Date of Award

12-2007

Degree Type

Thesis

Degree Name

Master of Science

Major

Animal Science

Major Professor

Jun Lin

Committee Members

Kelly R. Robbins, Barton W. Rohrbach, Stephen P. Oliver

Abstract

Previous studies on macrolide resistance in Campylobacter were primarily focused on the isolates from various origins using in vitro systems. In this study, both in vitro and in vivo experiments were conducted to examine the development, stability, and genetic basis of macrolide resistance in C. jejuni. All in vitro and in vivo selected EryR mutants were derived from the same parent strain C. jejuni NCTC 11168. To determine if long-term exposure of low-level EryR C. jejuni to low-dose tylosin selects for high-level EryR mutants (MIC > 512 μg/ml), two low-level EryR mutants (MIC = 32 or 64 μg/ml) were used to inoculate chickens at 15 days of age in two independent experiments. Total and EryR C. jejuni populations in swabs collected at different time points were determined by differential plating and MIC test. The in vitro stability was tested by repeated subculturing of EryR mutants in Ery-free broth medium. The in vivo stability was tested by inoculating 3-day-old chickens (12-13 birds/group) receiving nonmedicated feed with EryR mutants and collecting cloacal swabs from each chickens at 12, 22, 38, and 47 days of age. Total and EryR C. jejuni populations in culture (after 10, 20 and 33 passages) or swab were determined by differential plating and MIC test. Genomic DNA from each of 63 selected EryR mutants was used for PCR amplification and sequence analysis of 23S rRNA gene and ribosomal proteins L4 and L22. Mutation in CmeABC multidrug efflux pump was transferred to EryR strains to determine the role of CmeABC efflux pump in Ery resistance. Chicken studies showed that the length of exposure time to subtherapeutic level of tylosin is not a sole factor contributing to the emergence of highly EryR Campylobacter. Prolonged exposure of low-level EryR C. jejuni (MIC = 32 or 64 μg/ml) to tylosin did not select for highly EryR mutants. The low- level Ery resistance (MIC = 32μg/ml) was stable after 10 passages in vitro but majority of C. jejuni were sensitive to Ery after 20 passages. The instability of low-level Ery resistance was also observed in chickens as early as 9 days postinoculation and EryR mutants were rarely isolated 35 days postinoculation. However, high-level Ery resistance (MIC > 512μg/ml) displayed remarkably stability in vitro and in vivo. All high-level EryR mutants selected in vivo displayed the A2074G mutation in 23S rRNA gene, distinct from the specific mutation (A2074C) observed in all highly EryR mutants selected in vitro. No mutations were observed in ribosomal proteins L4 for all in vitro selected EryR mutants but specific mutations in L4 (G74D or G57D) were widely found in low level EryR mutants selected in vivo (Ery MIC = 8-64 μg/ml). Insertion of three amino acids TSH at position 98 in L22 was only observed in mutants selected in vitro with Ery MIC ranging from 32-512 μg/ml. The CmeABC efflux pump worked synergistically with other mechanisms to confer Ery resistance in C. jejuni. Together, these findings indicated that C. jejuni utilize complex and different mechanisms to develop Ery resistance in vitro and in vivo.

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