Expression of the efflux pump genes cmeB, cmeF and the porin gene porA in multiple-antibiotic-resistant Campylobacter jejuni

Lilian Pumbwe1, Luke P. Randall2, Martin J. Woodward2 and Laura J. V. Piddock1,*

1 Antimicrobial Agents Research Group, Division of Immunity and Infection, University of Birmingham, Birmingham B15 2TT; 2 Department of Food and Environmental Safety, Veterinary Laboratory Agency (Weybridge), Woodham Lane, Addlestone, Surrey KT13 3NB, UK

Received 9 March 2004; returned 6 April 2004; revised 18 May 2004; accepted 18 May 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Aims: In Escherichia coli, increased expression of efflux pumps and/or decreased expression of porins can confer multiple antibiotic resistance (MAR), causing resistance to at least three unrelated classes of antibiotics, detergents and dyes. It was hypothesized that in Campylobacter jejuni, the efflux systems CmeABC, CmeDEF and the major outer membrane porin protein, MOMP (encoded by porA) could confer MAR.

Methods: The expression of cmeB, cmeF and porA in 32 MAR C. jejuni isolated from humans or poultry was determined by comparative (C)-reverse transcriptase (RT)-PCR and denaturing DHPLC. A further 13 ethidium bromide-resistant isolates and three control strains were also investigated. Accumulation of ciprofloxacin±carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) was also determined for all strains.

Results: Although resistance to ethidium bromide has been associated with MAR, expression of all three genes was similar in the ethidium bromide-resistant isolates. These data indicate that CmeB, CmeF and MOMP play no role in resistance to this agent in C. jejuni. Six MAR isolates over-expressed cmeB, 3/32 over-expressed cmeB and cmeF. No isolates over-expressed cmeF alone. Expression of porA was similar in all isolates. All nine isolates that over-expressed cmeB contained a mutation in cmeR, substituting glycine 86 with alanine. All cmeB over-expressing isolates also accumulated low concentrations of ciprofloxacin, which were restored to wild-type levels in the presence of CCCP.

Conclusions: These data indicate that over-expression of cmeB is associated with MAR in isolates of C. jejuni. However, as cmeB was over-expressed by only one-third (9/32) of MAR isolates, these data also indicate other mechanisms of MAR in C. jejuni.

Keywords: transporter , antibiotic resistance


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Campylobacter spp. are the most common cause of bacterial human gastro-enteritis worldwide.1,2 Most infections are due to C. jejuni (95–98%) and C. coli (2–5%).1,2 Human infection with Campylobacter is usually self-limiting but there can be serious sequelae that can be fatal.3 When therapy is indicated, the antibiotics of choice are usually erythromycin (a macrolide) or ciprofloxacin (a fluoroquinolone).1,2

Treatment of bacterial infection can be hindered by antibiotic resistance and multiple antibiotic resistance (MAR) is of increasing concern. MAR bacteria typically show resistance to at least three antibiotic classes, disinfectants, detergents and dyes.4 Efflux pumps in bacteria limit the access of antibiotics to their intracellular targets.4 CmeB, an RND efflux pump with homology to E. coli AcrB was identified in C. jejuni NCTC11168.5 This pump belonged to the CmeABC efflux system, where CmeA is the outer membrane channel protein and CmeC the integral membrane protein. The CmeABC system was shown to confer intrinsic resistance to antibiotics including ciprofloxacin, detergents and dyes, and enhanced expression was proposed to confer MAR in isolates of C. jejuni.5 Lin et al.6 confirmed that CmeABC conferred MAR and also bile resistance, and showed that CmeABC is essential for colonization of the chicken gut.7 A second RND efflux pump, CmeF, belonging to the CmeDEF system (where CmeD is the outer membrane protein and CmeE the integral membrane protein), has also been identified (Pumbwe & Piddock, submitted for publication). This system also confers MAR but apparently does not transport ciprofloxacin.

In E. coli, MAR has also been attributed to decreased expression of the outer membrane porin OmpF.8 Two outer membrane porin proteins have been characterized in Campylobacter spp., major outer membrane protein (MOMP), which is encoded by the gene porA, and Omp50.9,10 MOMP is a large channel protein (45 kDa) belonging to the trimeric porin family of which E. coli OmpF and OmpC are members, and has been implicated in intrinsic antibiotic resistance.11

In E. coli, MAR can arise after mutation in the local efflux pump regulators (e.g. acrR) or the global regulatory marRAB or soxRS loci. The mar and sox regulons regulate several different genes including those involved with antibiotic resistance such as ompF and acrB.8 The marA locus can reduce the level of OmpF by enhancing the expression of micF, an antisense RNA motif which interferes with translation of ompF mRNA.8 The marRAB operon is regulated by MarR, a repressor protein, which inhibits the expression of marA. MarA is a DNA binding protein, which binds a consensus sequence, the mar box, found upstream of genes responsive to marA. Similarly, the soxRS regulon is regulated by SoxR, with SoxS being the activator protein of the sox box.8 Inactivation of marR or soxR leads to the over-expression of marA or soxS, respectively. However, soxS is also induced by agents such as paraquat and menadione.8

We hypothesized that MAR isolates of C. jejuni over-express cmeB and/or cmeF, and that this over-expression would be mediated by a global regulatory gene such as marA or soxS as found for E. coli and other Gram-negative bacteria. Therefore, the aims of this study were (i) to determine whether changes in the expression of efflux pumps or porins were associated with ciprofloxacin resistance and/or MAR in C. jejuni isolated from humans or poultry, (ii) to determine whether any enhanced expression occurred simultaneously with mutations in gyrA and, (iii) to identify any homologues of marA or soxS global regulator genes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bacterial strains and growth media

Fifty-two strains of C. jejuni, including NCTC 11168 (P270), a ß-lactamase positive control (P1313), and tet(M) (P1312) and tet(O) (P1313) positive control strains were used in this study (Table 1).12 The remaining isolates were chosen as follows: from our collection of Campylobacter spp. obtained from a wide variety of sources comprising 700 isolates, 239 strains were ciprofloxacin-resistant of which 32 (16 human and 16 poultry) were also MAR. A further 13 (non-MAR ciprofloxacin-susceptible) ethidium bromide-resistant poultry isolates were investigated, as resistance to this agent in other bacteria has been associated with enhanced efflux. The remaining three strains P1038 (cmeB::KanR);5 P1043 (cmeF::kanR) and P1048 (selected on cefotaxime and over-expressed cmeB) were laboratory-created mutants. All strains were routinely cultivated under microaerophilic conditions (5% O2, 7.5% CO2, 85% N2) on Mueller–Hinton (MH) agar plates supplemented with 5% sterile horse blood.


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Table 1. Strains and isolates used in this study

 
Determination of antibiotic susceptibility profiles

The MICs of the antibiotics ampicillin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, tetracycline, the detergents sodium dodecyl sulphate (SDS) and sodium deoxycholate, and the dyes acridine orange and ethidium bromide were determined with the agar dilution method recently recommended for susceptibility testing of Campylobacter.13 The MIC was the lowest concentration of antibiotic (in mg/L agar) that inhibited growth. All agents were made up and used according to the manufacturers' instructions. All plates were incubated in 7.5% CO2 at 37°C for 48 h. The MIC was defined as the lowest concentration of antibiotic (in mg/L agar) at which no more than 10 colonies were detected.14 MAR was defined as resistance to at least three antibiotic classes, detergents and dyes. As there are no internationally recommended breakpoint concentrations available for Campylobacter, an MIC reproducibly greater than two-fold that for wild-type was considered resistant.

Measurement of accumulation of ciprofloxacin

The concentration of ciprofloxacin accumulated was measured as previously described.12 Accumulation experiments were carried out with and without the protonophore carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) added to a final concentration of 150 µM at the 5 min time point. All experiments were carried out in duplicate on at least three separate occasions. Data were analysed by Student's t-test, where a P value ≤0.05 was considered significant.

Detection of ß-lactamase, tet(O) and tet(M) genes

ß-Lactamase was detected by the nitrocefin spot test. A 100 mL liquid culture was grown to an OD600 of 0.2–0.4. The cultures were centrifuged, and the pellet washed twice in 10 mL ice-cold phosphate buffer (pH 7.0) and finally resuspended in 1 mL of buffer. The suspension was sonicated and then centrifuged for 30 min at 10 000 g. In a microtitre plate, a 50 µL aliquot of the supernatant was added to 10 µL of nitrocefin. An immediate colour change from yellow to red was interpreted as positive for ß-lactamase production. The presence of tet(M) and tet(O) was determined by PCR using conditions described previously.12

Genomic and sequence analysis of cmeR (Cj0368c), Cj1042c and the QRDR of gyrA or gyrB

In order to investigate the possibility of global regulation of multiple antibiotic resistance in Campylobacter spp., the genome sequence (http://www.sanger.ac.uk/projects/C-jejuni/) was explored for protein homologues of MarA and SoxS using tBLASTn (WU BLAST 2.0).15

DNA was isolated from 3 mL overnight culture using the DNAce spin cell mini kit (Bioline). PCR of the quinolone resistance determining regions (QRDRs) of gyrA (codons 38–126) and gyrB (codons 387–499) was carried out as previously described,14 and mutations in these regions detected by denaturing (D) HPLC (Transgenomic Inc.).16 Briefly, PCR products of the QRDRs from different isolates were mixed at a 1:1 ratio with those of wild-type (P270, NCTC 11168). The mixtures were denatured and then allowed to slowly re-anneal by ramping the temperature down to 35°C in 1°C/min steps. The products (5 µL) were immediately loaded on the DNasep cartridge (Transgenomic Inc.) and analysed at 58°C. DNA elution profiles were captured online and analysed using Navigator software (Transgenomic Inc.). Amplimers corresponding to novel DHPLC patterns were confirmed by sequencing using the ABI prism Big Dye Terminator version 3.0 cycle sequencing kit (Applied Biosystems). Cycle sequence products were analysed on an ABI Prism 3700 sequencer (Functional Genomics Laboratory, University of Birmingham). The coding region plus 100 bases upstream and downstream of cmeR was also sequenced as above with two sets of primers to cover the entire region (Table 2).


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Table 2. Oligonucleotide primers used in this study

 
Expression of cmeB, cmeF and porA

Bacteria were cultured overnight in MH broth. Each culture was diluted to give a 4% inoculum in 10 mL, and incubated further to OD600 0.2–0.4. The cultures were harvested by centrifugation at 4°C. Total RNA was isolated using the RNAce spin cell mini kit (Bioline). Four independent RNA samples were prepared from each strain. Residual DNA was removed by treatment with RNase-free DNase I (Roche) at 37°C for 15 min, and RNA samples confirmed to be DNA-free by PCR. RNA concentration and purity were measured using GeneTools software (SynGene) against standards of known concentrations. Four sets of complementary DNA (cDNA) were synthesized from each DNA-free RNA template using Superscript II H-Reverse transcriptase (Invitrogen) and random hexamer primers (Invitrogen) according to the manufacturers’ instructions. Second strand PCR amplification of the cDNA was carried out with primers designed to amplify 100–600 bp products within the coding regions of the respective genes (Table 2). All primer sets amplified the cDNA independently of each other. Comparative RT–PCR was used to determine expression of each gene essentially as described by Eaves et al.17 first relative to the housekeeping gene 16S rRNA of each isolate, and then compared with expression by P270 (NCTC 11168).17 To amplify 16S rRNA, cDNA was diluted 1/1000, and 2 µL used in a 50 µL reaction containing master mix (1.5 mM MgCl2) (ABgeneR), primers (0.25 µM each). cmeB and cmeF were amplified in a multiplex PCR reaction (50 µL) containing master mix, neat cDNA (2 µL), cmeB primers (0.25 µM each) and cmeF primers (0.5 µM each). porA was amplified in 50 µL PCR reaction containing master mix, cDNA diluted 1/2 (2 µL) and porA primers (0.25 µM each). Amplification was carried out in a Techne thermal cycler at 95°C for 5 min (initial denaturation), 30 cycles of 95°C (1 min), 52°C (1 min), 72°C (1 min) and a final extension of 72°C (10 min). After amplification, the 16S rRNA PCR products were mixed 1:1 with the cmeB/cmeF multiplex PCR products or the porA products and 20 µL loaded on a DNASep cartridge and quantified by DHPLC analysis, using the Wave DNA fragment analysis system (Transgenomic Inc., USA). DNA fragment elution profiles were captured online and quantified as peak areas using Navigator software (Transgenomic Inc.). 16S rRNA was amplified 10 times from C. jejuni NCTC 11168 and a reference mean ± S.D. of the peak area determined. Differential gene expression by different isolates was normalized by comparison to the reference. Expression data were calculated as mean ± S.D. from four independent RNA isolations and cDNA PCR amplifications. A change of greater than 50% compared to wild-type was considered different. Comparisons were validated by Student's t-test, where a P value ≤0.05 was considered significant.

The expression of porA was confirmed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) from cells grown to late logarithmic phase (OD600 0.2–0.4).18


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Antibiotic susceptibility and analysis of the QRDRs of gyrA or gyrB

Compared with C. jejuni NCTC 11168 (P270) and P1038 (cmeB::kanR), all MAR isolates and the cefotaxime-selected MAR mutant (P1048) were resistant to at least three different antibiotics plus disinfectants, detergents and dyes. The MIC patterns are exemplified by the CmeB-over-expressing isolates (Table 3). All the MAR human and animal isolates were resistant to ciprofloxacin and the dyes acridine orange and ethidium bromide. The ciprofloxacin MICs were generally high (64–128 mg/L). Four animal isolates (P283, P284, P301 and P327) were resistant to ampicillin (≥64 mg/L). All these isolates also produced a ß-lactamase. Eight human isolates (P68-P72, P88, P129 and P174) were resistant to tetracycline (128 mg/L). None of the isolates were positive for tet(M). However, tet(O) was detected in all these isolates. Six isolates (P153, P161, P174, P281, P282 and P338) were highly resistant to ampicillin and tetracycline (MICs 64–128 mg/L). Of these, only P281, P282 and P338 produced a ß-lactamase; all were positive for tet(O). Only three human isolates (P153, P16 and P174) produced a ß-lactamase at a level similar to that seen for the eight animal isolates. Two animal isolates (P271, P301) were resistant to kanamycin (MICs 128 mg/L), possibly due to an aminoglycoside-modifying enzyme.


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Table 3. Susceptibility of cmeB over-expressing isolates to antibiotics, detergents and dyes

 
A third (6/19) of ethidium bromide-resistant isolates also had a MAR phenotype but for these isolates, the phenotype did not include ciprofloxacin or tetracycline resistance (data not shown).

DHPLC mutation detection and DNA sequencing showed that 24/32 of MAR human and animal isolates contained a mutation in gyrA. This conferred the substitution of threonine 86 with isoleucine in the QRDR of GyrA. Eight isolates (P88, P129, P271, P1157-P1160 and P1170) were MAR and ciprofloxacin-resistant but had no mutation in gyrA. None of the isolates had a mutation in gyrB. P1048 did not have a mutation in either gyrA or gyrB (data not shown).

Expression of cmeB, cmeF and porA

Of the MAR isolates, 6/32 (P71, P72, P88, P129, P271, P1157) over-expressed cmeB alone, three (P71, P72, P153) of these also had a gyrA mutation. The fold-increase compared with cmeB expression by NCTC 11168 (P270) ranged from 1.7 to 2.2: P71 (1.8-fold), P72 (1.9-fold), P88 (2.2-fold), P129 (2.2-fold), P271 (1.7-fold) and P1157 (1.9-fold) (Figure 1a). Three isolates over-expressed both cmeB and cmeF and also had a mutation in the gyrA QRDR. These were P153 (1.9-fold increase in cmeB expression and 1.7-fold increase in cmeF expression, respectively), P161 (2.9-fold and 2.1-fold, respectively) and P174 (2.5-fold and 2.1-fold, respectively) (Figure 1a). Twenty-two isolates had similar levels of expression of cmeB or cmeF to NCTC 11168 (P270). None of the ethidium bromide-resistant isolates over-expressed cmeB or cmeF (data not shown). P1048, the laboratory-derived MAR mutant over-expressed cmeB alone (2.35-fold) (Figure 1a). porA expression in all MAR isolates, ethidium bromide-resistant isolates, and laboratory-created mutants was similar to NCTC 11168 (P270) (wild-type) (data not shown).



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Figure 1. (a) Expression of cmeB (black bars), and cmeF (white bars), measured by comparative RT-PCR. PCR products were analysed by denaturing HPLC and peak areas quantified. Data show only those strains and isolates where changes were observed compared to NCTC 11168 (P270). Values shown are the mean±S.D. from eight independent experiments. All except P279, P280 and P301 expressed statistically significantly higher levels of cmeB than P270 (P<0.05). Three isolates (P153, P161 and P174) also expressed significantly higher levels of cmeF than P270. (b) Accumulation of ciprofloxacin–CCCP (black bars) or + CCCP (white bars) by NCTC 11168 (P270), cmeB over-expressing isolates (P71, P72, P88, P129, P153, P161, P174 and P1157) and three MAR isolates (P279, P280 and P301) that do not over-express cmeB. Values are from five independent experiments expressed as mean±S.D. All isolates shown, except three (P279, P280 and P301) accumulated statistically significantly lower levels of ciprofloxacin than P270 (P<0.05). The effect of CCCP was also significantly increased in these isolates compared to P270.

 
Relationship between gyrA mutation, cmeB/cmeF over-expression and MICs

Four phenotypes were observed from the susceptibility testing and efflux pump expression data. The first phenotype comprised MAR isolates (19/32) that had a gyrA mutation but did not over-express cmeB or cmeF. The ciprofloxacin MICs for these isolates ranged from 8 to 64 mg/L. The second phenotype comprised MAR isolates (6/32) with a gyrA mutation and also over-expressed cmeB or cmeB plus cmeF. These isolates were inhibited by 64–128 mg/L ciprofloxacin. The third phenotype comprised isolates (4/32) that had no mutation in gyrA but over-expressed cmeB or cmeB and cmeF. The MICs of ciprofloxacin for these isolates were 2–32 mg/L. The fourth phenotype comprised isolates (4/32) that had no gyrA mutations or cmeB over-expression. These isolates were inhibited by similar ciprofloxacin MICs to wild-type (0.25–0.5 mg/L). The MAR phenotype of these isolates did not include ciprofloxacin as a substrate.

Accumulation of ciprofloxacin

All cmeB over-expressing isolates accumulated significantly less ciprofloxacin than wild-type (P<0.05). The protonophore CCCP restored these concentrations to those seen for NCTC 11168 (Figure 1b). Three MAR isolates (P279, P280 and P301) that did not over-express cmeB or cmeF accumulated similar concentrations of ciprofloxacin as did NCTC 11168.

marA, soxS and micF homologues in Campylobacter spp

tBLASTn amino acid sequence homology searches against the C. jejuni NCTC 11168 genome for homologues of MarA, SoxS and a genome search for micF gave no hits even at a low stringency. These data indicated that the Campylobacter genome did not possess homologues of these global regulators and that a micF homologue was unlikely to effect expression of porA into MOMP. However, a search of the C. jejuni NCTC 11168 genome sequence for putative regulators revealed an ORF Cj1042 located 12 665 bases upstream of the cmeDEF operon. The predicted protein product of this gene belongs to the same family of proteins as MarA (AraC-family), although the amino acid sequence identity of the protein product is only 17% of that of MarA and 16% of that of SoxS.

Mutations in cmeR and Cj1042

Sequence analysis of the putative regulator cmeR of the laboratory-selected mutant that over-expressed cmeB (P1048) revealed that it contained two mutations, one leading to the substitution of glutamine 9 with proline and the other substituting glycine 86 with alanine. Upon comparison with E. coli AcrR, these substitutions were at conserved residues predicted on the basis of the AcrR structure and within the substrate-binding region. Of the nine cmeB over-expressing isolates, one (P161) had both the above substitutions, and eight (P71, P72, P88, P129, P153, P174, P271 and P1157) contained the glycine 86 to alanine substitution alone. No substitutions were detected in the Cj1042 protein product.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
As with E. coli, the number of ciprofloxacin-resistant and MAR isolates of Campylobacter spp. have increased over the past few years. Although the mechanisms of resistance in E. coli are considered well characterized, those in Campylobacter spp. are still not fully understood. In 2003, there were 43 243 laboratory confirmed cases of Campylobacter infection in England and Wales. Approximately 15% (6486) were resistant to ciprofloxacin (Health Protection Agency). From our laboratory collection, 32/239 (13.4%) such strains were MAR. An understanding of the mechanism(s) of MAR is important so that alternative eradication and/or therapeutic (should treatment be necessary) strategies can be devised. In E. coli, efflux pumps and porins have been shown to be major contributors to MAR. In Campylobacter, the efflux pump CmeB, and the major porin MOMP have been shown to be widely distributed in various wild-type strains from different sources.6,11 CmeB, CmeF and MOMP are involved in transport of a variety of antibiotics, detergents and dyes in wild-type Campylobacter spp.5,11 However, no investigation of their role in MAR had been carried out.

From this study, 6/32 MAR isolates over-expressed cmeB alone and 3/32 over-expressed both cmeB and cmeF. Three non-gyrA mutants had a MAR phenotype and over-expressed cmeB, showing that MAR and cmeB over-expression were not associated with mutations in gyrA. However, the MIC values showed that when CmeB over-expression and gyrA mutations occurred simultaneously, there was a greater decrease in ciprofloxacin susceptibility than when either was present alone. Occurrence of gyrA mutations was evenly distributed between human and poultry isolates, whereas occurrence of cmeB over-expression was greater in human than poultry isolates. ParC or ParE were not investigated as they have not been identified in Campylobacter spp.14 Overall, over-expression of cmeB was detected more frequently than that of cmeF, and over-expression of cmeF did not occur without that of cmeB. Although the changes in gene expression could be considered modest, recent publications on microarrays coupled with physiological data reveal that relatively modest gene expression changes can have huge physiological impact.19,20 Similar expression changes have also been observed for efflux pump mutants of Salmonella enterica.17 Compared with other bacteria, the Campylobacter genome is small and has very few local regulators, with most genes being co-regulated.15 The cmeABC operon has an upstream tetR-like regulator, cmeR (Cj0368),5,6 and mutations in this gene were detected in all 10 isolates that over-expressed cmeB. These data indicate that mutation in cmeR leads to a derepression of cmeABC, resulting in over-expression of cmeB. A recent conference report has shown that a cmeR knockout mutant also displays an increase in the expression of cmeB, supporting the hypothetical role of CmeR.21 No regulator has been identified for the cmeDEF locus.

Expression of porA was unaltered in all the MAR isolates, suggesting little or no role of MOMP in MAR in Campylobacter spp. The protein product of the putative regulator, Cj1042c belongs to the same family of AraC-type proteins as MarA and also has a predicted helix-turn-helix (HTH) motif. However, no mutations in Cj1042c were found for any of the isolates. In addition, the predicted amino acid sequence homology between the Cj1042c protein produced and MarA was poor. The overall HTH-motif structure of the Dj1042c protein product could suggest that it may have the same function as MarA, which binds DNA through the motif. In Salmonella, it has been shown that knocking out AcrB results in increased expression of AcrF.17 Disruption of cmeB did not affect expression levels of cmeF and vice versa, or porA expression, suggesting that these genes are not co-regulated by either local or global regulator genes (Pumbwe & Piddock, submitted for publication). There was also no association between the differential expression of cmeB, cmeF or porA in the different isolates, further suggesting that these genes were not co-regulated.

Randall et al.12 found that resistance to the dyes acridine orange and ethidium bromide was highly associated with MAR, suggesting that dyes were a good indicator for MAR. However, data from this study suggest that the overall resistance phenotype of some of the isolates was due to multiple mechanisms which was also associated on some occasions with increased expression of cmeB. Owing to the presence of multiple mechanisms of antibiotic resistance, identification of isolates of Campylobacter that over-express an efflux pump(s) cannot easily be made by analysis of MIC data. Agents that can indicate increased expression of efflux pumps are being sought. Data from this study also suggest that, in the absence of multiple mechanisms, the two RND efflux pumps of C. jejuni are not the sole contributors to MAR or dye resistance in this species. As the C. jejuni genome contains genes encoding at least eight other efflux pumps, their role in ciprofloxacin resistance and MAR now warrants further investigation.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Dr D. Griggs for help revising this manuscript. We are grateful to the Department of Environment, Food and Rural Affairs (formerly The Ministry for Agriculture, Fisheries and Food) for project grant OD2004. The Functional Genomics Laboratory is funded by a BBSRC grant (6/JIF13209). LJVP is a recipient of the Bristol-Myers Squibb Non-restricted Grant in Infectious Diseases.


    Footnotes
 
* Corresponding author. Tel: +44-121-414-6966; Fax: +44-121-414-6815; Email: l.j.v.piddock{at}bham.ac.uk


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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