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

Yusuke Hashimoto,Takahiro Nomura,Koichi Tanimoto,Kiyoko Tamai,Kazuma Uesaka,Makoto Taniguchi,Genjie Ruan,Bo Zheng,Hidetada Hirakawa,Haruyoshi Tomita

doi:10.3389/fmicb.2019.02568

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.

Highlights

Enterococci, a type of bacteria that are part of the human intestinal microflora, can cause diseases in certain clinical settings
During the evaluation of the multiplex PCR assay for vancomycin-resistant enterococci (VRE) in our previous reports, we had identified a VanA/VanM-type vancomycin-resistant E. faecium strain from a > 60 year-old patient in Japan, which was identified as a VanA-type VRE strain (Nomura et al, 2018)
We identified the transfer of pELF1 to FA2-2 (E. faecalis), BM4105RF (E. faecium), ATCC9790RF (E. hirae), and KT06RF (E. casseliflavus). pELF1 transferred at frequencies of 10−5, 10−8, 10−8, and 10−3 per donor cell to FA2-2, BM4105RF, ATCC9790RF, and KT06RF, respectively (Supplementary Table 2). pELF1 conferred VAN resistance and other drug resistances to these laboratory strains (Table 1)

Summary

Introduction

Enterococci, a type of bacteria that are part of the human intestinal microflora, can cause diseases in certain clinical settings. VanA-type and VanB-type VREs are predominant among the nine types of vancomycin resistance. These VanA-type and VanB-type VREs produce a peptidoglycan precursor ending in D-alanyl-D-lactate, which confers a higher level of vancomycin resistance to Enterococcus spp. VanM-type vancomycin-resistant bacteria, which produce the peptidoglycan precursor ending in D-alanyl-D-lactate, was first reported in 2010. VREs that harbored both vanA and vanM gene clusters were identified (Sun H.L. et al, 2019). These doublepositive strains exhibited high level resistance to vancomycin. VanM-type VREs were only isolated in China and Singapore (Teo et al, 2011)

Methods

Results

Conclusion