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

ABSTRACT 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. IMPORTANCE Methanogens 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.

in animal models to investigate the role of methanogens in hypersensitivity diseases.The administration of M. stadtmanae extract aerosol but not M. smithii aerosol in the airways of mice yielded a hypersensitivity response with an accumulation of eosinophils and neutrophils in the lungs (17,18), laying the groundwork for the potential pathogenic role of M. stadtmanae.The proinflammatory capacity of M. stadtmanae was next used to induce hypersensitivity pneumonitis disease in mice models (19,20).Moreover, further studies focused on human cells' response exposition to methanogens, M. smithii and M. stadtmanae are both able to activate mononuclear cells but M. stadtmanae induced a higher release of proinflammatory cytokines than M. smithii (21,22).M. stadtmanae phagocytosis is crucial for cell activation (21).At the molecular level, M. stadtmanae RNA was identified as the microbe-associated molecular pattern (MAMP) that can trigger an NLRP3 inflammasome activation through the toll-like receptor 8 (TLR8) (23).Interestingly, M. stadtmanae was more prevalent in patients with inflammatory bowel syndrome which developed a specific IgG response to this methanogen (22).More recently, a decrease in the Methanosphaera abundance was noticed in healthy to long-time diabetic patients, whereas an increase in M. smithii was observed (24).
Although M. stadtmanae was for a long time the sole Methanosphaera member cultured from human feces (5) and further detected by PCR-based methods in human dental plaque (25), a new human isolate, Methanosphaera sp.PA5 was provided by Hoedt et al. in 2018 (26).Furthermore, Methanosphaera has been cultured from mammal guts including Methanosphaera cuniculi (M.cuniculi) in rabbits (27), Methanosphaera sp.WGK6 in Western grey kangaroo (Macropus fuliginosus) (28), and Methanosphaera sp.BMS in cow guts (26).Recently, Hoedt et al. (26) and Chibani et al. (9) obtained, respectively, 7 and 20 Methanosphaera spp.genomes from metagenomic data of mammals (including humans), some of them not corresponding to known isolates.Particularly, among the 20 Methanosphaera spp.genomes provided by Chibani et al. from the human gut, 17 were related to M. stadtmanae, two to M. cuniculi, and one to a genome obtained from cow rumen, suggesting possible host transfers and adaptation from animals to human gut.
These cumulative data suggested that human health interest, host-associated, and yet-uncultured Methanosphaera members remained to be discovered by isolation and culture in the human gut microbiota, subject to the opportunity to apply renewed culture approaches to suitable clinical materials.Using a recently designed hydrogenfree and carbon dioxide-free medium named GG medium (29, patent FR 23 01404), combined with molecular approaches, our investigations of mammal and human feces led to the discovery of a novel Methanosphaera representative isolate.

Isolation and microscopic observations
Methane was detected from one methanogen 16S rRNA gene PCR-positive stool sample culture.Successive transfers and subcultures on a solid medium yielded colonies forming a translucent carpet (Fig. S1), which was further subcultured in a liquid medium and produced methane after a 15-day incubation at 37°C.Colonies comprised of Gram-pos itive diplococci and clumps, with the individual diameter measured at 850 nm using electron microscopy.Gram variability was observed after 15 days of culture.These non-motile cocci showed autofluorescence at 420 nm using confocal microscopy.The purity of the final culture was established as only autofluorescent cocci were observed in confocal microscopy from several fields as well and only cocci were observed in electron microscopy.Furthermore, we checked that no bacteria grew on a COS agar plate (Fig. 1)

MALDI-TOF mass spectrometry
MALDI-TOF mass spectrometry yielded reproducible peptide spectra but no identifica tion by spectra comparison with our bacterial database.The cell mass was first con firmed through PCR sequencing to match with BB6 isolate before MALDI-TOF analysis.Comparison with M. stadtmanae DSM3091 peptidic profile did not show significant differences within the two spectra (Fig. S2).

pH susceptibilily
pH susceptibility testing showed that isolate BB6 is also able to grow at pH 6 and pH 7.3 but not out of this range.Subcultures from these two pH conditions yielded to methane production after a 7-day culture.The pH of 7.3 in the GG medium was considered the reference pH for the methanogen culture (Table S4).

Substrate affinity testing and methanogenesis pathways
Ca. M. massiliense was able to grow on GG0 medium (basal GG medium) with meth anol under H 2 /CO 2 /N 2 atmosphere, GG0 medium with methanol and ethanol under nitrogen atmosphere, and GG medium (containing acetate, formate, and methanol) supplemented with ethanol as the reference condition (Table S4).Proxi-growth curves of methane production and optical density from day 0 to day 7 are also available in Table S4.Slow growth was observed on methanol alone.No methane was detected with no substrate, under the H 2 /CO 2 /N 2 atmosphere and no other substrate, on formate, acetate, ethanol alone, or ethanol under the H 2 /CO 2 /N 2 atmosphere (Table S4).Genome annotation revealed the presence of a NADPH-butanol dehydrogenase and an aldehyde dehydrogenase closely related to Methanosphaera sp.SHI1033 and Methanosphaera sp.WGK6 (Figures S3a through d), which allows growth in a hydrogen-free atmosphere in a medium containing methanol and ethanol.The absence of formate dehydrogenase and the presence of the incomplete enzymatic pathway for acetoclastic methanogenesis, only the acetyl-CoA synthetase is encoded, are consistent with the previous experimen tal results.

Feces screening by specific RT-PCR and amplicon sequencing
The specificity of the Ca.M. massiliense RT-PCR system was confirmed as all methano gen strains including Methanobrevibacter and Methanosphaera species and negative controls remained negative but was positive for Ca.M. massiliense, while the Methano brevibacter and Methanosphaera strains yielded positivity for the 16S rRNA gene (Table S5).Moreover, the RT-PCR system was in silico positive for Ca.M. massiliense, Methanos phaera sp.Mb5 and RUG761 but not for Methanosphaera sp.SHI1033 and WGK6 nor M. stadtmanae.Then, a total of 150 leftover human stool samples and 313 ground-collec ted feces from six mammal species located in four French departments (Fig. S4) were investigated using a Ca.M. massiliense specific RT-PCR system, with a Ct value <37 considered as positive.One of 150 human fecal samples (0.67%), 14/64 (22%) pig fecal samples, and 6/50 (12%) red kangaroo fecal samples were positive by RT-PCR for Ca.M. massiliense while the other mammal samples remained negative (Table S6a).The one positive human feces sample was the same as Ca.M. massiliense had been isolated by culture as above.Ca.M. massiliense identity in all positive samples was confirmed as the sequences showed an identity average of 96.28% ± 0.82 and 100% coverage with Ca.M. massiliense whole-genome sequence and no other correspondence was found in the GenBank database (Table S6b).Moreover, Ca.M. massiliense load was comparable in the human sample (3,77E+07 CFU/mL) and in pig and kangaroo samples (average load 2.68E+07 CFU/mL ± 1.25E+07) (Table S6a).

DISCUSSION
Ca. Methanosphaera massiliense is the second Methanosphaera species isolated in humans and described using a polyphasic approach combining morphology by light and electron microscopy, peptide fingerprinting by mass spectrometry, antibiotics, pH susceptibility, substrate affinity testing, and whole-genome sequence-derived analyses, with the negative results of controls introduced in all experiments confirming the authenticity of the results.Whole-genome sequence and 16S rRNA gene sequence data confirmed the originality of Ca.M. massiliense's identity among isolated species but Ca.M. massiliense might be the cultured representative of Methanosphaera sp.RUG761 and Methanos phaera sp.M5, a methanogen detected in bovine and a man with colorectal cancer (9).
Among isolated species, Ca.M. massiliense was found to be most closely metabol ically related to Methanosphaera sp.WGK6, a methanogen isolated by culture in one Western grey kangaroo (28).Both methanogens can grow on methanol and hydro gen but also in a hydrogen-free medium containing methanol and ethanol, as they share NADPH-butanol dehydrogenase and aldehyde dehydrogenase (28), GG medium supplemented with ethanol should be suitable for growing these methanogens.A distinctive feature of M. stadtmanae, M. cuniculi, and Methanosphaera sp.BMS is that they have only a hydrogen-dependent methanol-fueled metabolism (26,27,30).
The enzymes identified through genome sequencing and annotation play a crucial role in characterizing the population susceptible to the presence of Ca.M. massiliense in the gut microbiota.This population may consist of individuals who have had exposure to ethanol and methanol, both of which can originate from either bacterial and fungal sources or consumable items like beverages and food products (31)(32)(33).Indeed, ethanol and methanol are both chemical compounds found in various sources that can be consumed by humans.Ethanol is primarily found in alcoholic beverages such as beer, wine, and spirits (31).In addition, ethanol can be used as an additive or solvent in certain foodstuffs (32).Methanol can be present in foods due to natural fermentation processes or the breakdown of pectin in certain fruits and vegetables (33).Alcoholic beverages containing ethanol may also contain low levels of methanol, although these are generally considered safe for human consumption (34,35).Also, the fact that Ca.M. massiliense only remained alive in a narrow neutral pH further suggesting that individuals lacking gastric acidity may favor the implantation of Ca.M. massiliense in their gut, as well as individuals exposed to large Ca.M. massiliense inoculum (36).
Considering the findings from this study, we propose a hypothesis suggesting that potential sources of Ca.M. massiliense may be of mammalian zoonotic origin, as indicated by the observed prevalence of Ca.M. massiliense in pigs destined to slaugh terhouse.This species has also been identified in other animals, including those with rumen, cow, sheep, and human hosts (9).Fundamentally, our research suggests that this methanogenic archaeal species is not confined to a single host type and does not exhibit hostspecific lineages.This implies that food consumption and other contact with animals could serve as pathways for the transmission of these microorganisms to the human intestinal microbiota (37).The success of crossing implantation in individuals of other mammal species, including humans, may depend on the density of contact between species and individualspecific factors.Notably, other methanogens, particu larly Methanobrevibacter spp.and Methanomassiliicoccus spp., have been detected in both humans and other mammals (38)(39)(40), such as pigs: M. smithii, Methanobrevibacter millerae (M.millerae), and M. luminyensis (38).This finding underscores the intricate nature of microbial interactions within diverse host environments, emphasizing the need for additional research to comprehend the dynamics of microbiota exchange and its potential implications for human and animal health.
In conclusion, we are reporting the second isolation by culture of a Methanosphaera species from human feces referred to as Ca.M. massiliense sp.nov., which was obtained using a home-made specific culture GG medium under a nitrogen atmosphere.It is genetically more closely related to mammalian animals Methanosphaera than to M. stadtmanae.Our findings raise the question of potential zoonotic sources and the conditions of adaptation to a minority of individuals of Ca.M. massiliense and metha nogens in general and their possible consequences to human health.

Specimen collection
This prospective study was conducted from August 2021 to September 2021 at the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection in Marseille, France.In this study, only leftover stool samples were studied after the microbiological diagnosis laboratory work was completed and after the investigators had verified that no patient objected to the use of leftover clinical samples for research purposes (Article L1211-2 of the French Public Health Code).According to French law, the research on anony mized leftover stool samples used in this study was considered non-interventional research (Article L1221-1.1 of the French Public Health Code) and did not require any ethics committee approval.In addition, we used animal fecal samples collected on the ground.No ethical approval was required for these samples, for which the mam mal owners' oral consent was obtained by two of us (B.D. and B.L.).Samples were screened for methanogens presence using RT-PCR (primers: Metha_16S_2_MBF, 5′-C GAACCGGATTAGATACCCG-3′ and Metha_16S_2_MBR, 5′-CCCGCCAATTCCTTTAAGTT-3′; probe: FAM_Metha_16S_2_MBP, [FAM]-5′-CCTGGGAAGTACGGTCGCAAG-3′) as previously described (41).

Culture and isolation of the new methanogen species
A 0.5 g 16S rRNA RT-PCR-positive stool sample was suspended in 10 mL of Dulbecco's phosphatebuffered saline (DPBS) 1× water (Thermo Fisher Scientific, Waltham, MA, USA) and vortexed for a few seconds.A 200 µL volume of this suspension was inoculated into a Hungate tube containing GG medium (29, patent FR 23 01404) that is derived from SAB medium (42), containing acetate, formate, and methanol as standards, previously degassed for 3 minutes with 2 bar nitrogen.The inoculated tube was incubated for 7 days at 37°C.On day 7, methane was detected by gas chromatography using a Clarus 580 chromatograph (Perkin Elmer, Waltham, MA, USA) confirming methanogen growth as previously described (42) and optical density was measured by putting the Hungate tubes in a spectrophotometer cell density meter model 40 (Thermo Fisher Scientific, Waltham, MA, USA).Then, 200 µL of growing culture was inoculated into a fresh GG medium containing fresh antibiotics, 100 mg/L amoxicillin, 100 mg/L vancomycin, 100 mg/L imipenem, 50 mg/L daptomycin, and 50 mg/L amphotericin B (Sigma Aldrich, Saint Quentin Fallavier, France) and supplemented with ethanol at 2% (vol/vol).Then, 100 µL of the growing culture was inoculated on the same home-made solid GG medium obtained by adding 15 g/L agar (Condalab, Madrid, Spain) to the broth formulation of the GG medium with ethanol which was then incubated at 37°C in an anaerobic atmosphere using BD Pouch Gaspak Ez Anaerobe System (Becton, Dickinson and Company, Franklin Lakes, USA) for 9 days.Negative controls consisted in the inoculation of a solid medium with PBS, the liquid medium previously inoculated with PBS for 7 days (Fig. 1).The isolate was sub-cultured 10 times to ensure the purity of the culture, checked by autofluores cence using a confocal light microscope LSM 900 (Carl Zeiss Microscopy GmbH, Jena, Germany) at the 63× objective with immersion oil as previously described (43), electron microscopy with the Scanning Electron Microscope TM4000 plus (Hitachi, Tokyo, Japan), and inoculation of 100 µL on Columbia agar 5% sheep blood (COS) media (bioMérieux, Marcy-Étoile, France) plate.

Light microscopy
Gram staining was performed as previously described (44).For confocal microscopy, a 10 µL volume from the pure culture was placed on a glass slide and sealed with a coverslip under an anaerobic chamber.Autofluorescence was observed under a confocal light microscope LSM 900 (Carl Zeiss Microscopy GmbH, Jena, Germany) at the 63× objective with immersion oil as previously described (43).

Electron microscopy
A 100 µL volume of inoculated culture broth was mixed with 100 µL of 4× glutaralde hyde and then stirred for 30-60 minutes at room temperature.A 20 µL volume of 10% phosphotungstic acid was added and stirred again for 5 minutes, deposited on a Cytospin (Thermo Fisher Scientific, Waltham, MA, USA), and centrifugated for 7 minutes at 800 rpm.Slides were read with the Scanning Electron Microscope TM4000 plus (Hitachi, Tokyo, Japan).Micrographs were acquired at high magnifications ranging from 2,500× to 10,000× with an accelerating voltage of 10 kV using the Backscatter Electron detector under the high vacuum mode.

Whole genome sequencing
DNA was extracted from 200 µL of culture as previously described (45) using the EZ1 DNA Tissue Kit (Qiagen, Courtaboeuf, France) after overnight incubation at 56°C with 20 µL proteinase K (Qiagen).Glass powder was added to the mix, incubated for 20 minutes at 100°C and immediately vortexed 90 s at 6.5 m/s with the FastPrep instru ment (MP Biomedical Europe, Illkirch, France).The mix was centrifuged for 5 minutes at 17,000 × g and DNA was extracted from 200 µL of supernatant and eluted in a 50 µL volume.The methanogen identification was first done by partial 16S rRNA gene PCRamplification followed by Sanger sequencing (primers: SDArch0333aS15, 5′-TCCA GGCCCTACGGG-3′ and SDArch0958aA19, 5′-YCCGGCGTTGAMTCCAATT-3′) as previously described (46).Generated reads were assembled by ChromasPro software (version 1.34), and then aligned to the NCBI GenBank database using the Blast platform.DNA was then engaged for WGS using the MiSeq Illumina pair-end protocol (Illumina, San Diego, CA, USA) and Oxford Nanopore single-long reads (Oxford Nanopore Technologies, Oxford, UK) platforms as previously described (47,48).Quality control of NGS data was done using the fastQC command on the Galaxy Europe online platform (https://usegalaxy.eu/).Illumina and Nanopore reads were concatenated and de novo assembled using Spades software version (version 3.15.4)and generated contigs were blasted against the NCBI GenBank database.The quality of genome assemblies was checked using CheckM (49).The ANI analysis was performed with the 38 genomes included in this study (Table S3) using PYANI software version (0.2.7) with standard parameters, and genome sequences with >95% identity corresponding to the same species (50).dDDH (DNA-DNA hybridization) values were calculated using the TYGS [Type (Strain) Genome Server] online tool (51) based on the 38 included genome sequences.The threshold for DDH values indicating membership in the same species was above 70% (52).Phylogenetic analysis based on WGS-derived 16S rRNA gene sequences with the 100 hit-blasts was performed using Neighbor-Join and BioNJ algorithms standard parameters on MEGA software (version 7.0.26).The evolutionary history was inferred using the Maximum Likelihood method based on the JTT matrix-based model and 1,000 replicates boot strap consensus.Branches corresponding to partitions reproduced in less than 90% of bootstrap replicates are collapsed.The initial tree was automatically obtained by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, then selecting the topology with the superior log likelihood value.All positions containing gaps and missing data were eliminated, and a total of 1138 positions were included in the final data set (Fig. 3).Genome representation was performed using the Proksee platform with standard parameters (version 1.0.0;https:// proksee.ca/).The genome was annotated on the DDBJ Fast Annotation and Submis sion Tool online platform (https://dfast.ddbj.nig.ac.jp/) and putative enzymes involved in the utilization of different substrates for methanogenesis were sought.Phyloge netic trees of NADPH-dependent butanol dehydrogenase and aldehyde dehydrogenase gene sequences and protein sequences were obtained using Neighbor-Join and BioNJ algorithms standard parameters on MEGA software (version 7.0.26).The evolutionary history was inferred using the maximum likelihood method based on the JTT matrixbased model (Figures S3a through d).Blastp was used against the COG database to find putative encoded protein functions for Ca.M. massiliense and other methanogens (Table S1).

MALDI-TOF mass spectrometry
Protein extraction was performed as previously described ( 53) from 1 mL of pure liquid cultures of the new isolate and M. stadtmanae DSMZ3091 (CSUR P9634).Two deposits were made for each sample and peptide profiles were determined using an Autoflex II mass spectrometer equipped with a 337 nm nitrogen laser (Brüker Daltonics, Bremen, Germany).Protein extract of E. coli DH5α (Brüker Daltonics) was used as a positive control and non-inoculated medium as a negative control.Spectra were recorded in the positive linear mode for masses ranging from 2 to 20 kDa (parameter settings: ion source 1 [IS1], 20 kV; IS2, 18.5 kV; lens, 7 kV).A spectrum was obtained after 675 shots with a varia ble laser power.The software tool AutoXecute acquisition control was applied for the automated data acquisition.Peptidic spectra analysis and comparison were performed using FlexAnalysis 2.4 software (Brüker Daltonics).

Antibiotic susceptibility testing
Antibiotic susceptibility was tested using a previously described macrodilution technique and based on M. stadtmanae susceptibility (54).Amoxicillin, imipenem, metronidazole, bacitracin, fusidic acid, and chloramphenicol (Sigma Aldrich, Saint Quentin Fallavier, France) were dissolved in sterile water and added separately to the broth medium to obtain a final concentration of 100 mg/L for amoxicillin and imipe nem, 1 mg/L for metronidazole, 4 mg/L for chloramphenicol and 4 mg/L for bacitracin and fusidic acid.For each antibiotic, two Hungate tubes containing 4.5 mL of liquid medium were inoculated with 10 5 colony-forming units (CFU)/mL of methanogen, and one control tube was inoculated with 500 µL of PBS.Two Hungate tubes containing 4.5 mL of liquid medium without antibiotics inoculated with 10 5 CFU/mL of methanogen were used as positive controls.Culture tubes were incubated for 5 days at 37°C in an anaerobic atmosphere and were monitored using gas chromatography at day 0 (D0) and day 5 (D5).100 µL was sampled from the headspace of the tube and injected into a Clarus 580 chromatograph (Perkin Elmer, Waltham, MA, USA).The so-called "sensi tive" method was used and the resulting area under the curve values and their conver sion to molar concentration (mol/L) were used to quantify methane production.The minimum inhibitory concentration (MIC) was defined as the lowest antibiotic concentra tion that inhibited methane production.The following controls were performed for each antibiotic to verify its activity: E. coli CSUR (Collection Souches Unité des Rickettsies, Marseille, France) Q6942 and S. aureus CSUR Q7280 whose antibiotic susceptibility was previously determined, were each seeded on two Columbia agar 5% sheep blood (COS) media (bioMérieux, Marcy-Étoile, France) and two additional COS media were inoculated with the same antibiotic solution used for each experiment, two non-seeded COS media were inoculated with the antibiotic solution and two others with PBS.

pH susceptibility testing
Five pH conditions were tested after the pH of the liquid culture medium was adjusted to pH 2, 4, 6, 8, and 10 using HCl or KOH (Sigma Aldrich, Saint Quentin Fallavier, France).For each condition, two Hungate tubes containing 4.5 mL of liquid culture medium were inoculated with 10 5 CFU/mL of the methanogen, and one Hungate tube was inoculated with PBS as a negative control.Positive controls consisted of two Hungate tubes containing liquid medium at pH 7.3 inoculated with 10 5 CFU/mL.The culture tubes were incubated for 7 days at 37°C in an anaerobic atmosphere and were monitored using gas chromatography at day 0 (D0) and day 7 (D7) as described above.The methanogen was considered susceptible to a pH value when no methane was detected after a 7-day incubation.Methane-positive cultures were then subcultured in GG medium at pH 7.3 and 37°C for 7 days to ensure the viability of the methanogen.

Substrate affinity testing
A basal GG medium (GG0) derived from the original GG medium but without acetate, formate, methanol or ethanol was used, and each substrate or substrate combinations were added separately in the medium GG0.Nine conditions were tested using GG0 medium, six under nitrogen atmosphere: no substrate, acetate (2 g/L), formate (2 g/L), methanol (2% vol/vol), ethanol (2% [vol/vol]), methanol and ethanol (2% [vol/vol]), and three under 5% H 2 /20% CO 2 /75% N 2 atmosphere without any substrate, with methanol or with ethanol.For each condition, three Hungate tubes containing 4.5 mL of liquid culture medium were inoculated with 10 5 CFU/mL of the methanogen, and one Hungate tube was inoculated with PBS as a negative control.The culture tubes were incubated for 7 days at 37°C and were monitored using gas chromatography at day 0 (D0) and day 7 (D7) as described above.

Human and animal faeces molecular screening
The prevalence of the methanogen was assessed in 150 human and 313 animal stool samples using a real-time (RT) PCR assay, with the fecal samples coming from 52 dogs, 20 sheep, 75 horses, 52 cows, 50 red kangaroos, and 64 pigs (Fig. S4).DNA was extracted as described above from 0.5 g put in 500 µL of G2 buffer (Qiagen, Courtaboeuf, France).We used phylogeny and annotation of the methanogen genome sequence to investigate and identify a candidate sequence for RT-PCR screening of a large stool panel from humans and animals.Because the 16S rRNA-encoding gene was not informative for the screening, the DNA polymerase encoding gene was targeted for primer design.Using the Primer3 program (https://primer3.org/), an in-house RT-PCR system was designed, targeting 124 bp of Ca.M. massiliense polymerase encoding gene including a 6carboxyfluorescein [FAM]-5′-AGCACGTATGACTACAGGACA-3′ probe, forward primer (5′-AACACCGCTTACTGTTGCAC-3′) and reverse primer (5′-ACTCGTCCGTAT CTGGCTTTA-3′).RT-PCR specificity was in silico tested by the NCBI BLAST program (http:// www.ncbi.nlm.nih.gov/BLAST) using each primer alone and in-silico amplified using both primers and the specific probe, particularly against Methanosphaera sp.Mb5, RUG761, SHI1033, WGK6 genome sequences, using Amplify4 software (version 1.0, Bill Engels, University of Wisconsin).The RT-PCR amplification program was 95°C for 15 minutes, followed by 39 cycles of 95°C for 30 s and 60°C for 1 minutes, and a final step of 40°C for 30 s. RT-PCR validation was done in a 20 µL volume containing 5 µL DNA, 20 µM of each primer, and 10 µM of the specific probe, using the Roche Master Mix according to the manufacturer's protocol (Roche, Indianapolis, IN, USA).To disclose any cross-contamination, 5 µL of DNase/RNase-free distilled water was used as a negative control.The specificity was tested with M. smithii Q5488, M. oralis Q6268 preserved in the CSUR, and M. stadtmanane DSM3091 (CSUR P9634).As a supplementary control, the methanogens 16S rRNA gene was also amplified as previously described to ensure the presence of methanogens (38).A calibration curve was obtained using serial dilution from 10 8 CFU/mL (0.5 McFarland turbidity standard) to 10 1 CFU/mL and was used to quantify the load of Ca.M. massiliense in each sample.

FIG 2
FIG 2 Circular representation of Candidatus methanosphaera massiliense sp.nov.genome.Genome representation was performed using the Proksee platform