Involvement of the CmeABC efflux pump in the macrolide resistance of Campylobacter coli

Cédric Cagliero, Christian Mouline, Sophie Payot* and Axel Cloeckaert

Institut National de la Recherche Agronomique, UR086 BioAgresseurs, Santé, Environnement, 37380 Nouzilly, France


* Corresponding author. Tel: +33-2-47-42-79-88; Fax: +33-2-47-42-77-74; E-mail: payot{at}tours.inra.fr

Received 6 June 2005; returned 20 June 2005; revised 26 July 2005; accepted 27 July 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: This study was conducted to examine the role of the CmeABC efflux pump in decreasing the susceptibility of Campylobacter coli to macrolides and ketolides in the context of absence or presence of mutations in the 23S rRNA genes.

Methods: The cmeB gene was inactivated in strains of C. coli showing two different patterns of erythromycin resistance (low or high level of resistance) associated with the absence or presence of a A2075G mutation in the 23S rRNA genes. MICs of erythromycin, azithromycin, tylosin, telithromycin and ciprofloxacin were compared for wild-type (with or without efflux pump inhibitor) and mutant strains.

Results: The cmeB gene inactivation (or addition of efflux pump inhibitor) led to the restoration of susceptibility of the low-level-resistant strains (no A2075G mutation in the 23S rRNA genes). In the highly resistant strains (A2075G mutation in the 23S rRNA genes), the MICs of erythromycin decreased 128- to 512-fold upon inactivation of the cmeB gene. MICs of azithromycin, tylosin and telithromycin were also affected by both addition of efflux pump inhibitor and cmeB gene inactivation, revealing these molecules as substrates of the CmeABC efflux pump. Compared with azithromycin, MICs of telithromycin drastically decreased upon cmeB gene inactivation even in the presence of a A2075G mutation in 23S rRNA genes.

Conclusions: The CmeABC efflux pump acts synergically with 23S rRNA mutations to drastically increase the MICs of erythromycin and tylosin in C. coli. In contrast, azithromycin was less affected by efflux and telithromycin, although being a good substrate for the CmeABC efflux pump, was less affected by an A2075G mutation in 23S rRNA genes.

Keywords: transporters , gene inactivation , mutations , 23S rRNA , ketolides


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Campylobacter is a major cause of food-borne acute gastroenteritis. Among the species incriminated, Campylobacter coli is associated with up to 15% of human campylobacteriosis cases.1 Campylobacter is a zoonotic pathogen and poultry and pigs can act as sources of infection.1 Macrolides are the drug of choice to treat Campylobacter gastroenteritis in humans since until now the rate of erythromycin resistance was reported to be low worldwide.2 However, recent reports indicate higher rates of resistance in Northern Ireland and Bulgaria3,4 and resistance to macrolides is usually higher in C. coli than in Campylobacter jejuni.2

Macrolides are potent inhibitors of protein synthesis that act by binding to 23S rRNA in the 50S ribosomal subunit. In Campylobacter, mutations (A to G at position 2075 in most cases) in the three 23S rRNA gene loci have been associated with a high level of resistance to erythromycin.5 An efflux system (CmeABC) consisting of three components, a periplasmic fusion protein CmeA, an inner membrane drug transporter CmeB, and an outer membrane protein CmeC, has been described in C. jejuni.6 It was found to contribute to the intrinsic resistance of C. jejuni to antibiotics including erythromycin, ciprofloxacin, detergents and dyes since susceptible strains became hypersusceptible to these compounds upon inactivation of the cmeB gene.6 However, the role of this system has not been studied in C. coli, and its involvement in resistance to macrolides (erythromycin, azithromycin and tylosin) and ketolides has not been examined.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Human isolates (named 2002/0116 and 2002/0697) were obtained from the National Centre of Reference for Campylobacter (Bordeaux, France). Poultry and pig (named O1611 and 12) strains were isolated, respectively, by the Agence Française de Sécurité Sanitaire des Aliments (Ploufragan, France) at slaughterhouse, and by the Ecole Nationale Vétérinaire of Nantes from fattening pigs or from piglets. Routine growth and detection of mutations in the 23S rRNA were performed as described previously.7 Susceptibility testing was done using agar dilution as described previously.7 Concentrations tested were: erythromycin, 0.125–2048 mg/L; tylosin, 0.5–2048 mg/L; azithromycin, 0.125–1024 mg/L; telithromycin, 0.06–256 mg/L and ciprofloxacin, 0.06–64 mg/L. Breakpoints used were >4 mg/L for erythromycin and >1 mg/L for ciprofloxacin as recommended by the French Antibiogram Committee (CA-SFM, available at http://www.sfm.asso.fr/). There are no approved breakpoints for tylosin, azithromycin and telithromycin and therefore resistance was not interpreted for these antibiotics. The efflux pump inhibitor (Phe-Arg-ß-naphthylamide) was incorporated in the plates at a concentration of 20 mg/L. The cmeB::kan mutants were constructed by natural transformation with genomic DNA (1 µg) of a 81176 cmeB::kan mutant provided by Q. Zhang (University of Iowa, Ames, IA, USA) using the biphasic method.8 Before storage at –80°C, mutants were confirmed by PCR, using primers Kan-2TNP-Rev (5'-CTGAAGCTTGCATGCCTGC-3') located in the kanamycin cassette and Cj0366a (5'-GACCTGTTTTTGCTTCAGTT-3') located in the cmeB gene. Genomic DNA was extracted from cultures grown for 24 h at 42°C using the QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France). PCR was performed in a 25 µL volume containing 100 ng of template genomic DNA, 2 µM of each primer, 200 µM deoxynucleotide triphosphates, 1.5 mM MgCl2, and 0.5 U of Taq polymerase (Promega, Charbonnières, France). After an initial denaturation step of 5 min at 95°C, amplification was performed over 30 cycles, with 30 s at 95°C, 30 s at 50°C, and 2.5 min at 72°C, and a final extension step of 10 min at 72°C. Mutants were also confirmed by western blot using anti-CmeB antibodies as previously described.9

Before determining the MICs, mutants were confirmed again by PCR since we have shown that the –80°C storage could lead to the loss of the transposon-carried kanamycin resistance cassette in some mutants.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Knockout cmeB::kan mutants were constructed in different C. coli strains chosen among collections of human, poultry and pig isolates for their erythromycin susceptibility pattern. Seven isolates had a low level of resistance (LLR) to erythromycin (a 14C-membered macrolide) (MIC = 8–16 mg/L) (Table 1) and four strains had a high level of resistance to erythromycin (HLR) (MIC ≥ 1024 mg/L). As previously described,7 only the HLR strains carried mutated copies of 23S rRNA genes (with an A to G mutation at position 2075 in the three copies of 23S rRNA). No mutation was found at position 2074 of the 23S rRNA genes in all the strains examined. Interruption of the cmeB gene in LLR strains led to a restoration of susceptibility to erythromycin (Table 1). A similar decrease in the MIC could be obtained when using an efflux pump inhibitor specific for RND efflux systems (Phe-Arg-ß-naphthylamide) (Table 1) as previously reported.7,10 This suggests that the LLR phenotype is imputable to an efflux mechanism mediated mainly by the CmeABC efflux pump. The efflux pump inhibitor only enabled a decrease of four- to eightfold of the MICs of the HLR strains, probably because of a limited competitive inhibition (Table 1). In contrast, the MIC of erythromycin of the HLR strains greatly decreased upon cmeB gene inactivation (128- to 512-fold) (Table 1). Only one strain remained resistant to erythromycin but with an LLR phenotype (MIC = 8 mg/L). This suggests that the HLR phenotype observed in the C. coli strains is due to a synergy between 23S rRNA mutations and efflux mediated by the CmeABC efflux pump. In most of the strains examined, mutations in the 23S RNA genes were not sufficient to confer resistance to erythromycin when the CmeABC pump was inactive, highlighting the major contribution of this pump for erythromycin resistance in C. coli. As expected, the MIC of ciprofloxacin decreased upon inactivation of the cmeB gene in all the strains examined. The efflux pump inhibitor was poorly effective in competing with ciprofloxacin as previously observed.7 MICs of azithromycin (15C-macrolide), tylosin (16C-macrolide) and telithromycin (ketolide) were also affected by the addition of the efflux pump inhibitor and by the inactivation of the cmeB transporter gene, indicating that these molecules are also substrates of the CmeABC efflux pump. However, tylosin and telithromycin appeared to be better substrates for this pump than azithromycin, as MICs decreased 16-fold and 32- to 64-fold compared with four- to eightfold, respectively, upon inactivation of the cmeB gene in LLR strains (Table 1). This difference was also seen for HLR strains since MICs of azithromycin remained high even in cmeB-inactivated mutants whereas MICs of tylosin and telithromycin were greatly affected (>128-fold decrease of MIC upon cmeB gene inactivation). In addition, MICs of telithromycin were 8- to 64-fold lower than MICs of erythromycin, azithromycin and tylosin in HLR strains, suggesting that this molecule was less affected by the A2075G mutation in the 23S rRNA genes than the other related molecules. This enhanced activity of telithromycin is probably due to a higher binding affinity to the 50S ribosomal unit enabled by the chemical structure of the molecule.11


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Table 1. Susceptibility to erythromycin (ERY), tylosin (TYL), azithromycin (AZM), telithromycin (TEL) and ciprofloxacin (CIP) and mutations in the 23S rRNA genes of C. coli wild-type strains and their derivative cmeB::kan knockout mutant

 

    Acknowledgements
 
We thank Francis Mégraud from the National Reference Center of Campylobacter (INSERM ERI 10, Laboratoire de Bacteriologie, C.H.U. Pellegrin, Bordeaux, France), Isabelle Kempf from the Agence Française de Sécurité Sanitaire des Aliments (Ploufragan, France) and Catherine Magras from the Ecole Nationale Vétérinaire of Nantes (UMR INRA/ENV 1014 SECALIM, Nantes, France) for providing the strains used in this study. CmeB antibodies and genomic DNA of the 81176 cmeB::kan mutant were kindly provided by Qijing Zhang (Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, USA). The technical assistance of Alice Château was also greatly appreciated.


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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3. Boyanova L, Gergova G, Spassova Z et al. Campylobacter infection in 682 Bulgarian patients with acute enterocolitis, inflammatory bowel disease, and other chronic intestinal diseases. Diagn Microbiol Infect Dis 2004; 49: 71–4.[CrossRef][ISI][Medline]

4. Rao D, Rao JR, Crothers E et al. Increased erythromycin resistance in clinical Campylobacter in Northern Ireland—an update. J Antimicrob Chemother 2005; 55: 395–6.[Free Full Text]

5. Taylor DE, Tracz DM. Mechanisms of antimicrobial resistance in Campylobacter. In: Ketley JM, Konkel ME, eds. Campylobacter: Molecular and Cellular Biology. Norfolk, UK: Horizon Bioscience, 2005; 193–204.

6. Lin J, Akiba M, Zhang Q. Multidrug efflux systems in Campylobacter. In: Ketley JM, Konkel ME, eds. Campylobacter: Molecular and Cellular Biology. Norfolk, UK: Horizon Bioscience, 2005; 205–18.

7. Payot S, Avrain L, Magras C et al. Relative contribution of target gene mutation and efflux to fluoroquinolone and erythromycin resistance, in French poultry and pig isolates of Campylobacter coli. Int J Antimicrob Agents 2004; 23: 468–72.[CrossRef][ISI][Medline]

8. van Vliet AH, Wood AC, Henderson J et al. Genetic manipulation of enteric Campylobacter species. In: Norris JR, Ribbons DW, eds. Methods in Microbiology. London: Academic Press, 1998; 407–19.

9. Payot S, Cloeckaert A, Chaslus-Dancla E. Selection and characterization of fluoroquinolone-resistant mutants of Campylobacter jejuni using enrofloxacin. Microb Drug Resist 2002; 8: 335–43.[CrossRef][ISI][Medline]

10. Mamelli L, Amoros JP, Pages JM et al. A phenylalanine-arginine ß-naphthylamide sensitive multidrug efflux pump involved in intrinsic and acquired resistance of Campylobacter to macrolides. Int J Antimicrob Agents 2003; 22: 237–41.[CrossRef][ISI][Medline]

11. Franceschi F, Kanyo Z, Sherer EC et al. Macrolide resistance from the ribosome perspective. Curr Drug Targets Infect Disord 2004; 4: 177–91.[CrossRef][Medline]





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