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

Abstract

SUMMARY Bacterial culture was the first method used to describe the human microbiota, but this method is considered outdated by many researchers.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.

Culture of previously uncultured members of the human gut microbiota by culturomics

“…The pure culture of prokaryotes, neglected in recent decades, remains essential to elucidating the role of these organisms 2 . We recently introduced microbial culturomics, a culturing approach that uses multiple culture conditions and matrix-assisted laser desorption/ionization-time of flight and 16S rRNA for identification 2 .…”

“…The pure culture of prokaryotes, neglected in recent decades, remains essential to elucidating the role of these organisms 2 . We recently introduced microbial culturomics, a culturing approach that uses multiple culture conditions and matrix-assisted laser desorption/ionization-time of flight and 16S rRNA for identification 2 . Here, we have selected the best culture conditions to increase the number of studied samples and have applied new protocols (fresh-sample inoculation; detection of microcolonies and specific cultures of Proteobacteria and microaerophilic and halophilic prokaryotes) to address the weaknesses of the previous studies [3][4][5] .…”

“…Before we initiated microbial culturomics 13 of the approximately 13,410 known bacterial and archaea species, 2,152 had been identified in humans and 688 bacteria and 2 archaea had been identified in the human gut. Culturomics consists of the application of high-throughput culture conditions to the study of the human microbiota and uses matrix-assisted laser desorption/ ionization-time of flight (MALDI-TOF) or 16S rRNA amplification and sequencing for the identification of growing colonies, some of which have been previously unidentified 2 . With the prospect of identifying new genes of the human gut microbiota, we extend here the number of recognized bacterial species and evaluate the role of this strategy in resolving the gaps in metagenomics, detailing our strategy step by step (see Methods).…”

“…Beginning in 2014, to reduce the culturomics workload and extend our stool-testing capabilities, we analysed previous studies and selected the 18 best culture conditions 2 . We performed cultures in liquid media in blood culture bottles, followed by subcultures on agar (Supplementary Table 2b).…”

“…We designed these culture conditions by analysing our first studies. The results of those studies indicated that emphasizing three components was essential: preincubation in a blood culture bottle (56% of the new species isolated), the addition of rumen fluid (40% of the new species isolated) and the addition of sheep blood (25% of the new species isolated) [2][3][4][5] . We applied this strategy to 37 stool samples from healthy individuals with different geographic provenances and from patients with different diseases (Supplementary Table 1).…”