Identification of a multiresistant mosaic plasmid carrying a new segment of IS1216E-flanked optrA with integrated Tn551-ermB element in linezolid-resistant Enterococcus faecalis human isolate

概要

Linezolid, the prototype of oxazolidinones, is recognized as an important therapeutic option for infections caused by drug- resistant Gram-positive pathogens including MRSA and VRE. Linezolid inhibits initiation of protein synthesis by binding to domain V of the 23S rRNA in the 50S ribosomal subunit. Since the first description of linezolid resistance among MRSA and VRE clinical isolates in 2001, linezolid-resistant staphylococci and enterococci have been increasingly reported throughout the world. Linezolid resistance in those isolates is attributed majorly to specific nucleotide substitutions in the domain V region of the 23S rRNA gene, and less frequently to amino acid substitutions in the L3, L4, and L22 50S ribosomal proteins encoded by altered rplC, rplD and rplW genes, respectively [1]. In addition, transferable plasmid- mediated linezolid-resistance genes cfr, optrA, and more recently poxtA have been identified in enterococci from food animals and humans [2]. We report here a multiresistant plasmid carrying the optrA from a linezolid-resistant Enterococcus faecalis clinical isolate.

　E. faecalis isolate (strain S7316) was recovered in 2019 from a bile sample of a patient in his 70 s suffering from cholangitis. MICs were determined by broth microdilution method, in accordance with CLSI 2019 guidelines. The MICs of linezolid, chloramphenicol, erythromycin and minocycline were 8, 64, >4 and >8 mg/L, respectively, but the S7316 was susceptible to penicillins, glycopeptides, and levofloxacin. Detection of optrA gene was conducted by PCR. WGS analysis was performed using illumina HiSeq and de novo assembled by A5-miseq pipeline. The assembled genome was subjected to annotation (DFAST) and characterization (Center for Genomic Epidemiology). In-depth exploration of virulence genes was performed manually on WGS sequence data.

　The genome sequences of E. faecalis S7316 were assembled into 41 contigs with a total length of 2,811,450 bp. Genome annotation identified probable 2614 protein-coding genes including antimi- crobial resistance genes optrA, fexA, ermB, tetL, tetM, dfrG, and lsaA. No substitutions, deletions, and insertions were found in the nucleotide and amino acid alignments in both 23S rRNAs and L3, L4 and L22 ribosomal proteins. The optrA was located on a plasmid pS7316optrA (GenBank LC499744) with the size of 68,368 bp, GC content of 35.2 % harbouring probable 75 protein-coding genes (Fig. 1A). The pS7316optrA, which encodes RepA_N protein also carried other resistance genes including fexA located in close proximity upstream of the optrA, ermB, tetL and tetM. The complete sequence of the plasmid exhibited no significant structural homology to other plasmids available in the GenBank database (accessed February 2020) except for several regions. Namely, an 18,713-bp region containing Tn551-ermB, optrA and fexA shared 99.2% nucleotide sequence identity with that of the optrA-positive plasmid pN60443F-2 in an E. faecalis from cattle in the USA (GenBank CP028725). A 19,595-bp region containing Tn6247-like- associated tetL and tetM shared 99.9% nucleotide sequence identity with that of the plasmid p6742_2 in an E. faecalis from human in Poland (GenBank KY513281). A 4,642-bp region containing Tn551- ermB, and a 15,815-bp region containing pcfD to pcfJ, zntA, mazF, mazE had high nucleotide sequence homology (99%) with the corresponding regions in the plasmid pN60443F-1 of an E. faecalis from cattle in the USA (GenBank CP028726). The pS7316optrA carried prgZ and pcfD to pcfJ genes but lacked prgX and prgQ, which are associated with RepA_N pheromone-responsive pCF10 plasmid and needed for the regulation of pheromone-inducible conjuga- tion. E. faecalis S7316 belonging to a rare sequence type ST634 had several virulence genes, including adhesins and biofilm-associated genes (ace, srtA, srtC, fsrA, fsrB, fsrC, gelE, sprE, ebpABC and efaAfs), survival genes (tpx and elrABCDE), hyaluronidase genes (hylA and hylB), and sex pheromone-associated genes (cad, camE, cCF10 and cOB1).

　The pS7316optrA had a unique feature in that its entire sequence showed no significant matches to the DNA sequences deposited in the GenBank database. Our analyses revealed that the plasmid displayed a mosaic structure probably evolved from pCF10, with containing several regions composed of mobile genetic elements and antimicrobial resistance genes optrA, fexA, ermB, tetL and tetM of different plasmid origins. The presence of DIS1216E-fexA-optrA with the integration of Tn551-ermB element, which is unique among plasmids harbouring optrA, fexA and ermB (Fig. 1B), is likely to be of particular concern because the IS1216E has been implicated in the wide spread of optrA among enterococcal plasmids and streptococcal chromosomes [3,4]. Tn6247 containing tetL and tetM has only been identified in E. faecium of swine origin in China. Macrolides, tetracyclines and florphenicol are frequently used in farm animals, while oxazoli- dinones are not. Thus, the pS7316optrA carrying the resistance genes to those antimicrobials simultaneously, including optrA that confers cross-resistance to phenicols and oxazolidinones might be associated with livestock sources to which phenicols have been used for a longtime as veterinary drugs and feed additives. The emergence of such multiresistant optrA-positive mosaic plasmid in E. faecalis human clinical isolate would pose a future health risk in Japan where only one study has reported optrA [5].

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