Aurelia Caputo

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.

Identification of Algerian Field-Caught Phlebotomine Sand Fly Vectors by MALDI-TOF MS

Background Phlebotomine sand flies are known to transmit Leishmania parasites, bacteria and viruses that affect humans and animals in many countries worldwide. Precise sand fly identification is essential to prevent phlebotomine-borne diseases. Over the past two decades, progress in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as an accurate tool for arthropod identification. The objective of the present study was to investigate the usefulness of MALDI-TOF MS as a tool for identifying field-caught phlebotomine. Methodology/Principal Findings Sand flies were captured in four sites in north Algeria. A subset was morphologically and genetically identified. Six species were found in these areas and a total of 28 stored frozen specimens were used for the creation of the reference spectrum database. The relevance of this original method for sand fly identification was validated by two successive blind tests including the morphological identification of 80 new specimens which were stored at -80°C, and 292 unknown specimens, including engorged specimens, which were preserved under different conditions. Intra-species reproducibility and inter-species specificity of the protein profiles were obtained, allowing us to distinguish specimens at the gender level. Querying of the sand fly database using the MS spectra from the blind test groups revealed concordant results between morphological and MALDI-TOF MS identification. However, MS identification results were less efficient for specimens which were engorged or stored in alcohol. Identification of 362 phlebotomine sand flies, captured at four Algerian sites, by MALDI-TOF MS, revealed that the subgenus Larroussius was predominant at all the study sites, except for in M’sila where P. (Phlebotomus) papatasi was the only sand fly species detected. Conclusion The present study highlights the application of MALDI-TOF MS for monitoring sand fly fauna captured in the field. The low cost, reliability and rapidity of MALDI-TOF MS analyses opens up new ways in the management of phlebotomine sand fly-borne diseases.

Whole-genome assembly of Akkermansia muciniphila sequenced directly from human stool

BackgroundAlterations in gut microbiota composition under antibiotic pressure have been widely studied, revealing a restricted diversity of gut flora, including colonization by organisms such as Enterococci, while their impact on bacterial load is variable. High-level colonization by Akkermansia muciniphila, ranging from 39% to 84% of the total bacterial population, has been recently reported in two patients being treated with broad-spectrum antibiotics, although attempts to cultivate this microorganism have been unsuccessful.ResultsHere, we propose an original approach of genome sequencing for Akkermansia muciniphila directly from the stool sample collected from one of these patients. We performed and assembly using metagenomic data obtained from the stool sample. We used a mapping method consisting of aligning metagenomic sequencing reads against the reference genome of the Akkermansia muciniphila MucT strain, and a De novo assembly to support this mapping method. We obtained draft genome of the Akkermansia muciniphila strain Urmite with only 56 gaps. The absence of particular metabolic requirement as possible explanation of our inability to culture this microorganism, suggests that the bacterium was dead before the inoculation of the stool sample. Additional antibiotic resistance genes were found following comparison with the reference genome, providing some clues pertaining to its survival and colonization in the gut of a patient treated with broad-spectrum antimicrobial agents. However, no gene coding for imipenem resistance was detected, although this antibiotic was a part of the patient’s antibiotic regimen.ConclusionsThis work highlights the potential of metagenomics to facilitate the assembly of genomes directly from human stool.ReviewersThis article was reviewed by Eric Bapteste, William Martin and Vivek Anantharaman.

Pan-genomic analysis to redefine species and subspecies based on quantum discontinuous variation: the Klebsiella paradigm

BackgroundVarious methods are currently used to define species and are based on the phylogenetic marker 16S ribosomal RNA gene sequence, DNA-DNA hybridization and DNA GC content. However, these are restricted genetic tools and showed significant limitations.ResultsIn this work, we describe an alternative method to build taxonomy by analyzing the pan-genome composition of different species of the Klebsiella genus. Klebsiella species are Gram-negative bacilli belonging to the large Enterobacteriaceae family. Interestingly, when comparing the core/pan-genome ratio; we found a clear discontinuous variation that can define a new species.ConclusionsUsing this pan-genomic approach, we showed that Klebsiella pneumoniae subsp. ozaenae and Klebsiella pneumoniae subsp. rhinoscleromatis are species of the Klebsiella genus, rather than subspecies of Klebsiella pneumoniae. This pan-genomic analysis, helped to develop a new tool for defining species introducing a quantic perspective for taxonomy.ReviewersThis article was reviewed by William Martin, Pierre Pontarotti and Pere Puigbo (nominated by Dr Yuri Wolf).

Non-contiguous finished genome sequence and description of Nosocomiicoccus massiliensis sp. nov.

Nosocomiicoccus massiliensis strain NP2T sp. nov. is the type strain of a new species within the genus Nosocomiicoccus. This strain, whose genome is described here, was isolated from the fecal flora of an AIDS-infected patient living in Marseille, France. N. massiliensis is a Gram-positive aerobic coccus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,645,244 bp long genome (one chromosome but no plasmid) contains 1,738 protein-coding and 45 RNA genes, including 3 rRNA genes.

Microvirga massiliensis sp. nov., the human commensal with the largest genome

Microvirga massiliensis sp. nov. strain JC119T is a bacteria isolated in Marseille from a stool sample collected in Senegal. The 16S rRNA (JF824802) of M. massiliensis JC119T revealed 95% sequence identity with Microvirga lotononidis WSM3557T (HM362432). This bacterium is aerobic, gram negative, catalase positive, and oxidase negative. The draft genome of M. massiliensis JC119T comprises a 9,207,211‐bp‐long genome that is the largest bacterial genome of an isolate in humans. The genome exhibits a G+C content of 63.28% and contains 8685 protein‐coding genes and 77 RNA genes, including 21 rRNA genes. Here, we describe the features of M. massiliensis JC119T, together with the genome sequence information and its annotation.

Non-contiguous finished genome sequence and description of Holdemania massiliensis sp. nov.

Holdemania massiliensis strain AP2T sp. nov. is the type strain of H. massiliensis sp. nov., a new species within the genus Holdemania. This strain, whose genome is described here, was isolated from the fecal flora of a 21-year-old French Caucasian female suffering from severe restrictive anorexia nervosa. H. massiliensis is a Gram-positive, anaerobic bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,795,625 bp-long genome (one chromosome but no plasmid) contains 3,461 protein-coding and 49 RNA genes, including 3 rRNA genes.

Noncontiguous finished genome sequence and description of Paenibacillus ihumii sp. nov. strain AT5

Paenibacillus ihumii sp. nov. strain AT5 (= CSUR 1981 = DSM 100664) is the type strain of P. ihumii. This bacterium was isolated from a stool sample from a morbidly obese French patient using the culturomics approach. The genome of this Gram-negative, facultative anaerobic, motile and spore-forming bacillus is 5 924 686 bp long. Genomic analysis identified 253 (5%) of 3812 genes as ORFans and at least 2599 (50.03%) of 5194 orthologous proteins not shared with the closest phylogenetic species.

Non-contiguous finished genome sequence and description of Gorillibacterium massiliense gen. nov, sp. nov., a new member of the family Paenibacillaceae

Strain G5T gen. nov., sp. nov. is the type strain of Gorillibacterium massiliense, a newly proposed genus within the family Paenibacillaceae. This strain, whose genome is described here, was isolated in France from a stool sample of a wild Gorilla gorilla subsp. gorilla from Cameroon. G. massiliense is a facultatively anaerobic, Gram negative rod. Here we describe the features of this bacterium, together with the complete genome sequence and annotation. The 5,546,433 bp long genome (1 chromosome but no plasmid) contains 5,145 protein-coding and 76 RNA genes, including 69 tRNA genes.

Non-contiguous finished genome sequence and description of Corynebacterium jeddahense sp. nov.

Corynebacterium jeddahense sp. nov., strain JCBT, is the type strain of Corynebacterium jeddahense sp. nov., a new species within the genus Corynebacterium. This strain, whose genome is described here, was isolated from fecal flora of a 24-year-old Saudi male suffering from morbid obesity. Corynebacterium jeddahense is a Gram-positive, facultative anaerobic, nonsporulating bacillus. Here, we describe the features of this bacterium, together with the complete genome sequencing and annotation, and compare it to other member of the genus Corynebacterium. The 2,472,125 bp-long genome (1 chromosome but not plasmid) contains 2,359 protein-coding and 53 RNA genes, including 1 rRNA operon.