Tn6349: a novel cfr- and poxtA-carrying transposon of enterococcal origin in a linezolid-resistant MRSA

1. Introduction The spread of methicillin-resistant S. aureus (MRSA) is a worldwide public health concern, and percentages from invasive infections in our country have been constantly above 30% during the past decade. Linezolid is among the last resort agents for the treatment of MRSA infections, and resistance to this antibiotic, despite being uncommon, should be carefully monitored. Different resistance mechanisms to linezolid were described, including chromosomal mutations affecting the ribosomal target and acquired resistance mediated by genes such as cfr, which encodes a 23S rRNA methyltransferase, and optrA and poxtA which encode ABC-F ribosomal protection proteins. In this study we characterized the genetic context and stability of the first described cfr- and poxtA-carrying transposon in a linezolid-resistant MRSA strain of clinical origin. 2. Materials and Methods The genetic context of cfr and poxtA was investigated by bioinformatic analysis of whole-genome sequencing (WGS) data of S. aureus AOUC-0915, in conjunction with a PCR and Sanger sequencing approach to solve repeated regions. Gene transfer experiments were carried out using laboratory strains of S. aureus and Enterococcus faecalis as recipients. Stability of the genetic context of selected resistance genes was evaluated with inverse PCR experiments and curing experiments. 3. Results WGS analysis of MRSA strain AOUC-0915 revealed the presence of a 48,350 bp composite transposon, named Tn6349. This transposon showed 99% of identity over 84% of its length (ca. 40 kb) with pEF12-0805, a pRE25-like plasmid from Enterococcus faecium carrying the cfr and optrA resistance genes. Tn6349 contains the erm(B), cfr, poxtA and fexB resistance genes and was inserted into a phiN315-like prophage element integrated in the bacterial chromosome. The cfr gene was bracketed by two identical ISEnfa5 insertion sequences that were in the same orientation. The poxtA and fexB resistance genes were flanked by IS1216 elements and were located in a region of enterococcal origin at the transposon 3’ end. Tn6349 and the genetic context of cfr gene proved capable of undergoing excision in circular form, despite not being transferable to S. aureus and/or E. faecalis hosts. 4. Discussion and Conclusions This study reports on the genetic structure of Tn6349, a novel chromosomal cfr- and poxtA- carrying composite transposon, and represents the first description of the genetic context of the recently described poxtA resistance gene. The high similarity of Tn6349 with regions of plasmids from Enterococcus spp. suggests the likely acquisition of such structure through a recombination event involving an ancestral enterococcal plasmid and the S. aureus AOUC-0915 chromosome.