Description of Agathobaculum massiliense sp. nov., a new bacterial species prevalent in the human gut and predicted to produce indole and tryptophan based on genomic analysis.

The novel bacterial strain Marseille-P4005T was isolated from the stool sample of a healthy donor. It is a Gram-stain negative, non-motile, non-spore-forming rod. It grew optimally at 37 °C and at pH 7.0 on 5% sheep blood-enriched Columbia agar after preincubation in a blood-culture bottle supplemented with rumen and blood. This strain does not ferment monosaccharides (except D-tagatose), disaccharides, or polymeric carbohydrates. The major cellular fatty acids were hexadecenoic (24.6%), octadecanoic (22.8%), and tetradecanoic (20.1%) acids. Next-generation sequencing revealed a genome size of 3.2 Mbp with a 56.4 mol% G + C. Phylogenetic analysis based on the 16S rRNA gene highlighted Agathobaculum desmolans strain ATCC 43058T as the closest related strain. The OrthoANI, AAI, and digital DNA-DNA hybridization values were below the critical thresholds of 95%, 95-96%, and 70%, respectively, to define a novel bacterial species. Antibiotic resistance genes APH(3')-IIIa, erm(B), and tet(W) were detected with high identity percentages of 100%, 98.78%, and 97.18% for each gene, respectively. The APH(3')-IIIa gene confers resistance to amikacin, erm(B) gene confers resistance to erythromycin, lincomycin, and clindamycin, while tet(W) gene confers resistance to doxycycline and tetracycline. Based on KEGG BlastKOALA analyses, the annotation results showed that our strain could use glucose to produce L-lactate and pyruvate but not acetate or ethanol. Also, strain Marseille-P4005T was predicted to use phenylalanine to produce indole, a major intercellular signal molecule within the gut microbial ecosystem. Through having a gene coding for tryptophan synthase beta chain (trpB), strain Marseille-P4005T could produce L-tryptophan (an essential amino acid) from indole. Strain Marseille-P4005T showed its highest prevalence in the human gut (34.19%), followed by the reproductive system (17.98%), according to a query carried out on the Integrated Microbial NGS (IMNGS) platform. Based on phylogenetic, phenotypic, and genomic analyses, we classify strain Marseille-P4005T (= CSUR P4005 = CECT 9669), a novel species within the genus Agathobaculum, for which the name of Agathobaculum massiliense sp. nov. is proposed.

Draft genomes and descriptions of Urmitella timonensis gen. nov., sp. nov. and Marasmitruncus massiliensis gen. nov., sp. nov., isolated from severely malnourished African children using culturomics.

Two strains, designated as Marseille-P2918T and Marseille-P3646T, were isolated from a 14-week-old Senegalese girl using culturomics: Urmitella timonensis strain Marseille-P2918T (= CSUR P2918, = DSM 103634) and Marasmitruncus massiliensis strain Marseille-P3646T (= CSUR P3646, = CCUG72353). Both strains were rod-shaped, anaerobic, spore forming motile bacteria. The 16S rRNA gene sequences of strains Marseille-P2918T (LT598554) and Marseille-P3646T (LT725660) shared 93.25% and 94.34% identity with Tissierella praeacuta ATCC 25539T and Anaerotruncus colihominis CIP 107754T, their respective phylogenetically closest species with standing in nomenclature. Therefore, strain Marseille-P2918T is classified within the family Tissierellaceae and order Tissierellales whereas strain Marseille-P3646T is classified within the family Oscillospiraceae and order Eubacteriales. The genome of strain Marseille-P2918T had a size of 2.13 Mb with a GC content of 50.52% and includes six scaffolds and six contigs, and that of strain Marseille-P3646T was 3.76 Mbp long consisting of five contigs with a 50.04% GC content. The genomes of both strains presented a high percentage of genes encoding enzymes involved in genetic information and processing, suggesting a high growth rate and adaptability. These new taxa are extensively described and characterised in this paper, using the concept of taxono-genomic description.

Anaerococcus jeddahensis sp. nov., a New Bacterial Species Isolated From Healthy Nomadic Bedouin Woman From Saudi Arabia.

An understanding of the microbial diversity of the human body has generated significant interest in recent years. With the advent of MALDI-TOF mass spectrometry, high-speed sequencing, and the rebirth of microbial culture, knowledge of human microbiota is growing. Using culturomics, a strategy to explore the microbial diversity of samples, coupled with a taxono-genomic strategy, we isolated a new bacterium named Anaerococcus jeddahensis sp. nov. strain SB3T. This strain was isolated from the stool sample of a healthy nomadic Bedouin woman from Saudi Arabia. Here, we describe the characteristics of this organism, and the complete genome sequence and annotation. Strain SB3T is a Gram-positive obligate anaerobic coccus which is non-motile and non-spore forming. Fatty acid analysis shows that the major fatty acid is by far hexadecanoic acid (C16:0; 52%). Its genome is 1,903,534 bp long and has 29.70 mol% of G+C content. It contains 1756 protein-coding genes and 53 RNA genes. These results show that strategy provides a better understanding of the microorganism and that is a good methodology for microbial identification and characterization.

Antimicrobial Susceptibility Testing for Colistin: Extended Application of Novel Quantitative and Morphologic Assay Using Scanning Electron Microscopy.

Background: Colistin (Polymyxin E) has reemerged in the treatment of MDR Gram-negative infections. Traditional Colistin AST methods have long turnaround times and are cumbersome for routine use. We present a SEM-AST technique enabling rapid detection of Colistin resistance through direct observation of morphological and quantitative changes in bacteria exposed to Colistin. Methods: Forty-four Gram-negative reference organisms were chosen based on their Colistin susceptibility profiles. Bacterial suspensions of ∼107 CFU/mL were exposed to Colistin at EUCAST-ECOFF, with controls not exposed, incubated at 37°C, and then sampled at 0, 15, 30, 60, and 120 minutes. Phosphotungstic Acid (PTA) staining was applied, followed by SEM imaging using Hitachi TM4000PlusII-Tabletop-SEM at ×2000, ×5000 and ×7000 magnifications. Bacterial viability analysis was performed for all conditions by quantifying viable and dead organisms based on PTA-staining and morphologic changes. Results: We identified a significant drop in the percentage of viable organisms starting 30 minutes after exposure in susceptible strains, as compared to nonsignificant changes in resistant strains across all tested organisms. The killing effect of Colistin was best observed after 120 minutes of incubation with the antibiotic, with significant changes in morphologic features, including bacterial inflation, fusion, and lysis, observed as early as 30 minutes. Our observation matched the results of the gold standard-based broth microdilution method. Conclusions: We provide an extended application of the proof of concept for the utilization of the SEM-AST assay for Colistin for a number of clinically relevant bacterial species, providing a rapid and reliable susceptibility profile for a critical antibiotic.

Rapid microbial viability assay using scanning electron microscopy: a proof-of-concept using Phosphotungstic acid staining.

Multiple stains have been historically utilized in electron microscopy to provide proper contrast and superior image quality enabling the discovery of ultrastructures. However, the use of these stains in microbiological viability assessment has been limited. Phosphotungstic acid (PTA) staining is a common negative stain used in scanning electron microscopy (SEM). Here, we investigate the feasibility of a new SEM-PTA assay, aiming to determine both viable and dead microbes. The optimal sample preparation was established by staining bacteria with different PTA concentrations and incubation times. Once the assay conditions were set, we applied the protocol to various samples, evaluating bacterial viability under different conditions, and comparing SEM-PTA results to culture. The five minutes 10% PTA staining exhibited a strong distinction between viable micro-organisms perceived as hypo-dense, and dead micro-organisms displaying intense internal staining which was confirmed by high Tungsten (W) peak on the EDX spectra. SEM-PTA viability count after freezing, freeze-drying, or oxygen exposure, were concordant with culture. To our knowledge, this study is the first contribution towards PTA staining of live and dead bacteria. The SEM-PTA strategy demonstrated the feasibility of a rapid, cost-effective and efficient viability assay, presenting an open-view of the sample, and providing a potentially valuable tool for applications in microbiome investigations and antimicrobial susceptibility testing.

Use of scanning electron microscopy and energy dispersive X-ray for urine analysis: A preliminary investigation.

Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) are powerful tools to study the ultrastructure of numerous specimens and to determine their elemental composition, respectively. However, results have not yet been reported on their application to urine samples in routine clinical laboratory practice. Herein we investigate urine sediment by using SEM and EDX to detect and identify different urine components. A total of 206 urine samples from patients with and without urinary tract infections were analyzed using SEM and EDX. Microorganisms, crystals, epithelial cells, leukocytes, and erythrocytes were targeted in urine sediment samples. The identification of urine components was based on their morphology, size, contrast, and elemental composition. SEM-analysis allowed us to identify and classify microorganisms in urine sediments into the categories of gram-negative bacilli, cluster cocci, chain cocci, gram-negative bacilli, gram-positive bacilli, and yeasts. In addition, various types of epithelial cells such as renal, transitional, and squamous epithelial cells were found. Furthermore, leukocytes and erythrocytes were well identified, with the detection of various morphological forms of erythrocytes, such as dysmorphic and isomorphic erythrocytes. Using SEM-EDX analysis, calcium oxalate was the most frequently-identified crystal (92.0%), with prominent peaks of C, O, and Ca elements, followed by struvite (6%), with peaks of Mg, P, O, and N. These preliminary data suggest that the two complementary SEM-EDX analyses can be used to detect and identify microorganisms and crystals in urine samples. Further studies are still needed to apply SEM-EDX to urine sediment analysis. SEM-EDX analyses provided comparative results with the routine results, with accurate identification, high resolution and deep focus compared to the routine urinalysis SEM-analysis allowed us to identify and classify microorganisms in urine sediments into the categories of gram-negative bacilli, cluster cocci, chain cocci, gram-negative bacilli, gram-positive bacilli and yeasts. SEM-EDX analysis enabled the accurate identification of crystals based on both morphology and elemental composition.

A Tetragenococcus halophilus human gut isolate.

Tetragenococcus halophilus (T. halophilus) is a facultative anaerobic, coccus-shaped halophilic lactic acid-producing bacterium previously detected and cultured in various salty foods and credited for beneficial effects on human health. In this study, we investigated the presence of T. halophilus in human samples using a polyphasic approach including scanning electron microscopy, molecular biology methods and microbial culture. This unique investigation yielded the unprecedented presence of T. halophilus in human feces samples, thus enriching the repertoire of halophilic microorganisms colonizing the human gastrointestinal tract with the isolation and culture of T. halophilus for the first time in humans. Using the E-test strips, the MIC was assessed for T. halophilus strain CSURQ6002: rifampicin (MIC at 0.002 µg/mL), benzylpenicillin (MIC at 0.094 µg/mL), amoxicillin (MIC at 0.5 µg/mL), erythromycin (MIC at 2 µg/mL), clindamycin (MIC at 4 µg/mL), and vancomycin (MIC at 8 µg/mL). However, this strain showed a MIC up to 256 µg/mL for ciprofloxacin, fosfomycin, doxycyclin, imipenem, and colistin. In-silico profiling derived from whole genome sequencing (NCBI accession number: PRJNA780809), was confirmed. This discovery suggested that T. halophilus was part of the human digestive microbiota and that its potential role on human health should be considered.

A preliminary investigation into bacterial viability using scanning electron microscopy-energy-dispersive X-ray analysis: The case of antibiotics.

The metabolic stages of bacterial development and viability under different stress conditions induced by disinfection, chemical treatments, temperature, or atmospheric changes have been thoroughly investigated. Here, we aim to evaluate early metabolic modifications in bacteria following induced stress, resulting in alterations to bacterial metabolism. A protocol was optimized for bacterial preparation using energy-dispersive X-ray (EDX) microanalysis coupled with scanning electron microscopy (SEM), followed by optimizing EDX data acquisition and analysis. We investigated different preparation methods aiming to detect modifications in the bacterial chemical composition at different states. We first investigated Escherichia coli, acquiring data from fresh bacteria, after heat shock, and after contact with 70% ethanol, in order to prove the feasibility of this new strategy. We then applied the new method to different bacterial species following 1 h of incubation with increasing doses of antibiotics used as a stress-inducing agent. Among the different materials tested aiming to avoiding interaction with bacterial metabolites, phosphorous-doped silicon wafers were selected for the slide preparation. The 15 kV acceleration voltage ensured all the chemical elements of interest were excited. A thick layer of bacterial culture was deposited on the silicon wafer providing information from multiple cells and intra-cellular composition. The EDX spectra of fresh, heat-killed, and alcohol-killed E. coli revealed important modifications in magnesium, potassium, and sodium. Those same alterations were detected when applying this strategy to bacteria exposed to antibiotics. Tests based on SEM-EDX acquisition systems would provide early predictions of the bacterial viability state in different conditions, yielding earlier results than culture.

Antimicrobial susceptibility testing for Gram positive cocci towards vancomycin using scanning electron microscopy.

The rapid detection of resistant bacteria has become a challenge for microbiologists worldwide. Numerous pathogens that cause nosocomial infections are still being treated empirically and have developed resistance mechanisms against key antibiotics. Thus, one of the challenges for researchers has been to develop rapid antimicrobial susceptibility testing (AST) to detect resistant isolates, ensuring better antibiotic stewardship. In this study, we established a proof-of-concept for a new strategy of phenotypic AST on Gram-positive cocci towards vancomycin using scanning electron microscopy (SEM). Our study evaluated the profiling of Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus after brief incubation with vancomycin. Sixteen isolates were analysed aiming to detect ultrastructural modifications at set timepoints, comparing bacteria with and without vancomycin. After optimising slides preparation and micrographs acquisition, two analytical strategies were used. The high magnification micrographs served to analyse the division of cocci based on the ratio of septa, along with the bacterial size. Susceptible strains with vancomycin showed a reduced septa percentage and the average surface area was consequently double that of the controls. The resistant bacteria revealed multiple septa occurring at advanced timepoints. Low magnification micrographs made it possible to quantify the pixels at different timepoints, confirming the profiling of cocci towards vancomycin. This new phenotypic AST strategy proved to be a promising tool to discriminate between resistant and susceptible cocci within an hour of contact with vancomycin. The analysis strategies applied here would potentially allow the creation of artificial intelligence algorithms for septa detection and bacterial quantification, subsequently creating a rapid automated SEM-AST assay.

Scanning Electron Microscope: A New Potential Tool to Replace Gram Staining for Microbe Identification in Blood Cultures.

Blood culture is currently the most commonly used method for diagnosing sepsis and bloodstream infections. However, the long turn-around-time to achieve microbe identification remains a major concern for clinical microbiology laboratories. Gram staining for preliminary identification remains the gold standard. We developed a new rapid strategy using a tabletop scanning electron microscope (SEM) and compared its performance with Gram staining for the detection of micro-organisms and preliminary identification directly from blood cultures. We first optimised the sample preparation for twelve samples simultaneously, saving time on imaging. In this work, SEM proved its ability to identify bacteria and yeasts in morphotypes up to the genus level in some cases. We blindly tested 1075 blood cultures and compared our results to the Gram staining preliminary identification, with MALDI-TOF/MS as a reference. This method presents major advantages such as a fast microbe identification, within an hour of the blood culture being detected positive, low preparation costs, and data traceability. This SEM identification strategy can be developed into an automated assay from the sample preparation, micrograph acquisition, and identification process. This strategy could revolutionise urgent microbiological diagnosis of infectious diseases.

Rapid Detection of Imipenem Resistance in Gram-Negative Bacteria Using Tabletop Scanning Electron Microscopy: A Preliminary Evaluation.

Background: Enabling faster Antimicrobial Susceptibility Testing (AST) is critical, especially to detect antibiotic resistance, to provide rapid and appropriate therapy and to improve clinical outcomes. Although several standard and automated culture-based methods are available and widely used, these techniques take between 18 and 24 h to provide robust results. Faster techniques are needed to reduce the delay between test and results. Methods: Here we present a high throughput AST method using a new generation of tabletop scanning electron microscope, to evaluate bacterial ultra-structural modifications associated with susceptibilities to imipenem as a proof of concept. A total of 71 reference and clinical strains of Gram-negative bacteria were used to evaluate susceptibility toward imipenem after 30, 60, and 90 min of incubation. The length, width and electron density of bacteria were measured and compared between imipenem susceptible and resistant strains. Results: We correlated the presence of these morphological changes to the bacterial susceptibility and their absence to the bacterial resistance (e.g., Pseudomonas aeruginosa length without [2.24 ± 0.61 µm] and with [2.50 ± 0.68 µm] imipenem after 30 min [p = 3.032E-15]; Escherichia coli width without [0.92 ± 0.07 µm] and with [1.28 ± 0.19 µm] imipenem after 60 min [p = 1.242E-103]). We validated our method by a blind test on a series of 58 clinical isolates where all strains were correctly classified as susceptible or resistant toward imipenem. Conclusion: This method could be a potential tool for rapidly identifying carbapenem-resistance in Enterobacterales in clinical microbiology laboratories in <2 h, allowing the empirical treatment of patients to be rapidly adjusted.

Running after ghosts: are dead bacteria the dark matter of the human gut microbiota?

The human gut microbiota has been explored by a wide range of culture-dependent and culture-independent methods, revealing that many microbes remain uncharacterized and uncultured. In this work, we aimed to confirm the hypothesis that some of the species present in the human gut microbiota remain uncultured not because of culture limitations, but because all members of such species are dead before reaching the end of the gastro-intestinal tract.We evaluate this phenomenon by studying the microbial viability and culturability of the human gut microbiota from the fresh fecal materials of eight healthy adults. For the first time, we applied fluorescence-activated cell sorting (FACS) combined with 16S metagenomics analysis and microbial culturomics.We identified a total of 1,020 bacterial OTUs and 495 bacterial isolates through metagenomics and culturomics, respectively. Among the FACS metagenomics results, only 735 bacterial OTUs were alive, comprising on average 42% of known species and 87% of relative abundance per individual. The remaining uncultured bacteria were rare, dead, or injured.Our strategy allowed us to shed light on the dark matter of the human gut microbiota and revealed that both metagenomics and culturomics approaches are needed for greater insight into the diversity and richness of bacteria in the human gut microbiota. Further work on culture is needed to enhance the repertoire of cultured gut bacteria by targeting low abundance bacteria and optimizing anaerobic sample conditioning and processing to preserve the viability of bacteria.

A new protectant medium preserving bacterial viability after freeze drying.

Freeze-drying technology has been widely considered for decades as a suitable technique to preserve microorganisms. However, protective agents must be added prior to freeze drying to improve the survival and storage stability of the bacteria. The objective of our study was to evaluate the effect of a new protectant medium containing sucrose (10 %), trehalose (10 %), skimmed milk (10 %) and antioxidants on the viability of gut bacteria under different storage conditions. Two strains were tested, Escherichia coli and Akkermansia muciniphila, as examples of facultative aerobic and anaerobic bacteria, respectively. We studied the cell viability and bacterial morphology in 5 fecal samples in the presence and absence of this protectant medium using plating technique, flow cytometry and scanning electron microscopy. The results of bacterial viability assessed by plating method showed that the protectant medium yielded higher survival rates for both strains whatever the storage conditions (85-93 %) compared to normal saline solution (0.36-37.50 %). It also showed its effectiveness on fecal samples, where bacterial viability after freeze-drying was 89.47 ± 7.63 % and 84.01 ± 7.44 %, as evidenced by flow cytometry analysis and plating method. However unprotected samples showed the lowest cell viability at 19.01 ± 12.88 % and 13.23 ± 9.56 %, as measured by flow cytometry and plating method. In addition, bacterial size and shape were conserved in the protectant medium. In contrast, storage without protectant medium severely damaged bacterial morphology. In conclusion, our study is the first to use morphological features as well as culture-dependant and culture-independent tests to evaluate the effectiveness of a new protectant medium.

Rapid Scanning Electron Microscopy Detection and Sequencing of Severe Acute Respiratory Syndrome Coronavirus 2 and Other Respiratory Viruses.

There is an urgent need for accurate and rapid testing methods to quickly identify infected patients as well as asymptomatic carriers, in order to prevent the spread of emerging viruses. Here, we developed a rapid testing strategy by scanning electron microscopy capable of detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses directly from patients. We evaluated our results by comparing them to real-time reverse transcription-polymerase chain reaction (RT-PCR) and metagenomic sequencing results. We correlated the presence of the SARS-CoV-2 to the viral load, where samples with Ct values lower than 18 were all detected by scanning electron microscopy (SEM). The sensitivity deacresed progressively with higher Ct values. In addition, we found a correlation with metagenomic sequencing, where all samples detected by SEM were sequenced and viral sequences were easily recovered. Following this study, SEM proved its efficiency as a frontline method for directly detecting previously unknown microorganisms that cannot be targeted by molecular methods and can cause potential outbreaks.

Alcohol pretreatment of stools effect on culturomics.

Recent studies have used ethanol stool disinfection as a mean of promoting valuable species' cultivation in bacteriotherapy trials for Clostridium difficile infections (CDI) treatment with a particular focus on sporulating bacteria. Moreover, the culturomic approach has considerably enriched the repertoire of cultivable organisms in the human gut in recent years. This study aimed to apply this culturomic approach on fecal donor samples treated with ethanol disinfection to evidence potential beneficial microbes that could be used in bacteriotherapy trials for the treatment of CDI. Thereby, a total of 254 bacterial species were identified, 9 of which were novel. Of these, 242 have never been included in clinical trials for the treatment of CDIs, representing potential new candidates for bacteriotherapy trials. While non-sporulating species were nevertheless more affected by the ethanol pretreatment than sporulating species, the ethanol disinfection technique did not specifically select bacteria able to sporulate, as suggested by previous studies. Furthermore, some bacteria previously considered as potential candidates for bacteriotherapy have been lost after ethanol treatment. This study, while enriching the bacterial repertoire of the human intestine, would nevertheless require determining the exact contribution of each of species composing the bacterial consortia intended to be administered for CDI treatment.

Optimization and standardization of the culturomics technique for human microbiome exploration.

Culturomics is a high-throughput culture approach that has dramatically contributed to the recent renewal of culture. While metagenomics enabled substantial advances in exploring the microbiota, culturomics significantly expanded our knowledge regarding the bacterial gut repertoire through the discovery and the description of hundreds of new taxa. While this approach relies on the variation of culture conditions and media, we have tested so far more than 300 conditions since the beginning of culturomics studies. In this context, we aimed herein to identify the most profitable conditions for optimizing culturomics approach. For this purpose, we have analysed a set of 58 culturomics conditions that were previously applied to 8 faecal specimens, enabling the isolation of 497 bacterial species. As a result, we were able to reduce the number of conditions used to isolate these 497 of more than a half (i.e. to 25 culture conditions). We have also established a list of the 16 conditions that allowed to capture 98% of the total number of species previously isolated. These data constitute a methodological starting point for culture-based microbiota studies by improving the culturomics workflow without any loss of captured bacterial diversity.

Among Live and Dead Bacteria, the Optimization of Sample Collection and Processing Remains Essential in Recovering Gut Microbiota Components.

The human gut microbiota plays an important role in human health. Accessing and culturing the gut microbes remains critical in microbiology. The culturomics approach, combined with sequencing strategies, provides new insights in the study of gut microbiota components. However, we are still far from having described all the microbes. Many factors are involved in recovering as much bacteria as possible. One of the most important factors is sample preparation and conservation. The aim of the present work was to evaluate three different types of stool sample conditioning by mainly studying the effect of atmospheric oxygen on bacterial viability and culturability. Quantitative analysis of fecal samples from eight different healthy individuals was carried out using a culture-independent method (flow cytometry) and a culture-dependent method (plating technique). We found that the cultured bacteria reached a 50% yield when the samples were exposed to oxygen for 120 min without any protectant medium, while the percentage of culturability increased to 67% in the presence of antioxidants. More importantly, when samples were exposed to oxygen for less than 2 min, combined with the work under the anaerobic chamber, no discordance was found between the two counting techniques and the culturability increased to 87%. Our study confirmed the importance of sample conditioning to preserve the bacterial viability in samples, especially for oxygen-sensitive intestinal bacteria.

Extensive culturomics of 8 healthy samples enhances metagenomics efficiency.

Molecular approaches have long led to the assumption that the human gut microbiota is dominated by uncultivable bacteria. The recent advent of large-scale culturing methods, and in particular that of culturomics have demonstrated that these prokaryotes can in fact be cultured. This is increasing in a dramatic manner the repertoire of commensal microbes inhabiting the human gut. Following eight years of culturomics approach applied on more than 900 samples, we propose herein a remake of the pioneering study applying a dual approach including culturomics and metagenomics on a cohort of 8 healthy specimen. Here we show that culturomics enable a 20% higher richness when compared to molecular approaches by culturing 1 archaeal species and 494 bacterial species of which 19 were new taxa. Species discovered as a part of previous culturomics studies represent 30% of the cultivated isolates, while sequences derived from these new taxa enabled to increase by 22% the bacterial richness retrieved by metagenomics. Overall, 67% of the total reads generated were covered by cultured isolates, significantly reducing the hidden content of sequencing methods compared to the pioneering study. By redefining culture conditions to recover microbes previously considered fastidious, there are greater opportunities than ever to eradicate metagenomics dark matter.

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

Metagenomics revolutionized the understanding of the relations among the human microbiome, health and diseases, but generated a countless number of sequences that have not been assigned to a known microorganism1. The pure culture of prokaryotes, neglected in recent decades, remains essential to elucidating the role of these organisms2. 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 identification2. 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 studies3-5. We identified 1,057 prokaryotic species, thereby adding 531 species to the human gut repertoire: 146 bacteria known in humans but not in the gut, 187 bacteria and 1 archaea not previously isolated in humans, and 197 potentially new species. Genome sequencing was performed on the new species. By comparing the results of the metagenomic and culturomic analyses, we show that the use of culturomics allows the culture of organisms corresponding to sequences previously not assigned. Altogether, culturomics doubles the number of species isolated at least once from the human gut.