Characterization of the cmeABC Efflux Pump Operon on Campylobacter Coli Strains Isolated from Turkey, Swine, and Cattle

NON-TECHNICAL SUMMARY: Despite Campylobacter being one of the most prevalent organisms associated with human illness worldwide and accounting for approximately 2.5 million cases in the US annually, the pathogenesis of this foodborne pathogen is not well understood. Campylobacter is reported to use efflux pumps as a means of promoting antimicrobial resistance and to facilitate its colonization of chicken. The main objective of this study is to characterize the multidrug efflux pump operon in Campylobacter coli strains isolated from turkey, swine and cattle. To achieve our objective, we will amplify and sequence this operon from various Campylobacter coli strains isolated from the above mentioned three different hosts. This will enrich the GeneBank and enhance our understanding of the role of efflux pumps in Campylobacter. As a long term goal, this will enable us to design strategies to control and manage this important food pathogen. <P>APPROACH: Twenty isolates of Campylobacter coli representing each host (Turkey, Swine and Cattle) will be chosen to amplify the ~ 7 kb cmeABC gene operon (a total of sixty isolates). By thoroughly checking the genome sequence available in the gene bank for Campylobacter coli, PCR primers will be designed to amplify the cmeABC operon. Long PCR protocols will be used to amplify the 7 Kb fragments where the CHextension time of the PCR protocol will be increased to 7 minutes in each cycle to amplify the relatively long fragment. PCR will be performed using the Qiagen PCR master mix with a proofread Taq polymerase. The amplified fragments will be then purified using the Qiagen PCR purification kit. In cases where amplifying the whole operon is not achievable, it will be amplified in two or three smaller pieces. The amplified cmeABC operon obtained from the sixty Campylobacter coli isolates from the three different hosts will be completely sequenced in both strands using primer walking strategies. Sequence assembly and alignment will be carried out using DNA Lasergene 7.2 software. Dendrograms will be created using the BioNumerics software. DNA sequence of the cmeABC operon from the different strains of Campylobacter coli will be compared to sequences in Gene Bank using the Blast services of the NCBI website. Particular attention will be given to comparing the genetic variation in the DNA sequence of the cmeABC operon of Campylobacter coli strains determined in this proposal and those published for Campylobacter jejuni in the Gene Bank. All unique new sequences will be deposited in the Gene Bank.