Are Uncultivated Bacteria Really Uncultivable?

Puspita, Indun Dewi; Kamagata, Yoichi; Tanaka, Michiko; Asano, Kozo; Nakatsu, Cindy H.

doi:10.1264/jsme2.me12092

Cited by 130 publications

(101 citation statements)

References 132 publications

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“…These techniques were originally used by environmental microbiologists to explore minority populations (131,132). These scientists were also pioneers in revitalizing techniques, facilitating the mimicking of the natural environment to increase the proportions of cultured bacterial species (129,133). The most famous example of empirical culture was recently observed.…”

Section: Environmental Microbiology As a Source Of Media For Clinicalmentioning

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SUMMARY Bacterial culture was the first method used to describe the human microbiota, but this method is considered outdated by many researchers.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: Environmental Microbiology As a Source Of Media For Clinicalmentioning

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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“…Investigators have been attempting to identify factors limiting cultivation of most microbes on agar plates for a long time (2,3), but more research is necessary since numerous phylogenetic groups of environmental microbes still lack cultivated representatives. With the advent of state-of-the-art sequencing technologies (4,5), researchers have been trying to circumvent the limitation and laboriousness of isolation work by sequencing genomes of entire communities to reveal potential novel functions and phylogenetic relationships (6)(7)(8).…”

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confidence: 99%

A Hidden Pitfall in the Preparation of Agar Media Undermines Microorganism Cultivability

Tanaka

Kawasaki

Daimon

et al. 2014

Appl Environ Microbiol

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210

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dMicrobiologists have been using agar growth medium for over 120 years. It revolutionized microbiology in the 1890s when microbiologists were seeking effective methods to isolate microorganisms, which led to the successful cultivation of microorganisms as single clones. But there has been a disparity between total cell counts and cultivable cell counts on plates, often referred to as the "great plate count anomaly," that has long been a phenomenon that still remains unsolved. Here, we report that a common practice microbiologists have employed to prepare agar medium has a hidden pitfall: when phosphate was autoclaved together with agar to prepare solid growth media (PT medium), total colony counts were remarkably lower than those grown on agar plates in which phosphate and agar were separately autoclaved and mixed right before solidification (PS medium). We used a pure culture of Gemmatimonas aurantiaca T-27 T and three representative sources of environmental samples, soil, sediment, and water, as inocula and compared colony counts between PT and PS agar plates. There were higher numbers of CFU on PS medium than on PT medium using G. aurantiaca or any of the environmental samples. Chemical analysis of PT agar plates suggested that hydrogen peroxide was contributing to growth inhibition. Comparison of 454 pyrosequences of the environmental samples to the isolates revealed that taxa grown on PS medium were more reflective of the original community structure than those grown on PT medium. Moreover, more hitherto-uncultivated microbes grew on PS than on PT medium.

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“…etagenomics provides a means for the discovery of entirely new enzymes in microorganisms and their communities without the need to culture these microorganisms as individual species, which is technically very difficult (1)(2)(3)(4)(5). Although the discovery of new enzyme activities has progressed considerably (6), it has not managed to go beyond the effective identification of enzymatic activities at a rather limited number of environmental sites.…”

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confidence: 99%

Identification and Characterization of Carboxyl Esterases of Gill Chamber-Associated Microbiota in the Deep-Sea Shrimp Rimicaris exoculata by Using Functional Metagenomics

Alcaide

Tchigvintsev

Martínez-Martínez

et al. 2015

Appl Environ Microbiol

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iThe shrimp Rimicaris exoculata dominates the fauna in deep-sea hydrothermal vent sites along the Mid-Atlantic Ridge (depth, 2,320 m). Here, we identified and biochemically characterized three carboxyl esterases from microbial communities inhabiting the R. exoculata gill that were isolated by naive screens of a gill chamber metagenomic library. These proteins exhibit low to moderate identity to known esterase sequences (<52%) and to each other (11.9 to 63.7%) and appear to have originated from unknown species or from genera of Proteobacteria related to Thiothrix/Leucothrix (MGS-RG1/RG2) and to the Rhodobacteraceae group (MGS-RG3). A library of 131 esters and 31 additional esterase/lipase preparations was used to evaluate the activity profiles of these enzymes. All 3 of these enzymes had greater esterase than lipase activity and exhibited specific activities with ester substrates (<356 U mg ؊1 ) in the range of similar enzymes. MGS-RG3 was inhibited by salts and pressure and had a low optimal temperature (30°C), and its substrate profile clustered within a group of low-activity and substrate-restricted marine enzymes. In contrast, MGS-RG1 and MGS-RG2 were most active at 45 to 50°C and were salt activated and barotolerant. They also exhibited wider substrate profiles that were close to those of highly active promiscuous enzymes from a marine hydrothermal vent (MGS-RG2) and from a cold brackish lake (MGS-RG1). The data presented are discussed in the context of promoting the examination of enzyme activities of taxa found in habitats that have been neglected for enzyme prospecting; the enzymes found in these taxa may reflect distinct habitat-specific adaptations and may constitute new sources of rare reaction specificities. Metagenomics provides a means for the discovery of entirely new enzymes in microorganisms and their communities without the need to culture these microorganisms as individual species, which is technically very difficult (1-5). Although the discovery of new enzyme activities has progressed considerably (6), it has not managed to go beyond the effective identification of enzymatic activities at a rather limited number of environmental sites. While an extensive search in the specialized literature and public databases indicated that microbial communities from approximately 1,800 different sites worldwide have been examined for their genomic content, only in approximately 200 of those locations (11% of the total) have new active clones or enzymes been identified and/or partially characterized. Thus, only a tiny fraction of the Earth's biosphere has been explored for the purpose of enzyme discovery, despite the fact that natural microbial diversity has been recognized to be the major source of new microbes (7). Such diversity also contains novel genes and functions (8) whose activities remain mostly unknown but whose potential to contribute to an innovation-based economy is recognized (9, 10).One of the habitats less explored to date in terms of examining enzyme repertoires is the deep-sea realm, where...

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Are Uncultivated Bacteria Really Uncultivable?

Cited by 130 publications

References 132 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

A Hidden Pitfall in the Preparation of Agar Media Undermines Microorganism Cultivability

Identification and Characterization of Carboxyl Esterases of Gill Chamber-Associated Microbiota in the Deep-Sea Shrimp Rimicaris exoculata by Using Functional Metagenomics

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