Abstract
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.
Highlights
The repertoire of methanogenic archaea in the oral cavity is limited to six species belonging to the genera Methanobrevibacter (Methanobrevibacter smithii and Methanobrevibacter oralis)[1], Methanosphaera[2] and Methanosarcina[3]
Both were detected by polymerase chain reaction (PCR) in periodontitis cases and at peri-implantitis sites
We investigated methanogens in oral fluid samples using specific PCR-sequencing
Summary
The repertoire of methanogenic archaea (methanogens) in the oral cavity is limited to six species belonging to the genera Methanobrevibacter (Methanobrevibacter smithii and Methanobrevibacter oralis)[1], Methanosphaera[2] and Methanosarcina[3]. These methanogens have all been detected in subgingival plaque specimens but none of them has ever been documented in the oral fluid[4]. A recent review pooling the data from several studies reported the isolation of M. oralis from the dental plaques of healthy subjects and the identification of M. oralis-like organisms in cases of endodontic infection Both were detected by polymerase chain reaction (PCR) in periodontitis cases and at peri-implantitis sites. In order to test this hypothesis, we undertook a non-randomized, cross-sectional observational study to assess the presence of living methanogens in the oral fluid of voluntary individuals who did not have periodontal disease
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