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. 2024 Apr 16:14:1368923.
doi: 10.3389/fcimb.2024.1368923. eCollection 2024.

The complete genome sequence of unculturable Mycoplasma faucium obtained through clinical metagenomic next-generation sequencing

Affiliations

The complete genome sequence of unculturable Mycoplasma faucium obtained through clinical metagenomic next-generation sequencing

Artur J Sabat et al. Front Cell Infect Microbiol. .

Abstract

Introduction: Diagnosing Mycoplasma faucium poses challenges, and it's unclear if its rare isolation is due to infrequent occurrence or its fastidious nutritional requirements.

Methods: This study analyzes the complete genome sequence of M. faucium, obtained directly from the pus of a sternum infection in a lung transplant patient using metagenomic sequencing.

Results: Genome analysis revealed limited therapeutic options for the M. faucium infection, primarily susceptibility to tetracyclines. Three classes of mobile genetic elements were identified: two new insertion sequences, a new prophage (phiUMCG-1), and a species-specific variant of a mycoplasma integrative and conjugative element (MICE). Additionally, a Type I Restriction-Modification system was identified, featuring 5'-terminally truncated hsdS pseudogenes with overlapping repeats, indicating the potential for forming alternative hsdS variants through recombination.

Conclusion: This study represents the first-ever acquisition of a complete circularized bacterial genome directly from a patient sample obtained from invasive infection of a primary sterile site using culture-independent, PCR-free clinical metagenomics.

Keywords: Mycoplasma faucium; ice; insertion sequence; lung transplant patient; metagenomics.

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Conflict of interest statement

Authors TD and SB are employees of the company DNASTAR, Inc. SB is a minor shareholder in DNASTAR, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Phylogenetic trees based on three different sets of genes: (A) entire 16S rRNA genes, (B) 11 genes present in all analyzed Mycoplasma species, and (C) 109 genes present in all species of the hominis cluster. The trees were constructed using the Maximum Likelihood method by comparing nucleotide sequences (A) or translated amino acid sequences of the universal Mycoplasma genes (B) or ubiquitous genes of the hominis cluster (C) generated with the MegAlign Pro module (DNASTAR). All branch lengths are drawn to a scale bar, reflecting evolutionary distances equivalent to one replacement per every 100 nucleotides or amino acids.
Figure 2
Figure 2
Genome structure of M. faucium prophage phiUMCG-1 and comparison with M. arthritidis MAV1 (GenBank accession number: NC_001942) and M. hyosynoviae MAV1-like (GenBank accession number: CP101129) prophages. The arrows indicate open reading frames and their orientations. Green arrows: genes shared by all prophages. Yellow arrows: genes shared by M. faucium and M. hyosynoviae prophages phiUMCG-1 and MAV1-like, respectively. Black, orange and red arrows: genes only present in prophages MAV1, phiUMCG-1 and MAV1-like, respectively. Blue arrow: the IS30 gene integrated into orf2 in one of the copies of phiUMCG-1. Locations and sizes are aligned on a scale showing 2 kb intervals. The genes in M. faucium phiUMCG-1 and M. hyosynoviae MAV1-like are designated, using the nomenclature published for their counterparts in M. arthritidis MAV1 (Röske et al., 2004), where applicable. The genes and their positions in the M. faucium UMCG-MFM1 genome are described further in Supplementary Table S7 .
Figure 3
Figure 3
Structural organization of MICE elements. Annotated sequences of the MICE regions were obtained from M. hominis SP10291 genome (GenBank accession number: NZ_CP055149, position 13153.31485) and M. faucium UMCG-MFM1 genome (GenBank accession number: CP088155) and aligned with MAFFT in MegAlign Pro version 18.0.0(781) (DNASTAR). Sequence identities of homologous proteins, estimated by MAFFT, are shown above the respective genes. A set of MICE core genes, represented by grey arrows, was defined based on a previous analysis of ICEHo-II in M. hominis SP10291 (Henrich et al., 2020). The cargo genes shared by both ICEHo-II and ICEFa-II are shown in blue. Genes that are exclusive to ICEHo-II or ICEFa-II are highlighted in red and green, respectively. The inverted repeats are represented by vertical bars. Complementary sequences of the respective inverted repeats are underlined. Nucleotides that are identical in corresponding repeats of both ICEHo-II and ICEFa-II are highlighted in yellow.
Figure 4
Figure 4
Restriction-modification system of M. faucium UMCG-MFM1. (A) Structural organization. Arrows represent the genes: hsdM (methyltransferase), hsdS, hsdS’, and hsdS’’ (specificity subunits), as well as hsdR (restriction endonuclease). Direct repeats (DR) are depicted as yellow bars. (B) Sequence comparison of DRs. The black shading indicates identical nucleotides within direct repeats. (C) Nucleotide sequence identity among analyzed hsdS genes and their respective DRs.

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