Preliminary landscape of Candidatus Saccharibacteria in the human microbiome.

Introduction: Candidate Phyla Radiation (CPR) and more specifically Candidatus Saccharibacteria (TM7) have now been established as ubiquitous members of the human oral microbiota. Additionally, CPR have been reported in the gastrointestinal and urogenital tracts. However, the exploration of new human niches has been limited to date. Methods: In this study, we performed a prospective and retrospective screening of TM7 in human samples using standard PCR, real-time PCR, scanning electron microscopy (SEM) and shotgun metagenomics. Results: Using Real-time PCR and standard PCR, oral samples presented the highest TM7 prevalence followed by fecal samples, breast milk samples, vaginal samples and urine samples. Surprisingly, TM7 were also detected in infectious samples, namely cardiac valves and blood cultures at a low prevalence (under 3%). Moreover, we observed CPR-like structures using SEM in all sample types except cardiac valves. The reconstruction of TM7 genomes in oral and fecal samples from shotgun metagenomics reads further confirmed their high prevalence in some samples. Conclusion: This study confirmed, through their detection in multiple human samples, that TM7 are human commensals that can also be found in clinical settings. Their detection in clinical samples warrants further studies to explore their role in a pathological setting.

Candidate Phyla Radiation, an Underappreciated Division of the Human Microbiome, and Its Impact on Health and Disease.

Candidate phyla radiation (CPR) is an emerging division of the bacterial domain within the human microbiota. Still poorly known, these microorganisms were first described in the environment in 1981 as "ultramicrobacteria" with a cell volume under 0.1 µm3 and were first associated with the human oral microbiota in 2007. The evolution of technology has been paramount for the study of CPR within the human microbiota. In fact, since these ultramicrobacteria have yet to be axenically cultured despite ongoing efforts, progress in imaging technology has allowed their observation and morphological description. Although their genomic abilities and taxonomy are still being studied, great strides have been made regarding their taxonomic classification, as well as their lifestyle. In addition, advancements in next-generation sequencing and the continued development of bioinformatics tools have allowed their detection as commensals in different human habitats, including the oral cavity and gastrointestinal and genital tracts, thus highlighting CPR as a nonnegligible part of the human microbiota with an impact on physiological settings. Conversely, several pathologies present dysbiosis affecting CPR levels, including inflammatory, mucosal, and infectious diseases. In this exhaustive review of the literature, we provide a historical perspective on the study of CPR, an overview of the methods available to study these organisms and a description of their taxonomy and lifestyle. In addition, their distribution in the human microbiome is presented in both homeostatic and dysbiotic settings. Future efforts should focus on developing cocultures and, if possible, axenic cultures to obtain isolates and therefore genomes that would provide a better understanding of these ultramicrobacteria, the importance of which in the human microbiome is undeniable.

Limosilactobacillus caccae sp. nov., a new bacterial species isolated from the human gut microbiota.

Strain Marseille-P3519T isolated from the fecal flora of a 25-year-old healthy French woman was a Gram-positive anaerobic bacterium, non-motile and non-spore forming. The 16S rRNA gene sequence of Marseille-P3519 showed 97.73% of sequence similarity with Limosilactobacillus reuteri DSM 20016, the closest species, phylogenetically. Furthermore, the average nucleotide identity of strain Marseille-3519 with its closest related species was 75.8% that was very below the recommended threshold (>95-96%). Its genome had 2 237 367 bp with 45.42 mol% of G + C content. Major fatty acids were C16:0 (50.8%), C18:1n9 (18.0%), C18:2n6 (9.8%) and C19:1n9 (8.9%). It was catalase negative and fermented glycerol, glucose, fructose, D-maltose, lactose and mannose. These findings support that strain Marseille-P3519 ( = CSURP3519 = CECT 30110) is a new member of the genus Limosilactobacillus for which the name Limosilactobacillus caccae sp. nov., is proposed.

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.

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.

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.

Metagenomic and culturomic analysis of gut microbiota dysbiosis during Clostridium difficile infection.

Recently, cocktail of bacteria were proposed in order to treat Clostridium difficile infection (CDI), but these bacteriotherapies were selected more by chance than experimentation. We propose to comprehensively explore the gut microbiota of patients with CDI compared to healthy donors in order to propose a consortium of bacteria for treating C. difficile. We compared stool samples composition from 11 CDI patients and 8 healthy donors using two techniques: metagenomics, 16S V3-V4 region amplification and sequencing and culturomics, high throughout culture using six culture conditions and MALDI-TOF identification. By culturomics, we detected 170 different species in the CDI group and 275 in the control group. Bacteroidetes were significantly underrepresented in the CDI group (p = 0.007). By metagenomics, 452 different operational taxonomic units assigned to the species level were detected in the CDI group compared to 522 in the control group. By these two techniques, we selected 37 bacteria only found in control group in more than 75% of the samples and/or with high relative abundance, 10 of which have already been tested in published bacteriotherapies against CDI, and 3 of which (Bifidobacterium adolescentis, Bifidobacterium longum and Bacteroides ovatus) have been detected by these two techniques. This controlled number of bacteria could be administrated orally in a non-invasive way in order to treat CDI.

Faecal microbiota transplantation shortens the colonisation period and allows re-entry of patients carrying carbapenamase-producing bacteria into medical care facilities.

BACKGROUND: Colonisation with carbapenemase-producing Enterobacteriaceae or Acinetobacter (CPE/A) is associated with complex medical care requiring implementation of specific isolation policies and limitation of patient discharge to other medical facilities. Faecal microbiota transplantation (FMT) has been proposed in order to reduce the duration of gut colonisation. OBJECTIVES: This study investigated whether a dedicated protocol of FMT could reduce the negativation time of CPE/A intestinal carriage in patients whose medical care has been delayed due to such colonisation. METHOD: A matched case-control retrospective study between patients who received FMT treatment and those who did not among CPE/A-colonised patients addressed for initial clustering at the current institute. The study adjusted two controls per case based on sex, age, bacterial species, and carbapenemase type. The primary outcome was delay in negativation of rectal-swab cultures. RESULTS: At day 14 post FMT, 8/10 (80%) treated patients were cleared for intestinal CPE/A carriage. In the control group, 2/20 (10%) had spontaneous clearance at day 14 after CPE/A diagnosis. Faecal microbiota transplantation led patients to reduce the delay in decolonisation (median 3 days post FMT for treated patients vs. 50.5 days after the first documentation of digestive carriage for control patients) and discharge from hospital (median 19.5 days post FMT for treated patients vs. 41 for control patients). CONCLUSION: Faecal microbiota transplantation is a safe and time-saving procedure to discharge CPE/A-colonised patients from the hospital. A standardised protocol, including 5 days of antibiotic treatment, bowel cleansing and systematic indwelling devices removal, should improve protocol effectiveness.

Early Fecal Microbiota Transplantation Improves Survival in Severe Clostridium difficile Infections.

Background: Severe Clostridium difficile infections (CDIs) are associated with a high mortality rate despite medical and/or surgical treatment. Fecal microbiota transplantation (FMT) prevents recurrences, but its effect on survival has been shown only in patients with O27 ribotype CDI. Here, we investigated whether early FMT could improve survival in hospitalized CDI patients, particularly those with severe infection. Methods: We performed a retrospective cohort study between May 2013 and April 2016 at the infectious diseases department of the North University Hospital of Marseille, France. Patients received either medical treatment alone or treatment with early FMT. The primary outcome was the 3-month mortality rate. Results: A total of 111 patients were included: 66 in the FMT group and 45 in the non-FMT group. No patient underwent surgery. The O27 ribotype (odds ratio [OR], 3.64 [95% confidence interval {CI}, 1.05- 12.6], P = .04), severe CDI (OR, 9.62 [95% CI, 2.16-42.8], P = .003), and FMT (OR, 0.13 [95% CI, .04-.44], P = .001) were independent predictors of 3-month mortality. FMT improved survival in severe cases (OR, 0.08 [95% CI, .016-.34], P = .001) but not in nonsevere cases (OR, 1.07 [95% CI, .02-56.3], P = .97), independent of age, sex, comorbidities (Charlson score), and ribotype. The number of severe patients who needed to be treated to save 1 life at 3 months was 2. Conclusions: Early FMT dramatically reduces mortality and should be proposed as a first-line treatment for severe CDI. Further studies are needed to clarify complications and contraindications. Surgery should be reassessed in this context.

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.

Isolation of Vermamoeba vermiformis and associated bacteria in hospital water.

To detect new potential pathogens in hospital water, we isolated free-living amoebae in water samples taken from three different hospitals in Marseille (France). The samples were inoculated in media containing saline buffer and various bacteria as nutrient sources. The isolated amoebae were identified by gene sequencing. Among the 105 water samples, taken from 19 sites, we isolated 14 amoebae, of which 9 Vermamoeba vermiformis and 5 Acanthamoeba sp. None of the amoebae showed the presence of obligate bacterial endosymbionts. Because V. vermiformis was most commonly isolated, we used an axenic collection strain to isolate amoeba-resistant bacteria from the same sites. The isolated bacterial species included Stenotrophomonas maltophilia and Legionella sp. Legionella taurinensis was isolated for the first time in association with amoebae. A strict intracellular bacterium was isolated, that may represent a new genus among the Chlamydiales. We propose that it be named "Candidatus Rubidus massiliensis". Our study shows that the isolation and identification of new pathogens associated with amoebae, which were previously performed using Acanthamoeba sp., should instead use V. vermiformis because this organism is more commonly associated with humans and is an essential complement of Acanthamoeba sp. co-culture to study the ecology of hospital water supplies.