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OBJECTIVE To compare the bacteriome of the oral cavity in healthy dogs and dogs with various stages of periodontal disease. ANIMALS Dogs without periodontal disease (n = 12) or with mild (10), moderate (19), or severe (10) periodontal disease. PROCEDURES The maxillary arcade of each dog was sampled with a sterile swab, and swabs were submitted for next-generation DNA sequencing targeting the V1–V3 region of the 16S rRNA gene. RESULTS 714 bacterial species from 177 families were identified. The 3 most frequently found bacterial species were Actinomyces sp (48/51 samples), Porphyromonas cangingivalis (47/51 samples), and a Campylobacter sp (48/51 samples). The most abundant species were P cangingivalis, Porphyromonas gulae, and an undefined Porphyromonas sp. Porphyromonas cangingivalis and Campylobacter sp were part of the core microbiome shared among the 4 groups, and P gulae, which was significantly enriched in dogs with severe periodontal disease, was part of the core microbiome shared between all groups except dogs without periodontal disease. Christensenellaceae sp, Bacteroidales sp, Family XIII sp, Methanobrevibacter oralis, Peptostreptococcus canis, and Tannerella sp formed a unique core microbiome in dogs with severe periodontal disease. CONCLUSIONS AND CLINICAL RELEVANCE Results highlighted that in dogs, potential pathogens can be common members of the oral cavity bacteriome in the absence of disease, and changes in the relative abundance of certain members of the bacteriome can be associated with severity of periodontal disease. Future studies may aim to determine whether these changes are the cause or result of periodontal disease or the host immune response.

International audience; Orthopedic prosthesis infection must be medically managed after appropriate microbiological documentation. While bacteria and fungi are acknowledged to be causative opportunistic pathogens in this situation, the potential role of methanogens in orthopedic prosthesis infections is still unknown. In a retrospective study, a total of 100 joint and bone samples collected from 25 patients were screened by specific PCR assays for the detection of methanogens. PCR-positive samples were observed by autofluorescence, electron microscopy and tentatively cultured under specific culture conditions. Methanogens were detected by quantitative PCR in 4/100 samples, in the presence of negative controls. Sequencing identified Methanobrevibacter oralis in two cases, Methanobrevibacter smithii in one case and Methanobrevibacter wolinii in one case. Microscopic methods confirmed molecular findings and bacterial culture yielded two strains of Staphylococcus aureus, one strain of Staphylococcus epidermidis and one strain of Proteus mirabilis. These unprecedented data highlight the presence of methanogens in joint and bone samples of patients also diagnosed with bacterial orthopedic prosthesis infection, questioning the role of methanogens as additional opportunistic co-pathogens in this situation.

Orthopedic prosthesis infection must be medically managed after appropriate microbiological documentation. While bacteria and fungi are acknowledged to be causative opportunistic pathogens in this situation, the potential role of methanogens in orthopedic prosthesis infections is still unknown. In a retrospective study, a total of 100 joint and bone samples collected from 25 patients were screened by specific PCR assays for the detection of methanogens. PCR-positive samples were observed by autofluorescence, electron microscopy and tentatively cultured under specific culture conditions. Methanogens were detected by quantitative PCR in 4/100 samples, in the presence of negative controls. Sequencing identified Methanobrevibacter oralis in two cases, Methanobrevibacter smithii in one case and Methanobrevibacter wolinii in one case. Microscopic methods confirmed molecular findings and bacterial culture yielded two strains of Staphylococcus aureus, one strain of Staphylococcus epidermidis and one strain of Proteus mirabilis. These unprecedented data highlight the presence of methanogens in joint and bone samples of patients also diagnosed with bacterial orthopedic prosthesis infection, questioning the role of methanogens as additional opportunistic co-pathogens in this situation.

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.

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.

This study aimed to determine the prevalence and diversity of archaea and select bacteria in the subgingival biofilm of patients with peri-implantitis in comparison to patients with unaffected implants and patients with periodontitis. Samples of subgingival biofilm from oral sites were collected for DNA extraction (n = 139). A 16S rRNA gene-based polymerase chain reaction assay was used to determine the presence of archaea and select bacteria. Seven samples were selected for direct sequencing. Archaea were detected in 10% of samples from peri-implantitis sites, but not in samples from the unaffected dental implant. Archaea were present in 53% and 64% of samples from mild and moderate/advanced periodontitis sites, respectively. The main representative of the Archaea domain found in biofilm from periodontitis and peri-implantitis sites was Methanobrevibacter oralis. The present results revealed that archaea are present in diseased but not healthy implants. It was also found that archaea were more abundant in periodontitis than in peri-implantitis sites. Hence, the potential role of archaea in peri-implantitis and periodontitis should be taken into consideration.

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.

Methanogens are anaerobic prokaryotes from the domain archaea that utilize hydrogen to reduce carbon dioxide, acetate, and a variety of methyl compounds into methane. Earlier believed to inhabit only the extreme environments, these organisms are now reported to be found in various environments including mesophilic habitats and the human body. The biological significance of methanogens for humans has been re-evaluated in the last few decades. Their contribution towards pathogenicity has received much less attention than their bacterial counterparts. In humans, methanogens have been studied in the gastrointestinal tract, mouth, and vagina, and considerable focus has shifted towards elucidating their possible role in the progression of disease conditions in humans. Methanoarchaea are also part of the human skin microbiome and proposed to play a role in ammonia turnover. Compared to hundreds of different bacterial species, the human body harbors only a handful of methanogen species represented by Methanobrevibacter smithii, Methanobrevibacter oralis, Methanosphaera stadtmanae, Methanomassiliicoccus luminyensis, Candidatus Methanomassiliicoccus intestinalis, and Candidatus Methanomethylophilus alvus. Their presence in the human gut suggests an indirect correlation with severe diseases of the colon. In this review, we examine the current knowledge about the methanoarchaea in the human body and possible beneficial or less favorable interactions.

Prokaryotes forming the domain of Archaea, named after their first discovery in extreme environments, are acknowledged but still neglected members of the human digestive tract microbiota. In this microbiota, cultured archaea comprise anaerobic methanogens: Methanobrevibacter smithii, Methanobrevibacter oralis, Methanobrevibacter massiliense, Methanosphaera stadtmanae, Methanobrevibacter arboriphilus, Methanobrevibacter millerae and Methanomassiliicoccus luminyensis; along with the non-methanogen halophilic Archaea Halopherax massiliense. Metagenomic analyses detected DNA sequences indicative of the presence of additional methanogenic and non-methanogenic halophilic Archaea in the human intestinal tract and oral cavity. Methanogens specifically metabolize hydrogen produced by anaerobic fermentation of carbohydrates into methane; further transforming heavy metals and metalloids into methylated derivatives, such as trimethylbismuth which is toxic for both human and bacterial cells. However, the role of Archaea as pathogens remains to be established. Future researches will aim to increase the repertoire of the human digestive tract Archaea and to understand their possible association with intestinal and extra-intestinal infections and diseases including weight regulation abnormalities. Keywords: Human-associated Archaea, Methanogens, Halophiles, Oral cavity, Intestinal tract

The authors report the cases of 9 patients eventually diagnosed with methanogenic archaea refractory or recalcitrant chronic rhinosinusitis, a condition known to involve various anaerobic bacteria but in which the role of methanogenic archaea is unknown. The authors retrospectively searched these microorganisms by PCR in surgically-collected sinusal pus specimens from patients diagnosed with refractory sinusitis, defined by the persistance of sinus inflammation and related-symptoms for more than 12 weeks despite appropriate treatment. Of the 116 tested sinus surgical specimens, 12 (10.3%) from 9 patients (six females, three males; aged 20–71 years) were PCR-positive. These specimens were further investigated by fluorescence in-situ hybridization, PCR amplicon-sequencing and culture. Methanobrevibacter smithii was documented in four patients and Methanobrevibacter oralis in another four, one of whom was also culture-positive. They were associated with a mixed flora including Gram-positive and Gram-negative bacteria. In the latter patient, “Methanobrevibacter massiliense” was the sole microorganism detected. These results highlight methanogenic archaea as being part of a mixed anaerobic flora involved in refractory sinusitis, and suggest that the treatment of this condition should include an antibiotic active against methanogens, notably a nitroimidazole derivative.

International audience; Culturing methanogenic archaea is fastidious, expensive, and requires an external source of hydrogen and carbon dioxide. Until now, these microorganisms have only been cultivated under strictly anaerobic conditions. We previously developed a single versatile culture medium containing sugars and anti-oxydants for cultivating all human known methanogens. Performing aerobic cultures in the presence of Bacteroides thetaiotaomicron, which produces hydrogen, allows for cultivation of Methanobrevibacter smithii which itself produces methane. To obtain colonies, we cultivated M. smithii in an agar plate in the upper part of a double chamber flask with a liquid culture of B. thetaiotaomicron in the lower compartment. We subsequently cultured four other methanogenic species for the first time and successfully isolated 13 strains of M. smithii and nine strains of Methanobrevibacter oralis from 100 stools and 45 oral samples. This procedure allows aerobic isolation and antibiotic susceptibility testing. This changes the ability to routinely study methanogens, which have been neglected in clinical microbiology laboratories and may be useful for biogas production.

International audience

Methanogens have already been described in periodontitis but not in peri-implantitis. Thirty peri-implantitis samples and 28 control samples were collected in 28 consenting peri-implantitis patients. PCR-sequencing of the 16S rRNA gene was used as a broad-spectrum screening method and results were further confirmed by real-time quantitative PCR targeting the mcrA genes. Results showed a methanogen community dominated by Methanobrevibacter oralis in 31/58 (51%) samples including 16/28 (57%) control samples and 15/30 (50%) peri-implantitis samples. Methanobrevibacter massiliense was detected in 5/58 (8.6%) samples including 3/28 (1%) control samples and 2/30 (6.7%) peri-implantitis samples. The prevalence of M. oralis or M. massiliense did not significantly differ in peri-implantitis and control samples (exact Fisher test, P = 0.61 and P = 0.67, respectively). Further ponderation of the methanogen load by the real-time quantitative PCR for actin human gene again indicated non-significant difference (Wilcoxon-Mann-Whitney test, P = 0.48 and P = 0.40, respectively). These data show that the prevalence of methanogens does not differ in peri-implantitis lesions and healthy sites, when individuals are their own control. These data do not allow assigning a specific pathogenic role to methanogens in peri-implantitis; methanogens rather are part of the commensal and normal flora of the oral cavity.

Archaea have been detected in several ecological niches of the human body such as the large intestine, skin, vagina as well as the oral cavity. At present, archaea are recognized as nonpathogenic microorganisms. However, some data indicate that they may be involved in the etiopathogenesis of several diseases, including intestinal diseases as well as oral diseases: periodontitis, peri-implantitis and endodontitis. In this study, on the basis of 16S rRNA gene sequence analysis, we examined whether archaea might be present in inflamed pulp tissues and contribute to the development of endodontic infection. In comparison, we also determined selected bacterial species associated with endodontitis. We detected archaea in 85% of infected endodontic samples. In addition, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola were present in inflamed pulp tissue samples and Treponema denticola occurred with the highest frequency (70%). Further analysis revealed the presence of methanogenic archaea in analyzed samples. Direct sequencing of archaeal 16S rRNA gene PCR products indicated the occurrence of methanogenic archaea in inflamed pulp tissues; phylogenetically most similar were Methanobrevibacter oralis and Methanobrevibacter smithii. Therefore, our results show that methanogenic archaea are present in inflamed pulp tissues and may participate in the development of endodontic infection.

A study was carried out in the odontology department of the Timone hospital in Marseille (France) with the aim of creating a diagnostic tool allowing an evaluation of the risk of peri-implantitis evolution according to the severity. Forty-three patients, 28 of whom were diagnosed with peri-implantitis on at least one implant and 15 control patients, were included in the study approved by the IHU Ethics Committee. Participants completed a questionnaire, underwent a thorough clinical examination and a systematic retro-alveolar intraoral radiography of the incriminated implant(s). The following criteria were observed: the number of faces of the implant showing bleeding and / or pus on probing, the probing depth on at least two faces of the implant, bone loss as a function of the length of the implant, the number of implant faces with dental plaque, the parameters required for the determination of excess cement (screwed or sealed prosthesis, situation in the gums of the sealed prosthesis), the periodontal status, glycemic status and tobacco use. 16/28 (57.1%) of cases were identified with high peri-implantitis risk compared to only 2/28 (7.2%) of cases with low risk. The remaining 10/28 (35.7%) correspond to patients with moderate peri-implantitis risk. Control patients: 15/15 (100%) had a low risk. In addition, peri-implant pocket samples were taken during the clinical observation to be analyzed by the URMITE (Research Unit on Infectious and Emerging Tropical Diseases) Mediterranean Infection. The aim was to highlight the relative involvement of methanogenic archaea, in particular Methanobrevibacter oralis, in pathogenesis of peri-implantitis, using samples from pathological implants and healthy control teeth. M. oralis and M. massiliense were found in the peri-implant pockets but also in the sulci of the healthy teeth. No correlation was found between the presence and concentration of these methanogens on the one hand, and the level of peri-implant risk provided by the proposed model.; Une étude a été menée dans le service d’odontologie de l’hôpital de la Timone à Marseille (France) dans le but de créer un outil diagnostique permettant une évaluation du risque d’évolution des péri-implantites en fonction de leur sévérité. Quarante-trois patients, dont 28 diagnostiqués avec une péri-implantite sur au moins un implant et 15 patients contrôles, ont été inclus dans l’étude. Les participants ont rempli un questionnaire, subi un examen clinique approfondi et une radiographie intra-orale rétro-alvéolaire systématique du (des) implant(s) incriminé(s). Les critères suivants ont été observés : le nombre de faces de l’implant présentant un saignement et/ou une suppuration au sondage, la profondeur de poche au sondage sur au moins deux faces de l’implant, la perte osseuse rapportée à la longueur de l’implant évaluée sur les radiographies, le nombre de faces implantaires présentant de la plaque, les paramètres requis pour la détermination des excès de ciment (prothèse vissée ou scellée, enfouissement des prothèses scellées), les statuts parodontal, glycémique et la tabacomanie. 16/28 (57,1%) des cas ont été identifiés à haut risque péri-implantaire contre seulement 2/28 (7,2%) des cas, à faible risque. Les 10/28 (35,7%) restants correspondent aux patients à risque péri-implantaire modéré. Les patients contrôles : 15/15 (100%) présentaient un risque faible. Par ailleurs, des prélèvements de poches péri-implantaires ont été effectués lors de l’observation clinique pour être analysés par l’URMITE (Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes) Méditerranée Infection. L’objectif était de mettre en évidence l’implication relative des archaea méthanogènes, notamment Methanobrevibacter oralis dans la pathogénèse des péri-implantites, à partir des prélèvements sur des implants pathologiques et des dents saines témoins. M. oralis et M. massiliense ont été retrouvées dans les poches péri-implantaires mais aussi dans les sulci des dents saines. Il n’a pas été mis en évidence de corrélation entre la présence et la concentration de ces méthanogènes d’une part, et le niveau de risque péri-implantaire fourni par le modèle proposé.

A PCR assay for the amplification of small subunit ribosomal DNA (SSU rDNA) of Euryarchaea was developed and used to detect archaeal rDNA in 37 (77%) out of 48 pooled subgingival plaque samples from 48 patients suffering from periodontal disease. One major group of cloned periodontal sequences was identical to Methanobrevibacter oralis and a second minor group to Methanobrevibacter smithii. These two groups and a third novel group were found to be more than 98% similar to each other over an 0.65-kb segment of the 16S rRNA gene sequenced. M. oralis was found to be the predominant archaeon in the subgingival dental plaque. Phylogenetic analysis of partial SSU rDNA sequences revealed evidence for a distinct cluster for human and animal Methanobrevibacter sp. within the Methanobacteriaceae family.

International audience; Background. Methanogens are antibiotic-resistant anaerobic archaea that escape routine detection in clinical microbiology. We hypothesized that methanogens are part of the anaerobic community that cause brain abscess. Methods. Methanogens were investigated in 1 index sample using specific polymerase chain reaction (PCR) sequencing and culture. The pathogenesis of a methanogen isolate was assessed in a mouse model. Archaea-specific quantitative (q) PCR and metagenomics were used to detect specific archaeal sequences in brain abscess samples and controls. Results. In 1 index sample, routine culture found Porphyromonas endodontalis and Streptococcus intermedius, and specific culture found Methanobrevibacter oralis susceptible to metronidazole and fusidic acid. Archaea-targeted PCR sequencing and metagenomics confirmed M. oralis along with 14 bacteria, including S. intermedius. Archaea-specific qPCR yielded archaea in 8/18 brain abscess specimens and 1/27 controls (P < .003), and metagenomics yielded archaea, mostly methanogens, in 28/32 brain abscess samples, and no archaea in 71 negative controls (P < 10(-6)). Infection of mice brains yielded no mortality in 14 controls and death in 17/22 M. oralis-inoculated mice (P < 10(-6)), 32/95 S. intermedius-inoculated mice (P < 10(-6)), and 75/104 mice inoculated with M. oralis mixed with S. intermedius (P < 10(-6)) 7 days post-inoculation. Conclusion. Methanogens belong to the anaerobic community responsible for brain abscess, and M. oralis may participate in the pathogenicity of this deadly infection. In mice, a synergy of M. oralis and S. intermedius was observed. Antibiotic treatment of brain abscess should contain anti-archaeal compounds such as imidazole derivatives in most cases.

International audience; Lovastatin is a prodrug that is hydrolysed in vivo to beta-hydroxy acid lovastatin, which inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-Co-A) reductase (HMGR), thereby lowering cholesterol in humans. A side effect of lovastatin is inhibition of isoprenoid synthesis and cell membrane formation in methanogenic Archaea, which are members of the human digestive tract microbiota and are emerging pathogens. In this study, the in vitro susceptibility of the human-associated methanogens Methanobrevibacter smithii, Methanobrevibacter oralis, Methanobrevibacter massiliense, Methanobrevibacter arboriphilus and Methanomassiliicoccus luminyensis to lovastatin (1-4 mu g/mL) was tested in the presence of five gut anaerobes aiming to metabolise lovastatin into alpha-hydroxy acid lovastatin as confirmed by ultra-high-performance liquid chromatography. Five days of incubation with lovastatin had no measurable effect on the growth of the five gut anaerobes but significantly reduced CH4 production and methanogen growth as measured by quantitative PCR (P < 0.01). Quantitative PCR analyses indicated that compared with controls, alpha-hydroxy acid lovastatin significantly increased the expression of the genes mta and mcrA implicated in methanogenesis and significantly decreased the expression of the fno gene implicated in methanogenesis. Expression of the HMGR gene (hmg) implicated in cell wall synthesis was significantly increased by alpha-hydroxy acid lovastatin (P < 0.01). These results strongly suggest that in the presence of gut anaerobes, lovastatin yields alpha-hydroxy acid lovastatin, which inhibits methane production and growth of methanogens by affecting their cell membrane biosynthesis. Lovastatin is the first licensed drug to exclusively affect the growth of methanogens whilst protecting the bacterial microbiota. (C) 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Weyrich, Laura S. et al., Recent genomic data have revealed multiple interactions between Neanderthals and modern humans1, but there is currently little genetic evidence regarding Neanderthal behaviour, diet, or disease. Here we describe the shotgun-sequencing of ancient DNA from five specimens of Neanderthal calcified dental plaque (calculus) and the characterization of regional differences in Neanderthal ecology. At Spy cave, Belgium, Neanderthal diet was heavily meat based and included woolly rhinoceros and wild sheep (mouflon), characteristic of a steppe environment. In contrast, no meat was detected in the diet of Neanderthals from El Sidrón cave, Spain, and dietary components of mushrooms, pine nuts, and moss reflected forest gathering2, 3. Differences in diet were also linked to an overall shift in the oral bacterial community (microbiota) and suggested that meat consumption contributed to substantial variation within Neanderthal microbiota. Evidence for self-medication was detected in an El Sidrón Neanderthal with a dental abscess4 and a chronic gastrointestinal pathogen (Enterocytozoon bieneusi). Metagenomic data from this individual also contained a nearly complete genome of the archaeal commensal Methanobrevibacter oralis (10.2× depth of coverage)—the oldest draft microbial genome generated to date, at around 48,000 years old. DNA preserved within dental calculus represents a notable source of information about the behaviour and health of ancient hominin specimens, as well as a unique system that is useful for the study of long-term microbial evolution., The Australian Research Council supported this work through the Discovery Project and Fellowship schemes.

Objective Chronic periodontitis is a chronic inflammatory disease of the periodontal tissues and is caused by invasion of certain types of bacteria and Archaea , with Methanobrevibacter oralis as the predominant archaeon. In this study, we investigated the prevalence and quantity of the newly discovered Archaea phylotype Thermoplasmata in patients with chronic periodontitis. Methods Subgingival plaque samples were obtained from 49 patients with chronic periodontitis and 45 periodontally healthy subjects. Qualitative analyses of Archaea and class Thermoplasmata were carried out by amplification of 16S rRNA genes in DNA extracts from plaque samples, and all the samples were quantitatively analyzed by real-time polymerase chain reaction (PCR). Results The prevalence of Archaea in patients with chronic periodontitis was 69.4% according to the conventional PCR results, but was 87.8% according to real-time PCR. In the control group, three samples were detected as positive, but none of these were confirmed in qualitative analyses. The prevalence of class Thermoplasmata was 18.4% by nested PCR and 24.5% by quantitative PCR in the chronic periodontitis group. The prevalence of Thermoplasmata was significantly lower than that of total Archaea. The relative abundances of Archaea and Thermoplasmata varied among samples. Thermoplasmata were not the predominant archaeons in the subgingival dental plaque. Among the clinical parameters of patients with periodontitis, probing depth was positively associated with Archaea detection. Conclusions The existence of Archaea was correlated closely with the presence of chronic periodontitis. Thermoplasmata represented a minor archaeon in periodontal infection.

Methanogenic archaea are involved in periodontitis in humans. They have also been implicated in digestive tract pathologies and obesity. These microorganisms are broadly resistant to antibiotics, except for metronidazole and ornidazole. In this study, eight imidazole derivatives were synthesised and their in vitro cytotoxicity and activity against six species of methanogenic archaea, including Methanobrevibacter smithii, Methanobrevibacter oralis, Methanosphaera stadtmanae, Methanobacterium beijingense, Methanosaeta concilii and Methanomassiliicoccus luminyensis, were tested. Whilst the effective half-maximum cytotoxic concentrations (EC50 values) of all compounds were ≤50 mg/L, minimum inhibitory concentrations (MICs) were 0.05-0.8 mg/L for most methanogenic archaea and 0.1-1mg/L for M. stadtmanae. These results indicated a >20-400 therapeutic index (EC50/MIC) for these compounds, which compared with metronidazole exhibited 1-log increased activity against methanogenic archaea cultured from the human microbiota. These compounds are therefore promising molecules for the treatment of methanogenic archaea-related infections.

Both group I (HSP60) and group II (CCT) chaperonins are targets of autoantibodies. Autoimmune reactions to HSP60 have been well characterized, while immune reactions to group II chaperonin have not been clarified. Methanobrevibacter oralis is a suspected periodontal pathogen with group II chaperonin. In this study, serum responses to M. oralis chaperonin, human HSP60, and CCT subunits were examined using sera from patients with periodontitis and autoimmune diseases. In comparison with healthy controls, periodontitis patients showed significantly higher responses to CCT4 and CCT8 on dot blot analysis. Signals for CCT3 and CCT8 in autoimmune disease patients were significantly higher than in controls. Significant differences were also demonstrated by Western blotting in anti-CCT4 response in both patient groups. All subjects showed strong reactivity to M. oralis chaperonin and faint signals to human HSP60. Autoantibodies were raised against CCT rather than HSP60; and CCT3, CCT4, and CCT8 were shown to be the main targets. Host immune systems may be frequently exposed to chaperonins of Archaea in various habitats. Although further studies of the cross-reactivity between M. oralis chaperonin and human CCT are required, anti-CCT autoantibodies may be involved in the pathogenesis of periodontitis and autoimmune diseases.

Here, the draft genome sequences of four different Methanobrevibacter species are presented. Three of the Methanobrevibacter species ( M. curvatus, M. cuticularis , and M. filiformis ) have been isolated from the termite hindgut, while M. oralis was isolated from human subgingival plaque.

International audience; no abstract

This work represents an update of knowledge regarding the detection methods for human microbiome-associated archaea. Despite the fact that, during the last three decades, only four methanoarchaeal species have been isolated from the human mucosa, including faeces, subgingival plaque, and vaginal mucosa (Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanobrevibacter oralis and, most recently, ‘Methanomassiliicoccus luminyensis’), molecular studies, including PCR and metagenomic analyses, have detected DNA sequences indicative of the presence of additional methanoarchaea, as well as non-methanogenic archaea, in the human intestinal tract. Opinion is divided on the roles (if any) of these organisms in human disease, and certainly the data are still unclear. Future research and recently reported data highlighting the antimicrobial susceptibility of the human methanoarchaea could help in the design of selective media to discover additional human mucosa-associated archaea and ascertain their role in human infections involving complex flora.

Dridi B, Raoult D, Drancourt M. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of Archaea: towards the universal identification of living organisms. APMIS 2012; 120: 85–91. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) identification of Archaea has been limited to some environmental extremophiles belonging to distant taxa. We developed a specific protocol for MALDI-TOF-MS identification of Archaea and applied it to seven environmental human-associated Methanobrevibacter smithii, Methanobrevibacter oralis, Methanosphaera stadtmanae, and the recently described Methanomassiliicoccus luminyensi Archaea. After mechanical lyse, we observed a unique protein profile for each organisms comprising 7–24 peaks ranging from 3 015 to 10 632 Da with a high quality score of 7.38 ± 1.26. Profiles were reproducible over successive experiments performed at 1, 2, and 3-week growth durations and unambiguously distinguished the Archaea from all of the 3 995 bacterial spectra in the Bruker database. After the incorporation of the determined profiles into a local database, archaeal isolates were blindly identified within 10 min with an identification score of 1.9–2.3. The MALDI-TOF-MS-based clustering of these archaeal organisms was consistent with their 16S rDNA sequence-based phylogeny. These data prove that MALDI-TOF-MS profiling could be used as a first-line technique for the identification of human Archaea. In complement to previous reports for animal cells, Bacteria and giant viruses, MALDI-TOF-MS therefore appears as a universal method for the identification of living unicellular and multicellular organisms.

Matarazzo F, Ribeiro AC, Feres M, Faveri M, Mayer MPA. Diversity and quantitative analysis of Archaea in aggressive periodontitis and periodontally healthy subjects. J Clin Peridontol 2011; doi: 10.1111/j.1600-051X.2011.01734.x. Abstract Aim: To investigate the diversity, levels and proportions of Archaea in the subgingival biofilm of generalized aggressive periodontitis (GAgP; n=30) and periodontally healthy (PH; n=30) subjects. Materials and methods: Diversity was determined by sequencing archaeal 16S rRNA gene libraries from 20 samples (10/group). The levels and proportions of Archaea were analysed by quantitative PCR (qPCR) in four and two samples/subject in GAgP and PH groups, respectively. Results: Archaea were detected in 27/28 subjects and 68% of the sites of the GAgP group, and in 26/30 subjects and 58.3% sites of the PH group. Methanobrevibacter oralis was found in all 20 samples studied, Methanobacterium curvum/congolense in three GAgP and six PH samples, and Methanosarcina mazeii in four samples from each group. The levels and proportions of Archaea were higher in GAgP than in PH, whereas no differences were observed between the two probing depth category sites from the GAgP group. Conclusion: Archaea were frequently found in subjects with periodontal health and GAgP, especially M. oralis. However, the higher levels and proportions (Archaea/total prokaryotes) of this domain observed in GAgP in comparison with PH subjects indicate a possible role of some of these microorganisms as an environmental modifier in GAgP.

Faveri M, Goncalves LFH, Feres M, Figueiredo LC, Gouveia LA, Shibli JA, Mayer MPA. Prevalence and microbiological diversity of Archaea in peri-implantitis subjects by 16S ribosomal RNA clonal analysis. J Periodont Res 2011; 46: 338–344.© 2011 John Wiley & Sons A/S Background and Objective: This study evaluated the prevalence and the molecular diversity of Archaea in the subgingival biofilm samples of subjects with peri-implantitis. Material and Methods: Fifty subjects were assigned into two groups: Control (n = 25), consisting of subjects with healthy implants; and Test (n = 25), consisting of subjects with peri-implantitis sites, as well as a healthy implant. In the Test group, subgingival biofilm samples were taken from the deepest sites of the diseased implant. In both groups, subgingival biofilm was collected from one site with a healthy implant and from one site with a periodontally healthy tooth. DNA was extracted and the 16S ribosomal RNA gene was amplified with universal primer pairs for Archaea. Amplified genes were cloned and sequenced, and the phylotypes were identified by comparison with known 16S ribosomal RNA sequences. Results: In the Control group, Archaea were detected in two and three sites of the implant and the tooth, respectively. In the Test group, Archaea were detected in 12, 4 and 2 sites of diseased implants, healthy implants and teeth, respectively. Diseased implants presented a significantly higher prevalence of Archaea in comparison with healthy implants and natural teeth, irrespective of group. Over 90% of the clone libraries were formed by Methanobrevibacter oralis, which was detected in both groups. Methanobacterium congelense/curvum was detected in four subjects from the Test group and in two subjects from the Control group. Conclusion: Although M. oralis was the main species of Archaea associated with both healthy and diseased implant sites, the data indicated an increased prevalence of Archaea in peri-implantitis sites, and their role in pathogenesis should be further investigated.

Methanobrevibacter oralis is an archaeal species frequently isolated from sites of severe periodontitis. However, its pathogenic roles remain unclear. Here, we aimed to isolate group II chaperonin from M. oralis and examine its antigenicity. The genes encoding two chaperonin subunits (Cpn-1 and Cpn-2) were cloned from M. oralis using polymerase chain reaction and genome walking procedures. Recombinant proteins Cpn-1 and Cpn-2 were generated, and the reactivities of sera from patients with periodontitis were examined by Western immunoblotting. The open reading frames of Cpn-1 and Cpn-2 genes consisted of 1641 and 1614 base pairs, respectively. Putative ATP-binding domains conserved among the chaperonin family were observed in both genes. The deduced amino acid sequences of the two genes showed 28.8-40.0% identity to each of the subunits of human CCT (CCT1-8). Thirty and 29 of 36 patients' sera reacted with the recombinant Cpn-1 and recombinant Cpn-2, respectively. Western immunoblotting using antiserum against human CCT subunits indicated that anti-CCT3 and anti-CCT8 antibodies recognized recombinant Cpn-1. In addition, anti-CCT1, CCT3, CCT6, and CCT8 antibodies recognized an antigen of approximately 60 kDa in M. oralis. The results suggested that the chaperonin subunits of M. oralis were antigenic molecules that were recognized by periodontitis patients and that may cross-react with human chaperonin CCT.

Phylogenetic analysis of bacterial and archaeal 16S rRNA was used to examine polymicrobial communities within infected root canals of 20 symptomatic and 14 asymptomatic patients. Nucleotide sequences from ∼750 clones amplified from each patient group with universal bacterial primers were matched to the Ribosomal Database Project II database. Phylotypes from 37 genera representing Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria were identified. Results were compared to those obtained with species-specific primers designed to detect Prevotella intermedia, Porphyromonas gingivalis, Porphyromonas endodontalis, Peptostreptococcus micros, Enterococcus sp., Streptococcus sp., Fusobacterium nucleatum, Tannerella forsythensis and Treponema denticola. Since members of the domain Archaea have been implicated in the severity of periodontal disease, and a recent report confirms that archaea are present in endodontic infections, 16S archaeal primers were also used to detect which patients carried these prokaryotes, to determine if their presence correlated with severity of the clinical symptoms. A Methanobrevibacter oralis-like species was detected in one asymptomatic and one symptomatic patient. DNA from root canals of these two patients was further analysed using species-specific primers to determine bacterial cohabitants. Trep. denticola was detected in the asymptomatic but not the symptomatic patient. Conversely, Porph. endodontalis was found in the symptomatic but not the asymptomatic patient. All other species except enterococci were detected with the species-specific primers in both patients. These results confirm the presence of archaea in root canals and provide additional insights into the polymicrobial communities in endodontic infections associated with clinical symptoms.

Our colons harbor trillions of microbes including a prominent archaeon, Methanobrevibacter smithii . To examine the contributions of Archaea to digestive health, we colonized germ-free mice with Bacteroides thetaiotaomicron , an adaptive bacterial forager of the polysaccharides that we consume, with or without M. smithii or the sulfate-reducing bacterium Desulfovibrio piger . Whole-genome transcriptional profiling of B. thetaiotaomicron , combined with mass spectrometry, revealed that, unlike D. piger , M. smithii directs B. thetaiotaomicron to focus on fermentation of dietary fructans to acetate, whereas B. thetaiotaomicron -derived formate is used by M. smithii for methanogenesis. B. thetaiotaomicron – M. smithii cocolonization produces a significant increase in host adiposity compared with monoassociated, or B. thetaiotaomicron – D. piger biassociated, animals. These findings demonstrate a link between this archaeon, prioritized bacterial utilization of polysaccharides commonly encountered in our modern diets, and host energy balance.

Archaea have been isolated from the human colon, vagina, and oral cavity, but have not been established as causes of human disease. In this study, we reveal a relationship between the severity of periodontal disease and the relative abundance of archaeal small subunit ribosomal RNA genes (SSU rDNA) in the subgingival crevice by using quantitative PCR. Furthermore, the relative abundance of archaeal small subunit rDNA decreased at treated sites in association with clinical improvement. Archaea were harbored by 36% of periodontitis patients and were restricted to subgingival sites with periodontal disease. The presence of archaeal cells at these sites was confirmed by fluorescent in situ hybridization. The archaeal community at diseased sites was dominated by a Methanobrevibacter oralis -like phylotype and a distinct Methanobrevibacter subpopulation related to archaea that inhabit the gut of numerous animals. We hypothesize that methanogens participate in syntrophic relationships in the subgingival crevice that promote colonization by secondary fermenters during periodontitis. Because they are potential alternative syntrophic partners, our finding of larger Treponema populations sites without archaea provides further support for this hypothesis.