Dissemination of the blaCTX-M-15 gene among Enterobacteriaceae via outer membrane vesicles.

Abstract

Bacterial outer membrane vesicles (OMVs) are an emerging source of antibiotic resistance transfer but their role in the spread of the blaCTX-M-15 gene encoding the most frequent CTX-M ESBL in Enterobacteriaceae is unknown. To determine the presence of blaCTX-M-15 and other antibiotic resistance genes in OMVs of the CTX-M-15-producing MDR Escherichia coli O104:H4 outbreak strain and the ability of these OMVs to spread these genes among Enterobacteriaceae under different conditions. OMV-borne antibiotic resistance genes were detected by PCR; OMV-mediated transfer of blaCTX-M-15 and the associated blaTEM-1 was quantified under laboratory conditions, simulated intraintestinal conditions and under ciprofloxacin stress; resistance to antibiotics and the ESBL phenotype were determined by the CLSI disc diffusion methods and the presence of pESBL by plasmid profiling and Southern blot hybridization. E. coli O104:H4 OMVs carried blaCTX-M-15 and blaTEM-1 located on the pESBL plasmid, but not chromosomal antibiotic resistance genes. The OMVs transferred blaCTX-M-15, blaTEM-1 and the associated pESBL into Enterobacteriaceae of different species. The frequencies of the OMV-mediated transfer were significantly increased under simulated intraintestinal conditions and under ciprofloxacin stress when compared with laboratory conditions. The 'vesiculants' (i.e. recipients that received the blaCTX-M-15- and blaTEM-1-harbouring pESBL via OMVs) acquired resistance to cefotaxime, ceftazidime and cefpodoxime and expressed the ESBL phenotype. They were able to further spread pESBL and the blaCTX-M-15 and blaTEM-1 genes via OMVs. OMVs are efficient vehicles for dissemination of the blaCTX-M-15 gene among Enterobacteriaceae and may contribute to blaCTX-M-15 transfer in the human intestine.