New Approaches for Isolation of Previously Uncultivated Oral Bacteria

Sizova, Maria V.; Hohmann, Tine; Hazen, Amanda; Paster, Bruce J.; Halem, S.; Murphy, Christina; Panikov, N. S.; Epstein, Slava S.

doi:10.1128/aem.06813-11

Cited by 93 publications

(80 citation statements)

References 56 publications

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“…Recently, Sizova et al extended the cultured bacterial repertoire of the human oral cavity specifically as a result of the growth of anaerobic species (155). Those authors concomitantly developed in vivo culture methods to specifically enrich for species belonging to the oral microbiota (minitrap method), single-cell long-term cultivation to decrease the effects of fast-growing microorganisms, and modifications of usual enrichment techniques, notably the use of culture conditions without sugar.…”

Section: From Environmental To Clinical Microbiologymentioning

confidence: 99%

The Rebirth of Culture in Microbiology through the Example of Culturomics To Study Human Gut Microbiota

et al. 2015

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SUMMARY Bacterial culture was the first method used to describe the human microbiota, but this method is considered outdated by many researchers. Metagenomics studies have since been applied to clinical microbiology; however, a “dark matter” of prokaryotes, which corresponds to a hole in our knowledge and includes minority bacterial populations, is not elucidated by these studies. By replicating the natural environment, environmental microbiologists were the first to reduce the “great plate count anomaly,” which corresponds to the difference between microscopic and culture counts. The revolution in bacterial identification also allowed rapid progress. 16S rRNA bacterial identification allowed the accurate identification of new species. Mass spectrometry allowed the high-throughput identification of rare species and the detection of new species. By using these methods and by increasing the number of culture conditions, culturomics allowed the extension of the known human gut repertoire to levels equivalent to those of pyrosequencing. Finally, taxonogenomics strategies became an emerging method for describing new species, associating the genome sequence of the bacteria systematically. We provide a comprehensive review on these topics, demonstrating that both empirical and hypothesis-driven approaches will enable a rapid increase in the identification of the human prokaryote repertoire.

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Section: From Environmental To Clinical Microbiologymentioning

confidence: 99%

The Rebirth of Culture in Microbiology through the Example of Culturomics To Study Human Gut Microbiota

et al. 2015

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“…The Wade group has grown previously unculturable cluster A Synergistetes isolates from subgingival plaque in coculture with other bacteria from the mouth (83). In another approach to access the oral microbiome, Epstein and coworkers developed a miniature version of the trap described above that could be carried in a volunteer's mouth (73). The diversity obtained from the trap was compared with dilution to extinction and direct pour plating in petri plates.…”

Section: Approaches To Culturing the Missing Bacterial Diversitymentioning

confidence: 99%

Growing Unculturable Bacteria

2012

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The bacteria that can be grown in the laboratory are only a small fraction of the total diversity that exists in nature. At all levels of bacterial phylogeny, uncultured clades that do not grow on standard media are playing critical roles in cycling carbon, nitrogen, and other elements, synthesizing novel natural products, and impacting the surrounding organisms and environment. While molecular techniques, such as metagenomic sequencing, can provide some information independent of our ability to culture these organisms, it is essentially impossible to learn new gene and pathway functions from pure sequence data. A true understanding of the physiology of these bacteria and their roles in ecology, host health, and natural product production requires their cultivation in the laboratory. Recent advances in growing these species include coculture with other bacteria, recreating the environment in the laboratory, and combining these approaches with microcultivation technology to increase throughput and access rare species. These studies are unraveling the molecular mechanisms of unculturability and are identifying growth factors that promote the growth of previously unculturable organisms. This minireview summarizes the recent discoveries in this area and discusses the potential future of the field. What is an unculturable bacterium? While at first glance, there appears to be a contradiction in the title of this review, in this context, "unculturable" indicates that current laboratory culturing techniques are unable to grow a given bacterium in the laboratory. That all organisms must be growing in their natural environment is axiomatic; that many we cannot currently grow will be cultured in the future is certain. Therefore, "unculturable" does not mean "can never be cultured" but, rather, signifies that we lack critical information on their biology, and this presents both challenges and opportunities. These opportunities are the chance to learn the molecular principles behind this recalcitrant growth, allowing us to add that information to our repertoire of microbiological techniques and gaining access to previously hidden metabolic diversity that will provide new natural products and reveal factors that can contribute to both ecological balance and host health. This review examines the recent approaches that microbiologists are employing to convert currently unculturable bacteria into cultured isolates in the laboratory while concurrently beginning to discover the mechanisms behind their apparent unculturability.

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“…All media were supplemented with L-cysteine-HCl as a reducing agent (Sizova et al, 2012). Cell morphology was observed with a Leica DMBL light microscope equipped with phase-contrast optics.…”

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confidence: 99%

“…On: Sat, 12 May 2018 09:31:13 L-cysteine-HCl as a reducing agent and isolated in pure culture on agar-BM under an anaerobic atmosphere (2 % H 2 , 1 % CO 2 , 97 % N 2 ) (Sizova et al, 2012). The Human Oral Microbiome Database (HOMD) classified the isolated strains as members of oral taxon 108 (Chen et al, 2010).…”

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confidence: 99%

“…The peptidoglycan structure was analysed in the hydrolysates (4 M HCl, 100 uC, 16 h) according to the method of Rhuland et al (1955) and by GC/MS analysis after isolation of the peptidoglycan and its hydrolysis (Schumann, 2011) at the Identification Service of the German Collection of Microorganisms and Cell Cultures. 16S rRNA gene sequences were compared with those available from GenBank; phylogenetic analysis was performed as described previously (Sizova et al, 2012). Whole genome sequencing of strains ACB1 T and ACB7 T was carried out by the Broad Institute of Harvard and MIT, and the data are available at http://www.broadinstitute.org/.…”

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confidence: 99%

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Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov., obligately anaerobic bacteria from the human oral cavity, and emended description of the genus Oribacterium

Sizova

Muller

Stancyk

et al. 2014

International Journal of Systematic and Evolutionary Microbiology

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Three strictly anaerobic, Gram-positive, non-spore-forming, rod-shaped, motile bacteria, designated strains ACB1 T , ACB7 T and ACB8, were isolated from human subgingival dental plaque. All strains required yeast extract for growth. Strains ACB1 T and ACB8 were able to grow on glucose, lactose, maltose, maltodextrin and raffinose; strain ACB7 T grew weakly on sucrose only. The growth temperature range was 30-42 6C with optimum growth at 37 6C. Major metabolic fermentation end products of strain ACB1 T were acetate and lactate; the only product of strains ACB7 T and ACB8 was acetate. Major fatty acids of strain ACB1 T were C 14 : 0 , C 16 : 0 , C 16 : 1 v7c dimethyl aldehyde (DMA) and C 18 : 1 v7c DMA. Major fatty acids of strain ACB7 T were C 12 : 0 , C 14 : 0 , C 16 : 0 , C 16 : 1 v7c and C 16 : 1 v7c DMA. The hydrolysate of the peptidoglycan contained meso-diaminopimelic acid, indicating peptidoglycan type A1c. Genomic DNA G+C content varied from 42 to 43.3 % between strains. According to 16S rRNA gene sequence phylogeny, strains ACB1 T , ACB8 and ACB7 T formed two separate branches within the genus Oribacterium, with 98.1-98.6 % sequence similarity to the type strain of the type species, Oribacterium sinus. Predicted DNA-DNA hybridization values between strains ACB1 T , ACB8, ACB7 T and O. sinus F0268 were ,70 %. Based on distinct genotypic and phenotypic characteristics, strains ACB1 T and ACB8, and strain ACB7 T are considered to represent two distinct species of the genus Oribacterium, for which the names Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov. are proposed. The type strains are ACB1 T (5DSM

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New Approaches for Isolation of Previously Uncultivated Oral Bacteria

Cited by 93 publications

References 56 publications

The Rebirth of Culture in Microbiology through the Example of Culturomics To Study Human Gut Microbiota

The Rebirth of Culture in Microbiology through the Example of Culturomics To Study Human Gut Microbiota

Growing Unculturable Bacteria

Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov., obligately anaerobic bacteria from the human oral cavity, and emended description of the genus Oribacterium

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