Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate

Abstract

Vancomycin-resistant enterococci are troublesome pathogens in clinical settings because of few treatment options. A VanA/VanM-type vancomycin-resistant Enterococcus faecium clinical isolate was identified in Japan. This strain, named AA708, harbored five plasmids, one of which migrated during agarose gel electrophoresis without S1 nuclease treatment, which is indicative of a linear topology. We named this plasmid pELF1. Whole genome sequencing (WGS) analysis of the AA708 strain revealed that the complete sequence of pELF1 was 143,316 bp long and harbored both the vanA and vanM gene clusters. Furthermore, mfold analysis and WGS data show that the left end of pELF1 presumably forms a hairpin structure, unlike its right end. The pELF1 plasmid was not digested by lambda exonuclease, indicating that terminal proteins were bound to the 5′ end of the plasmid, similar to the Streptomyces linear plasmids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results were also consistent with the exonuclease assay results. In retardation assays, DNAs containing the right end of proteinase K-untreated pELF1 did not appear to move as well as the proteinase K-treated pELF1, suggesting that terminal proteins might be attached to the right end of pELF1. Palindromic sequences formed hairpin structures at the right terminal sequence of pELF1; however, sequence similarity with the well-known linear plasmids of Streptomyces spp. was not high. pELF1 was unique as it possessed two different terminal structures. Conjugation experiments revealed that pELF1 could be transferred to E. faecalis, E. faecium, E. casseliflavus, and E. hirae. These transconjugants exhibited not only high resistance levels to vancomycin, but also resistance to streptomycin, kanamycin, and erythromycin. These results indicate that pELF1 has the ability to confer multidrug resistance to Enterococcus spp. simultaneously, which might lead to clinical hazards.

First Report of the Local Spread of Vancomycin-Resistant Enterococci Ascribed to the Interspecies Transmission of a vanA Gene Cluster-Carrying Linear Plasmid

“…Because clonal dissemination of VRE strains alone cannot fully explain the PFGE results, we proceeded to confirm the plasmid content of these isolates. According to the results of PFGE performed with S1 nuclease as previously described (16), all these isolates carried several plasmids (Fig. S3).…”

“…Pulsed-field gel electrophoresis. PFGE analysis was performed as previously described (16). Briefly, 1% agarose plugs containing embedded enterococcal strains were digested with lysozyme solution (Roche Diagnostics K.K., Minneapolis, MN, USA) (10 mg/ml) at 37°C for 6 h, followed by digestion of proteinase K (Merck Millipore, Darmstadt, Germany) solution (60 mAnson units/ml) at 50°C for 48 h. For analysis of genetic relatedness, enterococcal DNA embedded in agarose plugs was digested using SmaI (New England Biolabs, Ipswich, MA, USA) at 25°C for 12 h and then subjected to PFGE using a Chef Mapper system (Bio-Rad, Hercules, CA, USA).…”

“…1; see also Fig. S2) (16). On the basis of the patterns of SmaI-digested chromosomal DNA, isolates were categorized into three major groups.…”

“…Notably, the nucleotide sequences of both ends were shown to be highly conserved between pELF2 and pELF1, suggesting that pELF2 was a linear plasmid possessing two different terminal structures (Fig. S4) (16). The left end of pELF2 was presumed to be a hairpin structure harboring a 5=-TATA-3= loop.…”

“…is considered to be much less frequent than intraspecies transfer (15). Recently, we discovered a transferrable linear plasmid harboring the vanA and vanM gene clusters from E. faecium in Japan (16). This plasmid conferring vancomycin resistance was reported to show interspecies transferability in enterococci.…”