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.IMPORTANCEMethanogens 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.

Keywords: Candidatus Methanosphaera massiliense; Methanosphaera; host transfer; human gut microbiota; hydrogen-free culture; methanogens.

Fig 1

Microscopy features of Candidatus methanosphaera massiliense sp. nov. (A) Gram coloration, Ca. M. massiliense appears as Gram-positive cocci, diplococci, or clump. (B) Electron microscopy (TM4000 HITACHI, 10 KV, ×10,000), zoom on Ca. M. massiliense diplococcus. (C) Electron microscopy (TM4000 HITACHI, 10 KV, ×2,500), showing a large view of Ca. M. massiliense culture, the methanogen appears as cocci, diplococci, and clumps. (D) Confocal microscopy, brightfill mode view of Ca. M. massiliense culture (LSM 900 [Carl Zeiss Microscopy GmbH]). (E) Confocal microscopy, autofluorescence at 420 nm: all the cocci, diplococci, and clumps are autofluorescent (LSM 900 [Carl Zeiss Microscopy GmbH]). (F) Confocal microscopy merged view of autofluorescence at 420 nm and bright fill mode (LSM 900 [Carl Zeiss Microscopy GmbH]).

Fig 2

Circular representation of Candidatus methanosphaera massiliense sp. nov. genome. Genome representation was performed using the Proksee platform with standard parameters (version 1.0.0) (https://proksee.ca/). The two contigs are presented with color-coded highlights to distinguish important genomic features and provide information about the composition and structure of the Candidatus Methanosphaera massiliense sp. nov. genome. ORF regions are indicated in blue, rRNA genes in orange, GC content in black, and positive and negative GC skew in green and red, respectively. This analysis reveals the distribution of protein-coding genes and the variation in GC content across the genome.

Fig 3

Maximum likelihood phylogenetic tree based on 16S rRNA sequence analysis of Candidatus Methanosphaera massiliense sp. nov. and the first 100 hit blasts downloaded from NCBI GenBank database (03-01-2023). The 16S rRNA sequence-based phylogenetic tree showed that Candidatus Methanosphaera massiliense sp. nov. (red) first clustered with uncultured archaeon clones from pig feces (green) and Methanosphaera sp. from western grey kangaroo (purple).

Fig 4

Heatmap phylogeny of Candidatus Methanosphaera massiliense sp. nov. and closely related methanogen species generated with ANI values calculated using PYANI software version (0.2.7) with standard parameters. Ca. M. massiliense was distant from the cultured Methanosphaera species, Methanopshaera sp. WGK6 (Average Nucleotide Identity (ANI), 79%) isolated from Western grey kangaroo gut, followed by M. stadtmanae (ANI, 78%) isolated from human gut, M. cuniculi (ANI, 75%) isolated from rabbit gut and Methanosphaera sp. BMS, a bovine species (ANI, 75%) but it clustered with Methanosphaera sp. RUG761 (ANI, 99%) detected in bovine and Methanosphaera sp. M5 (ANI, 99%) obtained from a man with colorectal cancer, the two genomes clustered together (ANI, 99%), and with Methanosphaera sp. SHI1033 (ANI, 97%), from sheep.