Detection of a cfr(B) Variant in German Enterococcus faecium Clinical Isolates and the Impact on Linezolid Resistance in Enterococcus spp

Abstract

The National Reference Centre for Staphylococci and Enterococci in Germany has received an increasing number of clinical linezolid-resistant E. faecium isolates in recent years. Five isolates harbored a cfr(B) variant gene locus the product of which is capable of conferring linezolid resistance. The cfr(B)-like methyltransferase gene was also detected in Clostridium difficile. Antimicrobial susceptibility was determined for cfr(B)-positive and linezolid-resistant E. faecium isolates and two isogenic C. difficile strains. All strains were subjected to whole genome sequencing and analyzed with respect to mutations in the 23S rDNA, rplC, rplD and rplV genes and integration sites of the cfr(B) variant locus. To evaluate methyltransferase function, the cfr(B) variant of Enterococcus and Clostridium was expressed in both E. coli and Enterococcus spp. Ribosomal target site mutations were detected in E. faecium strains but absent in clostridia. Sequencing revealed 99.9% identity between cfr(B) of Enterococcus and cfr of Clostridium. The methyltransferase gene is encoded by transposon Tn6218 which was present in C. difficile Ox3196, truncated in some E. faecium and absent in C. difficile Ox3206. The latter finding explains the lack of linezolid and chloramphenicol resistance in C. difficile Ox3206 and demonstrates for the first time a direct correlation of elevated linezolid MICs in C. difficile upon cfr acquisition. Tn6218 insertion sites revealed novel target loci for integration, both within the bacterial chromosome and as an integral part of plasmids. Importantly, the very first plasmid-association of a cfr(B) variant was observed. Although we failed to measure cfr(B)-mediated resistance in transformed laboratory strains the occurrence of the multidrug resistance gene cfr on putatively highly mobile and/or extrachromosomal DNA in clinical isolates is worrisome with respect to dissemination of antibiotic resistances.

Fig 1. Whole genome comparison of C. difficile Ox3196 and Ox3206.

BRIG alignment of the concatenated genomic contigs obtained by WGS revealed two regions which are absent in C. difficile Ox3206 and harbor Tn916-like related genes. Likewise, the entire transposon Tn6218 (red), containing the cfr resistance locus, is present in C. difficile Ox3196 only.

Fig 2. Schematic representation of Tn6218 in clinical E. faecium isolates.

Different variants of Tn6218-like elements were detected in the five German cfr(B)-positive E. faecium clinical isolates (UW numbering). UW12712 exhibits a truncated element encoding solely for the cfr(B) variant and a hypothetical protein of the original composite transposon Tn6218 of C. difficile (HG002389). E. faecium UW10882, UW11590 and UW11733 all harbor highly similar or even complete identical (UW11590 and UW11733) Tn6218-like structures and gene contents. A comparison of Tn6218 of the clinical isolates and of C. difficile Ox3196 to the mobile element of the American E. faecium isolate 448-18961R (KR610408) revealed an almost entirely different gene composition. Numbers represent the length of the published sequence or obtained contig, respectively. Abbreviations: int, integrase; xis, excisionase; rep, putative topoisomerase; HTH Xre, putative transcriptional regulator; i, integrase core domain fragment; h and hypo, hypothetical protein; sigma 70, sigma 70 region 4; H, HTH motif coding sequence (CDS); C, CD31680 CDS; r, regulatory protein.

Fig 3. Maximum likelihood analysis of German cfr(B)-positive E. faecium.

hylogenetic analysis inferred from mapping of whole genome sequencing reads to reference genome E. faecium 64/3 (CP012522) did not indicate a close relationship between the German E. faecium isolates. For comparative reasons, the length of the putative transposon Tn6218 sequence is indicated by boxes. Strain characteristics such as year and federal state of isolation as well as sequence types are listed. Abbreviations: NRW, North Rhine-Westphalia; SA, Saxony-Anhalt; BW, Baden-Wuerttemberg; SLV, single locus variant.

Fig 4. Determination of plasmid content and localization of the cfr(B) variant in the five E. faecium isolates.

(A) Separation of whole and linearized plasmid content by S1-PFGE. (B) Hybridization using a cfr-specific probe was carried out after transfer of plasmids to a nitrocellulose membrane. The asterisks indicate a cfr-positive signal (in B) and the corresponding plasmid (in A). S. aureus 8325was used as a reference strain for estimation of plasmid sizes.