Molecular mechanisms involved in the emergence and fitness of fluoroquinolone-resistant Campylobacter jejuni

Campylobacter jejuni, a Gram-negative microaerobic bacterium, is one the most prevalent bacterial foodborne pathogens in humans, causing more than 2 million cases of diarrhea each year in the U.S. alone. Fluoroquinolone (FQ) antimicrobials are one of the antibiotics that are often prescribed for clinical treatment of diarrhea caused by enteric bacterial pathogens including Campylobacter. However, Campylobacter is increasingly resistant to FQ antimicrobials and FQ-resistant (FQR) Campylobacter developed in food producing animals can be transmitted to humans via the food chain, which has become a major concern for public health. In Campylobacter, the main targets of FQs in bacteria are DNA gyrases and the resistance to FQ antimicrobials is mediated by point mutation in the quinolone resistance-determining region (QRDR) of gyrA in conjunction with the function of the multidrug efflux pump CmeABC. The Thr-86-Ile mutation in GyrA confers a high-level-resistance to fluoroquinolone. One unique feature of FQ resistance development in Campylobacter is the rapid emergence of FQR mutants from a FQ-susceptible population when treated with FQ antimicrobials and C. jejuni possess a high mutation rate to FQ resistance. The rapidness and magnitude of FQ resistance development in Campylobacter in response to FQ treatment suggest that both selective enrichment of pre-existing spontaneous mutants and adaptive gene expression may contribute to the emergence of FQR Campylobacter, but how Campylobacter responds to FQ treatment is unknown. Once developmed, FQ R Campylobacter can persist in the absence of antibiotic selection pressure. Notably, FQR Campylobacter carrying the Thr-86-Ile substitution in the GyrA outcompeted the FQ S strains in chickens, suggesting that acquisition of FQ resistance enhances the in vivo fitness of FQR Campylobacter. How the resistance-conferring mutation affects Campylobacter fitness remains to be determined.;In this project, we conducted a series of studies to determine how Campylobacter responds to FQ treatment, what facilitates the emergence of FQR mutants in Campylobacter, and what are the molecular mechanisms contributing to the enhanced fitness in FQ R Campylobacter. In the first study, we examined the gene expression profiles of C. jejuni NCTC 11168 in response to treatment with CIPRO using microarray and found that 45 genes showed ≥1.5-fold (p<0.05) changes in expression when exposed to a suprainhibitory dose of CIPRO for 30 min. Most of the differentially expressed genes involved in cell membrane biosynthesis and DNA repair are up-regulated, while the genes associated with cellular processes and energy metabolism are down-regulated following exposure to CIPRO. One of the up-regulated genes was mfd (mutation frequency decline), which encodes a transcription-repair coupling factor involved in DNA repair. We found that in this study Mfd promotes the emergence of spontaneous FQR mutants and the development of FQR mutants under FQ treatments. These findings define a novel function of Mfd and significantly improve our understanding of the molecular mechanisms underlying the development of FQR Campylobacter. In the second study, we formally defined the role of the Thr-86-Ile mutation of GyrA in the enhanced fitness FQR Campylobacter in chickens by reverting the mutant allele. Then we conducted in vitro supercoiling assay using recombinant gyrases to assess the impact of various resistance-associated mutations on the enzymatic activities of DNA gyrase. We found that compared with that of the wild-type gyrase, the mutant gyrase with the Thr-86-Ile change showed greatly reduced supercoiling activity, while other GyrA mutations, although reduced the susceptibility to ciprofloxacin, did not affect the supercoiling activity of the gyrase. Subsequently we determined the impact of the GyrA mutations on in vivo supercoiling (within Campylobacter cells) using a reporter plasmid. The in vivo supercoiling result was consistent with the in vitro supercoiling findings and revealed that the Thr-86-Ile mutation altered the DNA supercoiling state in FQR Campylobacter . In the third study, we examined if the altered function of mutant gyrase affected gene and protein expression in Campylobacter. We compared the differences in transcriptomes and protein profiles between FQR and FQS strains using DNA microarray and 2D DIGE. The microarray data and 2D DIGE data showed that the expression of multiple genes was altered between the FQR strains and FQ S strains. Especially, the iron-response system and proteins involved in energy metabolism were upregulated in FQR Campylobacter , which may contributes to its enhanced fitness in the chicken host. Together, these findings from this project have significantly improved improve our understanding of the molecular mechanisms underlying the development of FQR Campylobacter and the fitness of the FQR Campylobacter.