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Background The spectrum of infections caused by the emerging opportunistic pathogens methanogens which escape routine detection remains to be described. To determine the prevalence of archaemia, we searched for methanogens in the blood of febrile patients using specific tools. Methods We conducted a prospective study at Institut Hospitalier Universitaire Méditerranée Infection, Marseille, France, September 2018 - April 2020, enrolling 7,716 blood culture samples routinely collected in patients with fever. Blood samples were screened by specific PCR assays for the presence of 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 that contained cultures collected from 26 patients. Sequencing confirmed Methanobrevibacter smithii associated with staphylococci in 14 patients, fermentative Enterobacteriaceae in nine patients and streptococci in three patients. Metagenomics confirmed M. smithii in five blood samples, and M. smithii was isolated via culture 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, all diagnosed with infectious mitral endocarditis, were 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 methods of detection, M. smithii is demonstrated as causing archaemia and endocarditis in febrile patients who are coinfected by bacteria.

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.