Abstract

ABSTRACTBacterial antibiotic efflux pumps are key players in antibiotic resistance. Although their role in conferring multidrug resistance is well documented, the emergence of “super” efflux pump variants that enhance bacterial resistance to multiple drugs has not been reported. Here, we describe the emergence of a resistance-enhancing variant (named RE-CmeABC) of the predominant efflux pump CmeABC in Campylobacter, a major zoonotic pathogen whose resistance to antibiotics is considered a serious antibiotic resistance threat in the United States. Compared to the previously characterized CmeABC transporters, RE-CmeABC is much more potent in conferring Campylobacter resistance to antibiotics, which was shown by increased MICs and reduced intracellular accumulation of antibiotics. Structural modeling suggests that sequence variations in the drug-binding pocket of CmeB possibly contribute to the enhanced efflux function. Additionally, RE-CmeABC expands the mutant selection window of ciprofloxacin, enhances the emergence of antibiotic-resistant mutants, and confers exceedingly high-level resistance to fluoroquinolones, an important class of antibiotics for clinical therapy of campylobacteriosis. Furthermore, RE-CmeABC is horizontally transferable, shifts antibiotic MIC distribution among clinical isolates, and is increasingly prevalent in Campylobacter jejuni isolates, suggesting that it confers a fitness advantage under antimicrobial selection. These findings reveal a new mechanism for enhanced multidrug resistance and an effective strategy utilized by bacteria for adaptation to selection from multiple antibiotics.

Highlights

  • IntroductionThe native cmeABC operon in C. jejuni 81-176 was deleted and replaced with a chloramphenicol resistance gene (cat) by the homologous-recombination method [19]

  • Introduction of REcmeABC into C. jejuni 81-176

  • NT161 showed increased resistance to other antibiotics, including chloramphenicol (16-fold), ciprofloxacin (8-fold), erythromycin (4-fold), and tetracycline (4-fold). These findings indicated that the transformation resulted in the transfer of multidrug resistance, suggesting that either multiple antibiotic resistance genes/mutations or a multidrug resistance mechanism was transferred from DH161 to NT161

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Summary

Introduction

The native cmeABC operon in C. jejuni 81-176 was deleted and replaced with a chloramphenicol resistance gene (cat) by the homologous-recombination method [19]. The intact RE-cmeABC operon from DH161 was amplified with primers cmeAKpnI-F and cmeCKpnI-R. The amplicon was digested with KpnI and ligated into the corresponding sites of plasmid pUC-cadFermB-cj1476c [38]. PUC-cadF-ermB-cj1476c contains a macrolide resistance gene (ermB) between the sequences homologous to the cadF and cj1476c genes. The constructed suicide plasmid pUC-cadF-RE-cmeABCermB-cj1476c was used to introduce RE-cmeABC into the 81-176 cmeABC deletion mutant strain. The transformants (81-176-V) were selected on MH agar plates containing erythromycin (10 mg/liter) and verified by PCR and DNA sequence analysis

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