Characterization of a multidrug resistance plasmid from Enterococcus faecium that harbours a mobilized bcrABDR locus.

Sir, Over the past two decades, Enterococcus faecalis and Enterococcus faecium have developed to be the leading causes of life-threatening hospital-acquired infection worldwide. Bacitracin, a polypeptide antibiotic, is used as a topical application in human medicine and also for prophylaxis and therapy in food animals in China. Previous studies showed that an ABC transporter gene locus bcrABDR conferred bacitracin resistance in Enterococcus spp. and Clostridium perfringens. – 5 In this study, 86 enterococcal strains were isolated from 196 faeces samples in Henan Province, China. Of the strains, 29 were from humans (E. faecalis, n1⁄419; E. faecium, n1⁄410) and 57 were from pigs (E. faecalis, n1⁄422; E. faecium, n1⁄435). The study was conducted in accordance with Good Clinical Practice and the Declaration of Helsinki. Antimicrobial susceptibility testing was performed as previously described. Bacitracin-resistant strains were subjected to PCR to identify the presence of the bcrB gene using the primers bcrB-F/ bcrB-R listed in Table S1 (available as Supplementary data at JAC Online). The results indicated that the bcrB gene was present in both human (E. faecalis, n1⁄42; E. faecium, n1⁄42) and swine (E. faecalis, n1⁄49; E. faecium, n1⁄418) strains. However, the presence of the bcrB gene in swine strains (47.4%, 27/57) is much higher than that in human strains (13.8%, 4/29), and this may be largely because bacitracin, especially because it is licensed as a feed additive in the swine industry, is more widely used in pigs than in human medicine. MLST (performed following the protocols detailed at http://www.mlst.net/) indicated that there was no genetic similarity between the bcrB-carrying strains derived from humans and pigs, with the exception of E. faecalis ST116, which was shared by the H7 (human) strain and the P33 (pig) strain. Using E. faecalis JH2-2 (Rif) as the recipient, conjugation and transformation experiments were performed as previously described. For screening of both transconjugants and transformants, brain heart infusion agar was supplemented with 50 mg/L rifampicin and 32 mg/L bacitracin. The randomly selected E. faecalis H9 (human) and E. faecium P21 (pig) strains were able to transfer their bacitracin resistance by conjugation, and transfer frequencies of (4.5+0.3)×10 and (6.0+0.5)×10, respectively, were observed. For swine E. faecium P26, which failed to transfer in the mating experiment, the plasmids were extracted and then transferred into E. faecalis JH2-2 by transformation. The MICs for both transconjugants and transformants are shown in Table S2. A bcrB-positive plasmid derived from the transformant TP26 was sequenced using the 454 Life Sciences (Roche) GS-FLX System and designated pXD5. The complete sequence of pXD5 revealed a size of 56 543 bp in length as shown in Figure S1, which has been deposited in GenBank (KJ645709). Interestingly, the bcrABDR locus was found to be bracketed by two copies of the insertion sequence ISEnfa1 located in the same orientation, and furthermore the adjacent zeta gene was completely disrupted due to the integration of the ISEnfa1bcrABDR-ISEnfa1 segment. The assembling sequence of this segment was confirmed by three individual PCRs using primers P1/P2, P3/P4 and P5/P6, respectively (Table S1 and Figure 1). The characteristic 8 bp target site duplications (5′-TTTAGGAA-3′) were found (Figure 1). Based on the knowledge of the ISEnfa5-related insertion sequence, the acquisition of the bcrABDR-carrying segment in E. faecium P26 may relate to either of the following two processes. The first option is a single-step process postulating that the ISEnfa1-bcrABDR-ISEnfa1 segment might form a composite transposon. The second option postulates a double-step process. First, a single copy of ISEnfa1 integrates into the zeta gene. Then a circular intermediate consisting of bcrABDR and another copy of ISEnfa1 undergoes recombination with the already present ISEnfa1. An inverse PCR was performed using the primers P7/P8 (shown in Table S1 and Figure 1). Sequence analysis of the amplicon revealed that it contained the bcrABDR and one intact copy of ISEnfa1, suggesting that the bcrABDR locus might mobilize by the ISEnfa1-mediated recombination. This inverse PCR was also applied to the remaining bcrB-carrying strains, with positive results for 14 of the 31 strains. Analysis of the antimicrobial resistance regions on the plasmid pXD5 also found two resistance gene clusters (Figure S1). One was erm(B)-aadE-spw-lsa(E)-lnu(B), which seemed to originate from