A Fluoroquinolone Resistance Associated Mutation in gyrA Affects DNA Supercoiling in Campylobacter jejuni

Jing Han,Orhan Sahin,Yang Wang,Baoqing Guo,Qijing Zhang,Jianzhong Shen,Zhangqi Shen

doi:10.3389/fcimb.2012.00021

Abstract

The prevalence of fluoroquinolone (FQ)-resistant Campylobacter has become a concern for public health. To facilitate the control of FQ-resistant (FQR) Campylobacter, it is necessary to understand the impact of FQR on the fitness of Campylobacter in its natural hosts as understanding fitness will help to determine and predict the persistence of FQR Campylobacter. Previously it was shown that acquisition of resistance to FQ antimicrobials enhanced the in vivo fitness of FQR Campylobacter. In this study, we confirmed the role of the Thr-86-Ile mutation in GyrA in modulating Campylobacter fitness by reverting the mutation to the wild-type (WT) allele, which resulted in the loss of the fitness advantage. Additionally, we determined if the resistance-conferring GyrA mutations alter the enzymatic function of the DNA gyrase. Recombinant WT gyrase and mutant gyrases with three different types of mutations (Thr-86-Ile, Thr-86-Lys, and Asp-90-Asn), which are associated with FQR in Campylobacter, were generated in E. coli and compared for their supercoiling activities using an in vitro assay. The mutant gyrase with the Thr-86-Ile change showed a greatly reduced supercoiling activity compared with the WT gyrase, while other mutant gyrases did not show an altered supercoiling. Furthermore, we measured DNA supercoiling within Campylobacter cells using a reporter plasmid. Consistent with the results from the in vitro supercoiling assay, the FQR mutant carrying the Thr-86-Ile change in GyrA showed much less DNA supercoiling than the WT strain and the mutant strains carrying other mutations. Together, these results indicate that the Thr-86-Ile mutation, which is predominant in clinical FQR Campylobacter, modulates DNA supercoiling homeostasis in FQR Campylobacter.

Highlights

Campylobacter jejuni, a Gram-negative microaerophilic bacterium, has emerged as a leading bacterial cause of foodborne gastroenteritis in the United States and other developed countries (Slutsker et al, 1998)
Luo et al (2005) showed that FQR Campylobacter carrying the Thr-86-Ile substitution in GyrA subunit was able to outcompete the FQ-susceptible (FQS) strains in the absence of antibiotic usage, suggesting that acquisition of FQR enhances the in vivo fitness of FQR Campylobacter
DIRECT ROLE OF THE Thr-86-Ile CHANGE IN THE ENHANCED FITNESS A previous study by Luo et al (2005) showed that FQR Campylobacter carrying the Thr-86-Ile substitution GyrA outcompeted its isogenic FQS strains in chickens, suggesting that acquisition of FQR enhances the in vivo fitness of FQR Campylobacter

Summary

Introduction

Campylobacter jejuni, a Gram-negative microaerophilic bacterium, has emerged as a leading bacterial cause of foodborne gastroenteritis in the United States and other developed countries (Slutsker et al, 1998). Campylobacter is increasingly resistant to FQ antimicrobials, which has become a major concern for public health (Engberg et al, 2001; White et al, 2002; Gupta et al, 2004). There has been a concern on the use of FQ antimicrobials in poultry production since the use selectively enriches FQ-resistant (FQR) Campylobacter that can be transmitted to humans via the food chain (Luangtongkum et al, 2009). This concern led to the withdrawal of FQ antimicrobials from poultry production in the U.S in 2005. FQR Campylobacter continues to persist on poultry farms (Price et al, 2005, 2007; Luangtongkum et al, 2009), suggesting that FQR Campylobacter does not show a fitness cost in the absence of antibiotic selection pressure

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Conclusion