Rapid identification of clinically interesting methanogens using an improved MALDI-TOF-MS assay

Amir, Lynda; Couderc, Carine; Drancourt, Michel; Grine, Ghiles

doi:10.1099/acmi.0.000372

Cited by 4 publications

(3 citation statements)

References 22 publications

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“…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).…”

Section: Methodsmentioning

confidence: 99%

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

Pilliol,

Morsli,

Terlier

et al. 2024

Microbiol Spectr

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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.

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Section: Methodsmentioning

confidence: 99%

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

Pilliol,

Morsli,

Terlier

et al. 2024

Microbiol Spectr

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“…One millilitre of M. massiliense co-cultures Q8282 and Q8283 and 1 mL of the five P. piscolens strains (P2428, Q3381, Q3403, Q8451 and Q8452, Collection Souches Unité des Rickettsies, CSUR, WDCM 875, Marseille, France), cultured in GG medium, as well as one 5 µL loop resuspended in 1 mL of DPBS 1× water (Thermo Fischer Scientific, Waltham, MA, USA) of the five P. piscolens strains (P2428, Q3381, Q3403, Q8451 and Q8452), cultured in COS medium, were collected for protein extraction, as previously described [ 38 ]. Two deposits were made for each sample and the protein extract of Escherichia coli ( E. coli ) DH5α™ (Brüker Daltonics, Bremen, Germany) was used as a positive control and free matrix as a negative control.…”

Section: Methodsmentioning

confidence: 99%

Methanobrevibacter massiliense and Pyramidobacter piscolens Co-Culture Illustrates Transkingdom Symbiosis

Pilliol,

Beye,

Terlier

et al. 2024

Microorganisms

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Among oral microbiota methanogens, Methanobrevibacter massiliense (M. massiliense) has remained less studied than the well-characterised and cultivated methanogens Methanobrevibacter oralis and Methanobrevibacter smithii. M. massiliense has been associated with different oral pathologies and was co-isolated with the Synergistetes bacterium Pyramidobacter piscolens (P. piscolens) in one case of severe periodontitis. Here, reporting on two additional necrotic pulp cases yielded the opportunity to characterise two co-cultivated M. massiliense isolates, both with P. piscolens, as non-motile, 1–2-µm-long and 0.6–0.8-µm-wide Gram-positive coccobacilli which were autofluorescent at 420 nm. The two whole genome sequences featured a 31.3% GC content, gapless 1,834,388-base-pair chromosome exhibiting an 85.9% coding ratio, encoding a formate dehydrogenase promoting M. massiliense growth without hydrogen in GG medium. These data pave the way to understanding a symbiotic, transkingdom association with P. piscolens and its role in oral pathologies.

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“…However, within the field of laboratory diagnostic testing, several commercially available platforms now provide rapid and accurate microbial identification from direct analysis of colony material using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology. MALDI-TOF MS methods have been developed and implemented for identification of various bacterial, fungal, and archaeal targets isolated from numerous sources including clinical specimens, food and dairy products, and environmental samples (Singhal et al 2015 ; Gong et al 2021 ; Guindo et al 2022 ). These methods benefit from simplified sample processing, rapid time-to-results, low cost of reagents, and amenability to use with automation tools, when compared with conventional identification methods (Chudejova et al 2017 ; Theparee et al 2018 ).…”

Section: Introductionmentioning

confidence: 99%

Application of MALDI-TOF MS for enumerating bacterial constituents of defined consortia

Coryell

Sava

Hastie

et al. 2023

Appl Microbiol Biotechnol

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Characterization of live biotherapeutic product (LBP) batches typically includes a measurement of viability, such as colony forming units (CFU). However, strain-specific CFU enumeration assays can be complicated by the presence of multiple organisms in a single product with similar growth requirements. To overcome specific challenges associated with obtaining strain-specific CFU values from multi-strain mixtures, we developed a method combining mass spectrometry-based colony identification with a traditional CFU assay. This method was assessed using defined consortia made from up to eight bacterial strains. Among four replicate batches of an eight-strain mixture, observed values differed from expected values by less than 0.4 log10 CFU among all strains measured (range of differences, -0.318 to + 0.267). The average difference between observed and expected values was + 0.0308 log10 CFU, with 95% limits of agreement from -0.347 to 0.408 (Bland–Altman analysis). To estimate precision, a single batch of eight-strain mixture was assayed in triplicate by three different users, for a total of nine measurements. Pooled standard deviation values ranged from 0.067 to 0.195 log10 CFU for the eight strains measured, and user averages did not differ significantly. Leveraging emerging mass-spectrometry-based colony identification tools, a novel method for simultaneous enumeration and identification of viable bacteria from mixed-strain consortia was developed and tested. This study demonstrates the potential for this approach to generate accurate and consistent measurements of up to eight bacterial strains simultaneously and may provide a flexible platform for future refinements and modifications. Key points • Enumeration of live biotherapeutics is essential for product quality and safety. • Conventional CFU counting may not differentiate between strains in microbial products. • This approach was developed for direct enumeration of mixed bacterial strains simultaneously.

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Rapid identification of clinically interesting methanogens using an improved MALDI-TOF-MS assay

Cited by 4 publications

References 22 publications

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

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

Methanobrevibacter massiliense and Pyramidobacter piscolens Co-Culture Illustrates Transkingdom Symbiosis

Application of MALDI-TOF MS for enumerating bacterial constituents of defined consortia

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