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Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland
Correspondence
Minna Hannula
minna.hannula{at}helsinki.fi
Received 13 December 2007
Accepted 29 February 2008
Abbreviations: EPI, efflux pump inhibitor; NMP, 1-(1-naphthylmethyl)-piperazine; PAβN, phenyl-arginine-β-naphthylamide; RND, resistance–nodulation–cell division.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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It has been established that efflux pumps, particularly the resistance–nodulation–cell division (RND) type CmeABC pump, have an important role in the antimicrobial resistance of Campylobacter jejuni (Lin et al., 2002). The same efflux pump has recently been characterized in Campylobacter coli (Corcoran et al., 2005). This pump is able to significantly decrease the susceptibility of Campylobacter to a number of antimicrobials (Lin et al., 2002).
Efforts have been made to find substances capable of reversing the action of the efflux pumps and which therefore have the potential to prevent antimicrobial resistance in vivo. A broad-spectrum efflux pump inhibitor (EPI), phenyl-arginine-β-naphthylamide (PAβN), was first characterized in Pseudomonas aeruginosa (Lomovskaya et al., 2001) and has since been studied in several bacteria, including Campylobacter (Cagliero et al., 2005; Gibreel et al., 2007; Payot et al., 2004). Another putative EPI is 1-(1-naphthylmethyl)-piperazine (NMP), characterized in Escherichia coli (Bohnert & Kern, 2005). It is able to partially reverse multidrug resistance in some members of the Enterobacteriaceae and in Acinetobacter baumannii (Bohnert & Kern, 2005; Pannek et al., 2006; Schumacher et al., 2006) but its effects on Campylobacter have not been studied previously.
In E. coli, salicylic acid binds to a repressor protein, MarR, which results in the overexpression of the global regulatory gene marA. This causes the down-regulation of the outer-membrane protein OmpF and an increase in the synthesis of the RND multidrug efflux pump AcrAB-TolC, which leads to decreased antimicrobial accumulation (Price et al., 2000). Salicylate has also been reported to increase the effect of antimicrobial aminoglycosides in E. coli (Aumercier et al., 1990). Finally, Lin et al. (2005) have shown that bile salts, such as deoxycholate, significantly induce the expression of cmeABC in C. jejuni by preventing the binding of the transcriptional repressor of cmeABC (CmeR). This can lead to increased resistance to several antibiotics (Lin et al., 2005).
The aim of this study was to analyse the effect of these two EPIs, as well as the two putative efflux pump inducers, on the susceptibility of C. jejuni and C. coli to erythromycin, ciprofloxacin, kanamycin, tetracycline and rifampicin.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Determination of MICs. MICs of ciprofloxacin, erythromycin, rifampicin and tetracycline (all Sigma-Aldrich) in the presence and absence of NMP (100 mg l–1) (Chess), PAβN (50 mg l–1) (Sigma-Aldrich), sodium salicylate (100 mg l–1) or sodium deoxycholate (1 mg ml–1) (both Merck) were determined by using the agar dilution method following the guidelines set by the CLSI (2006). All experiments were repeated at least three times. The growth of each strain was verified on control plates containing the indicated amounts of the studied efflux pump inhibitors and inducers.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Ciprofloxacin-resistant variants were obtained by selection on media containing 1 mg ciprofloxacin l–1 (Hänninen & Hannula, 2007). The resistant variants had 16- to 128-fold greater MIC values than the respective parent strains, which corresponded to MIC values from 2 to 64 mg l–1.
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PAβN also decreased rifampicin resistance 8- to 64-fold in the studied strains. C. jejuni and C. coli are intrinsically resistant to rifampicin, and PAβN's ability to reduce this resistance suggests that rifampicin resistance in Campylobacter is at least partly associated with efflux. A recently published work on tetracycline reported 1.3- to 2.6-fold decrease in tetracycline resistance in highly resistant C. jejuni strains in the presence of 30 µg PAβN ml–1 (Gibreel et al., 2007). Congruently, the two tetracycline-resistant strains (6590 and 49/7RAT CIP) included in our study also had a 2-fold reduction in the tetracycline MIC values, but the same phenomenon was not observed in tetracycline-susceptible strains. With regards to kanamycin and ciprofloxacin, there was no evidence of PAβN affecting the MICs of these antimicrobials for the bacteria.
The inactivation by insertional mutagenesis of the cmeB gene of C. jejuni has led to an 8-fold reduction in ciprofloxacin MIC levels, 4- to 16-fold reduction in erythromycin MIC levels, 64- to 128-fold decrease in rifampicin MIC levels and an 8-fold decrease in tetracycline MIC levels (Lin et al., 2002). The effects on the MIC levels obtained when the PAβN pump inhibitor was added to the growth media were less pronounced or absent, suggesting that the action of PAβN does not result in the complete inactivation of CmeABC and that this substance may have other modes of action. However, the strong decreasing effect of PAβN on the MIC levels of erythromycin and rifampicin warrants the use of PAβN in further studies involving these antimicrobials.
Effect of NMP
Studies conducted with NMP showed a 2- to 8-fold decrease in erythromycin and rifampicin MIC levels for C. jejuni and C. coli, but did not show an increase in the susceptibility of Campylobacter to tetracycline, kanamycin or ciprofloxacin. According to our results, NMP confers an increase in susceptibility to the same antimicrobials as PAβN but with less effectiveness, which is in agreement with the studies on members of the Enterobacteriaceae and Acinetobacter baumannii (Bohnert & Kern, 2005; Pannek et al., 2006; Schumacher et al., 2006), indicating that NMP confers effects on MIC levels that differ from those seen with PAβN, suggesting a different mode of action for these pump inhibitors.
Effect of sodium salicylate
Salicylate and acetylsalicylate are commonly used non-steroidal anti-inflammatory agents that are often used in combination with antimicrobials for the treatment of microbial diseases. However, salicylate has been shown to decrease the susceptibility of bacteria, such as E. coli, to antimicrobials by altering membrane proteins and inducing efflux pumps (Price et al., 2000). Since C. jejuni possesses an outer-membrane protein (MOMP) belonging to the same porin family as E. coli OmpF (Bolla et al., 1995) and a multidrug efflux pump (CmeABC) belonging to the same RND family as AcrAB-TolC (Lin et al., 2002), it has been speculated that salicylate might also decrease antimicrobial accumulation in C. jejuni. A previous study by Randall et al. (2003) showed a small but significant effect of salicylate on the ciprofloxacin resistance of Campylobacter, and our results confirm a 2- to 4-fold increase in MIC values for ciprofloxacin in almost all strains, with ciprofloxacin-susceptible strains and their respective variants reacting alike. In the study by Randall et al. (2003), no effect was observed on kanamycin MIC levels, which was confirmed by our study, but unlike the previous study, we were unable to observe changes in tetracycline or erythromycin MIC levels in the presence of sodium salicylate (Randall et al., 2003). In 7 out of 17 strains, a 2-fold increase in rifampicin resistance was found when salicylate was included in the medium.
Effect of sodium deoxycholate
In the study by Lin et al. (2005), the expression of cmeABC was significantly induced by various bile salts, such as deoxycholate. Cholate (1 mg ml–1) and taurocholate (25 mg ml–1) included in the MH broth produced, respectively, a 0- to 2-fold and a 2- to 4-fold increase in the MIC levels of various antimicrobials, such as ciprofloxacin and erythromycin, in C. jejuni. In our study, sodium deoxycholate (1 mg ml–1) used in the agar dilution experiments had little effect on the MIC values of the studied antimicrobials. Strains with 2-fold increases in rifampicin MIC values were variably found in each round of testing, suggesting that although deoxycholate may have had an increasing effect on rifampicin MIC values, this effect was too modest to be confirmed by these experiments.
In summary, PAβN induced an 8- to 32-fold and 8- to 64-fold reduction in the MIC values of erythromycin and rifampicin for the studied strains, respectively. NMP produced a smaller, 2- to 8-fold reduction, for the same antimicrobials. With regards to the putative efflux pump inducers, sodium deoxycholate had little or no effect on the MIC values of the studied antimicrobials, whereas sodium salicylate brought about a small (2- to 4-fold) but reproducible increase in the MIC values for ciprofloxacin, and in 7/17 of the studied strains, a 2-fold increase in the MIC values for rifampicin. These results further confirm the importance of efflux in Campylobacter resistance and highlight the value of agents that affect efflux pumps as tools for research aimed at the inhibition of CmeABC and the reversal of antimicrobial resistance in Campylobacter.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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