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Brucellosis is a bacterial zoonosis caused by bacteria of the genus Brucella spp. It is cosmopolitan and rages around the Mediterranean, particularly in Algeria where it continues to wreak havoc on livestock. We report here a review on brucellosis in Algeria, where human brucellosis was already discovered in 1895 and since then it remains a major concern of health services. There is a disparity in human cases depending on the region, in fact, the most affected region is the steppe region which alone has the average incidence (cases/100,000 inhabitants) of 65.87% followed by 9.89 in North- Is according to the Ministry of Health. The prevalence of bovine brucellosis fell from 5% to 0.76% respectively in the mid-1990s and in 2014 with a delicate improvement which, after seven years of the identification and screening program as well as vaccination with the REV vaccine -1 set up by the Ministry of Health, remains unable to eradicate the disease. It should also be noted that the evolution of the number of new human cases reported is like that observed in goats, which constitute the most important reservoir of infection for humans in Algeria.

Agzul: Tawla n Malṭa d aṭṭan ittenṭaḍen ɣer umdan i d-xeddment tbaktiriyin n tewsit Brucella spp. D aṭṭan amaḍlan u tella deg tmura n wagrakal am Lezzayer anda mazal txeddem axessar deg lmal. Ad d-nawi dagi tasɣunt tamatut ɣef tawla n Malṭa di Lezzayer, anda i tt-id-ufan yakan ɣer umdan seg 1895 u deg wass-nni mazal-itt d ugur ameqqran i yimeẓla n tdawsa. Tella tezqaft deg tejṛutin n yimdanen deg temnaḍt ɣer tayeḍ, tamnaḍt yennul ugar d tamnaḍt uzawaɣ i yesɛan weḥd-s aktamur alemmas (tajṛut/100 000 imezdaɣ) n 65.87 teḍfer-itt-id s 9.89 deg temnaḍt Agafa-Asamar ɣef wakken i d-iwekked uɣlif n tdawsa. Aktamur n tejṛutin n tawla n Malṭa n yizgaren iɛedda s uḍfar deg 5% ar 0,76% deg tlemmast n yiseggasen n 90 d 2014 s usileɣ imfeccec anda seld ṣa yiseggasen n wahil n takza d usnefren akked ucraḍ s tgezzayt REV-1 i d-ihegga uɣlif n tdawsa mazal yegguma ad isenger aṭṭan-agi. Ilaq daɣen ad negzu dakken anerni n umḍan n tejṛutin timaynutin n yimdanen i ittujerden kifkif-it akked tejṛutin ɣer tɣeṭṭen i yellan d aɛwin n temsalmit meqqren i umdan di Lezzayer.

Background: Nanoarchaeota, obligate symbiont of some environmental archaea with reduced genomes, have been described in marine thermal vent environments, yet never detected in hosts, including humans. Methods: Here, using laboratory tools geared towards the detection of nanoarchaea including PCR-sequencing, WGS, microscopy and culture. Results: We discovered a novel nanoarchaea, Nanopusillus massiliensis, detected in dental plate samples by specific PCR-based assays. Combining fluorescent in situ hybridization (FISH) with scanning electron microscopy disclosed close contacts between N. massiliensis and the archaea Methanobrevibacter oralis in these samples. Culturing one sample yielded co-isolation of M. oralis and N. massiliensis with a 606,935-bp genome, with 23.6% GC encoded 16 tRNA, 3 rRNA and 942 coding DNA sequences, of which 400 were assigned to clusters of orthologous groups. Conclusion: The discovery of N. massiliensis, made publicly available in collection, extended our knowledge of human microbiota diversity, opening a new field of research in clinical microbiology here referred to as clinical nanoarchaeology.

Untreated tonsillar phlegmon is a life-threatening condition commonly caused by Streptococcus pyogenes and Fusobacterium necrophorum, among other pathogens. Here, using specific laboratory tools, we detected Methanobrevibacter smithii in addition to S. pyogenes. This unprecedented observation questions the role of methanogens in phlegmon and the optimal treatment of this mixed infection.

Methanobrevibacter smithii ( M. smithii ), initially isolated from human feces, has been recognised as a distinct taxon within the Archaea domain following comprehensive phenotypic, genetic, and genomic analyses confirming its uniqueness among methanogens. Its diversity, encompassing 15 genotypes, mirrors that of biotic and host-associated ecosystems in which M. smithii plays a crucial role in detoxifying hydrogen from bacterial fermentations, converting it into mechanically expelled gaseous methane. In microbiota in contact with host epithelial mucosae, M. smithii centres metabolism-driven microbial networks with Bacteroides , Prevotella , Ruminococcus , Veillonella , Enterococcus , Escherichia , Enterobacter , Klebsiella , whereas symbiotic association with the nanoarchaea Candidatus Nanopusillus phoceensis determines small and large cell variants of M. smithii . The former translocate with bacteria to induce detectable inflammatory and serological responses and are co-cultured from blood, urine, and tissular abscesses with bacteria, prototyping M. smithii as a model organism for pathogenicity by association. The sources, mechanisms and dynamics of in utero and lifespan M. smithii acquisition, its diversity, and its susceptibility to molecules of environmental, veterinary, and medical interest still have to be deeply investigated, as only four strains of M. smithii are available in microbial collections, despite the pivotal role this neglected microorganism plays in microbiota physiology and pathologies., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 Published by Elsevier Ltd.)

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., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

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., Competing Interests: V.P., M.D., G.A., G.G., and E.T. are co-inventors of a patented culture medium referred to as GG culture medium in this report (patent FR 23 01404).

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.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Tchan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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.

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., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

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 (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 hydrogen‑carbon 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., Competing Interests: Declaration of Competing Interest VP, ET, GA, MD and GG are co-inventor of patent regarding this work entitled: “Milieu et procédé de culture des archaea methanogens”., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare that there are no conflicts of interest., (© 2022 The Authors.)

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.

Tawla n Malṭa d aṭṭan ittenṭaḍen ɣer umdan i d-xeddment tbaktiriyin n tewsit Brucella spp . D aṭṭan amaḍlan u tella deg tmura n wagrakal am Lezzayer anda mazal txeddem axessar deg lmal. Ad d-nawi dagi tasɣunt tamatut ɣef tawla n Malṭa di Lezzayer, anda i tt-id-ufan yakan ɣer umdan seg 1895 u deg wass-nni mazal-itt d ugur ameqqran i yimeẓla n tdawsa. Tella tezqaft deg tejṛutin n yimdanen deg temnaḍt ɣer tayeḍ, tamnaḍt yennul ugar d tamnaḍt uzawaɣ i yesɛan weḥd-s aktamur alemmas (tajṛut/100 000 imezdaɣ) n 65.87 teḍfer-itt-id s 9.89 deg temnaḍt Agafa-Asamar ɣef wakken i d-iwekked uɣlif n tdawsa. Aktamur n tejṛutin n tawla n Malṭa n yizgaren iɛedda s uḍfar deg 5% ar 0,76% deg tlemmast n yiseggasen n 90 d 2014 s usileɣ imfeccec anda seld ṣa yiseggasen n wahil n takza d usnefren akked ucraḍ s tgezzayt REV-1 i d-ihegga uɣlif n tdawsa mazal yegguma ad isenger aṭṭan-agi. Ilaq daɣen ad negzu dakken anerni n umḍan n tejṛutin timaynutin n yimdanen i ittujerden kifkif-it akked tejṛutin ɣer tɣeṭṭen i yellan d aɛwin n temsalmit meqqren i umdan di Lezzayer., (© 2022 The Author(s).)

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., Competing Interests: All the authors declare that there is no conflict of interest., (© 2022 The Authors. Published by Elsevier B.V.)

Background: Nanoarchaeota, obligate symbiont of some environmental archaea with reduced genomes, have been described in marine thermal vent environments, yet never detected in hosts, including humans., Methods: Here, using laboratory tools geared towards the detection of nanoarchaea including PCR-sequencing, WGS, microscopy and culture., Results: We discovered a novel nanoarchaea, Nanopusillus massiliensis , detected in dental plate samples by specific PCR-based assays. Combining fluorescent in situ hybridization (FISH) with scanning electron microscopy disclosed close contacts between N. massiliensis and the archaea Methanobrevibacter oralis in these samples. Culturing one sample yielded co-isolation of M. oralis and N. massiliensis with a 606,935-bp genome, with 23.6% GC encoded 16 tRNA, 3 rRNA and 942 coding DNA sequences, of which 400 were assigned to clusters of orthologous groups., Conclusion: The discovery of N. massiliensis, made publicly available in collection, extended our knowledge of human microbiota diversity, opening a new field of research in clinical microbiology here referred to as clinical nanoarchaeology., Competing Interests: All the authors declare that there are no conflicts of interest., (© 2021 The Authors. Published by Elsevier B.V.)

The aetiology of appendicular abscess is predominantly microbial with aerobic and anaerobic bacteria from gut flora. In this study, by using specific laboratory tools, we co-detected Methanobrevibacter oralis and Methanobrevibacter smithii among a mixture of enterobacteria including Escherichia coli, Enterococcus faecium and Enterococcus avium in four unrelated cases of postoperative appendiceal abscesses. These unprecedented observations raise a question on the role of methanogens in peri-appendicular abscesses, supporting antibiotics as an alternative therapeutic option for appendicitis, including antibiotics active against methanogens such as metronidazole or fusidic acid., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

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., Competing 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. We confirm that the manuscript has been read and approved by all named authors. We confirm that the order of authors listed in the manuscript has been approved by all named authors., (© 2021 Published by Elsevier B.V.)

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.

Methanogens are the archaea most commonly found in humans, in particular in the digestive tract and are an integral part of the digestive microbiota. They are present in humans from the earliest moments of life and represent the only known source of methane production to date. They are notably detected in humans by microscopy, fluorescent in situ hybridization, molecular biology including PCR-sequencing, metagenomics, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and culture. Methanogens present in the human digestive tract play major roles, in particular the use of hydrogen from the fermentation products of bacteria, thus promoting digestion. They are also involved in the transformation of heavy metals and in the use of trimethylamine produced by intestinal bacteria, thus preventing major health problems, in particular cardiovascular diseases. Several pieces of evidence suggest their close physical contacts with bacteria support symbiotic metabolism. Their imbalance during dysbiosis is associated with many pathologies in humans, particularly digestive tract diseases such as Crohn's disease, ulcerative colitis, diverticulosis, inflammatory bowel disease, irritable bowel syndrome, colonic polyposis, and colorectal cancer. There is a huge deficit of knowledge and partially contradictory information concerning human methanogens, so much remains to be done to fully understand their physiological role in humans. It is necessary to develop new methods for the identification and culture of methanogens from clinical samples. This will permit to isolate new methanogens species as well as their phenotypic characterization, to explore their genome by sequencing and to study the population dynamics of methanogens by specifying in particular their exact role within the complex flora associated with the mucous microbiota of human., (Copyright © 2020. Published by Elsevier Ltd.)

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.

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., (Copyright © 2019. Published by Elsevier Ltd.)

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.

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.

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.

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., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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.

Methanobrevibacter smithii is the main human methanogen almost always found in the digestive tract of adults. Yet, the age at which M. smithii establishes itself as part of the developing intestinal microflora remains unknown. In order to gain insight into this, we developed a polyphasic approach, including microscopic observation by fluorescence in situ hybridisation, polymerase chain reaction (PCR) sequencing detection, identification and culture, to isolate and genotype M. smithii in one-day-old newborns' gastric juice specimens. In the presence of negative controls, 50/50 (100%) prospectively analysed newborn gastric juice specimens were PCR-positive for methanogens, all identified as M. smithii by sequencing. We succeeded in cultivating M. smithii in 35/50 (70%) newborn gastric juice specimens, while 15/50 specimens remained sterile. Further, M. smithii was observed by direct microscopic investigation using fluorescence in situ hybridisation. Multispacer sequence typing found one of seven different genotypes per specimen, these genotypes having all been previously described in adult human stools. Methanobrevibacter smithii is an early inhabitant of the human stomach, colonising the gastric mucosa just after birth, and the mother's gut microbiota is a probable source of colonisation.