-associated MGE with the human host, and the role of the gut mobile metagenome in community function, development, and host-microbe interaction (Modified from Ogilvie et al. 2012a) MGE type/process Summary Study(s) Conjugative • CTn-1549 like family of elements designated CTnRINT Kurokawa et al., DNA Res. transposons observed to be enriched in the gut microbiomes of Japanese 2007;14:169–81 and American individuals examined. In addition, a high level of recombinases and integrases were also observed in gut microbiomes Plasmids • Plasmids or plasmid families enriched and potentially Jones, Gut Microbes. 2010;1:41–31 unique to the human gut microbiome identified, with Jones et al., BMC Genomics. homologous sequences detected in gut microbiomes of 2010;11:46 geographically isolated hosts (America, Europe, Japan). Enrichment of some functions encoded by plasmids also observed. In particular plasmid pTRACA22 was noted as widely distributed and potentially unique to the human gut microbiome, with ReIBE type toxin-antitoxin addiction modules enriched in terms of relative abundance in human gut microbiomes compared to other datasets examined • Plasmids infecting gut associated bacteria exhibit the same Zaneveld et al., Nucleic Acids Res. patterns of habitat-associated gene convergence observed for 2010;38:3869–79 bacterial chromosomes • Lactobacilli commonly harbor large megaplasmids Claesson et al., Proc. Natl. Acad. encoding genes proposed to be involved in adaptation to the Sci. U. S. A. 2006;103:6718–28 gut environment. Genes associated with the probiotic or Corr et al., Proc. Natl. Acad. Sci. protective effect of some species also appear to be encoded by U. S. A. 2007;104:7617–21 megaplasmids Li et al., Appl. Environ. Microbiol. 2007;189:6128–39 • Megaplasmids harbored by Enterococcus faecalis shown to Zhang et al., Environ. Microbiol. facilitate biochemical flexibility and utilization of diverse 2011;13:518–528 carbon sources, proposed to enhance fitness in the gut and other environments • In silico analysis of 980 plasmids of varying sizes suggested Slater et al., FEMS Microbiol. a trend towards smaller plasmids (<100 kb) among those Ecol. 2008;66:3–13 associated with animals, compared to non-animal habitats (terrestrial and aquatic) Bacteriophage/ • Isolation and characterization of bacteriophage infecting Ebdon et al., Water Res. virus like Bacteroides sp. GB-124, indicated these bacteriophage were 2007;41:3683–90 particles specific to the human gut, and carriage in the general Ogilvie et al., PLoS One. 2012, in population is high. Subsequent comparative genomic and press metagenomic analyses confirmed gut specific nature and indicated existence of distinct subset of gut associated bacteriophage • A large inter-individual variation in gut virome composition Reyes et al., Nature. has been documented, but viromes found to be relative stable 2010;466:334–8 over time within an individual. Functions associated with a wide range of processes in anaerobic gut bacteria found to also be encoded by gut viromes. A dominance of temperate phages observed and a general lack of predator-prey dynamics in host-virus interactions suggested, which is in contrast to other microbial ecosystems • Host associated bacteriophage communities, including those Caporaso et al., PLoS One. 2011;6: found in the human gut mobile metagenome are distinct from e16900 non-host associated bacteriophage communities (continued) Mobile Metagenome 445 M

Mobile Metagenome, Table 2 (continued) MGE type/process Summary Study(s) • Distinct increase in concentrations of virus-like practices, Lepage et al., Gut. 2008;57:424–5 predominantly bacteriophage, observed in patients with Crohn’s disease compared to healthy controls. Bacteriophage hypothesised to play a role in the dysbiosis of the gut microbiota observed in Crohn’s disease by destabilizing the community General HGT • Gain of functional capacity in the Japanese gut microbiome Hehemann et al., Nature. attributed to plasmid mediated HGT of genes conferring 2010;464:908–12 ability to utilize seaweed glycans, from marine Bacteroides spp. to gut commensal Bacteroides spp. • Habitat associated gene convergence of glycoside Lozupone et al., Proc. Natl. Acad. hydrolases and glycosyltransferases in gut associated bacteria Sci. U. S. A. 2008;105:15076–81 and archaea largely generated through HGT • Evidence that the human microbiome is connected by a large Smillie et al., Nature. 2011. network of gene exchange with rates of HGT up to 25-fold doi:10.1038/nature10571 higher between human associated bacteria than non-human associated isolates

In keeping with this theory are the observa- by one of these plasmids were subsequently tions that some MGEs appear to be enriched or found to be more prevalent in gut metagenomic potentially unique to this community and encode datasets than in non-gut metagenomes (Fig. 2), functions that are prevalent within the human and aside from their well-known role in plasmid gut microbiota as a whole (Table 2). Particular stabilization, TA systems have been implicated M examples of such MGEs come from studies of in a wide range of cellular processes relevant to plasmids resident in this ecosystem (Jones survival in the gut. These include tolerance of et al. 2010). Sequences with high homology environmental stress, biofilm formation, and, to several of these plasmids could be detected potentially, interaction with the human host in the gut microbiomes of geographically (Jones 2010). Although the role of these TA isolated individuals distributed across the globe systems, if any, in the gut microbiome is yet to (America, Europe, Japan) (Jones et al. 2010). In be determined, these observations confirm that contrast, sequences with homology to these plas- MGEscomprisingthemobilemetagenomemay mids were not detected in the environmental encode functions prevalent in the community as metagenomic datasets examined. This indicates a whole (Jones et al. 2010; Jones 2010). that certain plasmids or plasmid families exhibit Similar observations have also been made for a distinct association with the human gut other MGEs, including CTn and bacteriophage. microbiota and suggests a long-term relation- In the case of the former, a Tn1549-like element ship with human gut bacteria (Jones et al. 2010; designated CTnRINT (for conjugative transpo- Jones 2010). son rich in the intestine) has been observed to Analysis of the functions encoded by these be highly prevalent in the Japanese gut plasmids further supported this hypothesis and microbiome. CTnRINT includes accessory revealed that several of these functions were regions that differ from other Tn1549-like ele- more prevalent in the human gut microbiome, ments previously characterized in gut-associated compared with other microbial ecosystems pathogens such as Clostridium difficile and examined (Fig. 2)(Jonesetal.2010;Jones Enterococcus faecalis (Kurowkawa et al. 2006). 2010). In particular, homologues of a novel It has been proposed that the accessory regions of RelBE like toxin-antitoxin (TA) system encoded CTnRINT are advantageous to members of the M 446 Mobile Metagenome

Mobile Metagenome, Fig. 2 Analysis of plasmid- plasmid replication; purple, plasmid stabilization (toxin- encoded functions in the human gut microbiome (From antitoxin addiction module); blue, unknown function. (b) Jones et al. 2010). Sequences with high homology to Relative abundance of plasmid-encoded functions in plasmid pTRACA22 were found to be well represented human gut and nonhuman metagenomic datasets. within human gut metagenomic datasets, but absent from Sequences homologous to open reading frames encoded environmental datasets examined. Several functions by pTRACA22 were identified in each dataset and used to encoded by pTRACA22 were also found to be enriched calculate relative abundance as hits/Mb DNA. Symbols in human gut metagenomes compared with other datasets above bars indicate significant differences between analyzed. (a) Physical map of pTRACA22. Block arrows human gut metagenomes and other datasets corresponding indicate open reading frames, and colors of arrows indi- to color of symbol. P ¼ 0.01 or lower cate putative function: yellow, plasmid mobilization; red, gut microbiome and facilitate survival in this gut microbiome that remains largely unexplored environment, but at present this hypothesis (Ogilvie et al. 2012b;Fig.3a). In addition, a dis- remains untested. tinct divergence between bacteriophage sequences There is also extensive evidence for the exis- affiliated with the human gut microbiome and tence of gut-specific bacteriophage, and presence those affiliated with environmental habitats was of distinct phage communities associated with the evident,relative to FB124-14 and FB40-8 genome gut microbiome, and other host-associated micro- composition (Fig. 3a). bial ecosystems (Reyes et al. 2010;Caporaso Other studies also point to a distinct subset of et al. 2011; Ebdon et al. 2007;Ogilvie bacteriophage associated with the human gut, et al. 2012b). Bacteriophage infecting the human based on recent large-scale comparative analyses fecal indicator Bacteroides fragilis strain GB124, of viral metagenomes from various habitats a prevalent human gut commensal, have previ- (Caporaso et al. 2011). In these investigations, ously been isolated from human feces, but found bacteriophage populations derived from host- to be absent from fecal samples derived from associated microbial communities, such as the a wide range of common domestic and wild ani- human gut microbiome, were found to exhibit mals, and were not present in the general environ- distinct differences to those from nonhost- ment (Ebdon et al. 2007). Further analysis of one associated “free-living” environmental ecosys- such bacteriophage, designated FB124-14, and tems (Fig. 3b; Caporaso et al. 2011). Analysis of the comparison of its complete genome sequence the gut viral metagenomes has also pointed to the with other bacteriophage genomes and gut viral long-term stability of human gut-associated bac- metagenomic fragments not only reinforced its teriophage populations (at least in the distal gut-specific nature but also suggested that along colon), with a potential dominance of temperate with the closely related FB40-8, this phage phage and a lack of predator-prey dynamics char- occupies a distinct ecological landscape in the acteristic of phage communities associated with Mobile Metagenome 447 M

Mobile Metagenome, Fig. 3 Evidence for distinct with this community and primarily encompasses bacteri- host-associated bacteriophage communities. (a) From ophage infecting genera of environmental origin. Virome, Ogilvie et al. (2012b). Correlations of differences in the all large fragments (n ¼ 188, >10 Kb) assembled from frequency of each of the 256 possible tetranucleotide human gut viral metagenomic libraries (originally gener- combinations in the genomes of gut-specific bacterio- ated by Reyes et al. 2010). Figures in brackets provide the phage fB124-14 and fB40-8, with 540 complete bacte- Pearson correlation coefficient for each sequence cate- riophage genomes and 188 human gut viral metagenome gory. (b) From Caporaso et al. (2011). Comparative anal- fragments. Sequences were assigned to one of the three ysis of 130 bacteriophage metagenomes from distinct categories based on their relation to the human gut habitats using Shotgun UniFrac. The main figure shows microbiome: Gut, comprises bacteriophage infecting bac- a principal coordinates plot of weighted Shotgun UniFrac terial genera commonly forming part of the normal human distances between distinct bacteriophage metagenomes, gut microbiota. Gut associated, comprises bacteriophage with each point representing a single metagenome colored M genomes infecting bacterial genera whose member spe- according to host community type (as detailed in the cies are associated with the gut but not generally consid- legend). Inset shows the same plot with data points col- ered to be members of the normal gut microbiota (such as ored according to data source demonstrating no clustering primary invasive gut pathogens), or where member spe- based on source of datasets (i.e., no clustering according to cies are more commonly associated with environmental research group/sequencing center generating a particular habitats. Non-gut, contains bacteriophage infecting bac- dataset). For details of the Shotgun UniFrac approach, see terial genera with member species not considered to be Caporaso et al. (2011) (Figure 3b used with author’s part of the human gut microbiota or typically associated permission)

“free-living” environmental microbial communi- with the gut microbiome will be far less exclusive ties (Reyes et al. 2010). This is in keeping with than for many of their bacterial hosts (Jones the observed temporal stability of the gut 2010). Despite this, there is now accumulating microbiome in healthy adults, and it has been evidence that at least some MGEs in the gut suggested that shifts in distal gut phage commu- mobile metagenome are distinctly associated nities may also be related to the onset or progres- with this important ecosystem and encode func- sion of intestinal disorders (Reyes et al 2010; tions prevalent in this community, supporting the Lepage et al. 2008). concept of the mobile metagenome as an addi- However, with the exception of bacterio- tional human-associated pool of genetic material. phage, which usually exhibit host ranges restricted to specific species or strains of bacteria, the promiscuous nature of most MGEs and their Role of the Mobile Metagenome in propensity to move between diverse bacterial Community Function species makes the boundaries of the gut mobile metagenome far less easy to define than for the The human gut microbiome is believed to be core gut microbiome. For most MGEs, affiliation functionally stable over time in the healthy M 448 Mobile Metagenome adult, and this is at least in part ascribed to func- In addition, studies of the Japanese gut tional redundancy in this community, where microbiome have demonstrated how MGEs can many member species are capable of undertaking facilitate the acquisition of new abilities by the a particular function, albeit with variation in gut microbiota, which benefit both host and capability and specific activity. It is proposed microbe (Hehemann et al. 2010). Hehemann that this broad distribution of key activities and coworkers recently demonstrated the acqui- among many members confers stability by reduc- sition of porphyranse and agarase degrading abil- ing the chances of losing a particular trait from ity by the gut microbiomes of Japanese the community, which would necessitate loss of individuals. This was linked to the horizontal all species contributing to a given functional out- transfer of these genes from marine bacteria nat- put (Ley et al. 2006). urally colonizing dietary seaweeds that are typi- Although this functional redundancy may be cally consumed raw, to members of the Japanese achieved through recruitment of member species gut microbiota (Hehemann et al. 2010). Acquisi- with overlapping metabolic and functional pro- tion of these genes by gut commensals should files, HGT in the gut microbiota has been impli- permit the Japanese gut microbiota to utilize cated as an important factor contributing to the this dietary energy source, unlocking additional development of a functionally stable community calories from the diet to the benefit of both host through this mechanism (Ley et al. 2006; and microbe (Hehemann et al. 2010) (Table 2). In Lozupone et al. 2008; Jones and Marchesi light of the potential for functional upgrades to 2007). In this case, MGEs are thought to aid this community that may be accessed through the the dissemination of genes encoding core activ- mobile metagenome, it has also been proposed ities to a wide range of species in the gut that a major role of this mobile gene pool may be microbiome, guarding against loss of key activ- as a conduit to the vast reservoir of genetic infor- ities, and ensuring continuity of important com- mation extant in the wider prokaryotic world munity outputs. Evidence for HGT generated (Jones 2010). redundancy of key activities in this community In an ecosystem such as the human gut where comes from studies of genes involved in carbo- a large population of closely related microbes are hydrate utilization, which underlies the salvage in close spatial proximity, a high level of HGT of energy from the host diet by gut microbes; this would be expected. This is supported by recent is a primary function of this community that studies providing evidence for an extensive net- directly benefits the human host. It has been work of gene exchange in the human microbiome estimated that up to 10 % of our daily calories as a whole (Smilie et al. 2011). In this network, are derived from microbial fermentation of plant the human gut is indicated as possessing one of polysaccharides that we consume as a normal the highest rates of gene transfer of all body sites part of our diet, but are unable to digest ourselves analyzed (Smilie et al. 2011). MGEs and the (McNiel 1984). mobile metagenome in general are likely to The ability of gut microbes to ferment such form the foundations of this genetic interchange. carbohydrates and release energy to the host in In the context of host-microbe coevolution, this the form of short-chain fatty acids is distributed high level of gene flow should provide gut bacte- among a diverse array of species spanning the ria with access to an immense genetic resource, main bacterial divisions of the human gut facilitating adaptation to the gut environment, microbiota. While some of this redundancy and and the acquisition and dissemination of benefi- the origins of the genes involved are the result of cial traits throughout the community to the early events in the development of the gut advantage of both host and microbe (Jones microbiota, recent studies have also highlighted 2010). In contrast there is also scope for poten- the role of HGT in the convergence and expan- tially deleterious traits to be acquired and dissem- sion of the required gene sets within the gut inated in this way, as has already been described microbiome (Lozupone et al. 2008). for antibiotic resistance and virulence genes Mobile Metagenome 449 M mobilized in the gut microbiome by the this microbiome and with bacterial ecosystems in extensive network of gene exchange (Smilie general (Ogilvie et al. 2012a; Jones 2010). et al. 2011). A greater understanding of the contribution of the mobile metagenome to evolution and func- tion of the human gut microbiome will be impor- Summary tant in the development of strategies to manipulate this community for the benefit of The study of the mobile metagenome and the human health, as well as harnessing its full bio- theories surrounding this concept are still in technological potential. their infancy, but there is now growing evidence that at least some aspects of this mobile gene pool reflect the coevolution of host and microbe in this community. As such, a greater understanding of References gut-associated MGE will provide important Breitbart M, Hewson I, Felts B, et al. Metagenomic ana- insights into the development of the gut microbial lyses of an uncultured viral community from human ecosystem and its interaction with the human feces. J Bacteriol. 2003;185:6220–3. host. Furthermore, as our understanding of the Caporaso JG, Knight R, Kelley ST. Host-associated and human microbiome in general grows, we will free-living phage communities differ profoundly in phylogenetic composition. PLoS ONE. 2011;6: increasingly move toward strategies to manipu- e16900. late this community for therapeutic or prophylac- Claesson MJ, Li Y, Leahy S, et al. Multireplicon genome tic purposes. The mobile metagenome offers architecture of Lactobacillus salivarius. Proc Natl significant opportunities for translational Acad Sci U S A. 2006;103:6718–23. Corr SC, Li Y, Riedel CU, et al. Bacteriocin production as research and “bioprospecting” in this regard, pro- a mechanism for the antiinfective activity of Lactoba- viding the raw material for novel genetic tools as cillus salivarius UCC118. Proc Natl Acad Sci U S A. M well as a library of functions of potential phar- 2007;104:7617–21. maceutical or biotechnological potential. Croxen MA, Finlay BB. Molecular mechanisms of Escherichia coli pathogenicity. Nat Rev Microbiol. Particular examples include bacteriocin pro- 2010;8:26–38. duction, which may constitute a viable alternative Ebdon J, Muniesa M, Taylor H. The application of to antibiotics for some diseases with less collat- a recently isolated strain of Bacteroides (GB-124) to eral damage to beneficial microbes, and there is identify human sources of faecal pollution in a tem- perate river catchment. Water Res. 2007;41:3683–90. also considerable interest in bacteriophage from Hehemann J-H, Correc G, Barbeyon T, et al. Transfer of this perspective. The observation that some carbohydrate-active enzymes from marine bacteria to MGEs in this community encode functions Japanese gut microbiota. Nature. 2010;464:908–12. facilitating survival in this environment, is of Jones BV. The human gut mobile metagenome: a meta- zoan perspective. Gut Microbes. 2010;1(6):417–33. relevance to a wide range of potential biotechno- Jones BV, Marchesi JR. Accessing the mobile logical applications, and the development of metagenome of the human gut microbiota. Mol novel probiotics and bacterial-based drug deliv- Biosyst. 2007;3:749–58. ery platforms would especially benefit from Jones BV, Sun F, Marchesi JR. Comparative metagenomic analysis of plasmid encoded functions access to a library of such traits. In addition, in the human gut microbiome. BMC Genomics. a greater understanding of the forces that govern 2010;11:46. gene flow in this community will also be impor- Kurokawa K, Itoh T, Kuwahara T, Oshima K, Toh H, tant in the development of effective methods to Toyoda A, et al. Comparative metagenomics revealed commonly enriched gene sets in human gut limit the appearance and spread of undesirable microbiomes. DNA Res. 2007;14:169–81. traits, such as antibiotic resistance genes and vir- Lepage P, Colombet J, Marteau P, et al. Dysbiosis in ulence factors (Ogilvie et al. 2012a). inflammatory bowel disease: a role for bacterio- Despite this, we remain relatively ignorant of phages? Gut. 2008;57:424–5. Ley RE, Peterson DA, Gordon JI. Ecological and evolu- the true diversity, abundance, and functions tionary forces shaping microbial diversity in the encoded by MGEs associated with the gut human intestine. Cell. 2006;124:837–48. M 450 MRSA/MSSA, Antibiotic Resistance

Licht TN, Wilcks A. Conjugative gene transfer in the The increasing use of antibiotics has resulted in gastrointestinal environment. Adv Appl Microbiol. the acquisition and spread of resistance markers 2005;58:77–95. Lozupone CA, Hamady M, Cantral BL, et al. The conver- among S. aureus strains. Antibiotic resistance can gence of carbohydrate active gene repertoires in be conferred by point mutations in specific genes, human gut microbes. Proc Natl Acad Sci U S A. or resistance genes can be carried on conjugative 2008;105:15076–81. plasmids or bacteriophages and transferred McNiel NI. The contribution of the large intestine to energy supplies in man. Am J Clin Nutr. between organisms or even between species. 1984;39:338–42. Ochman H, Lawrence JG, Groisman EA. Lateral gene transfer and the nature of bacterial innovation. Nature. Introduction 2000;405:299–304. Ogilvie LA, Firouzmand S, Jones BV. Evolutionary, eco- logical and biotechnological perspectives on plasmids The objective of this review is to give a concise resident in the human gut mobile metagenome. Bioeng description of Staphylococcus aureus, focusing Bugs. 2012a;3(1):1–19. on the genetic mechanisms of antibiotic resis- Ogilvie LA, Caplin J, Dedi C, et al. Comparative (meta) genomic analysis and ecological profiling of human tance relevant to this bacterium. S. aureus is gut-specific bacteriophage B124-14. PLoS ONE. a ubiquitous organism that causes infections in 2012b;7:e35053. both adults and children. More than 95 % of Qin J, Li R, Raes J, et al. A human gut microbial gene infections involve the skin and soft tissues; how- catalogue established by metagenomic sequencing. Nature. 2010;464:59–65. ever, invasive infections such as osteomyelitis, Reyes A, Haynes M, Hanson N, et al. Viruses in the faecal septic arthritis, pneumonia, myositis, microbiota of monozygotic twins and their mothers. pyomyositis, and severe sepsis have been associ- Nature. 2010;466:334–8. ated with significant morbidity and mortality Salyers AA, Gupta A, Wang YP. Human intestinal bacte- ria as reservoirs for antibiotic resistance genes. Trends (Kaplan et al. 2009). First described in 1882 by Microbiol. 2004;12:412–6. Ogston, S. aureus was associated with abscess Smalla K, Osburne AM, Wellington EMH. Isolation and formation and purulent discharge (Ogston characterisation of plasmids from bacteria. In: CM 1882). It belongs to the Micrococcaceae family Thomas (ed) The horizontal gene pool, bacterial plas- mids and gene spread. Amsterdam: Harwood Aca- and is described as a Gram-positive coccus that demic Publishers; 2000. p. 207–48. forms irregular clusters, as observed under Smilie CD, Smith MB, Friedman J, et al. Ecology drives a microscope. S. aureus is a coagulase-positive a global network of gene exchange connecting the organism, whereas coagulase-negative staphylo- human microbiome. Nature. 2011. doi:10.1038/ nature10571. cocci (CoNS), such as Staphylococcus Sommer MOA, Dantas G, Church GM. Functional char- epidermidis, are abundant commensals of the acterisation of the antibiotic resistance reservoir in the human microflora that can also cause infections. human microflora. Science. 2009;325:1128–31. S. aureus has proven adaptable to challenges in its environment as evident by the response to the development and use of antibiotics. Penicillin-resistant S. aureus isolates were MRSA/MSSA, Antibiotic Resistance described in the 1940s, shortly after the introduc- tion of penicillin. Following the introduction of Kristina G. Hulten and J. Chase McNeil methicillin in the early 1960s to treat penicillin- Department of Pediatrics, Baylor College of resistant S. aureus, methicillin resistance was Medicine, Houston, TX, USA reported in hospitals in Denmark and the United Kingdom. For decades, methicillin-resistant S. aureus (MRSA) isolates were associated with Definition infections acquired in the hospital setting. The hospital-acquired strains had specific genomic Staphylococcus aureus causes infections in characteristics that included a cluster of genes humans ranging from mild to severe disease. referred to as the staphylococcal cassette MRSA/MSSA, Antibiotic Resistance 451 M

MRSA/MSSA, Antibiotic Resistance, Table 1 Antibiotic resistance mechanisms in Staphylococcus aureus Common Chromosome/ resistance plasmid Antibiotic class Drug target/mechanism genes location Mechanism of resistance Penicillin, other Penicillin-binding proteins blaZ Plasmid Beta-lactamase early generation (PBPs) b-lactams Anti- PBP mecA Chromosome Production of altered PBP, PBP2a staphylococcal penicillins Glycopeptides Binding to terminal D-Ala vanA Chromosome, Alteration of terminal D-Ala to D-Lac residue, terminates insertion transpeptidation, RNA sequence polymerase inhibition Oxazolidinones Binding to 23S rRNA of the cfr, 23S Plasmid or Methylation of 23S rRNA, conferring 50S ribosomal subunit rRNA chromosome resistance to chloramphenicol, gene lincosamides, pleuromutilins, and mutations oxazolidinones Macrolides, Binding to 23S rRNA of the ermA, Plasmid or Methylation of ribosomal target lincosamides 50S ribosomal subunit ermC chromosome Macrolides, Binding to 23S rRNA of the msr(A) Plasmid Drug efflux pump group 50S ribosomal subunit B streptogramins Trimethoprim- Dihydrofolate reductase and dfrA, dpsA Chromosome Point mutations resulting in altered sulfamethoxazole dihydropterate synthetase drug target Chlorhexidine Nonspecific disruption of cell qacA/B, Plasmid Efflux of compound by drug efflux gluconate, other membrane qacC, pump antiseptics qacD, M smr, norA

chromosome mec (SCCmec) (Ma et al. 2002). In Centers for Disease Control and Prevention, addition to the mecA gene, which confers methi- CDC), have been attributed to most of the cillin resistance, SCCmec frequently carries addi- community-acquired infections; other clones tional antibiotic resistance determinants. have been predominant on other continents Epidemic MRSA clones have disseminated (McDougal et al. 2003). With the expansion of worldwide. Epidemiological studies have MRSA infections in the community, clones that suggested that these developed in multiple events were initially unique to community infections where SCCmec was incorporated into were ultimately introduced into the hospital envi- methicillin-susceptible S. aureus (MSSA) of dif- ronment. As a result, distinct genetic traits of an ferent backgrounds (Enright et al. 2002). In the isolate are not useful in discriminating between 1980s, community-acquired MRSA isolates were community and hospital-acquired infections. increasingly associated with infections among both children and adults who were otherwise healthy. Community-acquired MRSA isolates Antibiotic Resistance Mechanisms carry fewer antimicrobial resistance markers than isolates acquired in the hospital setting, The major mechanisms of antibiotic resistance in likely a result of the differences in antibiotic S. aureus are summarized in Table 1. Antimicro- pressure. In the United States, two community bial resistance can be acquired through the accu- MRSA clones, USA300 and USA400 (pulsed mulation of mutations in genes encoding drug field gel electrophoresis classification by the targets or through the acquisition of resistance M 452 MRSA/MSSA, Antibiotic Resistance

Transglycosidase

NAG NAM NAG NAM NAGNAM NAG NAM L-Ala L-Ala L-Ala D-Gln D-Gln D-Gln L-Lys L-Lys L-Lys D-Ala D-Ala D-Ala Gly x 5 D-Ala D-Ala D-Ala femA, femB

Gly x5 Gly x5 Gly x5 Transpeptidase D-Ala D-Ala D-Ala D-Ala D-Ala D-Ala D-Ala D-Ala L-Lys L-Lys L-Lys PBP L-Lys D-Gln D-Gln D-Gln D-Gln L-Ala L-Ala L-Ala b-Lactams L-Ala Glycopeptides NAMNAG NAM NAG NAM NAG NAM NAG

Plasma Membrane

MRSA/MSSA, Antibiotic Resistance, Fig. 1 Schematic of S. aureus cell wall and sites of action of cell wall active antimicrobials. NAG N-acetylglucosamine, NAM N-acetylmuramic acid elements on plasmids or insertion sequences. common resistance mechanism of S. aureus to Below is a detailed description of the most com- b-lactam antibiotics is through the production of mon forms of antibiotic resistance in S. aureus. a beta-lactamase enzyme (sometimes referred to Many antimicrobials, notably the b-lactam and as a penicillinase), typically encoded by a glycopeptide antimicrobials, act on the bacterial cell plasmid-associated gene, which hydrolyzes the wall peptidoglycan. Peptidoglycan consists of beta-lactam ring. The penicillinase enzyme, repeating units of N-acetylglucosamine (NAG) and encoded by the blaZ gene, is under the control N-acetylmuramic acid (NAM, Fig. 1). The NAM of a regulatory blaI-blaR system. The majority of subunits have a 4–5 amino acid branch (L-alanine- contemporary S. aureus MRSA and MSSA iso- D-isoglutamine-L-lysine-D-alanine-[D-alanine]). The lates carry blaZ and thus are resistant to individual peptidoglycan chains are further penicillin. cross-linked between the penultimate D-alanine residue and the L-lysine residue on an adjacent Methicillin Resistance and PBP2a peptidoglycan chain. In S. aureus, this connec- Methicillin is a member of a family of semisyn- tion is formed by a chain of glycine residues. thetic penicillins with decreased susceptibility to This complex organization is at least in part b-lactamases. MRSA has emerged as a clinical due to the activity of the so-called penicillin- entity of tremendous importance. There is strong binding proteins (PBPs), which have clinical data that support increased severity of a transglycosidase domain that links NAG and outcomes associated with MRSA versus NAM subunits plus a transpeptidase domain methicillin-susceptible S. aureus infections that connects the D-alanine to the glycine (Shurland et al. 2007). The primary mechanism linker. The PBPs are the target of b-lactam of methicillin resistance is via the production of antimicrobials. an altered PBP, PBP2a (also called PBP2’), that has a decreased affinity for methicillin. PBP2a is Penicillin Resistance encoded by the mecA on SCCmec As previously mentioned, S. aureus resistance to (Ma et al. 2002). Importantly, PBP2a lacks penicillin emerged shortly after the introduction a transglycosidase domain, and thus expression of penicillin in clinical practice. The most of other PBPs is necessary in order for MRSA/MSSA, Antibiotic Resistance 453 M peptidoglycan synthesis to occur (Pinho S. aureus infections, in particular MRSA. et al. 2001). To date, eight SCCmec types have Daptomycin is able to cross the peptidoglycan been identified. Expression of mecA and cell wall, insert its lipid moiety into the cell a number of other factors that limit the membrane and disrupt membrane potential, methicillin-resistant phenotype is controlled by resulting in a rapid bactericidal effect. Isolates a repressor protein MecI and a signal transducer with increased MICs to this agent were recovered protein MecR1 that are also encoded by in clinical trials of this agent versus traditional SCCmec. Other accessory genes also influence therapies (Fowler et al. 2006). Studies of these the level of methicillin resistance depending on and similar strains have revealed that daptomycin their level of expression. For example, a decrease nonsusceptibility is associated with a hetero- in expression of the femA and femB genes GISA phenotype and a thickened bacterial cell (de Lencastre and Tomasz 1994), important in wall, suggesting commonalities in the mecha- adding glycine residues to the peptide side nisms of resistance. Further work has revealed chain, results in loss of the methicillin resistance the existence of RNA polymerase (rpoB) muta- phenotype. tions in these organisms (Cui et al. 2010). While these and related findings are currently under Glycopeptide Resistance investigation, the mechanisms of resistance in Vancomycin is a glycopeptide antibiotic that these organisms are not fully understood at prevents cell wall assembly by binding to the this time. terminal D-alanine residues, preventing transpep- tidation and transglycosylation. In the late 1990s, Oxazolidinone Resistance the first cases of glycopeptide intermediate Oxazolidinones (i.e., linezolid) exert antimicro- S. aureus (GISA) were described and now have bial effects against a large spectrum of Gram- been reported in multiple sites throughout the positive bacteria including MRSA and M world. Some studies have shown that GISA pro- vancomycin-resistant enterococci and strepto- duce a thickened cell wall with many uncrossed cocci. Linezolid has become an important alter- linked D-alanine residues (Pereira et al. 2007). native to vancomycin in treating invasive MRSA Thus, GISA may be overexpressing the target infections. Its antibacterial effect is mediated for vancomycin to overcome the antimicrobial through binding of the 23S rRNA of the 50S effect. ribosomal subunit; resistance conferred through Full glycopeptide resistance is well described mutations within the 23S rRNA gene has been in Enterococcus spp. and is most frequently asso- reported (Ikeda-Dantsuji et al. 2011). Recently, ciated with the transposon Tn 1546, which pro- resistance mediated by the cfr gene was duces the VanA phenotype. Expression of described. The cfr gene encodes the genes carried on Tn 1546 leads to the a methyltransferase that modifies the 23S rRNA modification of the D-Ala-D-Ala side chain to and thus confers resistance to chloramphenicol, D-Ala-D-lactate thus changing the targeted lincosamides, and pleuromutilins, in addition to peptidoglycan structure to a resistant form. oxazolidinones. In addition, cfr is located on an In the past 10 years, there have been several IS element so horizontal transfer is possible. reports of VRSA in adult patients expressing the A number of outbreaks have been described VanA phenotype (Chang et al. 2003). These find- with S. aureus carrying cfr (Morales ings are alarming and active surveillance and et al. 2010), underscoring the clinical impact of study of these isolates are ongoing. these organisms.

Lipopeptide Resistance/Tolerance Sulfonamide Resistance Daptomycin is currently the only drug of the The most common sulfonamide antibiotic in clin- lipopeptide class approved for use in humans ical use in the United States is sulfamethoxazole and is licensed for the treatment of complicated (SMX) in combination with trimethoprim (TMP). M 454 MRSA/MSSA, Antibiotic Resistance

MRSA/MSSA, Antibiotic Resistance, Fig. 2 Schematic a macrolide is hidden by the hairpin loop, preventing of erm mRNA and translation regulation. IR inverted translation. In the absence of an inducer, translation of repeat. Panel a Translation of a short proximal peptide a short peptide from the proximal end of the mRNA occurs in the absence of a macrolide inducer. In the occurs. Panel b In the presence of low concentrations of absence of an inducer, IR1 and IR2 interact to form a macrolide, translation of the proximal peptide stalls in a hairpin loop, while IR3 and IR4 create a second loop. such a way that IR1 is prevented from interacting with The ribosome-binding and translational start site of the IR2. IR2 then binds to IR3, opening the second hairpin methylase is contained within IR4 and in the absence of loop and facilitating translation of the methylase

TMP-SMX is frequently used as an outpatient Clindamycin Constitutive/Inducible treatment for MRSA infections. Both agents Resistance inhibit enzymes necessary in the nucleotide bio- Three classes of antibiotics have the same site of synthetic pathway. TMP inhibits dihydrofolate binding and action on the bacterial ribosome: reductase (dfrA), and SMX inhibits macrolides, lincosamides (i.e., clindamycin), dihydropteroate synthetase (dpsA). When each and group B streptogramins. Cross-resistance to agent is used alone, their effect is bacteriostatic; these classes is most frequently mediated through when combined however, they exert a strong bac- the MLSB phenotype by activity of the plasmid tericidal effect. Despite, their frequent use, resis- borne erm family of genes. The erm genes encode tance to these agents is uncommon in clinical a methylase system that alters the 23S rRNA of practice in the United States (McNeil the 50S ribosome (Roberts et al. 1999). When the et al. 2012) Resistance is most commonly asso- phenotype is under inducible expression, initial ciated with the development of point mutations in susceptibility testing reveals resistance to dfrA or dpsA (Dale et al. 1997), although efflux macrolides but susceptibility to clindamycin. pumps or the development of thymidine auxotro- The erm system is regulated at the translational phy may play a role in some cases (Besier step (Fig. 2). The leader region of the methylase et al. 2007). mRNA contains a short open reading frame and MRSA/MSSA, Antibiotic Resistance 455 M four inverted repeat sequences, which can form Antiseptic Nonsusceptibility alternate hairpin loop structures. In the absence of Antiseptics for topical use exert a powerful a macrolide antibiotic, two stem-loop structures microbicidal effect on a diverse variety of bac- are formed (Fig. 2), and the ribosomal binding teria and fungi. The use of a number of these site and translational start site for the methylase agents in the hospital setting, in particular chlor- are located within the second stem-loop. When an hexidine gluconate, is associated with a decrease inducing level of macrolide is present, translation in healthcare-associated infections (Popovich of the short upstream open reading frame et al. 2009). While incompletely understood, it becomes stalled by the activity of the macrolide is believed that these agents disrupt the bacterial that prevents elongation. The stalled ribosome cell wall and interfere with cellular membrane interferes with the secondary structure of the potential (McDonnell and Russell 1999). mRNA, leading to an alternate structure that A number of genes, most notably qacA/B, frees the ribosome-binding site and allows trans- qacC, qacD, smr,andnorA, encoding efflux lation of the methylase gene. This phenomena, pumps in S. aureus have been discovered that referred to as inducible MLSB resistance, can be confer higher MICs and MBCs to these agents. detected in the clinical laboratory through the use In addition, these pumps often confer resistance of the D-test (Steward et al. 2005). If genetic to other agents including fluoroquinolones mutations develop within the upstream portions and b-lactams (Nakaminami et al. 2010). of the gene at the site of the inverted repeats, S. aureus isolates carrying the qacA/B genes opening of the hairpins can lead to a readily are increasingly isolated from hospital-acquired translatable mRNA and thus a constitutive infections in both adults and children (McNeil

MLSB phenotype. Constitutive resistance to the et al. 2013). lincosamides and macrolides can also be medi- ated through a chromosomal mutation affecting M the 50S portion of the ribosome itself. Summary Clindamycin is a common drug in the treat- ment of community-acquired MRSA infections, S. aureus is one of the most common infectious both in children and adults. However, resistance agents encountered in clinical practice. The rates ranging from approximately 5 % to 30 % emergence of resistance to multiple different have been reported for different patient antibiotics is a challenge to both clinicians and populations and from different geographic the laboratory researcher. Surveillance of antimi- areas. Numerous clinical failures of clindamycin crobial resistance among S. aureus isolates is therapy have been documented in patients with necessary to document changes in resistance to infections due to organisms with inducible specific antibiotics over time and to identify new clindamycin resistance (Siberry et al. 2003), and resistance markers. Further research should be clindamycin susceptibility testing including the geared toward a better understanding of the D-test is routine in the clinical microbiology mechanisms of resistance of currently available laboratory. antimicrobials and toward the development of new drugs. Macrolide Resistance In addition to the mechanisms discussed above, staphylococci can be resistant to macrolides Cross-References through an energy-dependent efflux pump encoded by the msr(A) gene (Ross et al. 1990). ▶ Antibiotic Classes and Mechanisms of Notably, this gene confers resistance to Resistance macrolides and group B streptogramins, but not ▶ Beta-lactam Resistance to lincosamides. ▶ MRSA/MSSA, Antibiotic Resistance M 456 MRSA/MSSA, Antibiotic Resistance

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