oral

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Uncultured Members of the Oral

William Wade, BSc, PhD; Hayley Thompson, BSc, PhD; Alexandra Rybalka, BSc, PhD; and Sonia Vartoukian, BDS, FDS, PhD

ABSTRACT Around one-third of oral bacteria cannot be cultured using conventional methods. Some bacteria have specif c requirements for nutrients while others may be inhibited by substances in the culture media or produced by other bacteria. Oral bacteria have evolved as part of multispecies biof lms, and many thus require interaction with other bacterial species to grow. In vitro models have been developed that mimic these interactions and have been used to grow previously uncultivated organisms.

AUTHORS

William Wade, BSc, Alexandra Rybalka, he human mouth is heavily in pathogenesis and contribution to PhD, is a professor of BSc, PhD, is a postdoctoral colonized by antimicrobial resistance is unknown, oral at scientist with interests in with all of the different types which is why there is substantial Queen Mary University oral microbiology and the represented: bacteria, , interest in culturing uncultivated of London with interests in development of novel model the characterization of the systems to study uncultured fungi, and . This bacteria and subjecting them to detailed and oral bacteria. Treview will focus on the bacteria because phenotypic and genomic analysis. development and evaluation Confl ict of Interest of their importance in the common dental The aim of this review is to list of novel strategies for the Disclosure: None reported. diseases, dental caries and periodontal the uncultivated members of the oral prevention and treatment diseases, and because the phenomenon microbiome, discuss the reasons why some of microbiome-associated Sonia Vartoukian, BDS, disease. FDS, PhD, is a specialist of unculturability has been extensively bacteria are diff cult to culture in vitro Confl ict of Interest periodontist and lecturer in investigated in bacteria. It has long and describe recent advances in culturing Disclosure: None reported. oral microbiology at Queen been realized that not all bacteria that previously uncultivated oral bacteria. Mary University of London. can be seen under the microscope can Hayley Thompson, BSc, Confl ict of Interest be cultured in the laboratory. An early Defi nition of “Unculturability” PhD, is a postdoctoral Disclosure: None reported. scientist with interests in oral estimate of oral bacterial culturability Clearly all bacteria that can be microbiology, the virulence was that only around half could be detected on Earth have grown at some factors of pathogenic grown.1 Recent advances in culture time. Culturability is, therefore, a relative bacteria and uncultured have modif ed this estimate so that it is term and dependent on the conditions oral bacteria. now considered that around two-thirds used to encourage growth in a particular Confl ict of Interest 2 Disclosure: None reported. of oral bacteria can be cultivated. experiment. A distinction should also be Although uncultivated bacteria cannot made between growth in monoculture be grown on commonly used laboratory and as part of a mixed community. The media, they clearly compete well in explosion in culture-independent studies the bacterial communities found in has revealed huge numbers of novel the mouth and many are associated bacterial taxa, the majority of which with oral disease. At present, their role cannot be identif ed as belonging to

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Bergeyella HOT-900 Bergeyella HOT-907 Bergeyella HOT-322 Bergeyella HOT-931 Bergeyella HOT-422 Bergeyella zoohelcum Bergeyella HOT-319 Capnocytophaga HOT-901 Capnocytophaga ochracea previously cultivated and characterized Capnocytophaga HOT-903 species. This does not mean, however, Capnocytophaga HOT-334 that all of these taxa cannot be cultured. Capnocytophaga HOT-902 Indeed, a comprehensive cultural analysis of the of severe early childhood Bacteroidetes [G-6] HOT-516 caries revealed 45 species-level taxa that, Porphyromonas HOT-285 at that time, had not been cultivated.3 Porphyromonas asaccharolytica In addition, it is often forgotten that Porphyromonas HOT-275 in microbiome surveys, only DNA is Porphyromonas HOT-277 detected, not living cells, and the detection Porphyromonas HOT-930 of an organism’s DNA does not necessarily mean that the organism was viable at Tannerella HOT-808 the time of sampling. Additionally, Tannerella HOT-916 DNA extraction and PCR reagents are Bacteroidetes [G-7] HOT-911 frequently contaminated with DNA from Bacteroidetes [G3] HOT-899 environmental bacteria and can make Bacteroidetes [G-3] HOT-281 up a signif cant proportion of amplicon Bacteroidetes [G-3] HOT-503 libraries, particularly when samples are Bacteroidetes [G-3] HOT-280 taken from sites with low bacterial levels.4 Bacteroidetes [G-3] HOT-436 A practical def nition of unculturability fragilis will be used for this review. An organism Alloprevotella HOT-308 will be regarded as uncultivated if there Alloprevotella HOT-914 are no reports that it has been grown Alloprevotella HOT-912 in previous cultivation studies. Alloprevotella HOT-913 Alloprevotella tannerae Uncultured Oral Bacteria Prevotella HOT-315 When culture-independent methods Prevotella HOT-515 were f rst used to study the composition Prevotella HOT-526 of the oral microbiota and compared to Prevotella HOT-304 cultural analyses of the same samples, Prevotella HOT-942 it was clear that a substantial number Prevotella HOT-301 of bacterial taxa could not be readily Prevotella HOT-309 cultured.5,6 The Human Oral Microbiome Prevotella HOT-443 Database (HOMD, homd.org)7 lists Prevotella HOT-396 the bacteria found in the mouth. Many species-level taxa have yet to be named Prevotella melaninogenica and are, therefore, assigned human Prevotella HOT-305 oral taxon (HOT) numbers. HOMD Prevotella HOT-293 release 13.2 includes 210 species-level Prevotella HOT-292 taxa that have yet to be cultured. Many Prevotella HOT-300 uncultivated taxa belong to genera whose Bacteroidetes [G-4] HOT-509 members are predominantly cultivable. Bacteroidetes [G-5] HOT-507 Because still relatively few oral bacteria Bacteroidetes [G-5] HOT-505 have been cultured and identif ed by 0.05 Bacteroidetes [G-5] HOT-511 16S rRNA gene sequence analysis, it is FIGURE 1. Phylogenetic tree showing uncultivated species-level oral taxa within the phylum Bacteroidetes. Tree prepared possible that these taxa are cultivable but by the neighbor-joining method from a distance matrix constructed using the Jukes-Cantor algorithm and an alignment of representative strains have not yet been 998 bases. Sequences representing type species of relevant genera are included for reference and colored red.

448 JULY 2016 CDA JOURNAL, VOL 44, Nº7 Selenomonas HOT-388 Selenomonas HOT-478 Selenomonas HOT-936 Selenomonas HOT-937 Selenomonas HOT-501 Selenomonas HOT-134 Selenomonas HOT-442 Selenomonas sputigena Mitsuokella HOT-521 Mitsuokella multiacida Veillonellaceae [G-1] HOT-132 Veillonellaceae [G-1] HOT-150 Veillonellaceae [G-1] HOT-148 Veillonellaceae [G-1] HOT-135 Veillonellaceae [G-1] HOT-918 Veillonellaceae [G-1] HOT-145 Veillonellaceae [G-1] HOT-483 encountered. Thus, the common genera Dialister HOT-119 , Prevotella, Streptococcus Dialister pneumosintes and Veillonella all include such taxa. Veillonella HOT-917 In contrast, many groups of Megasphaera HOT-123 uncultured taxa cluster in deep Megasphaera HOT-841 branches of the phylogenetic tree Megasphaera elsdenii Paenibacillus HOT-048 with no or few cultivated neighbors. Paenibacillus polymyxa FIGURE 1 shows a phylogenetic tree Erysipelothrix rhusiopathiae of the uncultivated members of the Erysipelotrichaceae [G-1] HOT-904 Erysipelotrichaceae [G-1] HOT-905 phylum Bacteroidetes. It can be seen HOT-168 that Bacteroidetes genera G-3 and G-7 Peptococcus niger comprise a branch of six uncultured Syntrophomonadaceae [VIII][G-1] HOT-435 Ruminococcus fl avefaciens species-level taxa of which Bacteroidetes Ruminococcaceae [G-1] HOT-075 [G-3] HOT-281 is the most commonly Ruminococcaceae [G-2] HOT-085 detected taxon, while Bacteroidetes Ruminococcaceae [G-3] HOT-366 Ruminococcaceae [G-3] HOT-381 genera G-4 and G-5 constitute another Clostridiales [F-1][G-1] HOT-093 deep branch with four uncultivated Clostridiales [F-1][G-2] HOT-402 taxa. Similarly, the phylum Firmicutes Peptoniphilaceae [G-2] HOT-790 includes a number of deep-branching Peptoniphilus asaccharolyticus Peptostreptococcaceae [XI][G-7] HOT-922 lineages made up of uncultivated taxa anaerobius (FIGURE 2). These include a major Peptostreptococcaceae [XI][G-4] HOT-103 branch of the Peptostreptococcaceae Peptostreptococcaceae [XI][G-4] HOT-369 Peptostreptococcaceae [XI][G-5] HOT-493 with seven uncultivated taxa from Peptostreptococcaceae [XI][G-1] HOT-383 f ve genera, four uncultivated Peptostreptococcaceae [XI][G-2] HOT-091 taxa within the Ruminococcaceae Peptostreptococcaceae [XI][G-3] HOT-495 Peptostreptococcaceae [XI][G-3] HOT-950 and a number of uncultivated Lachnospiraceae [G-10] HOT-094 representatives of the Lachnospiraceae, Catonella HOT-164 Syntrophomonadaceae and Veillonellaceae. Catonella morbi Catonella HOT-451 The Fusobacteria phylum includes Lachnospiraceae [G-7] HOT-086 an uncultured branch consisting of Lachnospiraceae [G-7] HOT-163 Fusobacteria [G-1] with two taxa: Butyrivibrio HOT-080 Butyrivibrio HOT-455 HOT-210 and HOT-220, and one Butyrivibrio HOT-090 comprised of uncultured Leptotrichia Butyrivibrio fi brisolvens taxa HOT-212, HOT-215, HOT- Lachnospiraceae [G-9] HOT-924 217 and HOT-392. Interestingly, the Stomatobaculum HOT-910 Stomatobaculum longum majority of Leptotrichia taxa have yet to Stomatobaculum HOT-097 be cultured, but most branches of this Stomatobaculum HOT-373 include cultivated members. Johnsonella HOT-166 Johnsonella ignava Bdellovibrio HOT-039 represents Lachnospiraceae [G-2] HOT-088 a deep uncultured branch within the Lachnospiraceae [G-3] HOT-100 phylum Proteobacteria. Bdellovibrio are Lachnoanaerobaculum HOT-496 Lachnoanaerobaculum HOT-089 normally aerobic, predatory bacteria, Lachnoanaerobaculum HOT-083 feeding on other Gram-negative bacteria8 0.05 Lachnoanaerobaculum umeaense and it will be interesting to know if this FIGURE 2. Phylogenetic tree showing uncultivated species-level oral taxa within the phylum Firmicutes. Tree prepared taxon has a similar predatory lifestyle. by the neighbor-joining method from a distance matrix constructed using the Jukes-Cantor algorithm and an alignment A substantial number of spirochetes of 1,126 bases. Sequences representing type species of relevant genera are included for reference and colored red. have yet to be cultured. All oral

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spirochetes belong to the genus within candidate division OP11.17 They for organisms formerly described as TM7.25 , and of the 49 oral Treponema are widely distributed, being found in Saccharibacteria are found in a range taxa, only 14 have been cultured. In anaerobic habitats such as deep-sea of animals. In invertebrates, they particular, one branch of 10 taxa has no sediments, various extreme environmental have been detected in the microbiota cultivable representatives: HOT-250-256, sites, the cow rumen and the human associated with sponges and ,26-28 HOT-508, HOT-517 and HOT-518. mouth.18 SR1 comprises two lineages, termites29-31 and nematodes.32 They The recently described phylum BH1 and BD2-14, with the former found form part of the intestinal microbiota Synergistetes9 includes a large number only in geothermal habitats. Human of mammals and appear to be a of uncultured taxa. Oral members oral SR1 representatives are found in consistent member of the mammalian of the phylum form two clusters, subgroup III of the BD2-14 lineage. gut microbiome, having been found denoted A and B.10 The majority of Three species-level taxa belonging to in mice,33,34 cattle,35-37 dogs,38 pigs,39 Cluster A taxa have yet to be cultured, the same genus-level taxon are found elephants, gazelle, bighorn sheep, while Cluster B includes the recently in the mouth: HOT-345, HOT-874 and takin, buffalo, bonobo and gorillas.40 described species Jonquetella anthropi HOT-875. Like GN02, SR1 is a rare In humans, Saccharibacteria have been and Pyramidobacter piscolens.11 detected in several habitats, including Until recently, the phylum Chlorof exi the intestinal tract,41 skin,42 vaginal f uid43 has had no cultivated representatives and oral cavity44-46 although they typically among the oral microbiota, although Saccharibacteria appear to make up less than 1 percent of the environmental relatives have been community at a given site. Saccharibacteria cultivated.12 Three strains of Anaerolineae be associated with oral appear to be associated with oral disease, bacterium HOT-439, an important disease, particularly those particularly those conditions associated taxon thought to serve as a biomarker conditions associated with a with a mature anaerobic biof lm. For for periodontitis,13 have recently example, Paster et al.44 found 34 sequences been isolated from subgingival plaque mature anaerobic biofi lm. representing the Saccharibacteria division samples and found to grow with the among 2,522 cloned 16S rRNA genes help of nucleatum.14 from the subgingival plaque of healthy Three oral phyla have no, or very subjects and patients with periodontal few, cultivated representatives: GN02, member of the oral microbiome, and disease, which were later identif ed as oral TM7 and SR1. Candidate division typically makes up around 0.01 percent taxa HOT-346, HOT-347, HOT-349, 355 GN02 was f rst described to comprise a of clone libraries. At this level, it will be and HOT-356. Of these, only HOT-346 group of sequences identif ed in a study challenging to detect individual cells by was found in health, while HOT-356 of the Guerrero Negro hypersaline f uorescence in situ hybridization (FISH) (represented by phylotype I025, now .15 Three oral taxa are or attempt direct . Three species- recognized to belong to HOT-356) was found: HOT-871, HOT-872 and level taxa of SR1 have been identif ed associated with periodontitis, a f nding HOT-873, representing two class- among the canine oral microbiome, conf rmed in a study using oligonucleotide level taxa. Interestingly, four related but were only detected when a specif c probes specif c for HOT-356, in which the taxa were identif ed among the canine Bacteroidetes-TM7-SR1 primer was used.16 taxon was found in 50 percent of healthy oral microbiome.16 Little is known The TM7 candidate division was subjects and 83 percent of patients with regarding the genetic potential or named with reference to the Torf, mittlere periodontitis.47 The same trend was seen in functional capability of this group of Schicht, or peat, middle layer, in which a polymerase chain reaction (PCR)-based organisms, although its ubiquity, albeit was f rst detected in a German peat bog.19 study, with HOT-356 being detected in 91 at low levels, suggests that it deserves Subsequently, members of this division percent of diseased sites and 71 percent to be the target of future studies. have been isolated from a wide range of of controls, although the difference was Sequences representing candidate environments including waste water and not statistically signif cant.48 In refractory division SR1 were originally detected batch reactor sludges,20,21 fresh and sea periodontitis, Saccharibacteria HOT-346, in sediments from Sulphur River in water22-24 and soil. The name Candidatus HOT-356 and HOT-437 were detected Parkers Cave, and were f rst classif ed Saccharibacteria has recently been proposed in signif cantly higher proportions than

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in patients whose periodontal treatment and parasitic relationship with a strain of Atmospheric conditions, particularly was successful or healthy controls in a Actinomyces odontolyticus.55 It would appear the presence or absence of oxygen, study using the Human Oral Microbe then that the Saccharibacteria isolates as well as the availability of CO2 are Identif cation Microarray (HOMIM).49 studied thus far are only found in such obviously extremely important. Some Three Saccharibacteria phylotypes close associations with other bacteria. It bacteria require specif c nutrients were found in oral samples collected was further reported that association with for growth. Methanosaeta species, for from subjects with halitosis and one TM7x caused the A. odontolyticus host example, are obligately acetotrophic; of them, HOT-352, was signif cantly to change its morphology from relative hence, the addition of acetone to associated with the condition.45 short rods to f laments,56 although the media, which is slowly converted to The culture of members of the division growth phase and natural morphological acetate, will promote the growth of Saccharibacteria has long been a goal. It variation of both partners in the these otherwise slow-growing species.58 was reported that Saccharibacteria had interaction requires further investigation. Sometimes the medium itself been successfully cultured after 50-day The TM7x genome was found to be can be toxic. It has been shown that aerobic incubation of low-nutrient solid small at 705 kb and completing lacking autoclaving agar-containing culture media, with microcolonies visible to the media in the presence of phosphate can naked eye.50 No further detail of this generate inhibitory levels of hydrogen isolation has been reported, however. peroxide, an effect that can be avoided 59 There have been a number of reports of Strictly anaerobic bacteria by replacing agar with gellan gum. the successful isolation of Saccharibacteria Oral bacteria typically live as part bacteria in mixed culture. For example, coexist with oxygen-consuming of a multispecies community in densely microcolonies of Saccharibacteria from and -tolerant species and packed biof lms. Within the biof lm, there soil were obtained using a soil substrate cooperate to protect each are a variety of gradients of nutrients, membrane system.51 After seven days signaling molecules and gases, due to the incubation, several morphotypes were other from atmospheric stress. diffusion patterns of these substances and detected by means of FISH with the the metabolic activity of neighboring TM7-905 probe, although no pure cultures bacteria, such that conditions for were obtained. Using the same method, individual cells, and groups of cells, can Abrams et al.52 reported the isolation of in amino acid biosynthesis capability, vary markedly.60,61 Despite the mouth microcolonies that included cells that perhaps explaining its need to parasitize being exposed to the atmosphere, about reacted positively with the TM7 probe, other bacteria. Small genomes are a half of oral bacteria are obligate anaerobes. but also others that were negative with the feature of a number of other Divisions As oral biof lms develop, obligate aerobes specif c probe but positive for a universal yet to be cultured, including SR1, OD1 and facultative anaerobes rapidly reduce bacterial probe. Rybalka53 found that and WWE 3,57 suggesting that a limited the local oxygen concentration; four Saccharibacteria could be isolated in mixed metabolic repertoire and dependence on days of plaque formation in vivo in culture with a variety of other species, association with other organisms may be two subjects produced a mean redox including Slackia exigua and Atopobium common features of phylum-level taxa potential at the tooth surface of –127 parvulum, but could not be isolated in with no or few cultivable representatives. mV.62 Strictly anaerobic bacteria coexist pure culture or even maintained as a with oxygen-consuming and -tolerant mixture for more than a few subcultures. Reasons for Unculturability species and cooperate to protect each The successful isolation of a pure If bacteria are able to grow in a other from atmospheric stress.63 culture of a Saccharibacteria phylotype was particular environment but we are unable In a similar way, there will be a reported from a sample of to cultivate them in the laboratory, then concentration gradient within the biof lm but a culture was not deposited with a clearly at a basic level we are unable for nutrients with their concentration culture collection.54 A Saccharibacteria to reproduce the conditions that they decreasing with increasing depth of strain successfully isolated and maintained need for growth. Understanding these biof lm, while bacterial metabolic from saliva, TM7x, was an extremely small conditions is key to the cultivation of products will be increased. There can found in an exclusive physical previously uncultivated organisms. be direct interactions between species

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TABLE

Summary of Recently Described Methods for the Cultivation of Difficult-to-Culture Bacteria Approach to cultivation Examples Reference number Media supplementation, Addition of supplements to media e.g., , N-acyl homoserine lactones or growth 14, 59, 68, 73, 74, 91, customization or modifi cation factors; design of media selective for specifi c bacterial taxa using the SMART method; modifi ed 92 media preparation methods/substitution of agar with gellan gum as gelling agent, to limit growth inhibition by hydrogen peroxide.

Modifi cation of growth conditions Modifi ed temperature, pH, O2 presence/absence, incubation time, gravity. 93, 94 Modifi cation of sample handling Dilution-to-extinction to achieve single-cell isolation; small inoculum for reduced microbial 77, 78 competition. Simulated natural environment Diff usion chamber incubated within the natural environment allowing passage of growth- 89, 95, 96 stimulatory chemical compounds across a membrane; hollow-fi ber membrane chamber for in situ cultivation in the natural environment; I-tip in situ cultivation device permitting inward diff usion of natural chemical factors. Microfl uidic device Encapsulation of subsets of the microbial community to form microdroplets that are exposed to 97, 98 signals or nutrients from external bacteria. Community culture and co-culture Bacterial culture facilitated by chemical components produced by the main bacterial 14, 99–103 community separated from the target organism by a membrane, transwell insert or a well within the media plate; growth of bacteria in consortia, followed by detection and enrichment of specifi c bacterial targets using colony hybridization; co-culture of bacterial strains with “helper” species on which they depend for provision of growth factors or for environment modifi cation. High-throughput methods I chip: A device comprised of hundreds of miniature diff usion chambers, within each of which 96,104 a single cell is cultured; hollow-fi ber membrane chamber device (see above) comprised of 48–96 chamber units.

with one using the end products of peptide, thought to be a signaling molecule Methods for Culture of Uncultured another for growth. For example, stimulated the growth of a previously Bacteria Veillonella species use lactate produced by uncultivated Psychrobacter strain.68 A number of approaches have been streptococci as a major carbon source.64 Resuscitation-promoting factor (Rpf) is taken in attempts to culture previously Other possibly important factors in a protein that was identif ed as being able uncultured bacteria and these are growth-regulating interactions between to revive Micrococcus cells from dormancy.69 summarized in the TABLE. Perhaps the most bacteria are bacterial signaling molecules. Rpf is structurally similar to lysozyme70 and promising approach is the recognition Gram-negative bacteria communicate cleaves . Rpf is, therefore, that bacteria in nature frequently live in by means of acyl homoserine lactones likely to generate peptidoglycan fragments multispecies biof lms and are, therefore, in (AHLs),65 and Gram-positives use small from the cell walls of intact bacteria, chemical contact with other bacteria. If diffusible peptides,66 but both systems which might act as signaling molecules.71 chemical interactions can be maintained are primarily intraspecies. In contrast, Muropeptide fragments are known to have in vitro, then isolation of novel organisms autoinducer-2 (AI-2), the product of the signaling properties in subtilis, where should be possible. For example, D’Onofrio luxS gene, has homologues in a wide variety they bind to PrkC, a serine/threonine et al.73 grew seawater sediment bacteria of organisms, both Gram-positive and kinase on the cell surface.72 A specif c on agar plates in mixed culture at various Gram-negative and has been suggested to muropeptide, a disaccharide-tripeptide dilutions. Because disproportionally more act across taxonomic boundaries.67 Bacterial with a meso-diaminopimelic-acid residue, colonies were seen on plates that had signaling affects a number of functional typically found in Gram-positive bacteria been heavily inoculated, pairs of colonies aspects including expression of the biof lm is necessary for this activity. Rpf then may growing within 2 cm of each other were phenotype, production of virulence factors function by producing muropeptides from subcultured and then grown together. and growth itself. Bacteria accustomed peptidoglycan with signaling, possibly Around 10 percent of these pairs showed to growing in biof lms may thus require growth-stimulating properties. Further evidence of the growth of one organism the presence of exogenous signals for work is required to investigate if this is a being dependent on its pair. The growth growth. For example, a small, 5-amino acid general method of growth regulation. of many of the dependent isolates was

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stimulated not only by its co-culture which led to the successful isolation of in samples of biomass.79,80 Thus, even partner but also by Escherichia coli. A several previously uncultivated bacterial though an organism cannot be grown, panel of E. coli mutants was, therefore, strains, including Chlorof exi taxon its morphology can be determined. This constructed and tested to determine the Anaerolineae bacterium HOT-439.14 has been successfully performed for TM7, identity of the substance produced by E. One method of achieving a pure Tannerella BU063 and Synergistetes cluster coli that was stimulating the growth of culture of a slow-growing organism is the A.10,21,18 An alternative labeling method the dependent isolates. Enterobactin, a dilution-to-extinction method whereby is to use if it is possible to , was found to be responsible dilution ensures that single cells are select an appropriate specif c . and adding siderophores to culture media placed in a growth medium and have Because antibody labeling, unlike DNA allowed a number of novel bacteria to grow. time to grow without being inhibited by probing, is nonlethal, it can be possible A novel system has been developed other bacteria.75,76 A high-throughput to obtain viable cells for culture, after that explores genomic data for information version of the method was successful in sorting as described above; in this way on specif c carbon source requirements cultivating a number of novel strains f uorescent antibodies have been used to and antimicrobial resistance of particular of the seawater organism SAR11 as obtain viable cells after sorting.82 Flow bacteria, leading to the development cytometry can be used to isolate single of highly selective media designed by cells from mixtures from which whole SMART — selective medium-design genome sequences can be obtained; this algorithm restricted by two constraints.74 A range of compounds with approach has been successfully used Using this method, the authors prepared siderophore activity have to sequence genomes of the health- “selective” media for f ve plant-pathogenic been screened for their ability associated taxon related to Tannerella bacteria and demonstrated accurate forsythia, HOT-286 (BU063).83 selection for the target bacterial species to stimulate the growth of oral Mixed primary cultures frequently among a panel of 18 strains representing bacteria that are unable to include representatives of bacterial 10 species. The use of such systems grow in pure culture. species not yet cultured in isolation. may provide a rational basis for the Colony hybridization is a useful method development of novel culture media. for determining the location of specif c A range of compounds with taxa on solid media.84 By membrane siderophore activity have been screened well as representatives of the abundant, blotting and the use of specif c probes, for their ability to stimulate the growth but previously uncultured, SAR116 target organisms can be localized to of oral bacteria that are unable to grow clade.77 Dilution to extinction would specif c regions of replica plates, allowing in pure culture.14 Growth of Prevotella appear to be a method most suited to their subculture and enrichment. This HOT-376 was more strongly stimulated by samples such as seawater where bacterial method was used to culture the f rst the siderophore pyoverdines-Fe, than by a concentrations are relatively low and representative of Synergistetes cluster culture f ltrate of its helper Fusobacterium the bacterial cells are found primarily in A: Fretibacterium fastidiosum from nucleatum (the positive control); to a lesser planktonic suspension and interactions subgingival plaque in periodontitis.85,86 extent, it was also stimulated by ferric between bacteria are, therefore, limited. The isolation of previously citrate, desferricoprogen, ferrichrome-Fe- The oral microbiome, conversely, is uncultivated bacteria may be a multi- free and salicylic acid. Likewise, growth of primarily made up of dense biof lms stage process. Clearly, the agar plate is Fretibacterium fastidiosum of Synergistetes where this method may be less generally an alien environment for bacteria used cluster A was consistently stimulated applicable, although some novel taxa to living in biof lms associated with by desferricoprogen, salicylic acid and have been recovered using this method.78 mammalian tissues. One approach has ferrichrome-Fe-free. Consequently, Culture-independent surveys have been to establish biof lms in vitro, seeded media used for culture of heavily made available 16S rRNA gene sequences with natural inocula. The Calgary Diof lm diluted samples of subgingival plaque, for the studied. These Device, a microplate-based system with were supplemented with siderophores data can then be used to design specif c plastic pegs coated with hydroxyapatite pyoverdines-Fe or desferricoprogen, or oligonucleotide probes which can be used to mimic the tooth surface, has been a neat suspension of subgingival plaque, in FISH to visualize uncultured bacteria successfully used to produce dental

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87 plaque biof lms. Saliva was used as the promoting signaling molecules. The 17. Harris JK, Kelley ST, Pace NR. New perspective on uncultured inoculum and biof lms with a composition culture of an organism remains the key bacterial phylogenetic division OP11. Appl Environ Microbiol 2004;70(2):845-9. resembling dental plaque could be factor in determining its characteristics, 18. Davis JP, Youssef NH, Elshahed MS. Assessment of the reproducibly established. Next-generation including the production of virulence diversity, abundance and ecological distribution of members sequence analysis of the biof lms showed factors and resistance to antimicrobials. of candidate division SR1 reveals a high level of phylogenetic that they included representatives of diversity but limited morphotypic diversity. Appl Environ Microbiol REFERENCES 2009;75(12):4139-48. uncultured oral bacteria and one of these, 1. Socransky SS, Gibbons RJ, Dale AC, et al. The microbiota of 19. Rheims H, Sproer C, Rainey FA, Stackebrandt E. Molecular Lachnospiraceae HOT-500, was successfully the gingival crevice in man. 1. Total microscopic and viable counts biological evidence for the occurrence of uncultured members and counts of specifi c organisms. Arch Oral Biol 1963;8:275-80. of the actinomycete line of descent in diff erent environments isolated following colony hybridization and geographical locations. Microbiology 1996;142 88 2. Dewhirst FE, Chen T, Izard J, et al. The human oral microbiome. enrichment. Mimicking natural J Bacteriol 2010;192(19):5002-17. (Pt 10):2863-70. conditions in a similar fashion, Jung and 3. Tanner AC, Mathney JM, Kent RL Jr., et al. Cultivable 20. Bond PL, Hugenholtz P, Keller J, Blackall LL. Bacterial co-workers89 developed the I-tip method Anaerobic Microbiota of Severe Early Childhood Caries. J Clin community structures of phosphate-removing and nonphosphate- Microbiol 2011. removing activated sludges from sequencing batch reactors. Appl as an in vitro cultivation device using the 4. Salter SJ, Cox MJ, Turek EM, et al. Reagent and laboratory Environ Microbiol 1995;61(5):1910-6. natural environment as a source not only contamination can critically impact sequence-based microbiome 21. Hugenholtz P, Tyson GW, Webb RI, Wagner AM, Blackall of the bacterial community, but also of analyses. BMC Biol 2014;12:87. LL. Investigation of candidate division TM7, a recently recognized 5. Munson MA, Banerjee A, Watson TF, Wade WG. Molecular major lineage of the domain Bacteria with no known pure-culture the associated chemical compounds. They analysis of the microfl ora associated with dental caries. J Clin representatives. Appl Environ Microbiol 2001;67:411-19. cultivated from Baikalian sponges a greater Microbiol 2004;42(7):3023-9. 22. Connon SA, Tovanabootr A, Dolan M, et al. Bacterial range of bacterial strains using this method 6. Munson MA, Pitt Ford T, Chong B, Weightman AJ, Wade WG. community composition determined by culture-independent and Molecular and cultural analysis of the microfl ora associated with -dependent methods during propane-stimulated than by conventional plating. Bacterial endodontic infections. J Dent Res 2002;81:761-66. in trichloroethene-contaminated groundwater. Environ Microbiol communities can even be cultured in 7. Chen T, Yu WH, Izard J, et al. The Human Oral Microbiome 2005;7(2):165-78. vivo by means of a device where an Database: A web accessible resource for investigating oral 23. Neulinger SC, Gartner A, Jarnegren J, et al. Tissue-associated microbe taxonomic and genomic information. Database (Oxford) “Candidatus Mycoplasma corallicola” and fi lamentous bacteria agar substrate is placed in a chamber 2010;2010:baq013. on the cold-water Lophelia pertusa (Scleractinia). Appl separated from the oral environment by 8. Dashiff A, Kadouri DE. Predation of oral pathogens by Environ Microbiol 2009;75(5):1437-44. a membrane. Bacteria can grow on the Bdellovibrio bacteriovorus 109J. Mol Oral Microbiol 24. Newton RJ, Kent AD, Triplett EW, McMahon KD. Microbial 2011;26(1):19-34. community dynamics in a humic lake: Diff erential persistence agar while in chemical communication 9. Jumas-Bilak E, Roudiere L, Marchandin H. Description of of common freshwater phylotypes. Environ Microbiol with their natural environment.90 This ‘Synergistetes’ phyl. nov. and emended description of the phylum 2006;8(6):956-70. method was found to be of value for the ‘Deferribacteres’ and of the family Syntrophomonadaceae, 25. Albertsen M, Hugenholtz P, Skarshewski A, et al. Genome phylum ‘Firmicutes.’ Int J Syst Evol Microbiol 2009;59(Pt sequences of rare, uncultured bacteria obtained by diff erential culture of previously uncultivated oral 5):1028-35. coverage binning of multiple metagenomes. Nat Biotechnol bacteria and complementary to dilution 10. Vartoukian SR, Palmer RM, Wade WG. Diversity and 2013;31(6):533-8. to extinction and conventional plating.78 morphology of members of the phylum ‘Synergistetes’ in 26. Ceh J, Van Keulen M, Bourne DG. Coral-associated bacterial periodontal health and disease. Appl Environ Microbiol communities on Ningaloo Reef, Western Australia. FEMS 2009;75(11):3777-86. Microbiol Ecol 2011;75(1):134-44. Future Prospects 11. Downes J, Vartoukian SR, Dewhirst FE, et al. Pyramidobacter 27. Webster NS, Soo R, Cobb R, Negri AP. Elevated seawater A number of approaches for the piscolens gen. nov., sp. nov., a member of the phylum temperature causes a microbial shift on crustose coralline algae ‘Synergistetes’ isolated from the human oral cavity. Int J Syst Evol with implications for the recruitment of coral larvae. ISME J cultivation of previously uncultivated Microbiol 2009;59(5):972-80. 2011;5(4):759-70. oral bacteria have been developed and 12. Yamada T, Sekiguchi Y. Cultivation of uncultured Chlorofl exi 28. Webster NS, Taylor MW. Marine sponges and their microbial successfully used to isolate representative subphyla: Signifi cance and ecophysiology of formerly uncultured symbionts: Love and other relationships. Environ Microbiol Chlorofl exi ‘subphylum i’ with natural and biotechnological 2012;14(2):335-46. strains. Progress has been slow, however, relevance. Microbes Environ 2009;24(3):205-16. 29. Miyata R, Noda N, Tamaki H, et al. Infl uence of feed with only a small number of new species 13. Szafranski SP, Wos-Oxley ML, Vilchez-Vargas R, et al. High- components on symbiotic bacterial community structure in the gut cultivated. Efforts should be directed resolution taxonomic profi ling of the subgingival microbiome for of the wood-feeding higher termite Nasutitermes takasagoensis. biomarker discovery and periodontitis diagnosis. Appl Environ Biosci Biotechnol Biochem 2007;71(5):1244-51. toward developing high-throughput Microbiol 2015;81(3):1047-58. 30. Nakajima H, Hongoh Y, Noda S, et al. Phylogenetic and methods of detecting the growth of novel 14. Vartoukian SR, Adamowska A, Lawlor M, et al. In Vitro morphological diversity of Bacteroidales members associated organisms. The relative ease in obtaining Cultivation of ‘Unculturable’ Oral Bacteria, Facilitated by with the gut wall of termites. Biosci Biotechnol Biochem Community Culture and Media Supplementation With 2006;70(1):211-8. genome sequences both of individual Siderophores. PLoS One 2016;11(1):e0146926. 31. Nakajima H, Hongoh Y, Usami R, Kudo T, Ohkuma M. isolates and from shotgun metagenomic 15. Ley RE, Harris JK, Wilcox J, et al. Unexpected diversity and Spatial distribution of bacterial phylotypes in the gut of the termite analysis of communities should provide complexity of the Guerrero Negro hypersaline microbial mat. Reticulitermes speratus and the bacterial community colonizing the Appl Environ Microbiol 2006;72(5):3685-95. gut epithelium. FEMS Microbiol Ecol 2005;54(2):247-55. information to guide the provision of 16. Dewhirst FE, Klein EA, Thompson EC, et al. The canine oral 32. Ladygina N, Johansson T, Canback B, Tunlid A, Hedlund K. nutrient substrates and potential growth- microbiome. PLoS One 2012;7(4):e36067. Diversity of bacteria associated with grassland soil nematodes of

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[London, UK: King’s College from neighboring organisms promote the growth of uncultured group of mouse intestinal bacteria. Microbiology 2002;148(Pt London; 2013]. bacteria. Chem Biol 2010;17(3):254-64. 11):3651-60. 54. Soro V, Dutton LC, Sprague SV, et al. culture of a 74. Kawanishi T, Shiraishi T, Okano Y, et al. New detection 35. Brulc JM, Antonopoulos DA, Miller ME, et al. Gene-centric candidate division TM7 bacterium from the human oral cavity systems of bacteria using highly selective media designed by metagenomics of the fi ber-adherent bovine rumen microbiome and biofi lm interactions with other oral bacteria. Appl Environ SMART: Selective medium-design algorithm restricted by two reveals forage specifi c glycoside hydrolases. Proc Natl Acad Sci Microbiol 2014;80(20):6480-9. constraints. PLoS One 2011;6(1):e16512. U S A 2009;106(6):1948-53. 55. He X, McLean JS, Edlund A, et al. Cultivation of a 75. Button DK, Schut F, Quang P, Martin R, Robertson BR. 36. Fernando SC, Purvis HT, 2nd, Najar FZ, et al. Rumen human-associated TM7 phylotype reveals a reduced genome Viability and isolation of marine bacteria by dilution culture: microbial population dynamics during adaptation to a high-grain and epibiotic parasitic lifestyle. Proc Natl Acad Sci U S A Theory, procedures and initial results. Appl Environ Microbiol diet. Appl Environ Microbiol 2010;76(22):7482-90. 2015;112(1):244-9. 1993;59(3):881-91. 37. Kong Y, Teather R, Forster R. Composition, spatial distribution 56. Bor B, Poweleit N, Bois JS, et al. Phenotypic and physiological 76. Schut F, Gottschal JC, Prins RA. Isolation and characterisation and diversity of the bacterial communities in the rumen of cows characterization of the epibiotic interaction between TM7x and its of the marine ultramicrobacterium Sphingomonas sp. strain fed diff erent forages. FEMS Microbiol Ecol 2010;74(3):612-22. basibiont Actinomyces. Microb Ecol 2016;71(1):243-55. RB2256. FEMS Microbiol Rev 2006;20:363-69. 38. Xenoulis PG, Palculict B, Allenspach K, et al. Molecular- 57. Kantor RS, Wrighton KC, Handley KM, et al. Small genomes 77. Stingl U, Cho JC, Foo W, et al. Dilution-to-extinction culturing phylogenetic characterization of microbial communities and sparse metabolisms of sediment-associated bacteria from four of psychrotolerant planktonic bacteria from permanently ice- imbalances in the small intestine of dogs with infl ammatory bowel candidate phyla. MBio 2013;4(5):e00708-13. covered lakes in the McMurdo Dry Valleys, Antarctica. Microb disease. FEMS Microbiol Ecol 2008;66(3):579-89. 58. Janssen PH. Selective enrichment and purifi cation of cultures Ecol 2008;55(3):395-405. 39. Lowe BA, Marsh TL, Isaacs-Cosgrove N, et al. Defi ning the of Methanosaeta spp. J Microbiol Methods 2003;52(2):239- 78. Sizova MV, Hohmann T, Hazen A, et al. New approaches “core microbiome” of the microbial communities in the tonsils of 44. for isolation of previously uncultivated oral bacteria. Appl Environ healthy pigs. BMC Microbiol 2012;12:20. 59. Tanaka T, Kawasaki K, Daimon S, et al. A hidden pitfall Microbiol 2012;78(1):194-203. 40. Ley RE, Hamady M, Lozupone C, et al. Evolution of mammals in the preparation of agar media undermines 79. Amann RI, Ludwig W, Schleifer KH. Phylogenetic identifi cation and their gut microbes. Science 2008;320(5883):1647-51. cultivability. Appl Environ Microbiol 2014;80(24):7659-66. and in situ detection of individual microbial cells without 41. Krogius-Kurikka L, Kassinen A, Paulin L, et al. Sequence 60. Stewart PS, Franklin MJ. Physiological heterogeneity in cultivation. Microbiol Rev 1995;59(1):143-69. analysis of percent G+C fraction libraries of human faecal biofi lms. Nat Rev Microbiol 2008;6(3):199-210. 80. Amann RI, Stromley J, Devereux R, Key R, Stahl DA. bacterial DNA reveals a high number of Actinobacteria. BMC 61. Kolenbrander PE. Oral microbial communities: biofi lms, Molecular and microscopic identifi cation of sulfate-reducing Microbiol 2009;9:68. interactions and genetic systems. Annu Rev Microbiol bacteria in multispecies biofi lms. Appl Environ Microbiol 42. Gao Z, Tseng CH, Pei Z, Blaser MJ. Molecular analysis of 2000;54:413-37. 1992;58(2):614-23. human forearm superfi cial skin bacterial biota. Proc Natl Acad 62. Kenney EB, Ash MM Jr. Oxidation reduction potential of 81. Zuger J, Luthi-Schaller H, Gmur R. Uncultivated Tannerella Sci U S A 2007;104(8):2927-32. developing plaque, periodontal pockets and gingival sulci. J BU045 and BU063 are slim segmented fi lamentous rods of 43. Fredricks DN, Fiedler TL, Marrazzo JM. Molecular Periodontol 1969;40(11):630-3. high prevalence but low abundance in infl ammatory disease- identifi cation of bacteria associated with . N 63. Bradshaw DJ, Marsh PD, Allison C, Schilling KM. Eff ect of associated dental plaques. Microbiology 2007;153(Pt Engl J Med 2005;353(18):1899-911. oxygen, inoculum composition and fl ow rate on development 11):3809-16. 44. Paster BJ, Boches SK, Galvin JL, et al. Bacterial diversity in of mixed-culture oral biofi lms. Microbiology 1996;142 ( Pt 82. Porter J, Edwards C, Morgan JA, Pickup RW. Rapid, human subgingival plaque. J Bacteriol 2001;183:3770-83. 3):623-9. automated separation of specifi c bacteria from lake water 45. Kazor CE, Mitchell PM, Lee AM, et al. Diversity of bacterial 64. Marsh PD. Dental plaque: biological signifi cance of a biofi lm and sewage by fl ow cytometry and cell sorting. Appl Environ populations on the tongue dorsa of patients with halitosis and and community life-style. J Clin Periodontol 2005;32 Suppl Microbiol 1993;59(10):3327-33. healthy patients. J Clin Microbiol 2003;41(2):558-63. 6:7-15. 83. Beall CJ, Campbell AG, Dayeh DM, et al. Single cell 46. Bik EM, Long CD, Armitage GC, et al. Bacterial 65. Whitehead NA, Barnard AM, Slater H, Simpson NJ, genomics of uncultured, health-associated Tannerella BU063 diversity in the oral cavity of 10 healthy individuals. ISME J Salmond GP. Quorum-sensing in Gram-negative bacteria. FEMS (Oral Taxon 286) and comparison to the closely related 2010;4(8):962-74. Microbiol Rev 2001;25(4):365-404. pathogen Tannerella forsythia. PLoS One 2014;9(2):e89398. 47. Ouverney CC, Armitage GC, Relman DA. Single- 66. Sturme MH, Kleerebezem M, Nakayama J, et al. Cell to 84. Datta AR, Moore MA, Wentz BA, Lane J. Identifi cation cell enumeration of an uncultivated TM7 subgroup in cell communication by autoinducing peptides in gram-positive and enumeration of Listeria monocytogenes by nonradioactive the human subgingival crevice. Appl Environ Microbiol bacteria. 2002;81(1-4):233-43. DNA probe colony hybridization. Appl Environ Microbiol 2003;69(10):6294-8. 67. Bassler BL, Losick R. Bacterially speaking. Cell 1993;59(1):144-9. 48. Kumar PS, Griff en AL, Barton JA, et al. New bacterial 2006;125(2):237-46. 85. Vartoukian SR, Palmer RM, Wade WG. Cultivation of a species associated with chronic periodontitis. J Dent Res 68. Nichols D, Lewis K, Orjala J, et al. Short peptide induces Synergistetes strain representing a previously uncultivated lineage. 2003;82(5):338-44. an “uncultivable” microorganism to grow in vitro. Appl Environ Environ Microbiol 2010;12(4):916-28. 49. Colombo AP, Boches SK, Cotton SL, et al. Comparisons of Microbiol 2008;74(15):4889-97. 86. Vartoukian SR, Downes J, Palmer RM, Wade WG. subgingival microbial profi les of refractory periodontitis, severe 69. Mukamolova GV, Kaprelyants AS, Young DI, Young M, Fretibacterium fastidiosum gen. nov., sp. nov., isolated from periodontitis and periodontal health using the human oral microbe Kell DB. A bacterial cytokine. Proc Natl Acad Sci U S A the human oral cavity. Int J Syst Evol Microbiol 2013;63(Pt identifi cation microarray. J Periodontol 2009;80(9):1421-32. 1998;95(15):8916-21. 2):458-63. 50. Hugenholtz P. Exploring prokaryotic diversity in the genomic 70. Cohen-Gonsaud M, Barthe P, Bagneris C, et al. The structure 87. Kistler JO, Pesaro M, Wade WG. Development and era. Genome Biol 2002;3(2):REVIEWS0003. of a resuscitation-promoting factor domain from Mycobacterium pyrosequencing analysis of an in-vitro oral biofi lm model. BMC 51. Ferrari BC, Binnerup SJ, Gillings M. Microcolony tuberculosis shows homology to lysozymes. Nat Struct Mol Biol Microbiol 2015;15:24. cultivation on a soil substrate membrane system selects for 2005;12(3):270-3. 88. Thompson H, Rybalka A, Moazzez R, Dewhirst FE, Wade previously uncultured soil bacteria. Appl Environ Microbiol 71. Keep NH, Ward JM, Cohen-Gonsaud M, Henderson B. WG. In vitro culture of previously uncultured oral bacterial 2005;71(12):8714-20. Wake up! Peptidoglycan lysis and bacterial nongrowth states. phylotypes. Appl Environ Microbiol 2015;81(24):8307-14.

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89. Jung D, Seo EY, Epstein SS, et al. Application of a new cultivation technology, I-tip, for studying microbial diversity in freshwater sponges of Lake Baikal, Russia. FEMS Microbiol Ecol 2014;90(2):417-23. 90. Gavrish E, Bollmann A, Epstein S, Lewis K. A trap for in situ cultivation of fi lamentous actinobacteria. J Microbiol Methods 2008;72(3):257-62. 91. Chan KG, Yin WF, Sam CK, Koh CL. A novel medium for the isolation of N-acylhomoserine lactone-degrading bacteria. J Ind Microbiol Biotechnol 2009;36(2):247-51. Paul Maimone 92. Tamaki H, Hanada S, Sekiguchi Y, Tanaka Y, Kamagata Y. Broker/Owner It is a Great Time to Sell! Inventory & Eff ect of gelling agent on colony formation in solid cultivation

Rates are Still Low & Buyer Demand is of microbial community in lake sediment. Environ Microbiol 2009;11(7):1827-34. High! Call for a Free in Office Valuation! 93. Aoyagi H, Kuroda A. Eff ects of low-shear modeled microgravity on a microbial community fi ltered through a 0.2- ARCADIA – (4) op comput G.P. Located in a well known Prof. Bldg. on a main thoroughfare. mum fi lter and its potential application in screening for novel Cash/Ins/PPO pt base. Annual Gross Collect $300K+ on a (3) day week. REDUCED microorganisms. J Biosci Bioeng 2012;114(1):73-9. BAKERSFIELD #31 - Free Stand. Bldg. & Pract. (4) op comput G.P. w excell. exposure & 94. Stott MB, Crowe MA, Mountain BW, et al. Isolation of novel th signage. (3) ops eqt./4 plumbed. Annual Gross Collect $325K+ Cash/Ins/PPO. bacteria, including a candidate division, from geothermal soils in CLAIREMONT – (3) op comput G.P. w newer eqt. 2015 Collect $265K Cash/PPO. SOLD New Zealand. Environ Microbiol 2008;10(8):2030-41. GROVER BEACH - (3) op Turnkey Office w included charts (not guaranteed). (2) ops eqt’d w 95. Bollmann A, Palumbo AV, Lewis K, Epstein SS. Isolation and newer eqt. 3rd plmbed. Digital Pano & x-ray. Dentrix. In a strip ctr. LL incentives. PENDING MONTEBELLO - (4) op comput G.P. (2) ops eqt’d. Located in a busy shop. ctr. w exposure & physiology of bacteria from contaminated subsurface sediments. visibility. Annual Gross Collect. $200K on a p.t. schedule. Cash/Ins/PPO. Seller retiring. Appl Environ Microbiol 2010;76(22):7413-9. MONTEREY PARK – (6) op comput G.P. located in a street front suite on a main thoroughfare 96. Aoi Y, Kinoshita T, Hata T, et al. Hollow-fi ber membrane w exposure/visibility. Cash/Ins/PPO/Small % Denti-Cal. Gross Collect $250K+ p.t. REDUCED chamber as a device for in situ environmental cultivation. Appl OXNARD #9 - (3) op comput G.P. & a Prof Office Condo for sale. Located on a main Environ Microbiol 2009;75(11):3826-33. thoroughfare. (3) ops eqt’d. Annual Gross Collect $200K+ p.t. Cash/Ins/PPO/HMO $4.5K/mos 97. Ben-Dov E, Kramarsky-Winter E, Kushmaro A. An in situ method Cap Cks. Digital x-rays. Low overhead. Buy & Combine or open a satellite. NEW for cultivating microorganisms using a double encapsulation SANTA BARBARA COUNTY – (3) op comput G.P. & a 1,900 sq ft Bldg. that houses the technique. FEMS Microbiol Ecol 2009;68(3):363-71. practice & a residential unit that can be rented or lived in. “Fee for Service.” No PPO, HMO or 98. Park J, Kerner A, Burns MA, Lin XN. Microdroplet-enabled Denti-Cal. 2015 Gross Collections ~ $275K on a relaxed 3½ day week. Seller refers all O.S., highly parallel co-cultivation of microbial communities. PLoS One Perio, Ortho, Endo & implant placement. Seller retiring but will assist w transition. 2011;6(2):e17019. So. EAST KERN COUNTY - (5) op comput. G.P. located in a free stand bldg. w exposure/ 99. Moon J, Kim J. Isolation of Paenibacillus pinesoli sp. nov. from visibility & signage. VERY LIMITED COMPETITION. 2015 Collect $600K. Cash/Ins/PPO. Digital x-rays & CT Scan. (6) sensors, Bldg. also available. Seller retiring. NEW forest soil in Gyeonggi-Do, Korea. J Microbiol 2014;52(4):273-7. SAN FERNANDO VALLEY #9 - (8) op comput. G.P. w modern eqt. In a prof. bldg. on a main 100. Tanaka Y, Benno Y. Application of a single-colony co-culture thoroughfare. Cash/Ins/PPO/HMO. Cap Ck approx $7K/mos. 2015 Collect $1.4M+ PENDING technique to the isolation of hitherto unculturable gut bacteria. SAN FERNANDO VALLEY #10 - Located in an exclusive area of the Valley. (5) op comput Microbiol Immunol 2015;59(2):63-70. G.P. w high end buildout. Digital x-ray and CT Scan, Laser, Dentrix s/w & (5) year old eqt. Gross 101. Vartoukian SR, Palmer RM, Wade WG. Cultivation of a Collect $1M+/yr. Cash/Ins/PPO pts. Reasonable overhead, high Net! NEW Synergistetes strain representing a previously uncultivated lineage. SAN GABRIEL VALLEY - (4) op comput G.P. w newer P&C Chairs/Eqt, All the toys & Env Microbiol 2010;12(4):916-28. whistles. Paperless, Schick digital x-rays, Solaris Steril Ctr, Soprocare Intra Oral Camera, 102. Thompson H, Rybalka A, Moazzez R, Dewhirst F, Wade W. Velscope Screen, The Wand, Air Abrasion, Electric Hand Pieces, Laser, etc. FFS, 2015 In-vitro culture of previously uncultured oral bacterial phylotypes. Gross Collect. $881K+ on a 3½ day week. (4) days of Hygiene. Seller retiring. NEW Appl Env Microbiol 2015;Sept 25, Epub ahead of print. SANTA ANA - absentee owned (6) op fully eqt’d G.P. First floor street front location on a main 103. Morris JJ, Johnson ZI, Szul MJ, Keller M, Zinser ER. thoroughfare. Exposure/visibility/signage. Cash/Ins/PPO. No HMO & No Denti-Cal. Pano eqt’d & Comput. Annual Gross Collect. ~ $500K on a (3½) to (4) day week. BACK ON MARKET Dependence of the cyanobacterium Prochlorococcus on THOUSAND OAKS (4) ops/(2) eqt’d comput. Turnkey Office w included charts. Chart included hydrogen peroxide scavenging microbes for growth at the but not guaranteed. Sirona Eqt. Located in a condo in a Prof. Bldg. on a main thoroughfare. ocean’s surface. PLoS One 2011;6(2):e16805. UPCOMING PRACTICES: Bakersfield, Beverly Hills, Central Coast, Covina, Downey, 104. Nichols D, Cahoon N, Trakhtenberg EM, et al. Use of ichip Duarte, Goleta, Oxnard, Pomona, San Gabriel, Palm Desert, Van Nuys, Visalia & West L.A.. for high-throughput in situ cultivation of “uncultivable” microbial species. Appl Environ Microbiol 2010;76(8):2445-50. D&M SERVICES: Q Practice Sales and Appraisals Q Practice Search & Matching Services THE CORRESPONDING AUTHOR, William Wade, BSc, PhD, can be Q Practice and Equipment Financing Q Locate and Negotiate Dental Lease Space reached at [email protected]. Q Expert Witness Court Testimony Q Medical/Dental Bldg. Sales & Leasing Q Pre - Death and Disability Planning Q Pre - Sale Planning P.O. 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