Excision and integration of unconventional circularizable structures involving the erm(B) gene in enterococci

Traditionally, insertion sequences (ISs) play a major role in disseminating antimicrobial resistance genes (ARGs) in bacteria through transposition and translocation, forming regions that contain multiple ARGs flanked by single or multiple copies of IS. In addition, unconventional circularizable structures (UCSs), lacking recombinase genes but being surrounded by directly repeated sequences (DRs) of various sizes which do not contain transposase genes, were reported to be involved in the dissemination of ARGs. In this study, a novel UCS was identified on plasmid pE508-2 in E. faecalis E508, which carried a 24,411 bp multiresistance gene cluster, consisting of the resistance genes aphA3, lnu(B), lsa(E), spw, aac(A)-aph(D), lnu(B), dfrG, and two copies of aadE flanked by copies of erm(B). PCR assays revealed that three types of UCSs with lengths of 7235, 16,437, and 23,673 bp were formed, each of which contained the respective resistance genes and one copy of erm(B). Using erm(B)-negative and -positive strains, we demonstrated that erm(B)-carrying UCSs failed to transfer into an erm(B)-negative strain, but could integrate into an erm(B)-positive strain in a new site adjacent to a pre-existing erm(B) gene by natural transformation. Database searches revealed that erm(B)-flanked multiresistance gene regions, which might be able to form the respective UCSs, are present among various bacteria from different sources in various countries. In summary, this study experimentally demonstrated the excision and integration of UCS involving structures that include erm(B). The widespread presence of these UCSs in various Gram-positive bacteria highlights its role in the dissemination of ARGs among bacterial pathogens.