Degree Type

Dissertation

Date of Award

2012

Degree Name

Doctor of Philosophy

Department

Veterinary Microbiology and Preventive Medicine

First Advisor

Qijing Zhang

Abstract

Campylobacter jejuni is the leading bacterial cause of foodborne diseases in the United States and other developed countries. As a zoonotic pathogen, C. jejuni is well adapted in the food production environments, is prevalent in food producing animals, and is transmitted to humans via unpasteurized milk, contaminated water, and undercooked poultry meat. The extensive use of antibiotics for animal production and human medicine has resulted in increasing prevalence of antibiotic resistant Campylobacter. Previously it was shown that antimicrobial efflux transporters, such as CmeABC, play key roles in both the intrinsic and acquired resistance to structurally diverse antimicrobials. However, the roles of antimicrobial efflux systems in facilitating Campylobacter adaptation to various environments and the associated molecular mechanisms remain to be determined. In this project, we determined the role of salicylate-induced overexpression of cmeABC in promoting the emergence of fluoroquinlone-resistant mutants in C. jejuni and identified two new efflux transporters that are involved in Campylobacter resistance to arsenic compounds. The entire project contains three sets of experiments. In the first set of experiments, the induction of the CmeABC multidrug efflux pump by salicylate, which is commonly present in medicine and food, was consistently shown by transcriptional fusion assays, real time quantitative reverse transcription-PCR (RT-PCR), and immunoblotting. The induction not only decreased the susceptibility of Campylobacter to ciprofloxacin, but also resulted in an approximately 70-fold increase in the observed frequency of emergence of fluoroquinolone-resistant mutants under selection with ciprofloxacin. These findings suggest that exposure of C. jejuni to salicylate could conceivably influence the development of antibiotic resistance in this pathogenic organism. In the second set of experiments, we determined the role of ArsB, a putative membrane efflux transporter, in the resistance of C. jejuni to arsenic compounds, which exist in nature and are used as feed additives in poultry production. Inactivation of arsB in C. jejuni 11168 resulted in 8- and 4-fold reduction in the MICs of arsenite and arsenate, respectively, and complementation of the arsB mutant restored the MIC of arsenite to the wild-type level. Additionally, overexpression of arsB in C. jejuni 11168 resulted in a 16-fold increase in the MIC of arsenite. These results indicate that ArsB is a key player in mediating the resistance to inorganic arsenic in Campylobacter. In the third set of experiments, we discovered a novel membrane transporter (named ArsP) that contributes to Campylobacter resistance to roxarsone, organic arsenic used as an additive in poultry feed. ArsP is predicted to be a membrane permease containing eight transmembrane helices, a structural feature distinct from other known arsenic transporters. Analysis of multiple C. jejuni isolates from various animal species revealed that presence of arsP is associated with elevated resistance to roxarsone. In addition, inactivation of arsP in C. jejuni resulted in a 4-fold reduction in the MIC of roxarsone compared to the wild-type strain. Furthermore, cloning of arsP into a C. jejuni strain lacking a functional arsP led to 8-fold increase in the MIC of roxarsone. Neither mutation nor overexpression of arsP affected the MICs of inorganic arsenic including arsenite and arsenate in Campylobacter. Moreover, acquisition of arsP gene in NCTC 11168 accumulated less roxarsone than the wild type strain lacking arsP gene. These results indicate that ArsP functions as an efflux transporter specific for extrusion of roxarsone and contributes to the resistance to organic arsenic in C. jejuni. All together, findings in this project reveal important roles of antimicrobial efflux transporters in facilitating Campylobacter adaptation to various environments and provide new insights into the pathobiology of C. jejuni as a major foodborne pathogen.

Copyright Owner

Zhangqi Shen

Language

en

Date Available

2013-10-31

File Format

application/pdf

File Size

124 pages

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