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Home , Dysbiosis, Flora (microbiology), Gut microbiota, Microbiome, Microbiota

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Methods in research Namrata Iyer

From the freezing lakes of Antarctica to satisfied most of these requirements. The metametabolomics are more technically the thermal vents deep within the oceans, most widely used method in microbiome challenging, given the sheer diversity of microbes have managed to survive and analysis is 16s rRNA sequencing2. The proteins and metabolites that each microbe thrive in the most inhospitable of con- 16s rRNA is highly conserved in all can produce. While these techniques are ditions. Unsurprisingly, the relatively and sequencing of its regions of still in their infancy, they promise to reveal luxurious abode of the human body is hyper-­variability allows the identification unique insights into the communication chockablock with microbial life. We have of different bacterial . But this tech- and cooperation between the different coevolved over centuries with these micro- nique suffers from inaccuracies at species members of the , and also - organisms, collectively referred to as our level classification2. Furthermore, it limits microbiota interactions. microbiota, resulting in a relationship the analysis to only bacterial species while that is mutually beneficial. However, it has ignoring other members in the . Microbiota structure only been in the past few decades that the Platforms are now being developed that can Community structure is a of who importance of the microbiota to human couple this technique with other genetic is present in the microbiota community health has been uncovered. It is now well- markers, allowing detection of eukaryotic and how these members interact with each known that imbalance in the ­microbiota— members of the microbiota as well3. other. The behavior of each microbe is or —is strongly linked with a While 16s rRNA sequencing looks at determined by the microbes and host cells number of , such as Crohn’s a single gene in every bacterium, whole in its immediate vicinity. Several patholo- , inflammatory bowel disease, can- metagenome sequencing looks at the entire gies, such as cer and many others1. In order to under- genomic content of the microbiota in an (IBD) and Crohn’s disease, are now known stand the link between these microbes and unbiased manner. This provides accurate to arise not only from an imbalance in the various , it becomes imperative to species-level identity of the microbes as microbiota, but also in their spatial orga- find the answers to several important ques- well as their complete . Analysis nization within the host. Visualization

Nature America, Inc. All rights reserved. America, Inc. © 201 6 Nature tions: What are the specific microbes that can reveal the metabolic potential of these of this spatial organization is performed inhabit our body? What functions do these communities, giving a better idea of their using FISH (fluorescent in situ hybridiza- microbes perform? Where are they located function as a whole. tion). Using fluorescent probes against 16s

npg with respect to the host and each other? rRNA sequences, FISH can specifically And finally, how do changes in microbiota Community function label various bacterial communities in lead to disease? Community composition has been the host tissues and reveal how these different Recent times have seen vast leaps in primary tool for microbiome research microbes are organized within their niche modern technology that allow us to begin in the past decades. As more is revealed (Fig. 1)6. These imaging techniques can answering some of these key questions. about the microbiota in different dis- further be coupled to mass spectrometry ease states, researchers are now trying to imaging to reveal the spatial location of a The diversity of microbes understand how these communities con- bacterium as well as its metabolic status Any site in the body can be considered as an tribute to the physiological state of the at the time. , inhabited by different microbial host. Metagenome information does not species, ranging from bacteria and tell us which subsets of are expressed Mechanistic link between microbiota to fungi and . To accurately define at any given time. Keeping this in mind, and host health this ecosystem, assigning an identity to whole metagenome sequencing is now Studies have uncovered startling correla- its members is of paramount importance. being coupled with metatranscriptome tions between changes in the microbiome The methods employed for this purpose sequencing to reveal the community gene and diseases ranging from to must be accurate, capable of detecting expression profile4. This dynamic picture . While most of these studies reveal even rare species, fast and cost-effective. of community function can help identify correlation, causation is yet to be compre- ‘Next Generation’ sequencing has largely the consequences of dysbiosis. Studies are hensively established. now being taken to the next level, ana- Answers to these puzzles are now being Department of Molecular and lyzing not only but also found using in vitro models of microbiota , Brown University, Providence, RI. Correspondence should be addressed to N.I. and of the com- research. These models simulate condi- ([email protected]) munity as a whole5. and tions in the specific niche to dissect out

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host-microbe and microbe-microbe 7 Probe ­interactions . These models could provide valuable insight into the physico-chemical Sample processes that occur at the gut interface.

Fixation A comprehensive understanding of the Target (ribosomal RNA) link between microbiota and health, how- ever, requires the use of live animals. The + – gold standard for such studies is gnotobiot- + + ic mice (mice with defined microbiological Epifluorescence – + Fixed cells are – status). Germ-free mice, born and bred in permeabilized + Ribosome – + completely sterile isolators (Fig. 2), reveal how the complete absence of microbiota affects host physiology. The lack of micro- Fluorescently labelled Flow cytometry oligonucleotides (probes) biota can have systemic effects on these Hybridization Quantification animals, ranging from an inability to effi- ciently digest food to an under-developed Washing . Hybridized cells These mice can be further selectively colonized with defined microbiota to assess their effect. Such studies have made FIGURE 1 | Basic steps of fluorescence in situ hybridization (FISH). The sample is first fixed to stabilize important revelations about the influence the cells and to permeabilize the cell membranes. The labeled oligonucleotide probe is then added and allowed to hybridize to its intracellular targets before the excess probe is washed away. The sample is of our microbiota on our metabolic sta- then ready for single-cell identification and quantification by either epifluorescence microscopy or flow tus. Obese and lean body types are now cytometry. From Nat. Rev. Microbiol. 6, 339–348 (2008). known to be transmissible between mice via a transfer of just their microbiota8. microbiota function and host response. of linked reactors that mimic the human Gnotobiotic mice have also been instru- Many systems recreating the human intes- gut, starting from the to the colon. mental in disclosing the complex relation- tine are now available. Examples such A recent model, known as ‘Gut-on-a-chip’, ship between our microbiota, diet and as the SHIME (Simulator of the Human is a microfluidic system that co-cultures health. Diet is now thought to be a factor Intestinal Microbial Ecosystem) make use gut cells with microbes to study ­various that dominates over our genome in deter- mining which microbes colonize our gut9.

Nature America, Inc. All rights reserved. America, Inc. © 201 6 Nature The translational impact of these stud- ies has further improved with the use of humanized mice (mice colonized with

npg microbes from the human gut). Research integrating gnotobiotic mice, dietary vari- ations and mathematical modeling have allowed scientists to predict the effects of diet on the composition of our microbiota and thereby our health10. This has wide implications for the growing fields of pre- biotics and . The dynamic nature of the microbiota, however, also means that minor changes in diet and other environmental conditions can significantly affect the microbiome, causing groups of mice that are genetically identical to display different . The variability in husbandry practices at different animal care facilities can, there- fore, cause problems with the reproducibil- ity of these studies11. Greater uniformity in animal maintenance and comprehensive FIGURE 2 | Example of a typical gnotobiotic facility with sterile isolators. All components (such as reporting of these conditions are required food and bedding) have to be sterilized before being placed in each isolator, creating a strict set of to generate a stronger foundation for requirements for maintaining gnotobiotic colonies. Image from NIAID. microbiota research.

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Future perspectives the key to breakthroughs in therapeutics omic approach. Gut 62, 1591–1601 (2013). The Project started with for a gamut of diseases. 6. Amann, R. & Fuchs, B.M. Single-cell identification in microbial communities by improved a goal to gain complete knowledge of our fluorescence in situ hybridization techniques. genome; the vision being that our genomic 1. Turnbaugh, P.J. et al. The Nat. Rev. Microbiol. 6, 339–348 (2008). code, once unlocked, would demystify project: exploring the microbial part of 7. Kim, H.J., Huh, D., Hamilton, G. & Ingber, D.E. Human gut-on-a-chip inhabited by microbial human physiology. Despite now having ourselves in a changing world. Nature 449, 804–810 (2007). that experiences intestinal peristalsis-like motions the genomic code firmly in our grasp, this 2. Rajendhran, J. & Gunasekaran, P. Microbial and flow. Lab Chip 12, 2165–2174 (2012). vision is far from fulfilled. The microbiota phylogeny and diversity: Small subunit 8. Zhao, L. The and : from is one of those missing links; an entity out- ribosomal RNA sequence analysis and beyond. correlation to causality. Nat. Rev. Microbiol. 11, Microbiol. Res. 166, 99–110 (2011). 639–647 (2013). side of our genome that exerts a profound 3. Morgan, X.C. & Huttenhower, C. Meta’omic analytic 9. Carmody, R.N. et al. Diet dominates host effect on almost every aspect of our physi- techniques for studying the intestinal microbiome. genotype in shaping the murine gut microbiota. ology. Deciphering the human metage- Gastroenterology 146, 1437–1448 (2014). Cell Host Microbe 17, 72–84 (2015). 4. Bikel, S. et al. Combining , 10. Faith J.J. et al. Identifying gut microbe–host nome, while seemingly a herculean task, and viromics to explore relationships using combinatorial has the potential to significantly improve novel microbial interactions: towards a systems- communities in gnotobiotic mice. Sci. Transl. our understanding of health and homeo- level understanding of human microbiome. Med. 6, 220ra11 (2014). Comput. Struct. Biotechnol. J. 13, 390–401 11. Hufeldt, M.R. et al. Variation in the gut stasis. Combining the strengths of sophis- (2015). microbiota of laboratory mice is related to both ticated technology, gnotobiotic models, and 5. Pérez-Cobas A.E. et al. Gut microbiota genetic and environmental factors. Comp. Med. inter-disciplinary approaches might hold disturbance during therapy: a multi- 60, 336–347 (2010). Nature America, Inc. All rights reserved. America, Inc. © 201 6 Nature npg

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