Non-contiguous finished genome sequencing and description of Enterococcus timonensis sp. nov. isolated from human sputum.

Enterococcus timonensis sp. nov., strain Marseille-P2817T, is a facultatively anaerobic, motile and non-spore-forming Gram-positive coccus which was isolated from the sputum of a healthy adult man in Marseilles. We present herein its phenotypic description together with MALDI-TOF (matrix-assisted laser-desorption/ionization time-of-flight) mass spectrometry analysis and genome sequencing and comparison. The genome of Enterococcus timonensis is 2 123 933 bp long with 38.46 mol% of G+C content, and it contains 1983 protein-coding genes and 65 RNA genes (including nine rRNA genes).

A hospital qPCR-based survey of 10 gastrointestinal parasites in routine diagnostic screening, Marseille, France.

There is a scarcity of recent epidemiological data on intestinal parasitic infections in France. We conducted a prospective study aimed at estimating the prevalence of 10 enteric parasites in Marseille, France, using real-time polymerase chain reaction (PCR)-based diagnosis. A total of 643 faeces from 488 patients referred to the Parasitology-Mycology Laboratory of the University Hospital of Marseille over a 6 months period were included. DNA was extracted using a semi-automated method. Parasites of interest were detected using singleplex quantitative PCRs (qPCRs). For positive samples, the Blastocystis subtype was determined by sequence analysis. During the study, the overall prevalence of enteric parasites was 17%. Blastocystis sp. was the most frequent species (10.5%), followed by Dientamoeba fragilis (2.3%) and Giardia intestinalis (2.3%). The prevalence of other parasites was <1% each. The ST3 Blastocystis subtype was predominant (43.6%) and the other subtypes identified were ST1, ST2, ST4 and ST6. This is the first time that a qPCR-based diagnosis has been used to survey the prevalence of 10 enteric parasites in a French University Hospital. This study confirms that fast, specific, sensitive and simultaneous detection in a single stool sample by qPCR clearly outperforms conventional microscopy-based diagnosis. Furthermore, qPCR is particularly well suited to surveying gastroenteritis agents.

'Cellulomonas timonensis' sp. nov., taxonogenomics description of a new bacterial species isolated from human gut.

'Cellulomonas timonensis' sp. nov. strain sn7T is a new species within the Cellulomonas genus. We present the main phenotypic characteristics and provide a complete annotation of its genome sequence. This facultative anaerobic bacterium, isolated from the stool of 38-year-old obese Frenchman, is Gram-positive, has motile rods and is sporulating. The genome is 4 057 828 bp long with 72.42% G + C content. Of the 3732 predicted genes, 3667 were protein-coding genes and 65 were RNAs.

Massilioclostridium coli gen. nov., sp. nov., a new member of the Clostridiaceae family isolated from the left colon of a 27-year-old woman.

Massilioclostridium coli strain Marseille-P2976T (= CSUR P2976 = DSM 103344) is a new bacterial genus isolated from the left colon of a patient who underwent colonoscopy for colorectal cancer screening. Massilioclostridium coli is a Gram-negative bacillus, strict anaerobic, nonsporogenous and nonmotile organism. We describe here the strain Marseille-P2976T and provide its complete annotated genome sequence according to taxonogenomics concepts. Its genome is 2 985 330 bp long and contains 2562 predicted genes and 75 RNA genes.

Marseillibacter massiliensis gen. nov., sp. nov., a new bacterial genus isolated from the human gut.

In this manuscript, we report the main characteristics of Marseillibacter massiliensis gen. nov., sp. nov., strain Marseille-P2840T (CSUR P2840), a new member of the family Oscillospiraceae that was isolated from the stool of a healthy 29-year-old Senegalese woman.

Noncontiguous finished genome sequence and description of Fusobacterium massiliense sp. nov. isolated from human duodenum.

The strain Marseille-P2749T (= CSUR P2749 = DSM 103085) was isolated as part of culturomics study from a liquid duodenum sample from a French man. Bacterial cells were Gram-negative bacilli, fusiform shaped and non-spore forming, and they grew in microaerophilic and anaerobic atmosphere. Its genome is 1 809 169 bp long and contains 1646 protein-coding genes. The DNA G+C content was 27.33 mol%. This strain exhibited a 95.9% sequence similarity with Fusobacterium periodonticum, the phylogenetically closest species with standing in nomenclature. Strain Marseille-P2749T is suggested to be a novel species belonging to the genus Fusobacterium, for which the name Fusobacterium massiliense sp. nov. is proposed.

Noncontiguous finished genome sequence and description of Streptococcus timonensis sp. nov. isolated from the human stomach.

Strain Marseille-P2915T, a Gram-positive, facultative anaerobic and nonmotile coccus, was isolated from the gastric lavage of a patient with severe anaemia. The 16S rRNA and rpoB gene comparison exhibited a sequence identity of 98.7 and 92.6% with Streptococcus infantis strain JCM 10157T, respectively, collocating it within the 'Streptococcus mitis' group. On the basis of phenotypic and genomic analysis, we propose the validation of the type strain Streptococcus timonensis sp. nov. Marseille-P2915T (= DSM 103349 = CSUR P2915).

Discovery of a new species within Bacillus genus in a stool sample from Gabon: "Bacillus massiliogabonensis" sp. nov.

The discovery of new bacteria from the human gut using a culturomics method is a novel field of increasing interest in microbiology. Here the main characteristics of "Bacillus massiliogabonensis" strain Marseille P2639, a new Gram-negative bacterium isolated from the stool sample of a healthy 16-year-old Gabonese boy, are reported.

"Ruminococcus phoceensis," a new species identified from human stool from an obese patient before bariatric surgery.

We report here the main characteristics of a new bacterium species, "Ruminococcus phoceensis" strain AT10 (CSUR = P2086, DSM = 100837). This bacterium was isolated from the faeces of a 37-year-old woman from Marseille, France, with morbid obesity before bariatric surgery.

"Raoultibacter massiliensis" gen. nov., sp. nov., a new bacterium isolated from the human gut of a Saudi Bedouin.

We propose the description of a new bacterial genus and new bacterial species, "Raoultibacter massiliensis," isolated from a faecal specimen of a 19-year-old healthy Saudi Bedouin.

"Actinomyces ihumii," a new bacterial species isolated from the digestive microbiota of a HIV-infected patient.

We report the main properties of "Actinomyces ihumii" strain SD1 (= CSUR P2006) isolated from the stool of a 50-year-old HIV-infected man.

'Eisenbergiella massiliensis', a new species isolated from human stool collected after bariatric surgery.

We report the principal characteristics of 'Eisenbergiella massiliensis' sp. nov. strain AT11 (CSURP = P2120, DSM = 101499) that was isolated from a stool sample collected after bariatric surgery of a 56-year-old obese French woman.

Repertory of eukaryotes (eukaryome) in the human gastrointestinal tract: taxonomy and detection methods.

Eukaryotes are an important component of the human gut, and their relationship with the human host varies from parasitic to commensal. Understanding the diversity of human intestinal eukaryotes has important significance for human health. In the past few decades, most of the multitudes of techniques that are involved in the diagnosis of the eukaryotic population in the human intestinal tract were confined to pathological and parasitological aspects that mainly rely on traditionally based methods. However, development of culture-independent molecular techniques comprised of direct DNA extraction from faeces followed by sequencing, offer new opportunities to estimate the occurrence of eukaryotes in the human gut by providing data on the entire eukaryotic community, particularly not-yet-cultured or fastidious organisms. Further broad surveys of the eukaryotic communities in the gut based on high throughput tools such as next generation sequencing might lead to uncovering the real diversity of these ubiquitous organisms in the human intestinal tract and discovering the unrecognized roles of these eukaryotes in modulating the host immune system and inducing changes in host gut physiology and ecosystem.

Cutaneous and pulmonary dirofilariasis due to Dirofilaria repens.

Dirofilariasis is a worldwide zoonotic infection that accidentally affects humans. It is caused by filarial nematodes of the genus Dirofilaria, which are transmitted by mosquitoes. Cutaneous dirofilariasis appears as inflammatory lesions that could be consistent with Wells' cellulitis. We present a remarkable case of human infection with Dirofilaria repens, causing both subcutaneous and pulmonary nodules.

Pandoraea pulmonicola chronic colonization in a cystic fibrosis patient, France.

Pandoraea are considered emerging multidrug resistant pathogens in the context of cystic fibrosis. We report herein for the first time the case of a 30-year-old woman with cystic fibrosis, living in France, who was chronically infected with Pandoraea pulmonicola and who died of Pseudomonas aeruginosa sepsis 3 weeks after bilateral lung transplantation.

Microbial culturomics: paradigm shift in the human gut microbiome study.

Comprehensive determination of the microbial composition of the gut microbiota and the relationships with health and disease are major challenges in the 21st century. Metagenomic analysis of the human gut microbiota detects mostly uncultured bacteria. We studied stools from two lean Africans and one obese European, using 212 different culture conditions (microbial culturomics), and tested the colonies by using mass spectrometry and 16S rRNA amplification and sequencing. In parallel, we analysed the same three samples by pyrosequencing 16S rRNA amplicons targeting the V6 region. The 32 500 colonies obtained by culturomics have yielded 340 species of bacteria from seven phyla and 117 genera, including two species from rare phyla (Deinococcus-Thermus and Synergistetes, five fungi, and a giant virus (Senegalvirus). The microbiome identified by culturomics included 174 species never described previously in the human gut, including 31 new species and genera for which the genomes were sequenced, generating c. 10 000 new unknown genes (ORFans), which will help in future molecular studies. Among these, the new species Microvirga massiliensis has the largest bacterial genome so far obtained from a human, and Senegalvirus is the largest virus reported in the human gut. Concurrent metagenomic analysis of the same samples produced 698 phylotypes, including 282 known species, 51 of which overlapped with the microbiome identified by culturomics. Thus, culturomics complements metagenomics by overcoming the depth bias inherent in metagenomic approaches.

Detection and accurate identification of new or emerging bacteria in cystic fibrosis patients.

Respiratory infections remain a major threat to cystic fibrosis (CF) patients. The detection and correct identification of the bacteria implicated in these infections is critical for the therapeutic management of patients. The traditional methods of culture and phenotypic identification of bacteria lack both sensitivity and specificity because many bacteria can be missed and/or misidentified. Molecular analyses have recently emerged as useful means to resolve these problems, including molecular methods for accurate identification or detection of bacteria and molecular methods for evaluation of microbial diversity. These recent molecular technologies have increased the list of new and/or emerging pathogens and epidemic strains associated with CF patients. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of intact cells has also emerged recently as a powerful and rapid method for the routine identification of bacteria in clinical microbiology laboratories and will certainly represent the method of choice also for the routine identification of bacteria in the context of CF. Finally, recent data derived from molecular culture-independent analyses indicate the presence of a previously underestimated, complex microbial community in sputa from CF patients. Interestingly, full genome sequencing of some bacteria frequently recovered from CF patients has highlighted the fact that the lungs of CF patients are hotspots for lateral gene transfer and the adaptation of these ecosystems to a specific chronic condition.

Microbial diversity in the sputum of a cystic fibrosis patient studied with 16S rDNA pyrosequencing.

Recent studies using 16S rRNA gene amplification followed by clonal Sanger sequencing in cystic fibrosis demonstrated that cultured microorganisms are only part of the infecting flora. The purpose of this paper was to compare pyrosequencing and clonal Sanger sequencing on sputum. The sputum of a patient with cystic fibrosis was analysed by culture, Sanger clone sequencing and pyrosequencing after 16S rRNA gene amplification. A total of 4,499 sequencing reads were obtained, which could be attributed to six consensus sequences, but the length of reads leads to fastidious data analysis. Compared to clonal Sanger sequencing and to cultivation results, pyrosequencing recovers greater species richness and gives a more reliable estimate of the relative abundance of bacterial species. The 16S pyrosequencing approach expands our knowledge of the microbial diversity of cystic fibrosis sputum. The current lack of phylogenetic resolution at the species level for the GS 20 sequencing reads will be overcome with the next generation of pyrosequencing apparatus.

Unexpected enzyme TEM-126: role of mutation Asp179Glu.

The clinical isolate Escherichia coli CF884 exhibited low-level resistance to ceftazidime (4 mug/ml) by a positive double-disk synergy test and apparent susceptibility to cefuroxime, cefotaxime, cefepime, cefpirome, and aztreonam. The enzyme implicated in this phenotype was a novel 180-kb plasmid-encoded TEM-type extended-spectrum beta-lactamase designated TEM-126 which harbors the mutations Asp179Glu and Met182Thr. TEM-126 exhibited significant hydrolytic activity (k(cat), 2 s(-1)) and a K(m) value of 82 muM against ceftazidime. Molecular dynamics simulations suggested that the substitution Asp179Glu induces subtle conformational changes to the omega loop which may favor the insertion of ceftazidime in the binding site and the correct positioning of the crucial residue Glu166. Overall, these results highlight the remarkable plasticity of TEM enzymes, which can expand their activity against ceftazidime by the addition of one carbon atom in the side chain of residue 179.