JAC Advance Access originally published online on May 30, 2006
Journal of Antimicrobial Chemotherapy 2006 58(1):168-172; doi:10.1093/jac/dkl212
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High genetic variation in the multidrug transporter cmeB gene in Campylobacter jejuni and Campylobacter coli
Institut National de la Recherche Agronomique, UR1282 Infectiologie Animale Santé Publique (IASP-213) 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 3 March 2006; returned 3 April 2006; revised 2 May 2006; accepted 3 May 2006
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Abstract |
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Objectives: This study was conducted to examine the genetic variation occurring in the cmeB gene encoding the transporter component of the CmeABC efflux pump.
Methods: Expression of the CmeABC pump in 21 strains of Campylobacter jejuni and Campylobacter coli was studied by western-blot analysis. MIC determination was conducted in the presence or absence of an efflux pump inhibitor (EPI). Inactivation of the cmeB gene and sequencing of the cmeABC operon were performed for a single strain. The remaining strains were compared by RFLP analysis of the cmeB-specific PCR amplicon. The cmeB genes of two C. coli strains with different RFLP patterns were sequenced completely.
Results: Conflicting results were obtained in the western-blot analysis with anti-CmeB and anti-CmeC antibodies for one strain, whereas MIC determinations with EPI and cmeB gene inactivation confirmed the efflux pump's activity. The cmeB gene of this isolate showed only 78% nucleotide sequence identity with the sequence of reference strains. PCR–RFLP analysis identified 4 different patterns among the 5 C. jejuni and 14 different patterns among the 16 C. coli strains investigated. At the amino acid sequence level, variation was higher in the periplasmic loops of the transporter.
Conclusions: A total of 18 different cmeB-specific PCR–RFLP patterns were detected among the 21 C. jejuni and C. coli strains. These sequence variations might have an impact on the function and substrate recognition of this transporter. The sequence data obtained in this study will help to design suitable tools to study the presence or the expression of the gene cmeB.
Keywords: efflux , polymorphism , CmeABC , resistance
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Introduction |
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Campylobacter spp. are a leading cause of acute bacterial gastroenteritis throughout the world.1 Poultry and pigs are two known animal reservoirs of this zoonotic pathogen.
Macrolides and fluoroquinolones are the drugs of choice to treat severe Campylobacter infections in humans.1 However, the prevalence of fluoroquinolone-resistant Campylobacter strains is increasing worldwide2 and a rise of macrolide resistance was reported in human strains.3,4 In C. jejuni, Pumbwe and Piddock5 and Lin et al.6 both described a multidrug efflux system (CmeABC), belonging to the Resistance Nodulation Division (RND) family of transporters and conferring intrinsic resistance to various antimicrobials including fluoroquinolones and macrolides. This pump is widely distributed in Campylobacter, including C. coli, and is constitutively expressed.6–8
While studying CmeB expression by western blot in animal strains of Campylobacter, we surprisingly found one isolate giving a negative response. This prompted us to examine whether this isolate was deficient in the major efflux pump CmeABC or whether this negative response was due to a sequence variation in the transporter. The analysis was then extended to 20 other selected strains isolated from humans, pigs or poultry and to other sequences available in GenBank.
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Materials and methods |
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Bacterial strains, MIC determination and western-blot analysis
A total of 21 strains (including the NCTC 11168 and NCTC 81116 strains), with different levels of susceptibility for erythromycin, were included in the study (Table 1). Poultry strains were isolated in France by the Agence Française de Sécurité Sanitaire des Aliments, except the C356, C114 and 154KU strains isolated in The Netherlands. Pig strains were isolated by the Ecole Nationale Vétérinaire of Nantes. Strains NCTC 11168 and NCTC 81116 are human isolates (National Collection of Type Cultures, Colindale, London). Routine growth was performed as described previously.8
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MICs were determined by the agar dilution method as described previously.8 Breakpoints were those recommended by the French Antibiogram Committee (available at http://www.sfm.asso.fr/). The efflux pump inhibitor (EPI) Phe-Arg-ß-naphthylamide (Sigma Aldrich, St-Quentin-Fallavier, France) was incorporated in agar plates at a concentration of 20 mg/L.
Western-blot experiments were performed exactly as described previously.8
DNA manipulations and sequence analysis
Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France). The cmeAB, cmeB and cmeC genes were amplified using the Cj0366cRev (5'-CAAGCTGAAATCAAAAGAGT-3') and Cj0367cFwd (5'-TGGAATCAATAGCTCCAAAG-3') primers, the cmeA1078Fwd (5'-GAAGTTAAAGAAATTGGAGCACAATAA-3') and the cmeC47Rev (5'-TGGACTTAAAGAGCAAGCTGAAA-3') primers, and the CmeB 3009 (5'-TGGTGGAATGATCGCAGCATCAAC-3') and CmeC rev (5'-CCAGAAGAGGTATATAAGCAATTTTATC-3') primers, respectively. The pCR2.1 vector was used for cloning (TOPO TA cloning, Invitrogen, Cergy Pontoise, France). Inserts were sequenced by Genome Express (Meylan, France).
The cmeB::kan mutant of the 154KU strain was constructed by natural transformation with genomic DNA (1 µg) of a 81176 cmeB::kan mutant using the biphasic method.9
For PCR–RFLP experiments, the cmeB amplicons were analysed using the following restriction enzymes: HinfI, HindIII–PvuII, PvuII–XmnI, HaeII–HindIII, EcoRI–PstI, XmnI and EcoRV.
Transmembrane domains of the CmeB transporter were predicted through hydropathy analysis using the TMpred program (http://www.ch.embnet.org/cgi-bin/TMPRED_form_parser) and SOSUI (http://sosui.proteome.bio.tuat.ac.jp/sosui_submit.html). Antigenic index of the CmeB protein was calculated according to the Jameson–Wolf method using the 2DSweep program (http://genius.embnet.dkfz-heidelberg.de/menu/cgi-bin/w2h-open/w2h.open/w2h.startthis). Sequence variation at each amino acid position was quantified using the PsFind program.
Nucleotide sequence accession numbers
The gene sequences determined in the present study were deposited in GenBank under accession numbers DQ333454 (C. jejuni 154KU), DQ333455 (C. coli 596), DQ333456 (C. coli 275).
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Western-blot analysis of the CmeB and CmeC protein expression
Using polyclonal antibodies raised against peptides of the CmeB (amino acids 26–333) and CmeC (amino acids 41–248) proteins by Lin et al.,6 we analysed the expression of these proteins in 21 isolates. The CmeB protein was found to be expressed in all the strains examined, except in one isolate: the 154KU strain. Surprisingly, this strain expressed the CmeC outer membrane protein of the efflux system.
Involvement of the CmeABC efflux system in fluoroquinolone and macrolide resistance
Efflux was evaluated by measuring MICs in the presence of an EPI. MIC of erythromycin decreased 4- to 32-fold in the presence of EPI in the 21 strains examined (Table 1), even in the 154KU strain, suggesting the involvement of efflux in the intrinsic resistance to erythromycin in this strain. Inactivation of the cmeB gene in this strain led to a 16-fold decrease in the MIC of erythromycin and ciprofloxacin compared with the wild-type strain (Table 1), confirming the results obtained with the EPI.
Sequence analysis of the CmeABC operon of the 154KU strain
To explain the discrepancy observed between western-blot experiments and cmeB gene inactivation, we hypothesized that the CmeB sequence was different in the 154KU strain, affecting the epitopes targeted by the antibodies.
We therefore sequenced the cmeB gene of this strain and compared it with the cmeB sequence of strain 81–176 used for antibody production. Only 78% of identity was found, leading to 197 amino acid exchanges in the protein sequence for a total of 1040 amino acids (81% of identity) (Figure 1a). The modifications were distributed all over the sequence in particular in the large periplasmic loops (LPL) of the transporter, conferring substrate specificity in other RND transporters.10 In the first hydrophilic loop (308 amino acids, targeted by the antibodies used), 64 changes occurred, including one deletion at position 86, 3 changes in the characteristic A motif11 of the transporter and 42 changes in the most antigenic regions. Two changes also appeared in the characteristic RND C and D motifs and 41 changes in the second periplasmic loop (Figure 1a).
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The cmeA and cmeC genes of this strain were also sequenced. Only 7 amino acid changes (for a total of 368 amino acids) were found in the deduced CmeA sequence (98.1% identity with the NCTC 11168 and 81–176 CmeA sequences) and 16 (for a total of 493 amino acids) in the deduced CmeC sequence (96.6% identity with the reference CmeC sequences).
PCR–RFLP analysis of the cmeB polymorphism
The modifications in the cmeB sequence of the 154KU strain led to multiple changes in restriction sites. Based on these modifications, a PCR–RFLP was designed and used to study the cmeB polymorphism in 20 other strains (Table 1). Overall, 18 different patterns (Figure 1b) were observed. The major changes observed at positions 288, 920, 1273 of the cmeB sequence in the 154KU strain were found in other strains.
Comparison of the cmeB transporter sequence in C. jejuni and C. coli
The sequence of the cmeB gene was determined for two C. coli strains showing different restriction patterns (strains 275 and 596).
The deduced protein sequence was compared with the CmeB sequence (available in GenBank) of eight C. jejuni strains (NCTC 11168, 81–176, RM1221, 84–25, 260–94, CF93-6, HB 93–13 and the 154KU strain described above) and of two other C. coli strains (strains RM2228 and CIT 382). The pattern of sequence variation was different for the C. jejuni and C. coli groups. Diversity was observed all over the sequence but a greater variation was observed in the LPL of the transporter.
High sequence conservation was observed between the C. jejuni strains examined (98.5–100% of identity), except for the 154KU strain (only 80.3–81% of identity). Genetic variation of the CmeB protein sequence was higher between the C. jejuni and C. coli species than in the C. jejuni group (97.3% and 99.2% of identity, respectively). The 596 strain showed a higher sequence variation compared with the other C. coli strains (94.2% of amino acid sequence identity with the NCTC 11168 reference strain compared with 97.5%, 97.6% and 97.8% of identity for the RM2228, CIT382 and 275 strains, respectively).
Our results showed that sequence variation can exist in the cmeB gene of Campylobacter. This genetic variation could lead to false negatives when examining the presence of the cmeB gene by PCR or when studying the expression of the CmeABC efflux system by western blot or by RT–PCR. Indeed, recently, Olah et al.12 examined by PCR the prevalence of the cmeABC operon in Campylobacter strains isolated from turkeys. The primers located in the cmeB gene used for this study fell in regions subjected to modifications. Only 76% and 18% of the C. jejuni and C. coli isolates, respectively, tested positive in the present work and the authors concluded that the CmeABC efflux system appeared more prevalent in C. jejuni than in C. coli. The difference observed probably rather reflects a higher sequence divergence in the C. coli sequences and hence a higher number of false negative samples. Our results will thus be useful to design more appropriate tools for such studies.
In contrast to the results observed for the Campylobacter strains, the comparison of the sequences found in GenBank for the Escherichia coli and Salmonella AcrB proteins reveals high sequence conservation (99.9% of identity at the protein level). The polymorphism observed for the Campylobacter CmeB transporter could be related to the overall higher genetic diversity found in this bacterium, linked to its ability to acquire exogenous DNA by natural transformation and to undergo easily genetic recombination.13 This indicates at least that the CmeB transporter can tolerate sequence variation without being impaired in its function. The impact of the observed modifications on the function and on the substrate recognition of the efflux pump would thus be of interest for further investigation.
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None to declare.
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Acknowledgements |
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We thank Birgitta Duim and Dik Mevius (Institute for Animal Science and Health, Lelystad, The Netherlands), Isabelle Kempf from the Agence Française de Sécurité Sanitaire des Aliments (Ploufragan, France) and Catherine Magras from the Ecole Nationale Vétérinaire of Nantes for providing the strains used in the study. CmeB and CmeC 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 PsFind program used to analyse the CmeB sequence variation was kindly provided by Mark Achtman (Department of Molecular Biology, Max-Planck Institute of Infection Biology, Berlin, Germany). The technical assistance of Christian Mouline is also greatly appreciated.
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References |
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