The fosfomycin resistance gene fosB3 is located on a transferable, extrachromosomal circular intermediate in clinical Enterococcus faecium isolates

Abstract

Some VanM-type vancomycin-resistant Enterococcus faecium isolates from China are also resistant to fosfomycin. To investigate the mechanism of fosfomycin resistance in these clinical isolates, antimicrobial susceptibility testing, filter-mating, Illumina/Solexa sequencing, inverse PCR and fosfomycin resistance gene cloning were performed. Three E. faecium clinical isolates were highly resistant to fosfomycin and vancomycin with minimal inhibitory concentrations (MICs) >1024 µg/ml and >256 µg/ml, respectively. The fosfomycin and vancomycin resistance of these strains could be co-transferred by conjugation. They carried a fosfomycin resistance gene fosB encoding a protein differing by one or two amino acids from FosB, which is encoded on staphylococcal plasmids. Accordingly, the gene was designated fosB3. The fosB3 gene was cloned into pMD19-T, and transformed into E. coli DH5α. The fosfomycin MIC for transformants with fosB3 was 750-fold higher than transformants without fosB3. The fosB3 gene could be transferred by an extrachromosomal circular intermediate. The results indicate that the fosB3 gene is transferable, can mediate high level fosfomycin resistance in both Gram-positive and Gram-negative bacteria, and can be located on a circular intermediate.

Figure 1. Comparison of the sequences of fosB and tnpA genes.

Alignment of fosB/tnpA genes and the gel electrophoresis of inverse PCR products (A). Alignment of the deduced amino acid sequences of fosB genes (B). The junction region (underline sequence) found in inverse PCR consists of the left and right inverted repeats. Lanes: M, DL2000; 1–3, E. faecium clinical isolates of HS0661, HS0761 and HS07216, respectively; 4 and 5, transconjugants BM0661-1 and BM0661-2; 6 and 7, transconjugants BM0761-1 and BM0761-2; 8 and 9, BM07216-1 and BM07216-2; 10, E. faecium BM4105RF. The following sequences were used in this figure: fosB (AP006717) in S. haemolyticus, fosB (X54227) in S. epidermidis, fosB2 (NC_012581) in Bacillus anthracis, tnpA (AF403298) in E. faecium and fosB3/tnpA (HQ219726) in E. faecium HS0661. The residues which differ from the consensus sequence were boxed.

Figure 2. PFGE and Southern hybridization analysis of Enterococcus faecium clinical strains, recipient (E. faecium BM4105RF), and their respective transconjugants with a fosB3 probe.

Lanes: M, Lambda Ladder PFG Marker (NEB Inc., USA); 1–3, E. faecium clinical isolates of HS0661, HS0761 and HS07216, respectively; 4, E. faecium BM4105RF; 5 and 6, transconjugants BM0661-1 and BM0661–2; 7 and 8, transconjugants BM0761-1 and BM0761-2; 9 and 10, BM07216-1 and BM07216-2.