Outer membrane vesicles of E. coli-mediated resistance to ampicillin by carrying resistant genes and proteins

Background: Antibiotic-resistant bacteria are one of the biggest threats in modern medicine. Understanding the mechanisms of resistance as well as the transmission of resistance genes is crucial for the development of new classes of antibiotics. Outer membrane vesicles (OMVs), which are released by Gram-negative and -positive bacteria, have been found to play crucial roles in bacterial pathogenicity. Recent studies suggested that MVs are involved in the protection against antibioticmediated killing. Objectives: Here we hypothesize that OMVs contribute to antibiotic resistance. First, we aim to demonstrate the presence of resistant genes and functional enzymes within outer membrane vesicles. Then we will investigate whether OMVs can protect susceptible E. coli from antibiotics-meditated killing. Methods: One E. coli strain with plasmid encoding the beta-lactamase CTX-M-15 resistance gene and susceptible E. coli strain were used. Antibiotic susceptibility profiles of the strains were determined using a VITEK®2 system. OMVs were isolated from bacterial cultures by a combination of ultrafiltration and size-exclusion chromatography. The presence of OMVs was confirmed by tunable resistive pulse sensing in addition to the Bradford assay to determine the protein content. PCR and nitrocefin assays were used to detect resistance gene and active betalactamase, respectively. Disc diffusion test and microtiter plate test were used to investigate the efficacy of antibiotics and protection respectively when ampicillin was pre-incubated with OMVs derived from resistant or susceptible bacteria. Results: Our data show that E. coli releases a significant amount of OMVs during 18 h of culturing. Also, we could demonstrate the presence of DNA and most importantly resistance genes and functional beta-lactamase protein inside the MVs. Summary/Conclusion: Interestingly, OMVs derived from resistant bacteria decrease the efficacy of ampicillin and enhance the growth of susceptible E. coli when exposed to ampicillin comparing with OMVs derived from wild-type E. coli or phosphate-buffered saline. This finding emphasizes the contribution of OMVs in antibiotics resistance as an important virulence factor for bacterial surviving.