Molecular characterization of intrinsic and transferable genetic elements harbored by B-lactams and colistin resistant gram-negative bacterial strains

Abstract

The increasing prevalence of antimicrobial resistance among bacterial pathogens poses an enormous threat to human health. Although phenotypic resistance can be divided into two distinct categories, namely the intrinsic and acquired phenotypes conferred by intrinsic resistance genes or chromosomal mutations and extrinsic mobile resistance genes, the gravest threat is derived from the mobile genetic elements. However, data regarding the range of genetic elements causing a rapid increase in the prevalence of antibiotic resistance in several key pathogens in recent years are not available. In this study, we selected ß-lactam resistant Vibrio parahaemolyticus strains and colistin resistant Escherichia coli strains as two model systems to investigate the molecular structures of both intrinsic and mobile resistance elements harboured by these newly emerged resistant organisms. Molecular techniques including isolation and identification of bacteria, PCR, conjugation assay, transformation, S1-PFGE, hybridization, whole genome sequencing and comparative genomics analysis were employed to investigate the range of ß-lactam resistance genes harboured by V. parahaemolyticus, and characterize the genetic structures of the plasmid-mediated colistin resistance gene mcr-1 located in plasmids of E.coli. This work comprised three sections: First, the intrinsically ampicillin resistance gene blaCARB-17 was identified by genome comparison of all the whole genome sequence data in NCBI and characterized in V. parahaemolyticus to evaluate the function of this genetic element for the first time. A novel detection method targeting this intrinsic resistance gene was developed, conferring robust detection specificity. Second, the genetic features of extended-spectrum ß-lactamases (ESBLs) - producing V. parahaemolyticus was characterized, with results showing that the resistance elements blaVEB, blaCMY-2 and blaPER-1 were responsible for the resistance observed. The genetic environments of these genes were examined and various mobile elements were found to play a role in accelerating the transmission of the ESBLs genes in conjugative plasmids among V. parahaemolyticus. The novel blaPER-1 harboring plasmids were compared from the evolutional perspective and comparative genomics analyses were performed to illustrate the plasmid evolution process. In the third section of this study, a thorough study on mcr-1-bearing plasmids was conducted and mechanisms underlying the transmission of the mcr-1 gene between different genetic loci were elucidated. Based on findings in this work, several conclusions can be made. The intrinsic blaCARB-17 gene was responsible for causing the ampicillin resistance in V. parahaemolyticus by encoding a ß-lactamase to hydrolyze the antibiotic; this chromosomal gene is an ideal maker for differentiation between V. parahaemolyticus from V. alginolyticus. ESBLs genes were found to emerge in V. parahaemolyticus in recent years and our works showed that various types of plasmids and mobile resistance elements possessed the ability to transfer the ESBL genes to other species. Comparative studies of the plasmids concerned confirmed that they were the major facilitators of dissemination of resistance genes in V. parahaemolyticus. The types of plasmids harboring the mcr-1 gene were found to be diverse, and a circular intermediate with the structure ISApl1-mcr-1-orf-ISApl1(Tn6330) was found to play a potential role in the dissemination of mcr-1 between different genetic loci in plasmids. These findings provide important insight into the structures of genetic elements responsible for mediating an increasing prevalence of antibiotic resistance among Gram-negative bacterial pathogens, facilitating the prevention of emergence of new resistant strains through inhibition of transmission of specific resistance elements.