Salicylate functions as an efflux pump inducer and promotes the emergence of fluoroquinolone-resistant Campylobacter jejuni mutants

Abstract

Salicylate, a nonsteroidal anti-inflammatory compound, has been shown to increase the resistance of Campylobacter to antimicrobials. However, the molecular mechanism underlying salicylate-induced resistance has not yet been established. In this study, we determined how salicylate increases antibiotic resistance and evaluated its impact on the development of fluoroquinolone-resistant Campylobacter mutants. Transcriptional fusion assays, real-time quantitative reverse transcription-PCR (RT-PCR), and immunoblotting assays consistently demonstrated the induction of the CmeABC multidrug efflux pump by salicylate. Electrophoretic mobility shift assays further showed that salicylate inhibits the binding of CmeR (a transcriptional repressor of the TetR family) to the promoter DNA of cmeABC, suggesting that salicylate inhibits the function of CmeR. The presence of salicylate in the culture medium not only decreased the susceptibility of Campylobacter to ciprofloxacin but also resulted in an approximately 70-fold increase in the observed frequency of emergence of fluoroquinolone-resistant mutants under selection with ciprofloxacin. Together, these results indicate that in Campylobacter, salicylate inhibits the binding of CmeR to the promoter DNA and induces expression of cmeABC, resulting in decreased susceptibility to antibiotics and in increased emergence of fluoroquinolone-resistant mutants under selection pressure.

Fig. 1.

Effects of salicylate (100 μg/ml) on the growth of C. jejuni 11168 in the presence of various antibiotics, including ciprofloxacin (0.125 μg/ml), erythromycin (0.125 μg/ml), novobiocin (16 μg/ml), and tetracycline (0.031 μg/ml). The bars represent the means and standard deviations for triplicate samples from a single representative experiment.

Fig. 2.

Induction of the cmeABC operon in C. jejuni 11168 by salicylate. (A) Transcriptional fusion (β-galactosidase assay) demonstrating the increased expression of cmeABC in the presence of salicylate (100 μg/ml). The bars represent the means and standard deviations for triplicate samples from a single representative experiment. (B) qRT-PCR results showing the increased transcript levels of cmeB with salicylate (100 and 200 μg/ml). Bacterial cells grown in MH broth alone were used as the control for baseline expression. The bars represent the means and standard deviations for three independent experiments.

Fig. 3.

Immunoblotting analysis of CmeA, CmeB, CmeC, Cj0561c, and MOMP production in NCTC 11168 grown with 0 (lanes 1, 4, and 7), 100 (lanes 2, 5, and 8), or 200 (lanes 3, 6, and 9) μg/ml of salicylate. Whole-cell proteins were used for immunoblotting with specific antibodies against the indicated proteins. The position of each protein is indicated by an arrow. MOMP was used as an internal control.

Fig. 4.

Effects of salicylate, taurocholate, and ampicillin on the formation of CmeR-DNA complexes as determined by EMSA. Each reaction mixture contained 30 ng cmeABC promoter DNA and 120 ng rCmeR (except for that in lane 1). The amounts of chemicals used in the assay are indicated at the top.

Fig. 5.

Induction of cj0561c in C. jejuni 11168 by salicylate (100 μg/ml), as determined by transcriptional fusion (β-galactosidase assay). The bars represent the means and standard deviations for triplicate samples from a single representative experiment.

Fig. 6.

Diagram depicting the molecular basis of salicylate-mediated induction of CmeABC and Cj0561c. (A) Baseline expression of the genes in the absence of salicylate. Transcription of cmeABC and cj0561c is at a low level due to inhibition by CmeR. (B) Induction of the genes by salicylate. When salicylate is present, it inhibits the binding of CmeR and ameliorates the repression on cmeABC and cj0561c, leading to overexpression of CmeABC and Cj0561c.