Contribution of CmeG to antibiotic and oxidative stress resistance in Campylobacter jejuni

Abstract

Objectives: campylobacter jejuni is a leading foodborne pathogen worldwide and its resistance to antimicrobials is a major concern for public health. The cmeG (Cj1375) gene in C. jejuni encodes a putative efflux transporter of the major facilitator family, but its function in antimicrobial resistance has not been determined. This study aimed to characterize the function of CmeG in conferring resistance to antibiotics and oxidative stress.

Methods: the cmeG gene (Cj1375) in C. jejuni was inactivated by insertional mutagenesis and overexpressed by cloning with a shuttle vector. These constructs were compared with the wild-type strain using antimicrobial susceptibility tests and drug accumulation assays.

Results: the cmeG mutation reduced bacterial growth and rendered C. jejuni more susceptible to ciprofloxacin, erythromycin, gentamicin, tetracycline, rifampicin, ethidium bromide and cholic acid as well as hydrogen peroxide, and in trans complementation restored the susceptibility to near wild-type level. RT-PCR showed that cmeG is co-transcribed with its downstream gene cmeH (Cj1376) encoding a putative periplasmic protein, but mutation of cmeH alone did not affect the susceptibility to antibiotics. Notably, overexpression of the cmeGH operon in C. jejuni NCTC 11168 significantly increased its resistance to fluoroquinolones. In addition, the cmeG mutant accumulated more EtBr and ciprofloxacin than the wild-type strain.

Conclusions: these results indicate that CmeG functions as a multidrug efflux transporter contributing to antibiotic resistance and oxidative defence in Campylobacter.

Figure 1.

Genomic organization of the cmeGH operon and co-transcription of cmeGH as determined by RT–PCR. (a) Comparison of the cmeG locus and its flanking genes between C. jejuni NCTC 11168 and 81-176. cmeH is absent in 81-176. Boxed arrows depict open reading frames and gene orthologues are indicated with the same shading. (b) RT–PCR shows co-transcription of cmeG and cmeH. RT–PCR was performed with primers amplifying a region spanning both cmeG and cmeH. Lane 1, a PCR control performed with genomic DNA of C. jejuni 11168; lane 2, an RT–PCR negative control (the RT step was omitted); lane 3, a standard RT–PCR with the purified RNA.

Figure 2.

Susceptibility of various C. jejuni strains to hydrogen peroxide determined by disc inhibition assay. C. jejuni NCTC 11168 [wild-type (WT)], the cmeG mutant (JB401), the cmeH mutant (JB402) and the complemented cmeG mutant (JB401C) grown on MH agar plates were treated with the same amount of hydrogen peroxide. The experiment was repeated three times, and similar results were obtained each time.

Figure 3.

Intracellular accumulation of EtBr and ciprofloxacin. (a) EtBr accumulation in the wild-type (WT; filled circles), the cmeG mutant (JB401; open circles) and the complemented cmeG mutant (JB401C; filled triangles). Higher fluorescence units indicate more intracellular accumulation of EtBr. The results show the means and standard deviations of triplicate samples in one experiment. The assay was repeated four times and similar results were obtained. (b) Accumulation of ciprofloxacin in WT, JB401 and JB401C. Each bar represents the mean and standard deviation of triplicate samples in one experiment. Similar results were obtained in three independent experiments.