Involvement of efflux mechanisms in biocide resistance of Campylobacter jejuni and Campylobacter coli

Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
Journal of Medical Microbiology (Impact Factor: 2.25). 02/2012; 61(Pt 6):800-8. DOI: 10.1099/jmm.0.041467-0
Source: PubMed

ABSTRACT Active efflux has an important role in the antimicrobial resistance of Campylobacter jejuni and Campylobacter coli. The effects of two putative efflux pump inhibitors (EPIs), phenylalanine-arginine β-naphthylamide and 1-(1-naphthylmethyl)-piperazine, and the effects of inactivation of the cmeB,cmeF and cmeR genes on resistance to a broad range of antimicrobials were studied using the broth microdilution method. The antimicrobials tested in C. jejuni and C. coli were the biocides triclosan, benzalkonium chloride, chlorhexidine diacetate, cetylpyridinium chloride and trisodium phosphate, along with the anionic surfactant SDS and the antibiotics erythromycin and ciprofloxacin. Both EPIs partially reversed the resistance to all of these antimicrobials. Differences between these EPIs were seen for substrate preference and reductions in MIC. The MICs of the antimicrobials were reduced in the cmeB and cmeF mutants and increased in the cmeR mutant, with few exceptions. Both of these putative EPIs further decreased the MICs of the antimicrobials in these mutant strains. These data confirm that active efflux is an important mechanism in biocide resistance in C. jejuni and C. coli. At least one non-CmeABC efflux system or reduced uptake is responsible for resistance to biocides.

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    • "The minimum inhibitory concentrations (MICs) of erythromycin, ciprofloxacin, BC, CHA, CPC (≥98%, Sigma-Aldrich), TSP (96%, Sigma-Aldrich), TLN (99.5%, Merck) and SDS (99.5%, Invitrogen) were determined according to the broth microdilution method, in Mueller Hinton broth (MHB, Oxoid) with inocula of 10 6 bacteria/ml using 96-well microtiter plates, as described previously (Mavri and Smole Možina, 2012). The two-fold serial dilutions used included: for erythromycin , ciprofloxacin and TLN, concentrations of 0.016–512 μg/ml; for BC, CPC and CHA, concentrations of 0.016–4.0 "
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    ABSTRACT: The potential for adaptive resistance of Campylobacter jejuni and Campylobacter coli after step-wise exposure to increasing sub-inhibitory concentrations of five biocides as triclosan, benzalkonium chloride, cetylpyridinium chloride, chlorhexidine diacetate and trisodium phosphate, was investigated, to identify the mechanisms underlying resistance. The biocide resistance and cross-resistance to the antimicrobials erythromycin and ciprofloxacin, and to sodium dodecyl sulphate, were examined according to the broth microdilution method. The presence of active efflux was studied on the basis of restored sensitivity in the presence of the efflux pump inhibitors phenylalanine-arginine beta-naphthylamide, 1-(1-naphthylmethyl)-piperazine, cyanide 3-chlorophenylhydrazone, verapamil and reserpine. Changes in the outer membrane protein profiles and morphological changes in adapted strains were studied, as compared with the parent strains. Repeated exposure of C. jejuni and C. coli to biocides resulted in partial increases in tolerance to biocides itself, to other biocides and antimicrobial compounds. The developed resistance was stable for up to 10 passages in biocide-free medium. More than one type of active efflux was identified in adapted strains. These adapted strains showed different alterations to their outer membrane protein profiles, along with morphological changes. The data presented here suggest that different mechanisms are involved in adaptation to biocides and that this adaptation is unique to each strain of Campylobacter and does not result from a single species-specific mechanism.
    International journal of food microbiology 01/2013; 160(3):304-12. DOI:10.1016/j.ijfoodmicro.2012.11.006 · 3.08 Impact Factor
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    • "The involvement of efflux mechanisms in bacterial intrinsic and acquired resistance has been demonstrated mostly by the use of efflux-pump inhibitors (EPIs), such as 1-(1- naphthylmethyl)-piperazine (NMP) and the competitive inhibitor phenylalanine-arginine b-naphthylamide (PAbN). These EPIs enhance drug accumulation inside the bacterial cell, thereby increasing the bacterial susceptibility to antimicrobials (Kern et al., 2006; Martins et al., 2009b; Mavri & Smole Možina, 2012). Similarly, the use of effluxpump inducers, such as bile salts, can induce the expression of efflux genes (Hannula & Hänninen, 2008). "
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    ABSTRACT: Multidrug efflux pumps, such as CmeABC and CmeDEF, are involved in the resistance of Campylobacter to a broad spectrum of antimicrobials. The aim of our study was to analyse the effects of the putative efflux-pump inducers bile salts and sodium deoxycholate on the resistance of Campylobacter to biocides (triclosan, benzalkonium chloride, chlorhexidine diacetate, cetylpyridinium chloride and trisodium phosphate), sodium dodecyl sulphate and erythromycin. The involvement of the CmeABC and CmeDEF efflux pumps in this resistance was studied on the basis of the effects of the bile salts and sodium deoxycholate in Campylobacter cmeB, cmeF and cmeR mutants. The genetic variation in the cmeB gene was also examined, to see whether this polymorphism is related to the function of the efflux pump. In 15 Campylobacter jejuni and 23 Campylobacter coli strains, bile salts and sodium deoxycholate increased the MICs of benzalkonium chloride, chlorhexidine diacetate, cetylpyridinium chloride and sodium dodecyl sulphate, and decreased the MICs of triclosan, trisodium phosphate and erythromycin. Bile salts and sodium deoxycholate further decreased or increased the MICs of biocides and erythromycin in these cmeF and cmeR mutants. For cmeB polymorphism, 17 different cmeB-specific PCR-RFLP patterns were identified, as 6 within C. jejuni only, 9 within C. coli only, and two in both species. In conclusion, bile salts and sodium deoxycholate can increase or decrease bacterial resistance to structurally unrelated antimicrobials. The MIC increases in the cmeF and cmeR mutants indicate that at least one non-CmeABC efflux system is involved in resistance to biocides. This study indicates that the cmeB gene polymorphism identified is not associated with biocide and erythromycin resistance in Campylobacter.
    Journal of Medical Microbiology 11/2012; 62(Pt_3). DOI:10.1099/jmm.0.052316-0 · 2.25 Impact Factor
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    ABSTRACT: Importance of the field: Antibiotic resistance in bacterial pathogens has increased worldwide leading to treatment failures. Concerns have been raised about the use of biocides as a contributing factor to the risk of antimicrobial resistance (AMR) development. In vitro studies demonstrating increase in resistance have often been cited as evidence for increased risks. It is therefore important to understand the mechanisms of resistance employed by bacteria toward biocides used in consumer products and their potential to impart cross-resistance to therapeutic antibiotics. Areas covered: In this review, the mechanisms of resistance and cross-resistance reported in the literature toward biocides commonly used in consumer products are summarized. The physiological and molecular techniques used in describing and examining these mechanisms are reviewed and application of these techniques for systematic assessment of biocides for their potential to develop resistance and/or cross-resistance is discussed. Expert opinion: The guidelines in the usage of biocides in household or industrial purpose should be monitored and regulated to avoid the emergence of any MDR strains. The genetic and molecular methods to monitor the resistance development to biocides should be developed and included in preclinical and clinical studies.
    Expert Opinion on Investigational Drugs 12/2012; 22(2). DOI:10.1517/13543784.2013.748035 · 5.53 Impact Factor
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