Validation of a non-targeted method devoted to identification and quantitation of toxicologically relevant compounds in plasma with HRMS.

The objective of this study was to develop and validate a simple method using liquid chromatography hyphenated to high resolution mass spectrometry (HRMS) allowing both the performance of a non-targeted screening and the simultaneous quantification of 29 compounds of interest in clinical and forensic toxicology. Extraction was done with QuEChERS salts and acetonitrile, after addition of internal standard to 200 µL of human plasma samples. The mass spectrometer was an Orbitrap, with a heated electrospray ionization (HESI) probe. The analyses were carried out in full scan experiment with a nominal resolving power of 60,000 FWHM within the 125-650 m/z mass range, followed by four cycles of data dependent analysis (DDA) with a mass resolution of 16,000 FWHM. The untargeted screening was evaluated using 132 compounds, mean limit of identification (LOI) was 8.8 ng/mL (min = 0.05 ng/mL, max = 500 ng/mL) and mean limit of detection (LOD) was 0.25 ng/mL (min = 0.05 ng/mL, max = 5 ng/mL). The method was linear in the 5 to 500 ng/mL range (0.5 to 50 ng/mL for cannabinoids, 6-acetylmorphine and buprenorphine) with correlation coefficients > 0.99, intra- and inter-day accuracy and precision were < 15% for all compounds. The method was successfully applied to 31 routine samples.

Neglectibacter timonensis gen. nov., sp. nov. and Scatolibacter rhodanostii gen. nov., sp. nov., two anaerobic bacteria isolated from human stool samples.

Strains Marseille-P2265T (=CSUR P2265T =DSM 102082 T) and Marseille-P3890T (=CSUR P3890T =CCUG 72341 T) were isolated from stool samples using the culturomics approach. The 16S rRNA gene sequences of both strains were sequenced and compared by BLASTn to the NCBI database. Strains Marseille-P2265T and Marseille-P3890T were most closely related to Acutalibacter muris with identities of 94.3% and 91.5%, respectively. Between the two strains, the 16S rRNA gene sequence identity was 91.5%. Both strains are anaerobic Gram-positive, oxidase- and catalase-negative. The major fatty acid methyl esters (> 10%) in both strains are C16:0 and anteiso-C15:0. Additionally, strain Marseille-P2265T has iso-C15:0 and C14:0, and strain Marseille-P3890T, iso-C14:0. Strain Marseille-P2265T has a genome size of 3,671,396-bp with a G + C content of 52.8%. As for strain Marseille-P3890T, the genome is 2,702,024-bp-long with a 39.8% G + C content. The genomic comparison of closely related species with strains Marseille-P2265T and Marseille-P3890T showed that all digital DNA-DNA hybridization (dDDH), orthologous average nucleotide identity (OrthoANI) and average amino acid identity (AAI) values were lower than the published species thresholds (70% for dDDH, 95-96% for OrthoANI/AAI). Based on these results, it was concluded that strains Marseille-P2265T and Marseille-P3890T belong to two new genera in the family Oscillospiraceae. For these two genera, the names Neglectibacter gen. nov. and Scatolibacter gen. nov. were proposed, with strains Marseille-P2265T and Marseille-P3890T being the type strains of Neglectibacter timonensis gen. nov., sp. nov. and Scatolibacter rhodanostii gen. nov., sp. nov., respectively.

Vitreoscilla massiliensis sp. nov., Isolated From the Stool of an Amazonian Patient.

Strain SN6T is a non-motile and non-spore-forming gram-negative bacterium which was isolated from the stool sample of an Amazonian patient. The optimum growth was observed at 37 °C, pH 7, and 0-5 g/l of NaCl. Based on the 16S rRNA gene sequence similarity, the strain SN6T exhibited 97.5% identity with Vitreoscilla stercoraria strain ATCC_15218 (L06174), the phylogenetically closest species with standing in nomenclature. The predominant fatty acid was hexadecenoic acid (31%). The genomic DNA G + C content of the strain SN6T was 49.4 mol %. After analysis of taxonogenomic data, phenotypic and biochemical characteristics, we concluded that strain SN6T represents a new species of the genus Vitreoscilla for which the name Vitreoscilla massiliensis sp.nov is proposed. The type strain is SN6T (=CSUR P2036 = LN870312 = DSM 100958).

The Antioxidants Glutathione, Ascorbic Acid and Uric Acid Maintain Butyrate Production by Human Gut Clostridia in The Presence of Oxygen In Vitro.

Uncontrolled oxidative stress, reported in Salmonella and HIV infections, colorectal cancer or severe acute malnutrition, has been associated with anaerobic gut microbiome alteration, impaired butyrate production, mucosal immunity dysregulation and disruption of host-bacterial mutualism. However, the role of major antioxidant molecules in the human body, such as glutathione, ascorbic acid and uric acid, has been neglected in this context. Here, we performed an in vitro metabolomics study of the 3 most odorous anaerobic microbes isolated from the human gut in our laboratory (Clostridium sporogenes, Clostridium subterminale and Romboutsia lituseburensis) when grown in anaerobiosis or in aerobiosis with these 3 antioxidant molecules via gas and liquid chromatography-mass spectrometry (GC/MS and LC/MS). There was no growth or volatile organic compound production in aerobic cultures without the 3 antioxidant molecules. In anaerobiosis, the major metabolic products of the bacteria were thiols, alcohols and short-chain fatty acid esters. The production of alkanes, cycloheptatriene and, paradoxically, increased butyrate production, was observed in the cultures grown in aerobiosis with the 3 antioxidant molecules. The qualitative shift suggests specific molecular mechanisms that remain to be elucidated. The increased production of butyrate, but also isobutyrate and isovalerate in vitro suggests that these 3 antioxidant molecules contributed to the maintenance and active resilience of host-bacterial mutualism against mucosal oxygen and uncontrolled oxidative stress in vivo.

Gorillibacterium timonense sp. nov., isolated from an obese patient.

A Gram-negative and facultative anaerobic bacterium, designated strain SN4T, was isolated from the stool sample of an obese Amazonian patient. The new isolate was characterized by the taxonogenomics approach. The strain SN4T was beige-colored, circular and not haemolytic. Cells are rod shaped and motile with several flagella. Strain SN4T grows optimally at pH 7 and can survive in the presence of a saline concentration of up to 75 g/l NaCl. The 16S ribosomal RNA gene sequence analysis of the novel strain SN4T showed 95.28% similarity in nucleotide sequence with Gorillibacterium massiliense G5T, the phylogenetically closest neighbor and the type species of this genus. Anteiso-C15:0, iso-C15:0 and C16:0 were found as the major components in the cellular fatty acid analysis of this isolate. The genomic draft of strain SN4T is 5,263,742 bp long with 53.33% of G+C content. The differences in physiological, biochemical characteristics and phylogenetic and genomic data make it possible to clearly distinguish the strain SN4T from G. massiliense G5T. Based on the taxonogenomic description and the phenotypic and biochemical characteristics of this bacterium presented in this article, we propose the SN4T strain (= CSUR P2011 = DSM 100,698) as a new species, Gorillibacterium timonense sp. nov.

Description of Janibacter massiliensis sp. nov., cultured from the vaginal discharge of a patient with bacterial vaginosis.

Strain Marseille-P4121T was isolated from a vaginal sample of a 45-year-old French woman with bacterial vaginosis. It is a Gram-positive, asporogenous, non-motile and aerobic bacterium. Strain Marseille-P4121T exhibits 98.2% 16S rRNA sequence similarity with Janibacter alkaliphilus strain SCSIO 10480T, a phylogenetically closely related species with standing in nomenclature. Its major fatty acids were identified as C18:1ω9 (34.4%), C16:0 (30.1%), and C18:0 (19%). The draft genome size of strain Marseille-P4121T is 2,452,608 bp long with a 72.5% G+C content and contains 2351 protein-coding genes and 49 RNA genes including 3 rRNA genes. We propose that strain Marseille-P4121T (= CECT 9671T = CSUR P4121T) is the type strain of the new species Janibacter massiliensis sp. nov.

Description and genomic characterization of Massiliimalia massiliensis gen. nov., sp. nov., and Massiliimalia timonensis gen. nov., sp. nov., two new members of the family Ruminococcaceae isolated from the human gut.

Using the culturomics approach, we isolated two strains, Marseille-P2963 and Marseille-P3753, from the intestinal microbiota of a 19-year-old healthy Saudi Arabian Bedouin male and from a 32-year-old healthy Senegalese male faecal transplant donor. Here, we studied their phenotypic, phylogenetic and genomic characteristics. Both strains were phylogenetically related, but different from Ruminococcus species. Bacterial cells were anaerobic, rod-shaped, non-spore-forming and not motile, with neither catalase nor oxidase activities. Their growth temperatures ranged from 28 to 45 °C, with an optimal growth at 37 °C. The genomes are 2,842,720 bp- and 2,707,061 bp-long respectively. The G + C contents are 47.18% and 46.90%, respectively. Based on these characteristics, we propose the creation of a new genus within the family Ruminococcaceae named Massiliimalia gen. nov., that contains the new species Massiliimalia massiliensis gen. nov., sp. nov., and Massiliimalia timonensis gen. nov., sp. nov. Strains Marseille-P2963T (= CSUR P2963 = DSM 106837) and Marseille-P3753T (= CSUR P3753 = CCUG 71632) are their type strains, respectively.

Dysgonomonas massiliensis sp. nov., a new species isolated from the human gut and its taxonogenomic description.

Culturomics has allowed the isolation of a significant number of new bacterial species from the human gut microbiota and proved to be a valuable complement to culture-independent techniques. Using this culture-based approach, a new bacterial species has been isolated from a stool sample of a 39-year-old healthy Pygmy male and described using the taxonogenomic strategy. Cells of strain Marseille-P4356T are Gram-stain negative cocci. The strain grows optimally at 37 °C and is catalase positive but oxidase negative. Its 16S rRNA gene sequence exhibited 92.96% sequence similarity with Dysgonomonas gadei strain JCM 16698T (NR_113134.1), currently its phylogenetically closest species that has been validly named. The genome of strain Marseille-P4356T is 3,472,011 bp long with 37.3 mol% G+C content. Phenotypic, biochemical, proteomic, genomic and phylogenetic analyses, clearly demonstrate that strain Marseille-P4356T (= CCUG 71356T = CSUR P4356T) represents a new species within the genus Dysgonomonas, for which we propose the name Dysgonomonas massiliensis sp. nov.

Miniphocibacter massiliensis gen. nov., sp. nov., a new species isolated from the human gut and its taxono-genomics description.

With the aim of describing the human microbiota by the means of culture methods, culturomics was developed in order to target previously un-isolated bacterial species and describe it via the taxono-genomics approach. While performing a descriptive study of the human gut microbiota of the pygmy people, strain Marseille-P4678T has been isolated from a stool sample of a healthy 39-year-old pygmy male. Cells of this strain were Gram-positive cocci, spore-forming, non-motile, catalase-positive and oxidase-negative, and grow optimally at 37°C under anaerobic conditions. Its 16S rRNA gene sequence exhibited 89.69% of sequence similarity with Parvimonas micra strain 3119BT (NR 036934.1), its phylogenetically closest species with standing in nomenclature. The genome of strain Marseille-P4678T is 2,083,161 long with 28.26 mol% of G+C content. Based on its phenotypic, biochemical, genotypic and proteomic profile, this bacterium was classified as a new bacterial genus and species Miniphocibacter massiliensis gen. nov., sp. nov. with the type strain Marseille-P4678T .

Description of three new Peptoniphilus species cultured in the vaginal fluid of a woman diagnosed with bacterial vaginosis: Peptoniphilus pacaensis sp. nov., Peptoniphilus raoultii sp. nov., and Peptoniphilus vaginalis sp. nov.

Three previously unidentified Gram-positive anaerobic coccoid bacteria, strains KhD-2T , KHD4T , and Kh-D5T , isolated from a vaginal swab, were characterized using the taxonogenomics concept. The phylogenic analysis, phenotypic characteristics, and genotypic data presented in this report attest that these three bacteria are distinct from previously known bacterial species with standing in nomenclature and represent three new Peptoniphilus species. Strain KhD-2T is most closely related to Peptoniphilus sp. DNF00840 and Peptoniphilus harei (99.7% and 98.2% identity, respectively); strain KHD4T to Peptoniphilus lacrimalis (96%) and strain Kh-D5T to Peptoniphilus coxii (97.2%). Strains KhD-2T , KHD4T , and Kh-D5T DNA G+C contents are, respectively, 34.23%, 31.87%, and 49.38%; their major fatty acid was C16:0 (41.6%, 32.0%, and 36.4%, respectively). We propose that strains KhD-2T (=CSUR P0125 = DSM 101742), KHD4T (=CSUR P0110 = CECT 9308), and Kh-D5T (=CSUR P2271 = DSM 101839) be the type strains of the new species for which the names Peptoniphilus vaginalis sp. nov., Peptoniphilus raoultii sp. nov., and Peptoniphilu pacaensis sp. nov., are proposed, respectively.

Correction to: Description of Mediterraneibacter massiliensis, gen. nov., sp. nov., a new genus isolated from the gut microbiota of an obese patient and reclassification of Ruminococcus faecis, Ruminococcus lactaris, Ruminococcus torques, Ruminococcus gnavus and Clostridium glycyrrhizinilyticum as Mediterraneibacter faecis comb. nov., Mediterraneibacter lactaris comb. nov., Mediterraneibacter torques comb. nov., Mediterraneibacter gnavus comb. nov. and Mediterraneibacter glycyrrhizinilyticus comb. nov.

Subsequent to the publication of the above article, it has been noticed that the designation of the type strain is not correct. The strain referred to throughout the article as strain AT7T should be designated as strain Marseille-P2086T (= CSUR P2086T = DSM 100837T). The corrected for protologue for the species Mediterraneibacter massiliensis, represented by strain Marseille-P2086T as type strain, is given below.

Description of Mediterraneibacter massiliensis, gen. nov., sp. nov., a new genus isolated from the gut microbiota of an obese patient and reclassification of Ruminococcus faecis, Ruminococcus lactaris, Ruminococcus torques, Ruminococcus gnavus and Clostridium glycyrrhizinilyticum as Mediterraneibacter faecis comb. nov., Mediterraneibacter lactaris comb. nov., Mediterraneibacter torques comb. nov., Mediterraneibacter gnavus comb. nov. and Mediterraneibacter glycyrrhizinilyticus comb. nov.

An anaerobic isolate, strain AT7T, was cultivated from a stool sample of a morbidly obese French woman using a microbial culturomics approach. The 16S rRNA gene sequence analysis showed that strain AT7T exhibited 96% nucleotide sequence similarity with Ruminococcus torques strain JCM 6553T (= ATCC 27756T = VPI B2-51T), currently the closest related species with a validly published name. The strain was observed to be a Gram-stain positive, non-motile, asporogenous and coccobacillary-shaped bacterium. It was found to be catalase positive and oxidase negative. Its major fatty acids were identified as C16:0 (54%) and C18:1n9 (30%). The draft genome of strain AT7T is 3,069,882 bp long with 42.4% G+C content. 2925 genes were predicted, including 2867 protein-coding genes and 58 RNAs. Based on phenotypic, biochemical, phylogenetic and genomic evidence, we propose the creation of the new genus Mediterraneibacter and species, Mediterraneibacter massiliensis, that contains strain AT7T (= CSUR P2086T = DSM 100837T), and the reclassification of Ruminococcus faecis, Ruminococcus lactaris, Ruminococcus torques, Ruminococcus gnavus, Clostridium glycyrrhizinilyticum as Mediterraneibacter faecis comb. nov., with type strain Eg2T (= KCTC 5757T = JCM15917T), Mediterraneibacter lactaris comb. nov., with type strain ATCC 29176T (= VPI X6-29T), Mediterraneibacter torques comb. nov., with type strain ATCC 27756T (= VPI B2-51T), Mediterraneibacter gnavus comb. nov., with type strain ATCC 29149T (= VPI C7-9T) and Mediterraneibacter glycyrrhizinilyticus comb. nov., with type strain ZM35T (= JCM 13368T = DSM 17593T), respectively.

Simultaneous UHPLC-UV analysis of hydroxychloroquine, minocycline and doxycycline from serum samples for the therapeutic drug monitoring of Q fever and Whipple's disease.

A fast UHPLC-UV method was developed for the simultaneous analysis of Hydroxychloroquine, Minocycline and Doxycycline drugs from 100µL of human serum samples. Serum samples were extracted by liquid-liquid extraction and injected into a phenyl hexyl reverse phase column. Compounds were separated using a mobile phase linear gradient and monitored by UV detection at 343nm. Chloroquine and Oxytetracycline were used as internal standards. Lower and upper limits of quantifications, as well as the other levels of calibration, were validated with acceptable accuracy (<15% deviation) and precision (<15% coefficient of variation) according to the European Medicines Agency guidelines. This new method enables cost and time reduction and was considered suitable for the clinical laboratory. It is the first published assay for the therapeutic drug monitoring of patients diagnosed with Q fever or Whipple's disease.

Hugonella massiliensis gen. nov., sp. nov., genome sequence, and description of a new strictly anaerobic bacterium isolated from the human gut.

The human gut is composed of a large diversity of microorganisms, which have been poorly described. Here, using culturomics, a new concept based on the variation in culture conditions and MALDI-TOF MS identification, we proceed to explore the microbial diversity of the complex ecosystem of the human gut. Using this approach, we isolated strain AT8T (=CSUR P2118 =  DSM 101782) from stool specimens collected from a 51-year-old obese French woman. Strain AT8T is a strictly anaerobic, nonmotile, nonspore-forming gram-positive coccus that do not exhibit catalase and oxidase activities. 16S rDNA-based identification of strain AT8T demonstrated 92% gene sequence similarity with Eggerthella lenta DSM 2243, the phylogenetically closed validly named type species. Here, we present a set of features for the strain AT8T and the description of its complete genome sequence and annotation. The 2,091,845 bp long genome has a G+C content of 63.46% and encodes1,849 predicted genes; 1,781 were protein-coding genes, and 68 were RNAs. On the basis of the characteristics reported here, we propose the creation of a new bacterial genus Hugonella gen. nov., belonging to the Eggerthellaceae family and including Hugonella massiliensis gen. nov., sp. nov., strain AT8T as the type strain.

Olegusella massiliensis gen. nov., sp. nov., strain KHD7T, a new bacterial genus isolated from the female genital tract of a patient with bacterial vaginosis.

Strain KHD7T, a Gram-stain-positive rod-shaped, non-sporulating, strictly anaerobic bacterium, was isolated from the vaginal swab of a woman with bacterial vaginosis. We studied its phenotypic characteristics and sequenced its complete genome. The major fatty acids were C16:0 (44%), C18:2n6 (22%), and C18:1n9 (14%). The 1,806,744 bp long genome exhibited 49.24% G+C content; 1549 protein-coding and 51 RNA genes. Strain KHD7T exhibited a 93.5% 16S rRNA similarity with Olsenella uli, the phylogenetically closest species in the family Coriobacteriaceae. Therefore, strain KHD7T is sufficiently distinct to represent a new genus, for which we propose the name Olegusella massiliensis gen. nov., sp. nov. The type strain is KHD7T.

Crystal structure of the phosphate-binding protein (PBP-1) of an ABC-type phosphate transporter from Clostridium perfringens.

Phosphate limitation is an important environmental stress that affects the metabolism of various organisms and, in particular, can trigger the virulence of numerous bacterial pathogens. Clostridium perfringens, a human pathogen, is one of the most common causes of enteritis necroticans, gas gangrene and food poisoning. Here, we focused on the high affinity phosphate-binding protein (PBP-1) of an ABC-type transporter, responsible for cellular phosphate uptake. We report the crystal structure (1.65 Å resolution) of the protein in complex with phosphate. Interestingly, PBP-1 does not form the short, low-barrier hydrogen bond with phosphate that is typical of previously characterized phosphate-binding proteins, but rather a canonical hydrogen bond. In its unique binding configuration, PBP-1 forms an unusually high number of hydrogen bonds (14) with the phosphate anion. Discrimination experiments reveal that PBP-1 is the least selective PBP characterised so far and is able to discriminate phosphate from its close competing anion, arsenate, by ~150-fold.