vanM, a new glycopeptide resistance gene cluster found in Enterococcus faecium

Abstract

Since glycopeptide-resistant enterococci (GRE) were reported in 1988, they have appeared in hospitals worldwide. Seven van gene cluster types (vanA, vanB, vanC, vanD, vanE, vanG, and vanL) are currently known. We investigated a clinical strain of Enterococcus faecium Efm-HS0661 that was isolated in 2006 from an inpatient with intra-abdominal infection in Shanghai. It was resistant to most antimicrobials, including vancomycin (MIC, >256 μg/ml) and teicoplanin (MIC, 96 μg/ml). Glycopeptide resistance could be transferred to E. faecium BM4105RF by conjugation. The donor and its transconjugant were negative by PCR for the known van genes. By cloning and primer walk sequencing, we discovered a novel van gene cluster, designated vanM. The vanM ligase gene was 1,032-bp in length and encoded a 343-amino-acid protein that shared 79.9, 70.8, 66.3, and 78.8% amino acid identity with VanA, VanB, VanD, and VanF, respectively. Although the vanM DNA sequence was closest to vanA, the organization of the vanM gene cluster was most similar to that of vanD. Upstream from the vanM cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Liquid chromatography-mass spectrometry analysis of peptidoglycan precursors extracted from the VanM-type strain Efm-HS0661 treated with vancomycin or teicoplanin revealed a modified precursor (UDP-N-acetylmuramic acid [MurNAc]-tetrapeptide-D-Lac), indicating that VanM, like VanA, confers glycopeptide resistance by the inducible synthesis of precursor ending in D-Ala-D-Lac.

FIG. 1.

Alignment of vancomycin resistance gene clusters of vanA (M97297), vanB (EFU35369), vanD (AF130997), vanF (AF155139), and vanM (FJ349556). The percent amino acid identities to vanRM, vanSM, vanYM, vanHM, vanM, and vanXM products are shown below the respective genes.

FIG. 2.

Analysis of peptidoglycan precursors by liquid chromatography-mass spectrometry. Shown is the XIC of protonated molecules at m/z 1,150 (MH⁺) and 1,151 (MH⁺), which correspond to UDP-MurNAc-tetrapeptide-d-alanine (peak 1) and UDP-MurNAc-tetrapeptide-d-lactate (peak 2), in the peptidoglycan precursors (A to C). The m/z value of the selected precursor ions for the MS/MS analysis is 1,151 (MH⁺). The protonated molecule could produce two protonated molecules at m/z 562 and 747, which is consistent with the UDP-MurNAc-tetrapeptide-d-lactate fragments tetrapeptide-d-lactate and MurNAc-tetrapeptide-d-lactate, respectively. The precursors were extracted from untreated E. faecium Efm-HS0661 (A), Efm-HS0661 treated with vancomycin (B), and Efm-HS0661 treated with teicoplanin (C).