A Study of Antibiotic Resistance Plasmids in Fresh Produce and In Wastewater Environments Using Functional Approaches, Comparative Genomics and Abundance Analysis

Abstract

Studies have shown that a major reservoir of clinically relevant antibiotic resistance genes (ARGs) is found in non-pathogenic environmental bacteria. Ciprofloxacin, meropenem and erythromycin are important clinical antibiotics due to their broad applications in the treatment of Gram-positive and Gram-negative infections. The capacity to capture, mobilise and control the expression of resistance genes is an important factor in the development of multidrug resistance and the mobilome (the community of mobile genetic elements) is responsible for its genetic plasticity. Plasmids are important constituents of the environmental mobilome. The capacity to replicate independently of the bacterial chromosome coupled with the ability to carry clinically relevant ARGs and other antibiotic resistance-encoding mobile genetic elements means a deep understanding of plasmids is necessary to fully appreciate their roles in the spread and evolution of antibiotic resistance determinants in human-impacted environments. The specific objectives of this study were to: 1) Characterize ciprofloxacin resistance plasmids isolated from fresh spinach using functional approaches and comparative genomics. 2) Investigate the diversity in ciprofloxacin and meropenem resistance plasmids isolated from three geographically-separated wastewater treatment plants (WWTPs) using functional approaches and comparative genomics. 3) Determine how conventional wastewater treatment processes impact the abundance of plasmids encoding ciprofloxacin, meropenem and erythromycin resistance genes, and also incF family incompatibility genes using culture-dependent and culture-independent approaches. During this research, I have isolated and characterized two ciprofloxacin resistanceencoding, multidrug resistance plasmids (pLGP4 and pHTP1) from fresh spinach. I also iii isolated six plasmids encoding ciprofloxacin and meropenem resistance genes from three WWTPs across Canada. The ciprofloxacin resistance plasmids included plasmids pFECG, pFECR, pPICR and pTIC. The meropenem resistance plasmids included plasmids pPIMR and pFEMG. Using enrichment culture and real-time quantitative PCR approaches respectively, it was determined that conventional wastewater treatment processes markedly reduced the abundance of plasmids encoding ciprofloxacin resistance genes and incF family incompatibility genes. Conventional wastewater treatment processes, however, seemed to select for and enrich the population of bacteria carrying a specific erythromycin resistance plasmid. The presence of plasmids encoding clinically relevant ARGs in non-clinical anthropogenic environments suggests there may be a link between the community of antibiotic resistance determinants in clinical and non-clinical environments. Keywords: antibiotic resistance plasmids, spinach, wastewater, ciprofloxacin, meropenem, erythromycin.