Molecular epidemiology of Campylobacter and Yersinia enterocolitica isolates from pigs reared in conventional and antibiotic free farms from different geographic regions.

摘要

This dissertation is composed of four studies conducted in two predominant bacterial pathogens; Campylobacter spp. and Yersinia enterocolitica 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 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 human campylobacteriosis. 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 mug/ml) and nalidixic acid (MIC ≥ 32 mug/ml), 19 were resistant to nalidixic acid only and the remaining forty were susceptible to both ciprofloxacin and nalidixic acid. Of the 21 ciprofloxacin and nalidixic acid resistant C. coli isolates, only six showed point mutations that resulted in Thr-86 to Ile (ACT to ATT) amino acid substitutions. These isolates had MICs ≥ 4 mug/ml for ciprofloxacin and = 128 mug/ml for nalidixic acid. No detectable gyrA mutation was detected among C. coli isolates with MIC ≤ 4 mug/ml to ciprofloxacin. Further investigation is required to define the mechanism of quinolone resistance among the remaining ciprofloxacin resistant C. coli isolates.;The genotypic diversity of 99 C. coli isolates from different geographic regions, production systems and with different resistance patterns was also investigated by multilocus sequence typing method in the third study of this dissertation. A total of 50 different sequence types (ST) were identified by sequencing seven housekeeping genes (aspA, glnA. gltA, glyA, pgm, tkt, and uncA). Seven new STs (ST-3813, 3814, 3815, 3816, 3817, 3818 and 3819) were identified in this study. Of these, three resulted from new allele sequence at tkt and uncA loci and the remaining resulted from new combinations of the previously described alleles. All STs belonged to ST-828 clonal complex with ST-828 putative founder ST. The most common ST was ST-854 (n=10) followed by ST-828 (n=5), ST-1096 (n=5) and ST-1100 (n=5). The C. coli population showed weak clonal structure with an IA of 0.46. The weak clonal structure of strains demonstrates the usefulness of MLST for investigation of the epidemiology of C. coli.;Yersinia enterocolitica is another important foodborne pathogen and pigs are recognized as a major reservoir and potential source of pathogenic strains to humans. A total of 172 Y. enterocolitica were investigated in the fourth study of this dissertation to determine their pathogenic significance to humans. Phenotypic and genotypic diversity of the isolates were assessed using antibiogram, serogrouping and amplified fragment length polymorphism (AFLP). Carriage of chromosomal and plasmid-borne virulence genes were investigated using PCR. A total of 12 antimicrobial resistance patterns were found. More than two-thirds of Y. enterocolitica were pan-susceptible and 27.9% were resistant against beta-lactams (either ampicillin or amoxicillin/clavulanic acid). The findings confirm that Y. enterocolitica strains in pigs could be an important source for human infection. (Abstract shortened by UMI.)