<p>Analysis of two pheromone-responsive conjugative multiresistance plasmids carrying the novel mobile <em>optrA</em> locus from <em>Enterococcus faecalis</em></p>

Abstract

Background: The acquired optrA gene, which encodes a ribosomal protection protein of the ABC-F family, can confer cross-resistance to linezolid and florfenicol, posing a serious therapeutic challenge to both human and veterinary medicine. Purpose: The objective of this study was to investigate the two Enterococcus faecalis ( E. faecalis ) plasmids for their fine structure, their transferability and the presence of mobile antimicrobial resistance loci. Methods: To elucidate their fine structure, the two plasmids were completely sequenced and the sequences analysed. Besides conjugation experiments, inverse PCR assays were conducted to see whether minicircles are produced from the mobile antimicrobial resistance loci. Results: Two pheromone-responsive conjugative optrA -carrying plasmids from E. faecalis , pE211 and pE508 were identified, which can transfer with frequencies of 2.6 ×10 −2 and 3.7 ×10 −2 (transconjugant per donor), respectively. In both plasmids, optrA was located on the novel mobile optrA locus with different sizes (12,834 bp in pE211 and 7,561 bp in pE508, respectively), flanked by two copies of IS 1216 genes in the same orientation. Inverse PCR revealed that circular forms can be generated, consisting of optrA and one copy of IS1216, indicating they are all active. The 77,562 bp plasmid pE211 also carried Tn 558 and a mobile bcr ABDR locus, and the 84,468 bp plasmid pE508 also harbored the genes fexA , tet (L), tet (O/W/32/O) and a mobile aac (A)- aph (D) locus. Conclusion: The presence of mobile genetic elements in these plasmids renders them flexible and these elements will aid to the persistence and dissemination of these plasmids among enterococci and potentially also other gram-positive bacteria.

Characterization of florfenicol resistance genes in the coagulase-negative Staphylococcus (CoNS) isolates and genomic features of a multidrug-resistant Staphylococcus lentus strain H29

“…The gene cfr was initially found on the 17.1-kb plasmid pSCFS1 from an S. sciuri isolate and was shown to encode an rRNA methylase mediating resistance to phenicol by methylation of the 23S rRNA. In contrast, the gene fexA , which encodes an efflux protein within the major facilitator superfamily (MFS), was first identified on the 34-kb plasmid pSCFS2 [ 17 ] from S. lentus and was shown to be part of the Tn 554 -like transposon Tn 558 [ 18 ]. fexB , also a phenicol exporter gene, was first identified on the pEFM-1 (35 kb in size) of E. faecium and pEH-1 (25.3 kb in size) of E. hirae , both strains with swine origins [ 19 ].…”

Dissemination of linezolid-resistance through sex pheromone plasmid transfer in Enterococcs faecalis

“…Plasmids with such gene organization have not been reported previously from any optrA -carrying E. faecalis isolates. While there has been one report of two pheromone-responsive plasmids carrying optrA in E. faecalis (30), both plasmids were identified in E. faecalis isolates from pigs, with only one of them (pE508) containing a single sex pheromone gene ( prgB ) based on full plasmid genome sequencing. Fourth, our plasmid typing studies demonstrated, for the first time, the abundant presence of rep 9 -type plasmids (prototype pCF10) in optrA -carrying E. faecalis clinical isolates (17/26 or 65%, Table 1), implying a high prevalence of sex pheromone plasmids in clinical isolates.…”

“…Indeed, there have been reports of easy transfer of optrA -carrying plasmids in vitro between E. faecalis and E. faecium (13) and between human- and pig-derived E. faecalis (30). Our previous transcriptomics (31) and proteomics (32) analyses of a linezolid-resistant E. faecalis strain P10748 consistently revealed a significant co-upregulation of optrA gene with several genes involved in mating and pheromone response, implying a localization of this gene in a sex pheromone plasmid allowing highly efficient resistance transfer.…”

Genomic Insights Into Last-Line Antimicrobial Resistance in Multidrug-Resistant Staphylococcus and Vancomycin-Resistant Enterococcus

“…Genomics has provided critical insights into the genetic relatedness of resistant strains, the structure of MGEs and their role in the mobilization of linezolid resistance determinants. Examples include: an analysis of optrA -carrying plasmids from E. faecalis , which found optrA flanked by two active copies of IS 1216 that can facilitate its spread ( Shang et al, 2019 ); the widespread identification of optrA located in a Tn 6674 -like element in E. faecalis isolated from different countries and sources ( Freitas et al, 2020b ); IS 1216E elements that have been shown to mediate the insertion of poxtA into a pT-E1077-31 plasmid in E. faecium ( Shan et al, 2020 ); while in MRSA, a Tn 6349 composite transposon carrying both poxtA and cfr was found to possess a modular structure consisting of fragments from other previously described MGEs ( D’Andrea et al, 2019 ). The presence of these MGEs in cfr , poxtA , and optrA carrying multi-resistance plasmids likely aids in the persistence and dissemination of linezolid resistance genes among these Gram-positive pathogens.…”