Toxin-antitoxin systems are ubiquitous and plasmid-encoded in vancomycin-resistant enterococci

Abstract

Vancomycin-resistant enterococci (VRE) are common hospital pathogens that are resistant to most major classes of antibiotics. The incidence of VRE is increasing rapidly, to the point where over one-quarter of enterococcal infections in intensive care units are now resistant to vancomycin. The exact mechanism by which VRE maintains its plasmid-encoded resistance genes is ill-defined, and novel targets for the treatment of VRE are lacking. In an effort to identify novel protein targets for the treatment of VRE infections, we probed the plasmids obtained from 75 VRE isolates for the presence of toxin-antitoxin (TA) gene systems. Remarkably, genes for one particular TA pair, the mazEF system (originally identified on the Escherichia coli chromosome), were present on plasmids from 75/75 (100%) of the isolates. Furthermore, mazEF was on the same plasmid as vanA in the vast majority of cases (>90%). Plasmid stability tests and RT-PCR raise the possibility that this plasmid-encoded mazEF is indeed functional in enterococci. Given this ubiquity of mazEF in VRE and the deleterious activity of the MazF toxin, disruption of mazEF with pharmacological agents is an attractive strategy for tailored antimicrobial therapy.

Fig. 1.

PCR anaysis of VRE isolates. (A) The prevalence of genes encoding vancomycin resistance and plasmid stability systems as assessed by PCR analysis of the total plasmid preparation from 75 VRE isolates. (B) Analysis of the physical linkage between TA systems and vanA. Single plasmids containing the vanA gene cluster were isolated and probed by PCR with primers complementary to the genes encoding the various TA systems.

Fig. 2.

Antibiotic-resistance profiles of the 75 VRE isolates.

Fig. 3.

Plasmid stability assays. (A) Plasmid pS345RF, which has mazEF as the only detectable TA gene system, is stable for 265 generations in enterococci in the absence of any antibiotic selection. Plasmid stability was determined by replica plating onto selective media. (B) Plasmid pAM401 is highly unstable in the enterococcal strain OG1X (▴) in the absence of antibiotic selection. When the mazEF loci is cloned into pAM401, the stability of the plasmid is increased (■). Error bars represent standard deviation from the mean.

Fig. 4.

RT-PCR analysis of 10 clinical VRE isolates (lanes 1–10) and a plasmid-free enterococcal strain (BM4105-RF) (lane 11). The isolates used were U563, C27282, C31582, C531926, S177, S234, S345, D1, SL278, and SL518 (lanes 1–10, respectively). (A) RT-PCR with primers complementary to mazEF indicates that this transcript is present in the clinical isolates but not in plasmid-free E. faecium. (B) Controls for DNA contamination, in which the reverse transcriptase is left out of the reaction mix, give no amplification of mazEF. (C) RT-PCR with primers complementary to the enterococci tuf gene (encoding EF-tu) gives the expected products. Lane M contains the 100-bp DNA ladder.