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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-1β, IL-10, IL-6, and MA abundance. The latter was negatively associated with neonatal EDN, IL-1β, 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.

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.

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

Methanogens, the archaea uniquely detoxifying fermentative hydrogen into methane in the digestive tract, are increasingly 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 mass spectrometry (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. Data here reported found MALDI-TOF-MS as a first line identification method for methanogens recovered from microbiota and clinical samples.

Methanogens are detected in human gut from the first moments of life and there is a diversification of methanogens during infancy. However, the sources of acquisition of methanogens are not well elucidated. We therefore investigated 56 dairy products as potential sources of methanogens by applying molecular biology. In the presence of negative controls, we obtained an overall prevalence of methanogens in 85.7% (48/56) of samples by real-time PCR. Further PCR-sequencing identified 73.2% (41/56) of Methanobrevibacter smithii. We also found for the first time in dairy products 1.8% (1/56) of Methanobrevibacter oralis, 7.1% (4/56) of Methanobrevibacter millerae, 1.8% (1/56) of Methanobrevibacter ruminantium, 1.8% (1/56) of Methanocorpusculum sp. We observed a significant presence (p-value=0.001) of methanogens in fermented dairy products compared to unfermented dairy products. This study gives credit to the fact that dairy products could be considered as a source of methanogens for humans, especially for children.

Archaeal sequences have been detected in human colostrum and milk, but no studies have determined whether living archaea are present in either of these fluids. Methanogenic archaea are neglected since they are not detected by usual molecular and culture methods. By using improved DNA detection protocols and microbial culture techniques associated with antioxidants previously developed in our center, we investigated the presence of methanogenic archaea using culture and specific Methanobrevibacter smithii and Methanobrevibacter oralis real-time PCR in human colostrum and milk. M. smithii was isolated from 3 colostrum and 5 milk (day 10) samples. M. oralis was isolated from 1 milk sample. For 2 strains, the genome was sequenced, and the rhizome was similar to that of strains previously isolated from the human mouth and gut. M. smithii was detected in the colostrum or milk of 5/13 (38%) and 37/127 (29%) mothers by culture and qPCR, respectively. The different distribution of maternal body mass index according to the detection of M. smithii suggested an association with maternal metabolic phenotype. M. oralis was not detected by molecular methods. Our results suggest that breastfeeding may contribute to the vertical transmission of these microorganisms and may be essential to seed the infant’s microbiota with these neglected critical commensals from the first hour of life.

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 Mediterranee Infection, Marseille, France.

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.

Highlights • The first ever detection in the meconium of the methanogen M. smithii. • These data position M. smithii among the early inhabitants of the human gut. • These data suggest the contribution of methanogens to the perinatal development of intestinal microbiota and physiology., 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.

Background: Methanogens are part of the human microbiota, the intestinal and oral microbiota. They have even been found in colostrum and breast milk, in vagina only in case of vaginosis and in urinary tract during urinary tract infections. They are present in humans from birth. Accordingly, the detection of methanogens in the meconium has not been reported. The objective of this work was to investigate the presence of methanogens in meconium. Methods: We used a polyphasic approach including microscopy and PCR sequencing in 33 meconium samples taken from 33 premature newborns, in accordance with current ethical regulations. Results: 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. Conclusion: 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. Trial Registration: NCT02738411. Funding Statement: YS and COG benefit from PhD grants from the Fondation Mediterranee Infection, Marseille, France. This work was supported by the French Government under the «Investissements d’avenir» (Investments for the Future) program managed by the Agence Nationale de la Recherche (ANR, fr: National Agency for Research), (reference: Mediterranee Infection 10-IAHU-03). Declaration of Interests: The authors declare no competing interests in relation to this study. Outside this study, JV reports speaker and consultancy fees in the past 5 years from Meda Pharma (Mylan), Novartis, Sanofi, Thermo Fisher Scientific, outside the submitted work. Ethics Approval Statement: Written informed parental consent was obtained for each preterm. This research project was approved by the Ethics Committee on Clinical Research of Nimes and Montpellier University Hospitals.

Methanogens are being members of anaerobe microbiota of the digestive tract of both human and mammals. 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 which could be sources of methanogens for human, by exploring methanogen carriage in animals in contact with the general population or with some restricted populations; comparing the repertoire of animal methanogens with the one of human methanogens in order to question methanogens as zoonotic microorganisms. We used RT-PCR, PCR-sequencing and multispacer sequence typing to investigate the presence of methanogens in 407 fecal specimens collected from nine different mammalian species. We detected by RT-PCR, the presence of methanogen DNA in all mammals here investigated and none of the negative controls. We obtained by 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. We obtained 24 M. smithii by PCR-sequencing 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 know 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.

Methanogen cultures require hydrogen produced by fermentative bacteria such asBacteroides 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 ofMethanobrevibacter oralis, compared the method to the biological reference method with a reference strain ofMethanobrevibacter smithiiand 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 the pure strains ofM. oralisandM. smithii, colonies appeared three days postinoculation with the chemical method versus nine days with the biological method. The average number ofM. smithiicolonies 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 when using the biological method than when using 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 ofM. oralisand three strains ofM. smithiifrom the 50 saliva samples. Establishing the chemical method will ease the routine isolation and culture of methanogens.

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.

International audience; 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.

Archaea, which form one of four domains of life alongside Eukarya, Bacteria, and giant viruses, have long been neglected as components of the human microbiota and potential opportunistic infectious pathogens. In this review, we focus on methanogenic Archaea, which rely on hydrogen for their metabolism and growth. On one hand, methanogenic Archaea in the gut are functional associates of the fermentative digestion of dietary fibers, favoring the production of beneficial short-chain fatty acids and likely contributing to the weaning reaction during the neonatal window of opportunity. On the other hand, methanogenic Archaea trigger the activation of innate and adaptive responses and the generation of specific T and B cells in animals and humans. In mouse models, lung hypersensitivity reactions can be induced by inhaled methanogenic Archaea mimicking human professional exposure to organic dust. Changes in methanogenic Archaea of the microbiota are detected in an array of dysimmune conditions comprising inflammatory bowel disease, obesity, malnutrition, anorexia, colorectal cancer, and diverticulosis. At the subcellular level, methanogenic Archaea are activators of the TLR8-dependent NLRP3 inflammasome, modulate the release of antimicrobial peptides and drive the production of proinflammatory, Th-1, Th-2, and Th-17 cytokines. Our objective was to introduce the most recent and major pieces of evidence supporting the involvement of Archaea in the balance between health and dysimmune diseases, with a particular focus on atopic and allergic conditions.

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.

Since its launch 7 years ago, in 2013, New Microbes and New Infections (NMNI) has published a total of 773 of the 1300 submitted papers retrievable in PubMed Central (Fig. 1). NMNI aims to provide access to an international forum for authors reporting facts in infectious and primarily tropical diseases and clinical microbiology that are new in one place and country. Accordingly, NMNI acknowledges contributions from emerging research teams, offering authors the opportunity to publish in their own language, in addition to English; in 2020, two manuscripts have been submitted in non-English languages (French). In the meantime, NMNI has been included in PubMed Central, obviously increasing its visibility and the interest of authors and readers. Open in a separate window Fig. 1 Submitted manuscripts and editorial outcomes 2016–2020 (Elsevier data).

International audience; The oral fluid microbiome comprises an important bacterial diversity, yet the presence of archaea has not been reported so far. In order to quest for the presence of methanogenic archaea (methanogens) in oral fluid, we used a polyphasic approach including PCR-sequencing detection, microscopic observation by fluorescence in-situ hybridization, isolation and culture, molecular identification and genotyping of methanogens in 200 oral fluid specimens. In the presence of negative controls, 64/200 (32%) prospectively analysed oral fluid specimens were PCR-positive for methanogens, all identified as Methanobrevibacter oralis by sequencing. Further, fluorescence in-situ hybridization detected methanogens in 19/48 (39.6%) investigated specimens; with morphology suggesting M. oralis in 10 cases and co-infecting Methanobrevibacter smithii in nine cases. M. oralis was cultured from 46/64 (71.8%) PCR-positive specimens and none of PCR-negative specimens; and one M. smithii isolate was co-cultured with M. oralis in one specimen. Multispacer Sequence Typing found one M. oralis genotype per specimen and a total of five different genotypes with 19/46 (41%) of isolates all belonging to spacer-type four. Statistical analyses showed a significant correlation between the PCR-detection of methanogens in oral fluid and tobacco smoking. These data indicate that M. oralis and M. smithii are oral fluid-borne methanogens in tobacco smokers. Both methanogens could be transmitted during intimate contacts such as mother-to-child contacts and kissing.