Tetracycline Resistance Mediated by tet(M) Has Variable Integrative Conjugative Element Composition in Mycoplasma hominis Strains Isolated in the United Kingdom from 2005 to 2015

Abstract

A minimal genome and absent bacterial cell wall render Mycoplasma hominis inherently resistant to most antimicrobials except lincosamides, tetracyclines, and fluoroquinolones. Often dismissed as a commensal (except where linked to preterm birth), it causes septic arthritis in immunodeficient patients and is increasingly associated with transplant failure (particularly lung) accompanying immunosuppression. We examined antimicrobial susceptibility (AST) on strains archived from 2005 to 2015 submitted to the Public Health England reference laboratory and determined the underlying mechanism of resistance by whole-genome sequencing (WGS). Archived M. hominis strains included 32/115 from invasive infection (sepsis, cerebrospinal [CSF], peritoneal, and pleural fluid) over the 10-year period (6.4% of all samples submitted from 2010 to 2015 were positive). No clindamycin resistance was detected, while two strains were resistant to moxifloxacin and levofloxacin (resistance mutations S83L or E87G in gyrA and S81I or E84V in parC). One of these strains and 11 additional strains were tetracycline resistant, mediated by tet(M) carried within an integrative conjugative element (ICE) consistently integrated at the somatic rumA gene; however, the ICEs varied widely in 5 to 19 associated accessory genes. WGS analysis showed that tet(M)-carrying strains were not clonal, refuting previous speculation that the ICE was broken and immobile. We found tet(M)-positive and -negative strains (including the multiresistant 2015 strain) to be equally susceptible to tigecycline and josamycin; however, the British National Formulary does not include guidance for these. Continued M. hominis investigation and AST surveillance (especially immunocompromised patients) is warranted, and the limited number of therapeutics needs to be expanded in the United Kingdom.

Keywords: Mycoplasma; Mycoplasma hominis; United Kingdom; antibiotic resistance; antimicrobial activity; antimicrobial resistance; epidemiology; genome analysis; genomics; integral conjugative element; tetracyclines.

FIG 1

Alignment of ICE elements carrying the tet(M) gene, aligned relative to their insertion at the 3′ end of the rumA gene. Open reading frames for the PL5 reference gene consist of rumA; Tn916 conjugation genes (green) ORF17, ORF16, ORF15, ORF14, and ORF13; the tet(M) resistance gene (pink); Tn916 regulation genes (blue, or gray at 80% homology) ORF9 and ORF7; Tn916 excisase and integrase genes (red), and accessory transporter genes from ICESpy2905 (GenBank accession number FR691055 [yellow]), which also includes the serine recombinase (red) at the end of the mobile genetic element.

FIG 2

Nucleotide alignments for the contigs containing the tet(M) gene showing SNP locations identified when sequencing the same strain three independent times (A) and three independent isolations of M. hominis from the same patient at 20, 28, and 36 weeks gestation (B).

FIG 3

Neighbor-joining phylogenetic analysis of MLST genes for strains with tet(M) (light blue boxes) relative to strains without (white boxes). Additionally, the typing of major surface antigen (VAA) is shown next to each isolate with the VAA type (blue circle, type 1; red circle, type 2; green circle, type 4 or 4b [due to 1 or 2 copies of module III, respectively] and yellow circle for novel VAA type), and the total number of ICE genes [excluding tet(M)] included in the ICE is shown in the square at the end of the isolate identifier. Conserved SNP variation previously identified by Mardassi et al. (18) is indicated as the last entry per line for types A and B and subvariant B1.

FIG 4

Neighbor-joining phylogenetic analysis for the core genome (304 genes) identified by Roary analysis of the assembled contigs following annotation using the Sprott genome (which defined a total of 1,931 genes) across all genomes. Even distribution of positive and negative strains supports ICE mobility. Furthermore, no clustering of the associated number of accessory genes [number in box, or tet(M)-SNP type (A, B or B1)] was seen, further confirming no clonal origins of ICE subtypes. The closest association continues to be observed between serially isolated strains AH3-20, AH3-28, and AH3-36 (the latter 2 of which were identical). Low contig length and anomalous large genome deletions required the removal of tet(M)-positive strains MH08-5 and MH10-15, as well as tet(M)-negative strains MH15-9 and MH05-13, from the analysis (otherwise, the core genome dropped significantly to 128 genes with poor discriminatory power).

FIG 5

Antimicrobial susceptibility testing for 40 isolates [13 tet(M)-carrying and 27 susceptible controls] for antibiotics with CLSI-defined resistance thresholds.

FIG 6

Antimicrobial susceptibility testing for 40 isolates [13 tet(M)-carrying and 27 randomly selected susceptible controls] for glycyltetracyline tigecycline and macrolide josamycin. Note that, to date, no breakpoints have been assigned for these antimicrobials.