Abstract
Background Methanobrevibacter smithii, Methanobrevibacter oralis, Methanosphaera stadtmanae, Methanomassilicoccus luminyensis and Methanobrevibacter arboriphilicus have been cultured from human digestive microbiota. Each one of these fastidious methanogenic archaea requires a specific medium for its growth, hampering their routine isolation and the culture.Methodology/Principal FindingsA new culture medium here referred as SAB medium was optimized and tested to cultivate methanogens associated with human microbiota, as well as two mesophile methanogens Methanobacterium beijingense and Methanosaeta concilii. It was further tested for the isolation of archaea from 20 human stool specimens including 10 specimens testing positive for PCR detection of M. smithii. After inoculating 105 colony-forming-unit archaea/mL or 1 g stool specimen in parallel in SAB medium and reference DSMZ medium in the presence of negative controls, growth of archaea was determined by optical microscopy and the measurement of methane production by gas chromatography. While the negative controls remained sterile, all tested archaea grew significantly more rapidly in SAB medium than in reference medium in 1–3 days (P<0.05, Student test). Among PCR-positive stool specimens, 10/10 grew in the SAB medium, 6/10 in DSMZ 119 medium, 5/10 in DSMZ 322 medium and 3/10 in DSMZ 334 c medium. Four out of ten PCR-negative stool specimens grew after a 3-week incubation in the SAB-medium whereas no growth was detected in any of the reference media. 16S rRNA gene sequencing yielded 99–100% sequence similarity with reference M. smithii except for one specimen that yielded 99–100% sequence similarity with reference Methanobrevibacter millerae.Conclusions/SignificanceSAB medium allows for the versatile isolation and growth of methanogenic archaea associated with human gut microbiota including the archaea missed by inoculation of reference media. Implementation of the SAB medium in veterinary and medical microbiology laboratories will ease the routine culture-based detection of methanogenic archaea in clinical and environmental specimens.
Highlights
New laboratory techniques for cultivating strict anaerobes, including the technique of Hungate [1,2] have allowed isolation and further characterization of several new species of the methanogenic archaea associated with human microbiota [3,4,5,6,7,8]
Growth Delay Monitoring While negative controls remained sterile in all the experiments, all the methanogenic archaea grew as expected, in the recommended standard DSMZ culture medium
M. smithii DSMZ 2374, M. smithii DSMZ 2375 and M. smithii ATCC 35061T grew after 24-hour incubation with a 3-hour doubling time in SAB medium versus 72-hour incubation and a 9hour doubling time in standard 119 DSMZ medium (P,0.05, Student test)
Summary
New laboratory techniques for cultivating strict anaerobes, including the technique of Hungate [1,2] have allowed isolation and further characterization of several new species of the methanogenic archaea associated with human microbiota [3,4,5,6,7,8]. Mastering the techniques of anaerobic culture is not sufficient to isolate and cultivate methanogens and additional knowledge in nutrient requirements has proved useful to design methanogenic archaea culture media [11]. Some methanogenic archaea, such as Methanobrevibacter smithii [7], Methanobrevibacter oralis [4] and Methanobrevibacter arboriphilicus [12] that we recently isolated from two human stool specimens Methanobrevibacter smithii, Methanobrevibacter oralis, Methanosphaera stadtmanae, Methanomassilicoccus luminyensis and Methanobrevibacter arboriphilicus have been cultured from human digestive microbiota Each one of these fastidious methanogenic archaea requires a specific medium for its growth, hampering their routine isolation and the culture
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