State of the Art in the Culture of the Human Microbiota: New Interests and Strategies.

The last 5 years have seen a turning point in the study of the gut microbiota with a rebirth of culture-dependent approaches to study the gut microbiota. High-throughput methods have been developed to study bacterial diversity with culture conditions aimed at mimicking the gut environment by using rich media such as YCFA (yeast extract, casein hydrolysate, fatty acids) and Gifu anaerobic medium in an anaerobic workstation, as well as media enriched with rumen and blood and coculture, to mimic the symbiosis of the gut microbiota. Other culture conditions target phenotypic and metabolic features of bacterial species to facilitate their isolation. Preexisting technologies such as next-generation sequencing and flow cytometry have also been utilized to develop innovative methods to isolate previously uncultured bacteria or explore viability in samples of interest. These techniques have been applied to isolate CPR (Candidate Phyla Radiation) among other, more classic approaches. Methanogenic archaeal and fungal cultures present different challenges than bacterial cultures. Efforts to improve the available systems to grow archaea have been successful through coculture systems. For fungi that are more easily isolated from the human microbiota, the challenge resides in the identification of the isolates, which has been approached by applying matrix-assisted laser desorption ionization-time of flight mass spectrometry technology to fungi. Bacteriotherapy represents a nonnegligible avenue in the future of medicine to correct dysbiosis and improve health or response to therapy. Although great strides have been achieved in the last 5 years, efforts in bacterial culture need to be sustained to continue deciphering the dark matter of metagenomics, particularly CPR, and extend these methods to archaea and fungi.

Methanobrevibacter smithii Archaemia in Febrile Patients With Bacteremia, Including Those With Endocarditis.

BACKGROUND: The spectrum of infections caused by methanogens remains to be described. We searched for methanogens in the blood of febrile patients using specific tools. METHODS: Blood culture samples routinely collected in patients with fever were prospectively screened by specific PCR assays for methanogens. Positive samples were observed by autofluorescence and electron microscopy, analyzed by metagenomics and cultured using previously developed methods. Blood culture bottles experimentally inoculated were used as controls. The presence of methanogens in vascular and cardiac tissues was assessed by indirect immunofluorescence, fluorescent in situ hybridization and PCR-based investigations. RESULTS: PCR detection attempted in 7,716 blood samples, was negative in all 1,312 aerobic bottles and 810 bacterial culture-negative anaerobic bottles. PCRs were positive in 27/5,594 (0.5%) bacterial culture-positive anaerobic bottles collected from 26 patients. Sequencing confirmed Methanobrevibacter smithii associated with staphylococci in 14 patients, Enterobacteriaceae in nine patients and streptococci in three patients. Metagenomics confirmed M. smithii in five samples, and M. smithii was isolated in broth from two samples; the genomes of these two isolates were sequenced. Blood cultures experimentally inoculated with Enterobacteriaceae, Staphylococcus epidermidis or Staphylococcus hominis yielded hydrogen, but no methane, authentifying observational data. Three patients diagnosed with infectious mitral endocarditis, were indisputably diagnosed by microscopy, PCR-based detections and culture: we showed M. smithii microscopically and by a specific PCR followed by sequencing method in two of three cardiovascular tissues. CONCLUSIONS: Using appropriate laboratory methods, M. smithii is demonstrated as causing archaemia and endocarditis in febrile patients who are coinfected by bacteria.

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).

Intra- and inter-laboratory comparison of mDixon and FastFung broths for Malassezia antifungal susceptibility testing.

BACKGROUND: Malassezia spp. antifungal susceptibility testing (AFST) capacities are limited by the lack of efficient and standardised AFST procedure, mainly because of the fastidious cultivation of these yeast. OBJECTIVES: This study aimed to compare the FastFung broth (FFB) to modified Dixon broth (mDIXB) for the in vitro AFST of Malassezia spp. Fluconazole, ketoconazole, voriconazole and terbinafine MICs against a 19 Malassezia strains, including 6 M furfur, 4 M pachydermatis, 5 M sympodialis and 4 M slooffiae. METHODS: The essential agreement (EA) between the two assays, and the intra- and inter-laboratory agreement of each assay were assessed. RESULTS: The MIC data obtained in our study were comparable to those reported in the literature. FFB showed to enhance Malassezia growth and displayed 100% (±2-fold dilution) EAs demonstrating similar performances to mDIXB. In addition, the MIC data obtained by using the FFB were reproducible between laboratories with EAs ranging from 94.7% to 100%. CONCLUSIONS: Therefore, FFB is a suitable alternative to mDXB for Malassezia spp. AFST.

Draft genome and description of Negativicoccus massiliensis strain Marseille-P2082, a new species isolated from the gut microbiota of an obese patient.

Strain Marseille-P2082, an anaerobic, non-motile, asporogenous, Gram-negative, coccoid bacterium was isolated from the faeces of a 33 year-old obese French woman before bariatric surgery. The isolate exhibits 98.65% 16S rRNA gene nucleotide sequence similarity with Negativicoccus succinicivorans strain ADV 07/08/06-B-1388T, its current closest phylogenetic neighbour with standing in nomenclature. However, the dDDH relatedness between the new isolate and N. succinicivorans type strain ADV 07/08/06-B-1388T is 52.5 ± 2.7%. Strain Marseille-P2082 has a genome of 1,360,589 bp with a 51.1% G+C content. Its major fatty acids were identified as C18:1n9, C18:0 and C16:0. Based on its phenotypic, genomic and phylogenetic characteristics, strain Marseille-P2082T [= CSURP2082 (Collection de Souches de l'Unité des Rickettsies) = DSM 100853] is proposed as the type strain of the novel species Negativicoccus massiliensis sp. nov. The 16S rRNA gene sequence and whole-genome shotgun sequence have been deposited in EMBL-EBI under accession numbers LN876651 and LT700188, respectively.

Passive Filtration, Rapid Scanning Electron Microscopy, and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Treponema Culture and Identification from the Oral Cavity.

We present here a new passive-filtration-based culture device combined with rapid identification with a new electron microscope (Hitachi TM4000) for the detection and culture of Treponema species from the human oral cavity. Of the 44 oral samples cultivated, 15 (34%) were found to be positive for Treponema using electron microscopy and were also culture positive. All were subcultured on agar plates; based on genome sequencing and analyses, 10 were strains of Treponema pectinovorum and 5 were strains of Treponema denticola The 29 samples that were negative for Treponema remained culture negative. In addition, 14 Treponema species ordered from the DSMZ collection were cultured in the T-Raoult culture medium optimized here. Finally, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used and 30 novel spectra were added to the MALDI-TOF MS database. We have successfully developed a new and effective method for treponemal detection, culture, and identification.

Oceanobacillus timonensis sp. nov. and Oceanobacillus senegalensis sp. nov., two new moderately halophilic, Gram-stain positive bacteria isolated from stools sample of healthy young Senegalese.

Oceanobacillus timonensis Marseille-P3532T (CSUR P3532, CCUG 70981) and Oceanobacillus senegalensis Marseille-P3587T (CSUR P3587, CCUG 70613), are the type strains of O. timonensis sp. nov. and O. senegalensis sp. nov., respectively. They are moderately halophilic, aerobic, motile and Gram-stain positive bacteria. The strains P3532T and P3587T were isolated from stools with 3.8% and 2.1% sodium chloride (NaCl) of healthy 10 year old female and male 7-year-old children, respectively and living respectively at Dielmo and N'diop two villages in Senegal (West Africa). This study aimed to describe the genome and phenotypic characteristics of O. timonensis Marseille-P3532T and O. senegalensis Marseille-P3587T. The genomes are 4,485,335 bp long for O. timonensis and 4,300,331 bp for O. senegalensis with 38.78% and 36.92% G+C content, respectively. They contain 4306 and 3979 protein-coding and 87 and 273 RNAs genes, respectively.

Genome sequence and description of Haloferax massiliense sp. nov., a new halophilic archaeon isolated from the human gut.

By applying the culturomics concept and using culture conditions containing a high salt concentration, we herein isolated the first known halophilic archaeon colonizing the human gut. Here we described its phenotypic and biochemical characterization as well as its genome annotation. Strain Arc-HrT (= CSUR P0974 = CECT 9307) was mesophile and grew optimally at 37 °C and pH 7. Strain Arc-HrT was also extremely halophilic with an optimal growth observed at 15% NaCl. It showed gram-negative cocci, was strictly aerobic, non-motile and non-spore-forming, and exhibited catalase and oxidase activities. The 4,015,175 bp long genome exhibits a G + C% content of 65.36% and contains 3911 protein-coding and 64 predicted RNA genes. PCR-amplified 16S rRNA gene of strain Arc-HrT yielded a 99.2% sequence similarity with Haloferax prahovense, the phylogenetically closest validly published species in the Haloferax genus. The DDH was of 50.70 ± 5.2% with H. prahovense, 53.70 ± 2.69% with H. volcanii, 50.90 ± 2.64% with H. alexandrinus, 52.90 ± 2.67% with H. gibbonsii and 54.30 ± 2.70% with H. lucentense. The data herein represented confirm strain Arc-HrT as a unique species and consequently we propose its classification as representative of a novel species belonging to the genus Haloferax, as Haloferax massiliense sp. nov.

Sediminibacillus massiliensis sp. nov., a moderately halophilic, Gram-positive bacterium isolated from a stool sample of a young Senegalese man.

A Gram-positive, moderately halophilic bacterium, referred to as strain Marseille-P3518T, was isolated from a stool sample with 2% NaCl concentration from a healthy 15-year-old male living in Dielmo, a village in Senegal. Cells are aerobic, rod-shaped and motile and display endospore formation. Strain Marseille-P3518T can grow in a medium with 0-20% (w/v) sodium chloride (optimally at 5-7.5% w/v). The major fatty acids were 12-methyl-tetradecanoic acid (45.8%), 13-methyl-tetradecanoic acid (26.9%) and 12-methyl-tridecanoic acid (12.8%). The genome is 4,347,479 bp long with 42.1% G+C content. It contains 4282 protein-coding and 107 RNA genes. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that strain Marseille-P3518T is a member of the Bacillaceae family and is closely related to Sediminibacillus albus (97.4% gene sequence similarity). Strain Marseille-P3518T was clearly differentiated from its phylogenetic neighbors on the basis of phenotypic and genotypic features. Strain Marseille-P3518T is, therefore, considered to be a novel representative of the genus Sediminibacillus, for which the name Sediminibacillus massiliensis sp. nov. is proposed, and the type strain is Marseille-P3518T (CSUR P3518T, DSM69894).

Fournierella massiliensis gen. nov., sp. nov., a new human-associated member of the family Ruminococcaceae.

An anaerobic bacterium, strain AT2T, was isolated from the fresh stool sample of a healthy French man using the culturomics approach. The 16S rRNA gene sequence analysis showed that strain AT2T had 95.2 % nucleotide sequence similarity with Gemmiger formicilisATCC 27749T, the phylogenetically closest species with standing in nomenclature. Cells are Gram-stain-negative, catalase- and oxidase-negative, obligately anaerobic, non-motile, non-spore-forming, rod-shaped, and the bacilli were mesothermophilic. The major fatty acids were C16 : 0 (43.8 %) and C18 : 1n9 (20 %). The DNA G+C content of the strain based on its genome sequence was 56.8 mol%. Based on the phenotypic, biochemical and phylogenetic analysis, we propose the creation of the genus Fournierella gen. nov., which contains strain AT2T (=CSUR P2014T=DSM 100451T) as the type strain of the type species Fournierella massiliensis gen. nov., sp. nov.

The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota.

Bacterial culture was the first method used to describe the human microbiota, but this method is considered outdated by many researchers. Metagenomics studies have since been applied to clinical microbiology; however, a "dark matter" of prokaryotes, which corresponds to a hole in our knowledge and includes minority bacterial populations, is not elucidated by these studies. By replicating the natural environment, environmental microbiologists were the first to reduce the "great plate count anomaly," which corresponds to the difference between microscopic and culture counts. The revolution in bacterial identification also allowed rapid progress. 16S rRNA bacterial identification allowed the accurate identification of new species. Mass spectrometry allowed the high-throughput identification of rare species and the detection of new species. By using these methods and by increasing the number of culture conditions, culturomics allowed the extension of the known human gut repertoire to levels equivalent to those of pyrosequencing. Finally, taxonogenomics strategies became an emerging method for describing new species, associating the genome sequence of the bacteria systematically. We provide a comprehensive review on these topics, demonstrating that both empirical and hypothesis-driven approaches will enable a rapid increase in the identification of the human prokaryote repertoire.

Description of Gabonibacter massiliensis gen. nov., sp. nov., a New Member of the Family Porphyromonadaceae Isolated from the Human Gut Microbiota.

The identification of human-associated bacteria is very important to control infectious diseases. In recent years, we diversified culture conditions in a strategy named culturomics, and isolated more than 100 new bacterial species and/or genera. Using this strategy, strain GM7, a strictly anaerobic gram-negative bacterium was recently isolated from a stool specimen of a healthy Gabonese patient. It is a motile coccobacillus without catalase and oxidase activities. The genome of Gabonibacter massiliensis is 3,397,022 bp long with 2880 ORFs and a G+C content of 42.09 %. Of the predicted genes, 2,819 are protein-coding genes, and 61 are RNAs. Strain GM7 differs from the closest genera within the family Porphyromonadaceae both genotypically and in shape and motility. Thus, we propose that strain GM7T (=CSUR P2336 = DSM 101039) is the type strain of the new genus Gabonibacter gen. nov. and the new species G. massiliensis gen. nov., sp. nov.

Anaerococcus rubiinfantis sp. nov., isolated from the gut microbiota of a Senegalese infant with severe acute malnutrition.

Anaerococcus rubiinfantis sp. nov. strain mt16(T) is a new species within the genus Anaerococcus, which was isolated by the culturomics approach from the gut microbiota of an infant suffering from kwashiorkor. A phenotypic, biochemical and proteomic description of this strain is hereby presented alongside a complete annotation of its genome. This strictly anaerobic species forms Gram-positive non-sporeforming cocci. The major fatty acid was hexadecanoic acid. The phylogenetic analysis of strain mt16(T) showed a 97.9% similarity level with Anaerococcus vaginalis, the closest validly published species. Its genome is 1,929,161 bp long with 29.5% G + C content and contains 1808 protein-coding genes and 56 RNA genes, among which are six rRNA genes. Genomic analysis identified 41/1864 coding genes as ORFans (2.2%) and at least 620/1808 (34.9%) orthologous proteins which are not shared with the closest phylogenetic species. We believe that the extension of the human anaerobic gut compendium by culturomics is one of the first steps that will improve the understanding of the links between the microbiome and health or disease.

Microbial culturomics unravels the halophilic microbiota repertoire of table salt: description of Gracilibacillus massiliensis sp. nov.

BACKGROUND: Microbial culturomics represents an ongoing revolution in the characterization of environmental and human microbiome. METHODS: By using three media containing high salt concentration (100, 150, and 200 g/L), the halophilic microbial culturome of a commercial table salt was determined. RESULTS: Eighteen species belonging to the Terrabacteria group were isolated including eight moderate halophilic and 10 halotolerant bacteria. Gracilibacillus massiliensis sp. nov., type strain Awa-1T (=CSUR P1441=DSM 29726), is a moderately halophilic gram-positive, non-spore-forming rod, and is motile by using a flagellum. Strain Awa-1T shows catalase activity but no oxidase activity. It is not only an aerobic bacterium but also able to grow in anaerobic and microaerophilic atmospheres. The draft genome of G. massiliensis is 4,207,226 bp long, composed of 13 scaffolds with 36.05% of G+C content. It contains 3,908 genes (3,839 protein-coding and 69 RNA genes). At least 1,983 (52%) orthologous proteins were not shared with the closest phylogenetic species. Hundred twenty-six genes (3.3%) were identified as ORFans. CONCLUSIONS: Microbial culturomics can dramatically improve the characterization of the food and environmental microbiota repertoire, deciphering new bacterial species and new genes. Further studies will clarify the geographic specificity and the putative role of these new microbes and their related functional genetic content in environment, health, and disease.

Clostridium polynesiense sp. nov., a new member of the human gut microbiota in French Polynesia.

Strain MS1, a Gram-positive, obligately anaerobic, motile and spore-forming rod belonging to the Clostridium genus, was isolated from the feces of a healthy Polynesian male living in French Polynesia. The temperature range for growth was 30-45 °C. We sequenced its complete genome and studied its phenotypic characteristics. The 3,560,738-bp long genome (one chromosome, no plasmid, G + C content 34%) contained 3535 protein-coding and 70 RNA genes. Strain MS1 exhibited a 98.24% 16S rRNA similarity with Clostridium amylolyticum, the phylogenetically closest species. When compared with other Clostridium species with standing in nomenclature, it had an average genomic similarity of 68.8-70%, a unique MALDI-TOF spectrum, and differed in nitrate reduction, motility and L-arabinose and D-lactose metabolism with most of the closest species. Therefore, strain MS1 is sufficiently distinct from type strains of the genus Clostridium to represent a novel species within this genus, for which the name Clostridium polynesiense sp. nov. is proposed. The type strain of C. polynesiense is MS1(T) (= CSUR P630 = DSM 27072).

Real-time PCR quantification of Methanobrevibacter oralis in periodontitis.

A real-time PCR assay developed to quantify Methanobrevibacter oralis indicated that its inoculum significantly correlated with periodontitis severity (P = 0.003), despite a nonsignificant difference in prevalence between controls (3/10) and patients (12/22) (P = 0.2, Fisher test). The M. oralis load can be used as a biomarker for periodontitis.

Culturing the Human Oral Microbiota, Updating Methodologies and Cultivation Techniques.

Recent years have been marked by a paradigm shift in the study of the human microbiota, with a re-emergence of culture-dependent approaches. Numerous studies have been devoted to the human microbiota, while studies on the oral microbiota still remain limited. Indeed, various techniques described in the literature may enable an exhaustive study of the microbial composition of a complex ecosystem. In this article, we report different methodologies and culture media described in the literature that can be applied to study the oral microbiota by culture. We report on specific methodologies for targeted culture and specific culture techniques and selection methodologies for cultivating members of the three kingdoms of life commonly found in the human oral cavity, namely, eukaryota, bacteria and archaea. This bibliographic review aims to bring together the various techniques described in the literature, enabling a comprehensive study of the oral microbiota in order to demonstrate its involvement in oral health and diseases.

Desulfovibrio piezophilus sp. nov., a piezophilic, sulfate-reducing bacterium isolated from wood falls in the Mediterranean Sea.

A novel sulfate-reducing bacterium, designated C1TLV30(T), was isolated from wood falls at a depth of 1693 m in the Mediterranean Sea. Cells were motile vibrios (2-4 × 0.5 µm). Strain C1TLV30(T) grew at temperatures between 15 and 45 °C (optimum 30 °C) and at pH 5.4-8.6 (optimum 7.3). It required NaCl for growth (optimum at 25 g NaCl l(-1)) and tolerated up to 80 g NaCl l(-1). Strain C1TLV30(T) used as energy sources: lactate, fumarate, formate, malate, pyruvate and ethanol. The end products from lactate oxidation were acetate, H(2)S and CO(2) in the presence of sulfate as terminal electron acceptor. Besides sulfate, thiosulfate and sulfite were also used as terminal electron acceptors, but not elemental sulfur, fumarate, nitrate or nitrite. Strain C1TLV30(T) possessed desulfoviridin and was piezophilic, growing optimally at 10 MPa (range 0-30 MPa). The membrane lipid composition of this strain was examined to reveal an increase in fatty acid chain lengths at high hydrostatic pressures. The G+C content of the genomic DNA was 49.6 % and the genome size was estimated at 3.5 ± 0.5 Mb. Phylogenetic analysis of the SSU rRNA gene sequence indicated that strain C1TLV30(T) was affiliated to the genus Desulfovibrio with Desulfovibrio profundus being its closest phylogenetic relative (similarity of 96.4 %). On the basis of SSU rRNA gene sequence comparisons and physiological characteristics, strain C1TLV30(T) ( = DSM 21447(T) = JCM 1548(T)) is proposed to be assigned to a novel species of the genus Desulfovibrio, Desulfovibrio piezophilus sp. nov.

Evaluation of Culture Top transport systems for assessing the bacterial diversity of microbiota by culturomics as compared to a routine transport system.

In recent years, metagenomics and then culturomics, which consists of the multiplication of media and culture conditions and the rapid identification of all bacterial colonies, have generated renewed interest in the human microbiota, and diseases associated with modifications in its composition in particular. The sample transport media included in diverse swab transport systems and the storage conditions are among the factors that influence the results of the culturomics. In this study, we compared the results of culturomics from paired skin, oral and rectal swabs from intensive care unit (ICU) patients using Culture Top sample transport medium as compared to our routine one. From 152 clinical samples, we were able to isolate and identify 45 600 colonies, belonging to 338 different bacterial species. The transport system Culture Top identified 282 different bacterial species, while 244 were identified by our routine system. Of these, 188 different bacterial species were commonly identified using both transport systems, while 94 (27.8 %) and 56 (16.5 %) were only identified using Culture Top and our routine system, respectively (P<0.001), but there was no significant difference in bacterial diversity at the genus or phylum level, or in terms of their type of respiration and cell wall. In conclusion, the Culture Top transport system appears to be complementary to our routine system, although it seems slightly superior in terms of isolated bacterial species.

A review of in vitro attempts to develop the axenic culture of Treponema pallidum and genomics-based suggestions to achieve this elusive goal.

To date, the axenic culture of Treponema pallidum remains a challenge in the field of microbiology despite countless attempts. Here, we conducted a comprehensive bibliographic analysis using several databases and search engines, namely Pubmed, Google scholar, Google, Web of Science and Scopus. Numerous unsuccessful empiric studies have been conducted and evaluated using as criteria dark-field microscopic observation of motile spiral shaped cells in the culture and virulence of the culture through rabbit infectivity. All of these studies failed to induce rabbit infectivity, even when deemed positive after microscopic observation leading to the misnomer of avirulent T. pallidum. In fact, this criterion was improperly chosen because not all spiral shaped cells are T. pallidum. However, these studies led to the formulation of culture media particularly favourable to the growth of several species of Treponema, including Oral Microbiology and Immunology, Zürich medium (OMIZ), Oral Treponeme Enrichment Broth (OTEB) and T-Raoult, thus allowing the increase in the number of cultivable strains of Treponema. The predicted metabolic capacities of T. pallidum show limited metabolism, also exhibited by other non-cultured and pathogenic Treponema species, in contrast to cultured Treponema species. The advent of next generation sequencing represents a turning point in this field, as the knowledge inferred from the genome can finally lead to the axenic culture of T. pallidum.