MOLECULAR EPIDEMIOLOGY OF CAMPYLOBACTER AND

ENTEROCOLITICA ISOLATES FROM PIGS REARED IN CONVENTIONAL AND

ANTIBIOTIC FREE FARMS FROM DIFFERENT GEOGRAPHIC REGIONS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate School

of The Ohio State University

By

Daniel Alemayehu Tadesse, D.V.M

* * * * *

The Ohio State University

2009

Dissertation Committee: Approved by

Professor Wondwossen A. Gebreyes, Advisor

Professor Fred DeGraves Advisor

Professor Paivi Rajala-Schultz Graduate Program in

Professor Thomas E. Wittum Veterinary Preventive Medicine

Copyright by

Daniel Alemayehu Tadesse

2009

ABSTRACT

This dissertation is composed of four studies conducted in two predominant

bacterial pathogens; Campylobacter spp. and in swine production

systems. While the main scope was focused on molecular epidemiology, antimicrobial

resistance has also been a key component of the studies. Campylobacter are one of the

leading causes of foodborne bacterial infections. The first study was conducted to

determine the prevalence and antimicrobial resistance profile of Campylobacter in 34

farm-slaughter pairs from conventional and antimicrobial free (ABF) pig farms representing four different states classified into two regions (region 1 – Ohio and

Michigan; region 2 – Wisconsin and Iowa) . A total of 838 fecal and 1173 carcass samples were examined. Campylobacter isolation was done following recommended protocol and speciated using multiplex PCR targeting ceuE and hipO genes. The minimum inhibitory concentration was determined using agar dilution to a panel of six antimicrobials (chloramphenicol, erythromycin, gentamicin, ciprofloxacin, nalidixic acid and tetracycline). The overall Campylobacter prevalence was 56.3%. We found

Campylobacter prevalence of 58.9% and 53.7% among conventional and antimicrobial

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free farms, respectively (p=0.24). There was no significant difference between region 1

(54.1%) and region 2 (58.2%) (p=0.92). Carcass Campylobacter contamination was

found at slaughter followed by a significant reduction at post chill. Antimicrobial

resistance was found to all tested antimicrobials with different frequency. Higher proportions of Campylobacter were resistant to tetracycline (64.5%) and erythromycin

(47.9%). Resistance to chloramphenicol (4.4%), gentamicin (3.2%), nalidixic acid

(23.5%) and ciprofloxacin (4.9%) were also detected. Forty-six of 61 ciprofloxacin resistant (75.4%) Campylobacter coli were recovered from ABF and the remaining 15

(24.6%) from conventional production systems. Thirty-seven out of 1257 Campylobacter coli (2.9%) were resistant to both erythromycin and ciprofloxacin, drugs of choice for . High prevalence of Campylobacter coli and high proportion

of resistance in both conventional and ABF systems, regardless of antimicrobial use status, was found.

The emergence and spread of fluoroquinolone resistant Campylobacter poses a

great challenge to public health. In the second study, we evaluated the contribution of point mutations in the quinolone resistance determining region (QRDR) of the gyrA gene to quinolone resistance. A total of eighty Campylobacter isolates were selected based on minimum inhibitory concentration (MIC) of ciprofloxacin and nalidixic acid. Of these, 21 were resistant to both ciprofloxacin (MIC ≥ 4 μg/ml) and nalidixic acid (MIC ≥ 32

μg/ml), 19 were resistant to nalidixic acid only and the remaining forty were susceptible

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to both ciprofloxacin and nalidixic acid. Of the 21