Colorectal Cancer Archaeome: A Metagenomic Exploration, Tunisia.

Colorectal cancer (CRC) is a serious public health problem known to have a multifactorial etiology. The association between gut microbiota and CRC has been widely studied; however, the link between archaea and CRC has not been sufficiently studied. To investigate the involvement of archaea in colorectal carcinogenesis, we performed a metagenomic analysis of 68 formalin-embedded paraffin fixed tissues from tumoral (n = 33) and healthy mucosa (n = 35) collected from 35 CRC Tunisian patients. We used two DNA extraction methods: Generead DNA FFPE kit (Qiagen, Germantown, MD, USA) and Chelex. We then sequenced the samples using Illumina Miseq. Interestingly, DNA extraction exclusively using Chelex generated enough DNA for sequencing of all samples. After data filtering and processing, we reported the presence of archaeal sequences, which represented 0.33% of all the reads generated. In terms of abundance, we highlighted a depletion in methanogens and an enrichment in Halobacteria in the tumor tissues, while the correlation analysis revealed a significant association between the Halobacteria and the tumor mucosa (p < 0.05). We reported a strong correlation between Natrialba magadii, Sulfolobus acidocaldarius, and tumor tissues, and a weak correlation between Methanococcus voltae and healthy adjacent mucosa. Here, we demonstrated the feasibility of archaeome analysis from formol fixed paraffin-embedded (FFPE) tissues using simple protocols ranging from sampling to data analysis, and reported a significant association between Halobacteria and tumor tissues in Tunisian patients with CRC. The importance of our study is that it represents the first metagenomic analysis of Tunisian CRC patients' gut microbiome, which consists of sequencing DNA extracted from paired tumor-adjacent FFPE tissues collected from CRC patients. The detection of archaeal sequences in our samples confirms the feasibility of carrying out an archaeome analysis from FFPE tissues using a simple DNA extraction protocol. Our analysis revealed the enrichment of Halobacteria, especially Natrialba magadii, in tumor mucosa compared to the normal mucosa in CRC Tunisian patients. Other species were also associated with CRC, including Sulfolobus acidocaldarius and Methanococcus voltae, which is a methanogenic archaea; both species were found to be correlated with adjacent healthy tissues.

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.

Culture of salivary methanogens assisted by chemically produced hydrogen.

Methanogen cultures require hydrogen produced by fermentative bacteria such as Bacteroides thetaiotaomicron (biological method). We developed an alternative method for hydrogen production using iron filings and acetic acid with the aim of cultivating methanogens more efficiently and more quickly (chemical method). We developed this new method with a reference strain of Methanobrevibacter oralis, compared the method to the biological reference method with a reference strain of Methanobrevibacter smithii and finally applied the method to 50 saliva samples. Methanogen colonies counted using ImageJ software were identified using epifluorescence optical microscopy, real-time PCR and PCR sequencing. For cultures containing pure strains of M. oralis and M. smithii, colonies appeared three days postinoculation with the chemical method versus nine days with the biological method. The average number of M. smithii colonies was significantly higher with the chemical method than with the biological method. There was no difference in the delay of observation of the first colonies in the saliva samples between the two methods. However, the average number of colonies was significantly higher with the biological method than with the chemical method at six days and nine days postinoculation (Student's test, p = 0.005 and p = 0.04, respectively). The chemical method made it possible to isolate four strains of M. oralis and three strains of M. smithii from the 50 saliva samples. Establishing the chemical method will ease the routine isolation and culture of methanogens.

Methanogens as emerging pathogens in anaerobic abscesses.

Methanogens are strictly anaerobic archaea metabolising by-products of bacterial fermentation into methane by using three known metabolic pathways, i.e. the reduction of carbon dioxide, the fermentation of acetate or the dismutation of methanol or methylamines. Methanogens described in human microbiota include only Euryarchaeota, i.e. Methanobrevibacter smithii, Methanobrevibacter oralis, Methanobrevibacter arbophilus, Methanobrevibacter massiliensis, Methanomassiliicoccus luminyensis, Methanosphaera stadtmanae and Ca. Methanomethylophilus alvus and Ca. Methanomassiliicoccus intestinalis. Methanogens are emerging pathogens associated with brain and muscular abscesses. They have been implicated in dysbiosis of the oral microbiota, periodontitis and peri-implantitis. They have also been associated with dysbiosis of the digestive tract microbiota linked to metabolic disorders (anorexia, malnutrition and obesity) and with lesions of the digestive tract (colon cancer). Their detection in anaerobic pus specimens and oral and digestive tract specimens relies on microscopic examination by fluorescence in situ hybridisation, specific DNA extraction followed by polymerase chain reaction (PCR)-based amplification of the 16S rRNA and mcrA gene fragments and isolation and culture in the supporting presence of hydrogen-producing bacteria. Diagnostic identification can be performed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and can be further completed by genotyping through multi-spacer sequencing and, ultimately, whole genome sequencing (WGS). Ornidazole derivatives, fusidic acid and rifampicin are the compounds to be included in in vitro susceptibility testing to complete the clinical workflow. Clinical microbiology laboratories should work toward developing cheap and easy protocols for the routine detection and identification of methanogens in selected specimens in order to refine the diagnosis of infections, as well as to expand the knowledge about this group of intriguing microorganisms.

Detection of Methanobrevibacter smithii in vaginal samples collected from women diagnosed with bacterial vaginosis.

Vaginosis is a dysbiotic condition of the vaginal cavity that has deleterious effects during pregnancy. The role of methanogens in this disease is unknown since current methods of investigation are not appropriate for the search of methanogens. We prospectively investigated the presence of methanogens in vaginal specimens collected from 33 women thereafter diagnosed with bacterial vaginosis and 92 women thereafter diagnosed without bacterial vaginosis (control group) by direct microscopic examination and fluorescent in situ hybridization, PCR-sequencing, and real-time PCR and isolation and culture. These investigations found only one methanogen, Methanobrevibacter smithii, exclusively in 97% bacterial vaginosis specimens and in two intermediate microbiota specimens. M. smithii was detected microscopically in 2/20 specimens analyzed, by PCR-based observations in 34/125 specimens with 99% sequence similarity with the reference 16S rRNA and mcrA gene sequences and was cultured in 9/40 specimens. These data suggest that the detection of M. smithii could be used as a biomarker for the laboratory diagnosis of bacterial vaginosis.

The discovery of Candidatus Nanopusillus phoceensis sheds light on the diversity of the microbiota nanoarchaea.

To further assess the spectrum of nanoarchaea in human microbiota, we prospectively searched for nanoarchaea in 110 leftover stool specimens, using the complementary approaches of PCR-sequencing screening, fluorescent in situ hybridization, scanning electron microscopy and metagenomics. These investigations yielded a nanoarchaea, Candidatus Nanopusillus phoceensis sp. nov., detected in stool samples by specific PCR-based assays. Microscopic observations indicated its close contact with the archaea Methanobrevibacter smithii. Genomic sequencing revealed 607,775-bp contig with 24.5% G + C content encoding 30 tRNAs, 3 rRNA genes, and 1,403 coding DNA sequences, of which 719 were assigned to clusters of orthologous groups. Ca. Nanopusillus phoceensis is only the second nanoarchaea to be detected in humans, expanding our knowledge of the repertoire of nanoarchaea associated with the human microbiota and encouraging further research to explore the repertoire of this emerging group of nanomicrobes in clinical samples.

Methanobrevibacter massiliense and Pyramidobacter piscolens Co-Culture Illustrates Transkingdom Symbiosis.

Among oral microbiota methanogens, Methanobrevibacter massiliense (M. massiliense) has remained less studied than the well-characterised and cultivated methanogens Methanobrevibacter oralis and Methanobrevibacter smithii. M. massiliense has been associated with different oral pathologies and was co-isolated with the Synergistetes bacterium Pyramidobacter piscolens (P. piscolens) in one case of severe periodontitis. Here, reporting on two additional necrotic pulp cases yielded the opportunity to characterise two co-cultivated M. massiliense isolates, both with P. piscolens, as non-motile, 1-2-µm-long and 0.6-0.8-µm-wide Gram-positive coccobacilli which were autofluorescent at 420 nm. The two whole genome sequences featured a 31.3% GC content, gapless 1,834,388-base-pair chromosome exhibiting an 85.9% coding ratio, encoding a formate dehydrogenase promoting M. massiliense growth without hydrogen in GG medium. These data pave the way to understanding a symbiotic, transkingdom association with P. piscolens and its role in oral pathologies.

Candidatus Methanosphaera massiliense sp. nov., a methanogenic archaeal species found in a human fecal sample and prevalent in pigs and red kangaroos.

Methanosphaera stadtmanae was the sole Methanosphaera representative to be cultured and detected by molecular methods in the human gut microbiota, further associated with digestive and respiratory diseases, leaving unknown the actual diversity of human-associated Methanosphaera species. Here, a novel Methanosphaera species, Candidatus Methanosphaera massiliense (Ca. M. massiliense) sp. nov. was isolated by culture using a hydrogen- and carbon dioxide-free medium from one human feces sample. Ca. M. massiliense is a non-motile, 850 nm Gram-positive coccus autofluorescent at 420 nm. Whole-genome sequencing yielded a 29.7% GC content, gapless 1,785,773 bp genome sequence with an 84.5% coding ratio, encoding for alcohol and aldehyde dehydrogenases promoting the growth of Ca. M. massiliense without hydrogen. Screening additional mammal and human feces using a specific genome sequence-derived DNA-polymerase RT-PCR system yielded a prevalence of 22% in pigs, 12% in red kangaroos, and no detection in 149 other human samples. This study, extending the diversity of Methanosphaera in human microbiota, questions the zoonotic sources of Ca. M. massiliense and possible transfer between hosts.IMPORTANCEMethanogens are constant inhabitants in the human gut microbiota in which Methanosphaera stadtmanae was the only cultivated Methanosphaera representative. We grew Candidatus Methanosphaera massiliense sp. nov. from one human feces sample in a novel culture medium under a nitrogen atmosphere. Systematic research for methanogens in human and animal fecal samples detected Ca. M. massiliense in pig and red kangaroo feces, raising the possibility of its zoonotic acquisition. Host specificity, source of acquisition, and adaptation of methanogens should be further investigated.

Current knowledge and clinical perspectives for a unique new phylum: Nanaorchaeota.

Nanoarchaea measuring less than 500 nm and encasing an average 600-kb compact genome have been studied for twenty years, after an estimated 4193-million-year evolution. Comprising only four co-cultured representatives, these symbiotic organisms initially detected in deep-sea hydrothermal vents and geothermal springs, have been further distributed in various environmental ecosystems worldwide. Recent isolation by co-culture of Nanopusillus massiliensis from the unique ecosystem of the human oral cavity, prompted us to review the evolutionary diversity of nanaorchaea resulting in a rapidly evolving taxonomiy. Regardless of their ecological niche, all nanoarchaea share limited metabolic capacities correlating with an obligate ectosymbiotic or parasitic lifestyle; focusing on the dynamics of nanoarchaea-bacteria nanoarchaea-archaea interactions at the morphological and metabolic levels; highlighting proteins involved in nanoarchaea attachment to the hosts, as well metabolic exchanges between both organisms; and highlighting clinical nanoarchaeology, an emerging field of research in the frame of the recent discovery of Candidate Phyla radiation (CPR) in human microbiota. Future studies in clinical nanobiology will expand knowledge of the nanaorchaea repertoire associated with human microbiota and diseases, to improve our understanding of the diversity of these nanoorganims and their intreactions with microbiota and host tissues.

Culturing clinical Methanobrevibacter smithii using GG medium in a minimal anaerobe atmosphere.

Methanobrevibacter smithii (M. smithii), the most prevalent and abundant gut methanogen, detoxifies hydrogen into methane and is, therefore, of paramount importance for the equilibrium of the gut microbiota. The isolation by culture of M. smithii has routinely relied upon hydrogen­carbon dioxide-enriched, oxygen-deprived atmospheres. In this study, we developed a medium referred to as "GG", which allowed for M. smithii growth and isolation by culture in an oxygen-deprived atmosphere, with no supply of either hydrogen or carbon dioxide, making it easier to detect M. smithii by culture in clinical microbiology laboratories.

Mycobacterium ulcerans-Bordetella trematum chronic tropical cutaneous ulcer: A four-case series, Côte d'Ivoire.

BACKGROUND: Chronic tropical cutaneous ulcers remain a neglected medical condition in West Africa, particularly Buruli ulcer, which is caused by mycolactone cytotoxin-secreting Mycobacterium ulcerans (M. ulcerans). Medical management of this highly debilitating and necrotising skin infection may be modified by colonisation and co-infection of the ulcer by opportunistic and pathogenic microorganisms, which considerably delays and increases the cost of treatment. METHODOLOGY/PRINCIPAL FINDING: We diagnosed chronic tropical cutaneous ulcers in nine patients in Côte d'Ivoire using M. ulcerans-specific PCRs and culturomics. This revealed M. ulcerans in 7/9 ulcer swabs and 5/9 control swabs as well as an additional 122 bacterial species, 32 of which were specific to ulcers, 61 specifics to the controls, and 29 which were shared, adding 40 bacterial species to those previously reported. Whole genome sequencing of four Bordetella trematum (B. trematum) isolates in four Buruli ulcer swabs and no controls indicated cytolethal distending toxins, as confirmed by cytotoxic assay. CONCLUSIONS/SIGNIFICANCE: In four cases of Buruli ulcer in Côte d'Ivoire, B. trematum was a co-pathogen which was resistant to rifampicin and clarithromycin, unmatching M. ulcerans antibiotic susceptibility profile and counteracting the current treatment of Buruli ulcer in West Africa and Australia. Thus, we report here chronic mixed M. ulcerans-B. trematum chronic tropical ulcer as a specific form of Buruli ulcer in West Africa.

The Archaeome's Role in Colorectal Cancer: Unveiling the DPANN Group and Investigating Archaeal Functional Signatures.

BACKGROUND AND AIMS: Gut microbial imbalances are linked to colorectal cancer (CRC), but archaea's role remains underexplored. Here, using previously published metagenomic data from different populations including Austria, Germany, Italy, Japan, China, and India, we performed bioinformatic and statistical analysis to identify archaeal taxonomic and functional signatures related to CRC. METHODS: We analyzed published fecal metagenomic data from 390 subjects, comparing the archaeomes of CRC and healthy individuals. We conducted a biostatistical analysis to investigate the relationship between Candidatus Mancarchaeum acidiphilum (DPANN superphylum) and other archaeal species associated with CRC. Using the Prokka tool, we annotated the data focusing on archaeal genes, subsequently linking them to CRC and mapping them against UniprotKB and GO databases for specific archaeal gene functions. RESULTS: Our analysis identified enrichment of methanogenic archaea in healthy subjects, with an exception for Methanobrevibacter smithii, which correlated with CRC. Notably, CRC showed a strong association with archaeal species, particularly Natrinema sp. J7-2, Ferroglobus placidus, and Candidatus Mancarchaeum acidiphilum. Furthermore, the DPANN archaeon exhibited a significant correlation with other CRC-associated archaea (p < 0.001). Functionally, we found a marked association between MvhB-type polyferredoxin and colorectal cancer. We also highlighted the association of archaeal proteins involved in the biosynthesis of leucine and the galactose metabolism process with the healthy phenotype. CONCLUSIONS: The archaeomes of CRC patients show identifiable alterations, including a decline in methanogens and an increase in Halobacteria species. MvhB-type polyferredoxin, linked with CRC and species like Candidatus Mancarchaeum acidiphilum, Natrinema sp. J7-2, and Ferroglobus placidus emerge as potential archaeal biomarkers. Archaeal proteins may also offer gut protection, underscoring archaea's role in CRC dynamics.

A Tetragenococcus halophilus human gut isolate.

Tetragenococcus halophilus (T. halophilus) is a facultative anaerobic, coccus-shaped halophilic lactic acid-producing bacterium previously detected and cultured in various salty foods and credited for beneficial effects on human health. In this study, we investigated the presence of T. halophilus in human samples using a polyphasic approach including scanning electron microscopy, molecular biology methods and microbial culture. This unique investigation yielded the unprecedented presence of T. halophilus in human feces samples, thus enriching the repertoire of halophilic microorganisms colonizing the human gastrointestinal tract with the isolation and culture of T. halophilus for the first time in humans. Using the E-test strips, the MIC was assessed for T. halophilus strain CSURQ6002: rifampicin (MIC at 0.002 µg/mL), benzylpenicillin (MIC at 0.094 µg/mL), amoxicillin (MIC at 0.5 µg/mL), erythromycin (MIC at 2 µg/mL), clindamycin (MIC at 4 µg/mL), and vancomycin (MIC at 8 µg/mL). However, this strain showed a MIC up to 256 µg/mL for ciprofloxacin, fosfomycin, doxycyclin, imipenem, and colistin. In-silico profiling derived from whole genome sequencing (NCBI accession number: PRJNA780809), was confirmed. This discovery suggested that T. halophilus was part of the human digestive microbiota and that its potential role on human health should be considered.

Rapid identification of clinically interesting methanogens using an improved MALDI-TOF-MS assay.

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 Methanobrevibacter smithii and 14 Methanobrevibacter oralis isolates broth-cultured from human stool and oral fluid, respectively, with scores >2. In addition, MALDI-TOF-MS differentiated five Methanobrevibacter smithii genotypes incorporated in the study. The data reported here found MALDI-TOF-MS as a first-line identification method for methanogens recovered from microbiota and clinical samples.

A Non-Invasive Neonatal Signature Predicts Later Development of Atopic Diseases.

Background: Preterm birth is a major cause of morbidity and mortality in infants and children. Non-invasive methods for screening the neonatal immune status are lacking. Archaea, a prokaryotic life domain, comprise methanogenic species that are part of the neonatal human microbiota and contribute to early immune imprinting. However, they have not yet been characterized in preterm neonates. Objective: To characterize the gut immunological and methanogenic Archaeal (MA) signature in preterm neonates, using the presence or absence of atopic conditions at the age of one year as a clinical endpoint. Methods: Meconium and stool were collected from preterm neonates and used to develop a standardized stool preparation method for the assessment of mediators and cytokines and characterize the qPCR kinetics of gut MA. Analysis addressed the relationship between immunological biomarkers, Archaea abundance, and atopic disease at age one. Results: Immunoglobulin E, tryptase, calprotectin, EDN, cytokines, and MA were detectable in the meconium and later samples. Atopic conditions at age of one year were positively associated with neonatal EDN, IL-1ß, IL-10, IL-6, and MA abundance. The latter was negatively associated with neonatal EDN, IL-1ß, and IL-6. Conclusions: We report a non-invasive method for establishing a gut immunological and Archaeal signature in preterm neonates, predictive of atopic diseases at the age of one year.

Meconial Methanobrevibacter smithii suggests intrauterine methanogen colonization in preterm neonates.

To understand the dynamics of methanogens in the human intestinal microbiota, we investigated the presence of methanogens in meconium using a polyphasic approach including microscopy and PCR-sequencing in 33 meconium samples collected from 33 pre-term neonates, in accordance with current ethics regulation. In the presence of negative controls, 90.9% samples were real-time PCR-positive for methanogens and 69.7 % were PCR-sequencing positive, identified as Methanobrevibacter (M.) smithii. Further, auto-fluorescent analysis detected methanogens in the two meconium samples analyzed, with a morphology suggesting M. smithii. Multispacer Sequence Typing found M. smithii genotypes ST1 and ST2, previously described as intestinal microbiota inhabitants. C-section delivery and non-use of peripartum antibiotics significantly correlated with PCR-detection of methanogens in meconium. These data position M. smithii among the early inhabitants of the human gut, detectable immediately after birth and suggest the contribution of methanogens to the perinatal development of intestinal microbiota and physiology.

Diversity of Methanogens in Animals' Gut.

Methanogens are members of anaerobe microbiota of the digestive tract of mammals, including humans. However, the sources, modes of acquisition, and dynamics of digestive tract methanogens remain poorly investigated. In this study, we aimed to expand the spectrum of animals that could be sources of methanogens for humans by exploring methanogen carriage in animals. We used real-time PCR, PCR-sequencing, and multispacer sequence typing to investigate the presence of methanogens in 407 fecal specimens collected from nine different mammalian species investigated here. While all the negative controls remained negative, we obtained by PCR-sequencing seven different species of methanogens, of which three (Methanobrevibacter smithii, Methanobrevibacter millerae and Methanomassiliicoccus luminyensis) are known to be part of the methanogens present in the human digestive tract. M. smithii was found in 24 cases, including 12/24 (50%) in pigs, 6/24 (25%) in dogs, 4/24 (16.66%) in cats, and 1/24 (4.16%) in both sheep and horses. Genotyping these 24 M. smithii revealed five different genotypes, all known in humans. Our results are fairly representative of the methanogen community present in the digestive tract of certain animals domesticated by humans, and other future studies must be done to try to cultivate methanogens here detected by molecular biology to better understand the dynamics of methanogens in animals and also the likely acquisition of methanogens in humans through direct contact with these animals or through consumption of the meat and/or milk of certain animals, in particular cows.

Detection of Methanobrevobacter smithii and Methanobrevibacter oralis in Lower Respiratory Tract Microbiota.

Methanogens, the sole microbes producing methane, are archaea commonly found in human anaerobic microbiota. Methanogens are emerging as opportunistic pathogens associated with dysbiosis and are also detected and cultured in anaerobic abscesses. Their presence in the respiratory tract is yet unknown. As a preliminary answer, prospective investigation of 908 respiratory tract samples using polyphasic approach combining PCR-sequencing, real-time PCR, fluorescent in situ hybridization (FISH), and methanogens culture was carried out. Methanobrevibacter smithii and Methanobrevibacter oralis DNA sequences, were detected in 21/527 (3.9%) sputum samples, 2/188 (1.06%) bronchoalveolar lavages, and none of 193 tracheo-bronchial aspirations. Further, fluorescence in situ hybridization detected methanogens in three sputum investigated specimens with stick morphology suggesting M. oralis and in another one bronchoalveolar lavage sample investigated, diplococal morphology suggesting M. smithii. These observations extend the known territory of methanogens to the respiratory tract and lay the foundations for further interpretation of their detection as pathogens in any future cases of isolation from bronchoalveolar lavages and the lungs.

Co-culture of Methanobrevibacter smithii with enterobacteria during urinary infection.

BACKGROUND: Urinary tract infections are known to be caused by bacteria, but the potential implications of archaea have never been studied in this context. METHODS: In two different university hospital centres we used specific laboratory methods for the detection and culture of archaeal methanogens in 383 urine specimens prospectively collected for diagnosing urinary tract infection (UTI). FINDINGS: Methanobrevibacter smithii was detected by quantitative PCR and sequencing in 34 (9%) of the specimens collected from 34 patients. Escherichia coli, Klebsiella pneumoniae, Enterobacter sp., Enterococcus faecium and mixed cultures were detected along with M. smithii in eighteen, six, three, one and six urine samples, respectively. Interestingly, using our specific culture method for methanogens, we also isolated M. smithii in 31 (91%) of the 34 PCR positive urine samples. Genotyping the 31 isolates using multispacer sequence typing revealed three different genotypes which have been previously reported in intestinal microbiota. Antibiotic susceptibility testing found the 31 isolates to be in vitro susceptible to metronidazole (MIC: 1 mg/L) but resistant to fosfomycin, sulfamethoxazole-trimethoprim, amoxicillin-clavulanate and ofloxacin, commonly used to treat bacterial UTI. Finally, 19 (54%) of the 34 patients in whose urine samples M. smithii was detected were diagnosed with UTIs, including cystitis, pyelonephritis and prostatitis. INTERPRETATION: Our results show that M. smithii is part of the urinary microbiota of some individuals and could play a role in community-acquired UTI in association with enteric bacteria. FUND: This study was supported by IHU Méditerranée Infection, Marseille, France.

Tobacco Smoking Affects the Salivary Gram-Positive Bacterial Population.

The microbial communities of the oral fluid are in direct contact with tobacco smoke, which may thus affect these communities. Few culture-based studies have analyzed the effects of tobacco smoking on the oral fluid microbiota. Using bacterial culture we investigated whether tobacco smoking altered the microbial diversity of the oral fluid, focusing on aerobic and facultative anaerobic Gram-positive bacteria otherwise comprising of major pathogens. Among 90 oral fluid specimens collected in 19 tobacco-smokers and 71 controls, the diversity did not significantly differ with age and with sex. However, diversity was significantly lower in tobacco-smokers (nine different species) than in non-smokers (18 different species) with all the species cultured in tabocco-smokers being also cultured in non-smokers. We isolated the human pathogen Streptococcus australis for the first time from oral fluid. Tobacco smoking significantly alters the saliva Gram-positive bacterial microbiota, including pathogens with potential implication in the pathogenesis of tobacco-related diseases such as periodontitis and peri-implantitis.