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Methanogens, the archaea uniquely detoxifying fermentative hydrogen into methane in the digestive tract, are increasingly being detected in pathology situations, rendering their rapid identification mandatory. We improved the experimental protocol to identify broth-cultured methanogens by matrix-assisted laser desorption time-of-flight MS (MALDI-TOF-MS). A database incorporating 34 reference spectra derived from 16 methanogen reference strains representative of eight species supported further identification of 21

Truffles are edible mushrooms with similar morphological characteristics, that make it difficult to distinguish between highly prized truffles (such as the Périgord black T. melanosporum) and inexpensive truffles (such as the Asian Black T. indicum). These biological and economic features have led to several misidentifications and/or fraudulent profit in the truffle markets. In this paper, we investigate Matrix-assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) biotyping to identify 34 commercial fresh truffles from Europe and Asia. The MALDI-TOF MS clustering rapidly distinguished seven Tuber species identified by ITS phylogenetic analysis. The tasty T. melanosporum was clearly differentiated from the Chinese and less expensive truffles. These cheaper mushrooms were marketed as T. indicum but corresponded to a mix of three species. In total, the method confirmed misidentifications in 26% of commercial specimens. Several unknown blind-coded truffles were rapidly identified, with scores >= 2, using the Bruker Biotyper algorithm against MS databases. This study demonstrates that MALDI-TOF MS is a reliable, rapid and cheaper new tool compared with molecular methods for the identification of truffle species and could be used to control frauds in the truffle markets. It could also be useful for the certification of truffle-inoculated seedlings and/or diversity in forest ecosystems.

A new Actinobacterium, designated Tessaracoccus massiliensis type strain SIT-7T (= CSUR P1301 = DSM 29060), have been isolated from a Nigerian child with kwashiorkor. It is a facultative aerobic, Gram positive, rod shaped, non spore-forming, and non motile bacterium. Here, we describe the genomic and phenotypic characteristics of this isolate. Its 3,212,234 bp long genome (1 chromosome, no plasmid) exhibits a G+C content of 67.81% and contains 3,058 protein-coding genes and 49 RNA genes.

International audience; Butyricimonas phoceensis strain AT9 (= CSUR 1981 = DSM 100664) was isolated from a stool sample from a morbidly obese French patient living in Marseille using the culturomics approach. The genome of this Gram-negative-staining, anaerobic and non-spore forming rod bacillus is 4 736 949 bp long and contains 3947 protein-coding genes. Genomic analysis identified 173 genes as ORFans (4.5%) and 1650 orthologous proteins (42%) not shared with the closest phylogenetic species, Butyricimonas virosa. Its major fatty acid was the branched acid iso-C15:0 (62.3%).

Xanthomonas massiliensis strain SN6T is a Gram-negative bacterium which is aerobic, motile and nonsporulating. This new species isolated from human faeces exhibited the characteristic traits of members of this genus, such as yellow pigmentation and viscosity. Here we present the main phenotypic characteristics and the taxonogenomics description of this strain. The genome is 3 690 720 bp long with DNA G + C content of about 70.52%. Keywords: Culturomics, genome, human gut microbiota, taxonogenomics, Xanthomonas massiliensis

International audience; Culturomics has permitted discovery of hundreds of new bacterial species isolated from the human microbiome. Profiles generated by using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry have been added to the mass spectrometer database used in clinical microbiology laboratories. We retrospectively collected raw data from MALDI-TOF mass spectrometry used routinely in our laboratory in Marseille, France, during January 2012-March 2018 and analyzed 16S rDNA sequencing results from misidentified strains. During the study period, 744 species were identified from clinical specimens, of which 21 were species first isolated from culturomics. This collection involved 105 clinical specimens, accounting for 98 patients. In 64 cases, isolation of the bacteria was considered clinically relevant. MALDI-TOF mass spectrometry was able to identify the species in 95.2% of the 105 specimens. While contributing to the extension of the bacterial repertoire associated with humans, culturomics studies also enlarge the spectrum of prokaryotes involved in infectious diseases.

International audience; Microbial culturomics is a new subfield of postgenomic medicine and omics biotechnology application that has broadened our awareness on bacterial diversity of the human microbiome, including the human vaginal flora bacterial diversity. Using culturomics, a new obligate anaerobic Gram-stain-negative rod-shaped bacterium designated strain khD1(T) was isolated in the vagina of a patient with bacterial vaginosis and characterized using taxonogenomics. The most abundant cellular fatty acids were C-15:0 anteiso (36%), C-16:0 (19%), and C-15:0 iso (10%). Based on an analysis of the full-length 16S rRNA gene sequences, phylogenetic analysis showed that the strain khD1(T) exhibited 90% sequence similarity with Prevotella loescheii, the phylogenetically closest validated Prevotella species. With 3,763,057bp length, the genome of strain khD1(T) contained (mol%) 48.7 G+C and 3248 predicted genes, including 3194 protein-coding and 54 RNA genes. Given the phenotypical and biochemical characteristic results as well as genome sequencing, strain khD1(T) is considered to represent a novel species within the genus Prevotella, for which the name Prevotella lascolaii sp. nov. is proposed. The type strain is khD1(T) (=CSUR P0109,=DSM 101754). These results show that microbial culturomics greatly improves the characterization of the human microbiome repertoire by isolating potential putative new species. Further studies will certainly clarify the microbial mechanisms of pathogenesis of these new microbes and their role in health and disease. Microbial culturomics is an important new addition to the diagnostic medicine toolbox and warrants attention in future medical, global health, and integrative biology postgraduate teaching curricula.

International audience; Using the culturomics strategy, a wide range of anaerobic bacteria was discovered including Anaeromassilibacillus senegalensis strain mt9(T) (= CSUR P1511 = DSM 102954), isolated from the gut microbiota of a 1-year-old Senegalese patient with kwashiorkor. This Gram-negative strain is a strictly anaerobic, spore-forming rod motile by a polar flagellum. The 3 511 289 bp long genome of this strain contains 3046 protein-coding and 49 RNA genes, including 45 tRNA and four rRNA genes, and exhibits a G+C content of 52.94%. Here we describe the features of this organism, together with the complete genome sequence and annotation.

Collinsella massiliensis strain GD3(T) is the type strain of Collinsella massiliensis sp. nov., a new species within the genus Collinsella. This strain, whose genome is described here, was isolated from the fecal flora of a 53-year-old French Caucasoid woman who had been admitted to intensive care unit for Guillain-Barré syndrome. Collinsella massiliensis is a Gram-positive, obligate anaerobic, non motile and non sporulating bacillus. Here, we describe the features of this organism, together with the complete genome sequence and annotation. The genome is 2,319,586 bp long (1 chromosome, no plasmid), exhibits a G+C content of 65.8% and contains 2,003 protein-coding and 54 RNA genes, including 1 rRNA operon.

Bacteroides timonensis strain AP1(T) (= CSUR P194 = DSM 26083) is the type strain of B. timonensis sp. nov. This strain, whose genome is described here, was isolated from the fecal flora of a 21-year-old French Caucasoid female who suffered from severe anorexia nervosa. Bacteroides timonensis is a Gram-negative, obligate anaerobic bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 7,130,768 bp long genome (1 chromosome, no plasmid) exhibits a G+C content of 43.3% and contains 5,786 protein-coding and 59 RNA genes, including 2 rRNA genes.

Kallipyga massiliensis strain ph2(T) is the type strain of Kallipyga massiliensis gen. nov., sp. nov., the type species of the new genus Kallipyga within the family Clostridiales Incertae Sedis XI. This strain, whose genome is described here, was isolated from the fecal flora of a 26-year-old woman suffering from morbid obesity. K. massiliensis is an obligate anaerobic coccus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,770,679 bp long genome (1 chromosome but no plasmid) contains 1,575 protein-coding and 50 RNA genes, including 4 rRNA genes.

Enorma massiliensis strain phI(T) is the type strain of E. massiliensis gen. nov., sp. nov., the type species of a new genus within the family Coriobacteriaceae, Enorma gen. nov. This strain, whose genome is described here, was isolated from the fecal flora of a 26-year-old woman suffering from morbid obesity. E. massiliensis strain phI(T) is a Gram-positive, obligately anaerobic bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,280,571 bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 62.0% and contains 1,901 protein-coding and 51 RNA genes, including 3 rRNA genes.

Brevibacillus massiliensis strain phR(T) sp. nov. is the type strain of B. massiliensis sp. nov., a new species within the genus Brevibacillus. This strain was isolated from the fecal flora of a woman suffering from morbid obesity. B. massiliensis is a Gram-positive aerobic rod-shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5,051,018 bp long genome (1 chromosome but no plasmid) contains 5,051 protein-coding and 84 RNA genes, and exhibits a G+C content of 53.1%.

Peptoniphilus obesi strain ph1(T) sp. nov., is the type strain of P. obesi sp. nov., a new species within the genus Peptoniphilus. This strain, whose genome is described here, was isolated from the fecal flora of a 26-year-old woman suffering from morbid obesity. P. obesi strain ph1(T) is a Gram-positive, obligate anaerobic coccus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,774,150 bp long genome (1 chromosome but no plasmid) contains 1,689 protein-coding and 29 RNA genes, including 5 rRNA genes.

International audience; A novel filamentous bacterium, designated GD1(T), was isolated from the gut microbiota of a 38-year-old male who suffered from a Coxiella burnetii vascular for which he received multiple a broad-spectrum antibiotic cocktail at the time of the stool collection. The strain was isolated as a part of culturomics study by cultivation on 5 % sheep blood agar in aerobic condition at 28 A degrees C, after 14 days of incubation. Strain GD1(T) shows 16S rRNA gene sequence similarities of 98.01 % to the type strain of Risungbinella pyongyangensis. We describe here the features of this bacterium, together with the complete genome sequence and annotation. The G+C content of the genomic DNA was determined to be 40.1 mol %. The major fatty acids of strain GD1(T) were identified as iso-C15:0, iso-C17:0, anteiso-C15:0, iso-C14:0 and C16:0. The 3,440,191 bp long genome contains 3540 protein-coding and 67 RNA genes, including three rRNA genes. Strain GD1(T) (= DSM 46691 = CSUR P1082) sp. nov. is here classified as the type strain of a new species, Risungbinella massiliensis, within the family Thermoactinomycetaceae. To date, strain GD1(T) is the first member of the family Thermoactinomycetaceae isolated from human gut and the fourth from a human specimen.

International audience; In this paper, we evaluate a rapid and safe pretreatment procedure using glass beads for MALDI-TOF yeast identification in a routine clinical laboratory avoiding the use of formic acid. We created a new yeast database library using 1186 yeasts, including 11 references strains. The database was tested using 2131 clinical isolates allowing accurate species-level identification in 98.9% (2107/2131) of cases with a score over 1.9 and in 99% (2123/2131) of the strains at the genus level. The new protocol is a rapid, reliable and safe procedure for the accurate identification of pathogenic Candida strains and requires minimal handling.

RATIONALE It is recommended that harmful Biosafety Level 3 (BSL-3) bacteria be inactivated prior to identification by mass spectrometry, yet optimal effects of inactivation protocol have not been defined. METHODS Here, we compare trifluoroacetic acid inactivation (protocol A) with ethanol inactivation (protocol B) of Yersinia organisms prior to identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS The total number of peaks detected was 10.5 ± 1.7 for protocol A and 15.7 ± 4.2 for protocol B (ρ

Propionibacterium species are mostly environmental bacteria, some being commensal of mammals including humans, and sometimes pathogenic. These bacteria are poorly identified using routine laboratory methods. Recently, Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) has emerged as a rapid and efficient method to identify bacterial species. We evaluated the use of MALDI-TOF-MS for identification of all validated Propionibacterium species. Only four of the 15 tested reference strains (26.7%) were correctly identified at the species level, and P. acnes, the most common human pathogenic species was not identified. When applying MALDI-TOF-MS to 48 P. acnes strains, only 18.7% were correctly identified, suggesting an intraspecific variability of proteic profiles among Propionibacterium strains. However, by enriching the Bruker database with spectra from five of these strains and re-testing the other 43 strains against this new database, 93.0% were correctly identified. Our study demonstrates that MALDI-TOF-MS may be used for the identification of Propionibacterium isolates but requires a database enrichment in spectra from additional isolates.

International audience; Detection of autoantibodies, which are involved in tissue injury and/or the reporters from the immune system of various pathologic events, has an important potential for diagnosis, prognosis, disease staging and treatment selection. This explains the interest for new proteomics technologies, such as the high-density protein microarray used here, that allow a high-throughput, multiplexed and sensitive detection of specific autoantibodies. So far, most of the research has been performed on blood. In this note, we focus on the cerebrospinal fluid in an attempt to address autoimmune events associated with neurological disorders. Importantly, the cerebrospinal fluid is quite different from the blood in terms of protein composition and concentration. We had therefore to adapt the available blood protocols. We present here the result of our optimization that will be useful to carry out full scale immunological studies of the cerebrospinal fluid using high-density protein microarrays.

Virgibacillus senegalensis SK-1T (= CSUR P1101 = DSM 28585) is the type strain of V. senegalensis sp. nov. It is an aerobic, Gram positive, moderately halophilic, motile bipolar flagellum isolated from a healthy Senegalese man. Here we describe the genomic and phenotypic characteristics of this isolate. The 3 755 098 bp long genome (one chromosome, no plasmid) exhibits a G + C content of 42.9% and contains 3738 protein-coding and 95 RNA genes.

International audience; Strain SIT6(T) was isolated from the fecal flora of a severely malnourished child as part of a broad culturomics study aiming to maximize the culture conditions for the in-depth exploration of the human microbiota. An analysis of the 16S rRNA gene sequence showed that strain SIT6(T) shared 94.1% 16S rRNA gene sequence similarity with Bacillus thermoamylovorans DKPT (NR\₀29151), the phylogenetically closest type species. Colonies are creamy white, circular, 4-5 mm in diameter after cultivation at 37 degrees C for 24 hr on 5% sheep blood-enriched Colombia agar. Growth occurs at temperatures in the range of 25-56 degrees C (optimally at 37 degrees C). Strain SIT6(T) is a gram-positive, facultative anaerobic rod and motile by means of peritrichous flagella and sporulating; it is catalase and oxidase positive. The 2,784,637-bp-long genome, composed of 16 contigs, has a G+C content of 35.19%. Of the 2,646 predicted genes, 2,572 were protein-coding genes and 74 were RNAs. The major fatty acids are saturated species (15:0 iso, 16:0 and 17:0 anteiso). Of the 14 detected fatty acids, 11 are saturated, either linear or branched (iso and anteiso). Digital DNA-DNA hybridization (dDDH) estimation and average genomic identity of orthologous gene sequences (AGIOS) of the strain SIT6(T) against genomes of the type strains of related species ranged between 18.6% and 38.3% and between 54.77% and 65.50%, respectively. According to our taxonogenomics results, we propose the creation of Bacillus kwashiorkori sp. nov. that contains the type strain SIT6(T) (=CSUR P2452(T), =DSM 29059(T)).

Megasphaera massiliensis strain NP3(T) sp. nov. is the type strain of Megasphaera massiliensis sp. nov., a new species within the genus Megasphaera. This strain, whose genome is described here, was isolated from the fecal flora of an HIV-infected patient. M. massiliensis is a Gram-negative, obligate anaerobic coccobacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,661,757 bp long genome (1 chromosome but no plasmid) contains 2,577 protein-coding and 61 RNA genes, including 5 rRNA genes.

Peptoniphilus obesi strain ph1T sp. nov., is the type strain of P. obesi sp. nov., a new species within the genus Peptoniphilus. This strain, whose genome is described here, was isolated from the fecal flora of a 26-year-old woman suffering from morbid obesity. P. obesi strain ph1T is a Gram-positive, obligate anaerobic coccus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,774,150 bp long genome (1 chromosome but no plasmid) contains 1,689 protein-coding and 29 RNA genes, including 5 rRNA genes.

It is recommended that harmful Biosafety Level 3 (BSL-3) bacteria be inactivated prior to identification by mass spectrometry, yet optimal effects of inactivation protocol have not been defined.Here, we compare trifluoroacetic acid inactivation (protocol A) with ethanol inactivation (protocol B) of Yersinia organisms prior to identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).The total number of peaks detected was 10.5 ± 1.7 for protocol A and 15.7 ± 4.2 for protocol B (ρ0.001, ANOVA test). The signal-to-noise ratio for the m/z 6049 peak present in all of the tested Yersinia isolates was 9.7 ± 3.1 for protocol A and 18.1 ± 4.6 for protocol B (ρ 0.001). Compared with spectra in our local database containing 48 Yersinia spp., including 20 strains of Y. pestis, the identification score was 1.79 ± 0.2 for protocol A and 1.97 ± 0.19 for protocol B (ρ = 0.0024).Our observations indicate that for the identification of Yersinia organisms, ethanol inactivation yielded MALDI-TOF-MS spectra of significantly higher quality than spectra derived from trifluoroacetic acid inactivation. Combined with previously published data, our results permit the updating of protocols for inactivating BSL-3 bacteria.

BACKGROUND: Non-tuberculous mycobacteria recovered from respiratory tract specimens are emerging confounder organisms for the laboratory diagnosis of tuberculosis worldwide. There is an urgent need for new techniques to rapidly identify mycobacteria isolated in clinical practice. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) has previously been proven to effectively identify mycobacteria grown in high-concentration inocula from collections. However, a thorough evaluation of its use in routine laboratory practice has not been performed. METHODOLOGY: We set up an original protocol for the MALDI-TOF MS identification of heat-inactivated mycobacteria after dissociation in Tween-20, mechanical breaking of the cell wall and protein extraction with formic acid and acetonitrile. By applying this protocol to as few as 10(5) colony-forming units of reference isolates of Mycobacterium tuberculosis, Mycobacterium avium, and 20 other Mycobacterium species, we obtained species-specific mass spectra for the creation of a local database. Using this database, our protocol enabled the identification by MALDI-TOF MS of 87 M. tuberculosis, 25 M. avium and 12 non-tuberculosis clinical isolates with identification scores ≥2 within 2.5 hours. CONCLUSIONS: Our data indicate that MALDI-TOF MS can be used as a first-line method for the routine identification of heat-inactivated mycobacteria. MALDI-TOF MS is an attractive method for implementation in clinical microbiology laboratories in both developed and developing countries.

Legionella species are facultative, intracellular bacteria that infect macrophages and protozoa, with the latter acting as transmission vectors to humans. These fastidious bacteria mostly cause pulmonary tract infections and are routinely identified by various molecular methods, mainly PCR targeting the mip gene and sequencing, which are expensive and time-consuming. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has emerged as a rapid and inexpensive method for identification of bacterial species. This study evaluated the use of MALDI-TOF-MS for rapid species and serogroup identification of 21 Legionella species recognized as human pathogens. To this end, a reference MS database was developed including 59 Legionella type strains, and a blind test was performed using 237 strains from various species. Two hundred and twenty-three of the 237 strains (94.1 %) were correctly identified at the species level, although ten (4.2 %) were identified with a score lower than 2.0. Fourteen strains (5.9 %) from eight species were misidentified at the species level, including seven (3.0 %) with a significant score, suggesting an intraspecific variability of protein profiles within some species. MALDI-TOF-MS was reproducible but could not identify Legionella strains at the serogroup level. When compared with mip gene sequencing, MALDI-TOF-MS exhibited a sensitivity of 99.2 and 89.9 % for the identification of Legionella strains at the genus and species level, respectively. This study demonstrated that MALDI-TOF-MS is a reliable tool for the rapid identification of Legionella strains at the species level.

Bacteria of the genus Bartonella are emerging zoonotic bacteria recognized in a variety of human diseases. Due to their poor chemical reactivity, these fastidious bacteria are poorly characterized using routine phenotypic laboratory tests. Identification is usually achieved using molecular techniques that are time-consuming, expensive and technically demanding. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a new technique for bacterial species identification. This study evaluated the use of MALDI-TOF MS for rapid genus and species identification of Bartonella species. Reference strains representing 17 recognized Bartonella species were studied. For each species, MS spectra for four colonies were analysed. The consensus spectrum obtained for each species was unique among spectra obtained for 2843 bacteria within the Bruker database, including 109 alphaproteobacteria. Thirty-nine additional blind-coded Bartonella strains were correctly identified at the species level, including 36 with a significant score. Altogether, these data demonstrate that MS is an accurate and reproducible tool for rapid and inexpensive identification of Bartonella species.

Bacteroides neonati strain MS4(T), is the type strain of Bacteroides neonati sp. nov., a new species within the genus Bacteroides. This strain, whose genome is described here, was isolated from a premature neonate stool sample. B. neonati strain MS4(T) is an obligate anaerobic Gram-negative bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5.03 Mbp long genome exhibits a G+C content of 43.53% and contains 4,415 protein-coding and 91 RNA genes, including 9 rRNA genes.

Corynebacterium ihumii strain GD7(T) sp. nov. is proposed as the type strain of a new species, which belongs to the family Corynebacteriaceae of the class Actinobacteria. This strain was isolated from the fecal flora of a 62 year-old male patient, as a part of the culturomics study. Corynebacterium ihumii is a Gram positive, facultativly anaerobic, nonsporulating bacillus. Here, we describe the features of this organism, together with the high quality draft genome sequence, annotation and the comparison with other member of the genus Corynebacteria. C. ihumii genome is 2,232,265 bp long (one chromosome but no plasmid) containing 2,125 protein-coding and 53 RNA genes, including 4 rRNA genes. The whole-genome shotgun sequence of Corynebacterium ihumii strain GD7(T) sp. nov has been deposited in EMBL under accession number GCA_000403725.

Nocardioides massiliensis sp. nov strain GD13T is the type strain of N. massiliensis sp. nov., a new species within the genus Nocardioides. This strain was isolated from the faeces of a 62-year-old man admitted to intensive care for Guillain-Barré syndrome. Nocardioides massiliensis is a strictly aerobic Gram-positive rod. Herein we describe the features of this bacterium, together with the complete genome sequence and annotation. The 4 006 620 bp long genome contains 4132 protein-coding and 47 RNA genes.

Senegalemassilia anaerobia strain JC110(T) sp.nov. is the type strain of Senegalemassilia anaerobia gen. nov., sp. nov., the type species of a new genus within the Coriobacteriaceae family, Senegalemassilia gen. nov. This strain, whose genome is described here, was isolated from the fecal flora of a healthy Senegalese patient. S. anaerobia is a Gram-positive anaerobic coccobacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,383,131 bp long genome contains 1,932 protein-coding and 58 RNA genes.