1 Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB; 2 Antimicrobial Agents Research Group, Division of Immunity and Infection, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Received 16 April 2003; returned 2 June 2003; revised 16 June 2003; accepted 17 June 2003
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Abstract |
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Materials and methods: The MICs of ampicillin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, tetracycline, cetrimide, triclosan, acridine orange, paraquat and ethidium bromide were determined. Resistance to organic solvents and the effect of salicylate (known inducer of the marRAB operon in Escherichia coli and Salmonella) were also examined.
Results: Two C. coli and 13 C. jejuni isolates, mainly from pigs or poultry, were resistant to three or more antibiotics and 12 of these strains had reduced susceptibility to acridine orange and/or ethidium bromide. Strains (n = 20) that were less susceptible to acridine orange, ethidium bromide and triclosan were significantly more resistant (P < 0.05) to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, nalidixic acid and tetracycline, with two- to four-fold increases in MIC values compared with strains (n = 20) most susceptible to acridine orange, ethidium bromide and triclosan. Growth of strains with 1 mM salicylate caused a small (up to two-fold) but statistically significant (P 0.005) increase in the MICs of chloramphenicol, ciprofloxacin, erythromycin and tetracycline.
Conclusions: These data indicate that multiple antibiotic resistant (MAR)-like Campylobacter strains occur and it may be postulated that these may overexpress cmeABC or another efflux system.
Keywords: efflux, cmeABC, salicylate, ethidium bromide
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Introduction |
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A multidrug-resistant laboratory mutant of Campylobacter jejuni (resistant to erythromycin, chloramphenicol, tetracycline, ß-lactams and quinolones) was proposed to be an efflux pump mutant.4 In Escherichia coli and Salmonella enterica strains, the AcrAB efflux pump shows broad substrate specificity and overexpression is associated with resistance to multiple antibiotics, disinfectants, dyes, detergents and organic solvents.5 Additionally, AcrAB has been shown to be up-regulated by up-regulation of marRAB and/or soxRS.5 Strains with the phenotype of resistance to multiple antibiotics, disinfectants, dyes, detergents and organic solvents are generally referred to as MAR mutants.5 A multidrug efflux pump, CmeABC, has recently been identified and described in C. jejuni.6 The amino acid sequence of CmeB shows a 52.6% similarity to the amino acid sequence of AcrB in E. coli.6 In wild-type C. jejuni this pump was shown to mediate a two- to four-fold change in susceptibility to ampicillin, ciprofloxacin, erythromycin, tetracycline, ethidium bromide and acridine orange and to be involved in active efflux of ciprofloxacin and ethidium bromide.6,7
The aim of this study was to survey campylobacters from a range of sources for resistance to antibiotics, disinfectants, dyes and organic solvents to determine the frequency of strains with a multiple resistance phenotype that may be indicative of overexpression of an efflux pump.
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Materials and methods |
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C. jejuni (n = 391) and C. coli (n = 52) isolated in the UK between 1997 and 2000 were used throughout and these were of animal and human origin. Strains obtained from the National Collection of Type Cultures (NCTC), Public Heath Laboratory Service, Colindale, London, UK were used as controls for the MICs.
Antibiotics, disinfectants, dyes, salicylate and organic solvents
All antibiotics, disinfectants, dyes, paraquat, organic solvents and salicylate were obtained from SigmaAldrich (Poole, Dorset, UK) with the exception of ciprofloxacin, which was kindly donated by Bayer (Newbury, Berks, UK), and the disinfectant triclosan (Irgasan DP300), which was kindly donated by Ciba Speciality Chemicals (Macclesfield, Cheshire, UK).
Determination of the MICs of antimicrobials, disinfectants, dyes and paraquat
MICs were determined according to the NCCLS agar dilution method.8 MICs were determined for some strains (n = 45) with and without 1 mM salicylic acid already in the agar. To determine the percentage of strains resistant to each of the antimicrobials, breakpoint concentrations published by the British Society for Antimicrobial Chemotherapy (BSAC) were used except for erythromycin, for which a previously described breakpoint concentration was used.9 The following cut-off concentrations denoted resistance: ampicillin > 8 mg/L, chloramphenicol > 8 mg/L, ciprofloxacin > 1 mg/L, erythromycin > 8 mg/L, kanamycin > 16 mg/L, nalidixic acid > 16 mg/L, tetracycline > 8 mg/L.
Organic solvent resistance
The method for organic solvent resistance was based on that of Asako et al.10 adapted to the growth conditions of Campylobacter.
Statistical analysis
To determine whether growth in the presence of salicylate significantly increased antibiotic resistance, the log10 MIC values of strains grown with and without salicylate were compared using a paired t-test. To determine whether there was an association between resistance to disinfectants, dyes or detergents (which might indicate an efflux type resistance mechanism) and resistance to the antibiotics tested, the following analyses were carried out. Strains positive for a tet resistance gene or positive for ß-lactamase production were excluded from analysis. Strains were then ranked in ascending order of resistance to a specific non-antibiotic. The MICs of antibiotics of the 20 strains most susceptible to a non-antibiotic (e.g. ethidium bromide) were compared with the MICs of antibiotics of the 20 strains most resistant to that non-antibiotic by calculating the geometric mean MIC and carrying out a paired t-test on the log10 antibiotic MIC values. To determine whether C. coli isolates were more resistant than C. jejuni isolates for all the agents tested, the MIC values of each agent were compared using a WilcoxonMannWhitney non-parametric test. The test was carried out using the software package StatXact (Cytel Software Corporation, Cambridge, MA, USA).
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Results |
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Overall, more strains were resistant to ampicillin and tetracycline than to any other agent tested, with fewest resistant to kanamycin (data not shown). However, 34.6% of the C. coli strains were resistant to erythromycin (data not shown). As the emphasis of this study was the occurrence of the MAR-like phenotype in Campylobacter and as strains were collected from a limited number of sources, a detailed breakdown of resistances to individual antibiotics is not given.
The WilcoxonMannWhitney test for all agents (except for organic solvents) showed that the C. coli strains were significantly more resistant than C. jejuni to all the agents tested except cetrimide, ciprofloxacin and acridine orange. For ciprofloxacin and acridine orange, there was no significant difference between the MICs for Campylobacter species. For cetrimide, C. jejuni were significantly more resistant than the C. coli.
All isolates tested were susceptible to cyclohexane and hexane (data not shown). All, except one strain of C. jejuni, were resistant to nonane (data not shown). Only two strains of C. coli and four strains of C. jejuni were resistant to cyclo-octane (data not shown). C. fetus NCTC 10842 was also resistant to cyclo-octane.
Multiple antibiotic resistance
Two C. coli and 13 C. jejuni isolates, mainly of porcine or poultry origin, were multidrug-resistant (MDR) with resistance to three antibiotics or more in different combinations (Table 1). However, most of the multi-resistant isolates also showed resistance (resistance calculated as MIC values of MIC90 value of that compound for C. jejuni isolates) to acridine orange and/or ethidium bromide, but this was not determined for other non-antibiotics. Further analysis (see Materials and methods) showed strains that were most resistant to acridine orange, ethidium bromide and triclosan were also significantly (P < 0.05) more resistant to some antibiotics compared with strains that were most susceptible to acridine orange, ethidium bromide and triclosan (Table 2). However, the differences in mean antibiotic MICs were only two- to four-fold (Table 2).
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BLAST searches for marRAB and soxRS genes and their products in C. jejuni at both low and high stringency revealed no hits, suggesting that homologues do not exist in C. jejuni.
Effect of salicylate on antimicrobial susceptibility
The MICs of salicylate were 48 mM for most strains tested (data not shown). A final concentration of 1 mM was used, as this concentration did not have a significant inhibitory effect on growth. Growth of strains with salicylate caused a small (less than or equal to two-fold), but significant increase in MICs of chloramphenicol (P = 0.005), ciprofloxacin (P < 0.001), erythromycin (P < 0.001) and tetracycline (P < 0.001) (data not shown). No effect was seen for ampicillin and kanamycin (data not shown).
Presence of ß-lactamase and/or tet(O) and tet(M) genes
Strains resistant to >8 mg/L ampicillin (the breakpoint concentration) were tested for the presence of ß-lactamase. Overall, 131/169 (78%) strains with ampicillin MICs of >8 mg/L were positive for ß-lactamase (data not shown).
PCR was used to detect the presence of tet(O) and tet(M) in Campylobacter strains resistant to >8 mg/L tetracycline (the breakpoint concentration). Overall, 97/128 (76%) strains were positive for tet(O) and none of the strains was positive for tet(M).
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Discussion |
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Growth of Campylobacter with salicylate caused a slight, but statistically significant, increase in the MICs of chloramphenicol, ciprofloxacin, erythromycin and tetracycline. Salicylate is the most potent inducer of the marRAB operon in E. coli by binding to the repressor MarR, resulting in increased expression of marA, which in turn increases expression of soxRS and acrAB.5 Hence, these data indicate that a similar operon may exist in Campylobacter. Although searches showed no homologues between E. coli marRAB and soxRS genes in C. jejuni, the cmeABC gene in Campylobacter is a homologue of the acrAB efflux pump gene of E. coli and Salmonella, and the cmeABC gene has been shown to be involved in the MAR phenotype in Campylobacter.6,7 Alternatively, salicylate induced other unrelated factors in Campylobacter that were associated with reduced susceptibility or, less likely, this phenotype reflected subtle differences in growth rates only.
In this study, only 3.8% of C. coli and 3.3% of C. jejuni were found to be resistant to three or more antibiotics. Whilst the data indicate that the multiple mechanisms of resistance confer the MDR phenotype in Campylobacter, the intriguing data showing an association between decreased susceptibility to acridine orange, ethidium bromide and triclosan with a significant increase in resistance to some antibiotics suggest another mechanism, possibly efflux, may also play a role. As multi-resistant E. coli have been shown to possess multiple mechanisms of resistance and have enhanced efflux,11 current work is focusing on determining the overexpression of efflux pump genes.
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Acknowledgements |
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Footnotes |
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References |
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