A single nucleotide change in mutY increases the emergence of antibiotic-resistant Campylobacter jejuni mutants

Abstract

Objectives: Mutator strains play an important role in the emergence of antibiotic-resistant bacteria. Campylobacter jejuni is a leading cause of foodborne illnesses worldwide and is increasingly resistant to clinically important antibiotics. The objective of this study was to identify the genetic basis that contributes to a mutator phenotype in Campylobacter and determine the role of this phenotype in the development of antibiotic resistance.

Methods: A C. jejuni isolate (named CMT) showing a mutator phenotype was subjected to WGS analysis. Comparative genomics, site-specific reversion and mutation, and gene knockout were conducted to prove the mutator effect was caused by a single nucleotide change in the mutY gene of C. jejuni.

Results: The C. jejuni CMT isolate showed ∼ 100-fold higher mutation frequency to ciprofloxacin than the WT strain. Under selection by ciprofloxacin, fluoroquinolone-resistant mutants emerged readily from the CMT isolate. WGS identified a single nucleotide change (G595 → T) in the mutY gene of the CMT isolate. Further experiments using defined mutant constructs proved its specific role in elevating mutation frequencies. The mutY point mutation also led to an ∼ 700-fold increase in the emergence of ampicillin-resistant mutants, indicating its broader impact on antibiotic resistance. Structural modelling suggested the G595 → T mutation probably affects the catalytic domain of MutY and consequently abolishes the anti-mutator function of this DNA repair protein.

Conclusions: The G595 → T mutation in mutY abolishes its anti-mutator function and confers a mutator phenotype in Campylobacter, promoting the emergence of antibiotic-resistant Campylobacter.

Figure 1.

Ciprofloxacin susceptibility test for C. jejuni 11168, CMT, W7 and IA3902 utilizing a broth microdilution method in a 96-well plate. The concentration range of ciprofloxacin used for this assay was from 0.0075 to 8 mg/L. Each of the isolates was assayed in duplicate. Pictures were taken of the same plate at 24 and 48 h of incubation.

Figure 2.

Growth of C. jejuni under ciprofloxacin (1 mg/L) treatment. (a) Comparison of C. jejuni CMT (filled circles) with NCTC 11168 (filled triangles). (b) Comparison of C. jejuni CMT_W199G::Km (filled circles) with CMT::Km (filled triangles). For both panels, at each timepoint the number of FQ^R mutants among 50 randomly picked colonies from antibiotic-free enumerating plates is shown at the top. The broken line in both panels represents the detection limit of the plating method. NA, no colonies available.

Figure 3.

Spontaneous FQ^R mutation frequencies of various C. jejuni isolates. (a) Elevated mutation frequency in C. jejuni CMT compared with strains 11168, W7 and IA3902. (b) Reduced mutation frequency in the revertant of MutY of the CMT isolate. (c) Mutation of mutY increases spontaneous FQ^R frequencies in C. jejuni 11168. Each bar represents the mean value of triplicate experiments.