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Mechanisms of fluoroquinolone and macrolide resistance in Campylobacter spp.
Microbes and Infection 8 (2006) 1967e1971 www.elsevier.com/locate/micinf
Forum on antimicrobial resistance
Mechanisms of fluoroquinolone and macrolide resistance in Campylobacter spp. Sophie Payot a,*, Jean-Michel Bolla b, Deborah Corcoran c, Se´amus Fanning c, Francis Me´graud d, Qijing Zhang e a
Institut National de la Recherche Agronomique, UR086 BioAgresseurs, Sante´, Environnement, 37380 Nouzilly, France b EA 2197, IFR48, Faculte´ de Me´decine, 27 Bd Jean Moulin, F-13385 Marseille Cedex, France c Centre for Food Safety, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland d INSERM ERI 10, Centre National de Re´fe´rence des Campylobacters, Universite´ Victor Segalen, Bordeaux 2, 33076 Bordeaux, France e Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50011, USA Available online 29 March 2006
Abstract The incidence of human Campylobacter infections is increasing worldwide, as well as the proportion of isolates resistant to fluoroquinolones and/or macrolides, the drugs of choice to treat campylobacteriosis. In this review, we report recent developments in the understanding of the resistance mechanisms to fluoroquinolones and macrolides. In addition, we will discuss the recent findings on multidrug resistance in Campylobacter spp. Ó 2006 Elsevier SAS. All rights reserved. Keywords: Campylobacter; Antibiotic resistance mechanisms; Efflux pumps; Fluoroquinolones; Macrolides
1. Introduction
2. Mechanisms of fluoroquinolone resistance
Campylobacter jejuni and C. coli are recognized as major causes of human gastroenteritis worldwide [1,2]. Campylobacteriosis is usually a self-limiting disease, and antimicrobial therapy is required only for severe, prolonged or systemic infections or to control infection in high-risk groups. In these cases, macrolides and fluoroquinolones are the drugs of choice for treatment [2]. However, resistance to these antibiotics, especially to fluoroquinolones, is on the rise in many countries, and this could compromise future treatment (see accompanying review ‘‘The epidemiology of antibiotic resistance in Campylobacter’’ [3]). In this article, we will review the mechanisms of antimicrobial resistance developed by Campylobacter, focusing on fluoroquinolone and macrolide resistance as well as multidrug resistance.
Quinolones available for clinical use are classified into four generations, according to their spectrum of activity [4]. The second generation of quinolones, fluoroquinolones (including ciprofloxacin and levofloxacin), derived from the first compounds by a fluorine substitution at position 6, thereby increasing their activity against Gram-negative bacteria. A substantial improvement in activity against Gram-positive bacteria occurred with the introduction of third-generation molecules (including moxifloxacin) containing new substitutions at positions 7 and 8 [4]. Three mechanisms providing resistance to quinolones have been described in Gram-negative bacteria. These include target mutations, reduced antibiotic intracellular accumulation (by lowering outer membrane permeability or increasing efflux activity), and target protection mediated by the Qnr protein [5]. The plasmid-borne qnr gene, confers a low level of resistance to fluoroquinolones in other bacterial species, but has not been described in Campylobacter.
* Corresponding author. Tel.: þ33 2 47 42 79 88; fax: þ33 2 47 42 77 74. E-mail address: [email protected] (S. Payot). 1286-4579/$ - see front matter Ó 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2005.12.032
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2.1. Target mutations
2.2. Decrease in antibiotic intracellular concentration
In Gram-negative bacteria, DNA gyrase (also known as topoisomerase II) and DNA topoisomerase IV are the primary and secondary targets of fluoroquinolones [4]. The corresponding gene products are large enzymatic quaternary structures consisting of two pairs of subunits, named GyrA and GyrB (DNA gyrase), and ParC and ParE (topoisomerase IV), respectively. Resistance to this class of antimicrobial agents arises from amino acid(s) substitution(s) in a target region of the corresponding topoisomerase termed the ‘‘quinolone resistance determining region’’ (QRDR). The latter is located within the DNA-binding domain on the surface of these enzymes. In Escherichia coli and Salmonella spp., common mutations have been reported located at positions 83 and 87 in the QRDR region of the GyrA subunit. Once a first-step mutation has occurred, the organism exhibits a phenotype consistent with a reduction in susceptibility to these agents. Additional mutations in gyrA and in gyrB or parC can further increase the level of resistance [5]. In Campylobacter, there is increasing evidence that the secondary target (topoisomerase IV) is absent. Only one report of a parC homologue exists in the literature [6], and in this case the sequence was highly similar to the corresponding gene in E. coli (95% nucleotide identity) with a percentage of GC higher than expected for Campylobacter (52% GC compared to a mean percentage of GC from 29.6% to 34.5% for the current Campylobacter genomes [7,8]). Furthermore, several laboratories have reported experimental attempts to amplify parC; all were unsuccessful [9e13] even when using the same C. jejuni strain and primers previously described in the parC report (Payot et al., Corcoran et al., unpublished data). In addition, searches of the annotated genomes failed to identify a parC homologue, suggesting that this gene is absent from the sequenced genomes of C. jejuni NCTC 11168 [8], and RM1221 [7] and C. coli RM2228 [7] [a tBLASTn analysis on these genomes using amino acid sequence of the reported ParC sequence of C. jejuni or ParC of E. coli found no homologue (Payot, unpublished data)]. The absence of a secondary target for fluoroquinolones in Campylobacter leads to a situation where a unique modification in the GyrA subunit (Thr86Ile, corresponding to position 83 in GyrA of E. coli or Salmonella) is sufficient to confer a resistant phenotype to fluoroquinolones in C. jejuni and C. coli [9e14]. Other modifications of the GyrA subunit have also been reported to be associated with quinolone resistance. These include Asp90Asn [9,11e14] and Thr86Lys [13] associated with moderate resistance (MIC ¼ 8e16 mg/ml) and the less frequent Thr86Ala (associated with a MIC of 2 mg/ml for ciprofloxacin) [9], Thr86Val [12] and Asp90Tyr [9]. Double mutations resulting in two amino acid modifications were also reported: Thr86Ile-Pro104Ser [12] and Thr86Ile-Asp90Asn [11,15]). This latter conferred a higher resistance level to moxifloxacin, whereas a single Thr86Ile mutant was still susceptible to this third-generation molecule [9,15]. No mutations in the GyrB subunit have been described in Campylobacter at the time of writing [9e12].
Modification of the outer membrane permeability as a mechanism of resistance to antibiotics has not yet been described in Campylobacter. Two porins have been characterized in C. jejuni: the major outer membrane protein (MOMP) [16] and a minor one (Omp50) [17]. This latter appears to be absent in C. coli [18]. No modification in expression nor porin sequence has been found to be associated with antibiotic resistance in Campylobacter [11,13,19]. Lin et al. [20] and Pumbwe and Piddock [21] both described the first efflux system in C. jejuni (CmeABC), conferring intrinsic resistance to multiple antibiotics (including fluoroquinolones, erythromycin, ampicillin, tetracycline, chloramphenicol), detergents and dyes (including ethidium bromide). The CmeB protein is structurally and functionally similar to members of the resistance nodulation and cell division (RND) superfamily of transporters described in other Gram-negative bacterial pathogens [22]. The cmeABC operon is widely distributed in different Campylobacter strains, including C. coli [23,24] and is constitutively expressed in wild-type strains [11,20,25]. Luo et al. showed that this pump functions synergistically with the gyrA mutation to confer a high level of resistance to fluoroquinolones in in vivo selected isolates of C. jejuni [13]. Lin et al. [26] characterized the CmeR transcriptional regulator of the CmeABC efflux pump which is a TetR-like repressor that is highly conserved in nature. An insertional cmeR mutant of the 81e176 C. jejuni strain exhibited a higher resistance level to ciprofloxacin, and this correlated with the overexpression of the CmeABC pump components [26]. A similar correlation was observed in a multidrug-resistant mutant of the same strain, obtained after three rounds of selection on increasing amounts of ciprofloxacin [26]. This mutant contained a single nucleotide deletion in the binding region of CmeR located in the promoter region of the cmeABC operon [26]. Pumbwe et al. [19] measured the expression of the cmeB gene in multidrug-resistant isolates of C. jejuni and found that one third of the isolates overexpressed the cmeB gene. These isolates accumulated less ciprofloxacin [19], suggesting that efflux may be involved in the multidrug resistance. These isolates contained a Gly86Ala modification in the CmeR repressor. Additional mutations have been described in the cmeR gene (Gln9Pro [19], A22G and L25F [27]) but their functional significance is unknown at present. A second efflux pump, CmeDEF, was described in C. jejuni [26,28]. The cmeF gene encoding the RND-type transporter of this efflux system was uniformly distributed in multiple strains of C. jejuni examined [26]. Although the cmeDEF genes were transcribed, the expression level was substantially lower than that of cmeABC. Insertional mutagenesis of cmeF in the NCTC 11168 strain only resulted in modest changes in the susceptibility to a few antimicrobials (excluding ciprofloxacin and erythromycin) [28], while the cmeF mutation in C. jejuni 81e176 did not change its susceptibility to ciprofloxacin, erythromycin, tetracycline, and chloramphenicol [26,29]. It is likely that cmeDEF is regulated by a transcriptional factor,
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but the identity of the regulator and the impact of overexpression of this system with respect to resistance to antimicrobials in Campylobacter remain to be determined. Pumbwe et al. [19] investigated the expression of the CmeDEF efflux pump in multidrug-resistant isolates of C. jejuni. These authors showed that 3 out of 32 isolates overexpressed both the cmeB and cmeF genes, but no isolates overexpressed the cmeF gene alone [19]. Further work is thus needed to determine the precise role of CmeDEF in antimicrobial resistance of Campylobacter. Pumbwe et al. exposed cmeB and cmeF insertional mutants to increasing concentrations of ciprofloxacin [28]. In vitro multidrug-resistant mutants (resistant to ciprofloxacin and also to chloramphenicol and tetracycline) were obtained that expressed the cmeB and cmeF genes at the same levels compared to original genetic backgrounds used for selection [28]. This observation suggested the involvement of another efflux pump or a reduced uptake of these antibiotics. Many other genes encoding putative efflux systems of the different families of transporters (MFS, SMR, ABC and MATE) are present in the sequenced genomes of C. jejuni NCTC 11168 [8] and RM1221 [7] and C. coli RM 2228 [7]. Recently, Ge et al. [29] inactivated genes encoding 8 putative efflux pumps (4, 2 and 2 homologues of the MFS, MATE and DMT families of transporters, respectively) in addition to the cmeB and cmeF genes. They found that, except for the cmeB gene, inactivation of the genes encoding these putative efflux pumps has no effect on susceptibility to chloramphenicol, ciprofloxacin, erythromycin and tetracycline [29]. The role of these putative efflux systems in antimicrobial resistance of Campylobacter needs further investigation. 3. Mechanisms of macrolide resistance Macrolide compounds inhibit bacterial growth by binding to bacterial ribosomes and interfering with protein synthesis. Erythromycin, a 14C member of the macrolide family, binds at a site on the ribosome that includes 23S rRNA and ribosomal proteins and causes early release of peptidyl-tRNA by physical blockage of the tunnel entrance of the ribosome [30]. Three mechanisms of macrolide resistance have been described in bacteria: (i) modification of the antibiotic, (ii) modification of the antibiotic target by methylation or mutation, and (iii) efflux of the antibiotic from the bacterial cell [31]. Modification of antibiotics through the activity of esterases and/or phosphotransferases has only been reported in staphylococci [31]. The two remaining mechanisms have been described in Campylobacter [26,32]. 3.1. Modification of the ribosomal target Modification of the ribosomal target of macrolides can occur by enzyme-mediated methylation. This mechanism gives rise to cross-resistance to macrolides, lincosamides and streptogramins B (MLSB phenotype). This is mediated by methylases encoded by erm (erythromycin ribosome methylase) genes. Methylation occurs at the A2058 (E. coli numbering) position
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of the 23S rRNA. This modification probably sterically hinders the proper positioning of the desosamine sugar of the macrolides and disrupts the alignment of the hydrogen bonding between the A2058 nucleotide and the sugar moiety of macrolide [30]. In Campylobacter, mobile erm determinants have been described only in C. rectus (previously Wolinella recta) [33]. Modification of the ribosomal target of macrolides is the more common mechanism, and this occurs by mutation. The two 23S rRNA nucleotides known to be targeted by this mechanism are in close proximity to the desosamine sugar of macrolides, and these mutations probably reduce the interaction between the tunnel wall of the ribosome and the macrocyclic ring of the macrolide [30]. Point mutation at position 2075 (A2075G, corresponding to position 2059 in E. coli) of the 23S rRNA was found to be associated with a high level of erythromycin-resistance phenotype (MIC > 128 mg/ml) in clinical strains of C. jejuni and C. coli [32,34e40]. In most of the cases, the three copies of 23S rRNA gene were all mutated [35e37,39], but a genetic mosaic of wild-type and mutated copies has also been described [37e39]. Vacher et al. described transversion mutations at position 2074 (A2074C or A2074T, equivalent to position 2058 in E. coli) of the 23S rRNA along with a double mutation A2074C/A2075G in one isolate [34,35]. The A2074T conferred a lower level of resistance and was not present in all copies of the 23S rRNA gene [35]. Recently, Gibreel et al. [39] described a A2074G mutation also in a clinical isolate of C. jejuni. The latter was associated with a very high level of resistance to erythromycin (MIC ¼ 512 mg/ml) but appeared to be less stable after successive subculturing than the A2075G mutation [39]. In order to circumvent macrolide resistance in Grampositive bacteria, new molecules of the macrolide family, ketolides, were developed. Telithromycin, for example, displayed greater efficiency against macrolide-resistant streptococci [41]. However, as recently reported by Mamelli et al. [42] and Cagliero et al. [23], Campylobacter strains that were resistant to erythromycin were also affected in their susceptibility to telithromycin (with MIC of 32 to >128 mg/ml for strains exhibiting mutations in 23S rRNA). The affinity of telithromycin was lower in mutated ribosomes than for the wild-type sensitive control [42]. Mutations affecting macrolide binding were also identified in the ribosomal proteins L4 and L22. Both are thought to form portions of the polypeptide exit tunnel within 70S bacterial ribosomes [30]. These mutations have been described in several bacterial species including Streptococci and Haemophilus influenzae [30]. Although no mutations in either the L4 or L22 proteins in Campylobacter were detected in a recent study [39], Corcoran et al. [40] described 13 Campylobacter isolates (6 C. jejuni and 7 C. coli) all possessing the characteristic A2075G polymorphism along with one or more amino acid substitutions in the L4 protein together with two or more substitutions in L22. In some cases these substitutions were associated with in-frame insertions or deletions of six consecutive amino acids. Interestingly a unique A103V substitution was identified in the L22 protein in each of two high-level erythromycin-resistant C. jejuni and C. coli. This
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substitution may be linked to the resistant phenotype in these isolates. Other substitutions in L4 and L22 described [40] do not appear to influence resistance to erythromycin, as these occur in both resistant and susceptible isolates. However, their role (if any) requires further investigation. 3.2. Efflux of macrolides The CmeABC pump that was proved to be involved in the intrinsic and acquired resistance to fluoroquinolones also includes erythromycin in its panel of expelled antimicrobials [20,21]. This pump could thus also be involved in macrolide resistance in Campylobacter. This hypothesis was examined by Mamelli et al. [43] by testing an efflux pump inhibitor (EPI) specific for RND transporters (Phe-Arg-b-naphthylamide, PAbN) on four macrolideresistant isolates of Campylobacter (2 C. jejuni isolates and 2 C. coli isolates) in association with erythromycin and clarithromycin. The EPI restored the susceptibility to erythromycin of three of the four isolates tested, suggesting that efflux may contribute to macrolide resistance in C. jejuni and C. coli. The contribution of efflux to macrolide resistance was also examined by Payot et al. [37] in 38 animal isolates of C. coli resistant to both fluoroquinolones and erythromycin. The EPI PAbN, tested in association with erythromycin, restored the susceptibility to erythromycin of the 26 isolates with a low level of resistance to erythromycin (MIC ¼ 8e16 mg/ ml). By contrast, it was not efficient to restore the susceptibility of the 12 isolates with a high level of resistance to erythromycin (MIC 512 mg/ml) that had mutated copies of the 23S rRNA gene. These results suggested that efflux played a role not only in intrinsic resistance but also in acquired low level of resistance to erythromycin in Campylobacter [37]. The cmeB gene, encoding the transporter component of the CmeABC efflux pump, was inactivated in strains of C. coli from various sources (human, poultry and pig), with different patterns of resistance to erythromycin [23]. The inactivation of the CmeABC efflux pump led to the restoration of the susceptibility of the intermediate- and low-level resistant strains (context of absence of mutations in the 23S rRNA genes). The same finding was also observed by other laboratories ([42]; Zhang, unpublished study). In the highly resistant strains (context of presence of mutations in the 23S rRNA genes), the erythromycin MIC decreased 128 to 256 fold upon interruption of the cmeB gene (with MIC 8 mg/ml) [23]. The CmeABC efflux pump thus acts synergically with 23S rRNA mutations to confer a high level of resistance to erythromycin in C. coli [23] as previously shown for the synergy between the Thr86Ile modification in the GyrA subunit and efflux by the CmeABC efflux pump leading to fluoroquinolone resistance [25]. Macrolide efflux mechanism in Campylobacter was shown to be also efficient on the ketolide telithromycin [23,42]. By contrast, azithromycin appears to be less affected by efflux [23]. Mamelli et al. [42] found that PAbN was still efficient in the cmeB mutants (also observed by another laboratory, Payot, unpublished results). This suggested either that another
PAbN-sensitive efflux pump is active in these cmeB mutants or that these strain derivatives are susceptible to PAbN due to the toxicity of this compound that could be substrate of the CmeABC efflux pump. The cmeR mutant and the mutant selected in vitro on ciprofloxacin by Lin et al. [26] exhibited a multidrug-resistance pattern that included erythromycin resistance. This was also the case for the human and poultry multidrug isolates examined by Pumbwe et al. [19]. These results indicated that cmeB overexpression may be associated with erythromycin resistance and more largely with multidrug resistance in C. jejuni. 4. Conclusions In C. jejuni and C. coli, the most frequently reported mechanism of resistance to fluoroquinolones and macrolides is target mutation in the gyrA gene or in the 23S rRNA gene, respectively. However, efflux mediated by the CmeABC system is also required and acts in synergy with these mutations to confer a high level of resistance to fluoroquinolones or macrolides. Overexpression of this efflux system can confer a multidrug pattern to Campylobacter, and mutations associated with this overexpression have been described in the repressor and the operating region of this repressor. In addition, other target mutations and efflux systems likely play a role in Campylobacter resistance to antimicrobials, which remains to be examined in future studies. Understanding of the mechanisms responsible for antimicrobial resistance will facilitate the design of strategies to control antibiotic-resistant Campylobacter. Acknowledgements Except for the corresponding author, who played a major and leading role in preparing the manuscript, all other authors are listed in alphabetical order as each of them played an equal role in the preparation of the manuscript. References [1] C.R. Friedman, J. Neimann, H.C. Wegener, R.V. Tauxe, Epidemiology of Campylobacter jejuni infections in the United States and other industrialized nations, in: I. Nachamkin, M.J. Blaser (Eds.), Campylobacter, American Society for Microbiology, Washington, 2000, pp. 121e138. [2] J.E. Moore, D. Corcoran, J.S.G. Dooley, S. Fanning, B. Lucey, M. Matsuda, D.A. McDowell, F. Me´graud, B.C. Millar, R. O’Mahony, L. O’Riordan, M. O’Rourke, J.R. Rao, P.J. Rooney, A. Sails, P. Whyte, Campylobacter, Vet. Res. 36 (2005) 351e382. [3] J.E. Moore, M.D. Barton, I.S. Blair, D. Corcoran, J.S.G Dooley, S. Fanning, I. Kempf, A.J. Lastovica, C.J. Lowery, M. Matsuda, D.A. McDowell, A. McMahon, B.C. Millar, J.R. Rao, P.J. Rooney, B.S. Seal, W.J. Snelling, O. Tolba, The epidemiology of antibiotic resistance in Campylobacter, Microb. Infect. in press. [4] F. Van Bambeke, J.M. Michot, J. Van Eldere, P.M. Tulkens, Quinolones in 2005: an update, Clin. Microbiol. Infect. 11 (2005) 256e280. [5] G.A. Jacoby, Mechanisms of resistance to quinolones, Clin. Infect. Dis. 41 (Suppl.2) (2005) S120eS126. [6] A. Gibreel, E. Sjogren, B. Kaijser, B. Wretlind, O. Skold, Rapid emergence of high-level resistance to quinolones in Campylobacter jejuni associated with mutational changes in gyrA and parC, Antimicrob. Agents Chemother. 42 (1998) 3276e3278.
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[25] J. Lin, O. Sahin, L.O. Michel, Q. Zhang, Critical role of multidrug efflux pump CmeABC in bile resistance and in vivo colonization of Campylobacter jejuni, Infect. Immun. 71 (2003) 4250e4259. [26] J. Lin, M. Akiba, Q. Zhang, Multidrug efflux systems in Campylobacter, in: J.M. Ketley, M.E. Konkel (Eds.), Campylobacter: molecular and cellular biology, Horizon Bioscience, Norfolk, 2005, pp. 205e218. [27] D. Corcoran, T. Quinn, L. Cotter, S. Fanning, Relative contribution of target gene mutation and efflux to varying quinolone resistance in Irish Campylobacter isolates, FEMS Microbiol. Lett. 253 (2005) 39e46. [28] L. Pumbwe, L.P. Randall, M.J. Woodward, L.J. Piddock, Evidence for multiple-antibiotic resistance in Campylobacter jejuni not mediated by CmeB or CmeF, Antimicrob. Agents Chemother. 49 (2005) 1289e1293. [29] B. Ge, G. McDermott, D.G. White, J. Meng, Role of efflux pumps and topoisomerase mutations in fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli, Antimicrob. Agents Chemother. 49 (2005) 3347e3354. [30] F. Franceschi, Z. Kanyo, E.C. Sherer, J. Sutcliffe, Macrolide resistance from the ribosome perspective, Curr. Drug Targets Infect. Disord. 4 (2004) 177e191. [31] R. Leclercq, Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications, Clin. Infect. Dis. 34 (2002) 482e492. [32] D.E. Taylor, D.M. Tracz, Mechanisms of antimicrobial resistance in Campylobacter, in: J.M. Ketley, M.E. Konkel (Eds.), Campylobacter: molecular and cellular biology, Horizon Bioscience, Norfolk, 2005, pp. 193e204. [33] D.E. Roe, A. Weinberg, M.C. Roberts, Mobile rRNA methylase genes in Campylobacter (Wolinella) rectus, J. Antimicrob. Chemother. 36 (1995) 738e740. [34] S. Vacher, A. Menard, E. Bernard, F. Megraud, PCR-Restriction fragment length polymorphism analysis for detection of point mutations associated with macrolide resistance in Campylobacter spp, Antimicrob. Agents Chemother. 47 (2003) 1125e1128. [35] S. Vacher, A. Menard, E. Bernard, A. Santos, F. Megraud, Detection of mutations associated with macrolide resistance in thermophilic Campylobacter spp. by real-time PCR, Microb. Drug Resist. 11 (2005) 40e47. [36] H. Niwa, T. Chuma, K. Okamoto, K. Itoh, Rapid detection of mutations associated with resistance to erythromycin in Campylobacter jejuni/coli by PCR and line probe assay, Int. J. Antimicrob. Agents 18 (2001) 359e364. [37] S. Payot, L. Avrain, C. Magras, K. Praud, A. Cloeckaert, E. ChaslusDancla, 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 23 (2004) 468e472. [38] L.B. Jensen, F.M. Aarestrup, Macrolide resistance in Campylobacter coli of animal origin in Denmark, Antimicrob. Agents Chemother. 45 (2001) 371e372. [39] A. Gibreel, V.N. Kos, M. Keelan, C.A. Trieber, S. Levesque, S. Michaud, D.E. Taylor, Macrolide resistance in Campylobacter jejuni and Campylobacter coli: molecular mechanism and stability of the resistance phenotype, Antimicrob. Agents Chemother. 49 (2005) 2753e2759. [40] D. Corcoran, T. Quinn, L. Cotter, S. Fanning, An investigation of the molecular mechanisms contributing to high-level erythromycin resistance in Campylobacter, Int. J. Antimicrob. Agents 27 (2006) 40e45. [41] D.J. Farrell, I. Morrissey, S. Bakker, S. Buckridge, D. Felmingham, In vitro activities of telithromycin, linezolid, and quinupristin-dalfopristin against Streptococcus pneumoniae with macrolide resistance due to ribosomal mutations, Antimicrob. Agents Chemother. 48 (2004) 3169e3171. [42] L. Mamelli, V. Prouzet-Maule´on, J.M. Pages, F. Me´graud, J.M. Bolla, Molecular basis of macrolide resistance in Campylobacter: role of efflux pumps and target mutations, J. Antimicrob. Chemother. 56 (2005) 491e497. [43] L. Mamelli, J.P. Amoros, J.M. Pages, J.M. Bolla, A phenylalaninearginine beta-naphthylamide sensitive multidrug efflux pump involved in intrinsic and acquired resistance of Campylobacter to macrolides, Int. J. Antimicrob. Agents 22 (2003) 237e241.
Forum on antimicrobial resistance
Mechanisms of fluoroquinolone and macrolide resistance in Campylobacter spp. Sophie Payot a,*, Jean-Michel Bolla b, Deborah Corcoran c, Se´amus Fanning c, Francis Me´graud d, Qijing Zhang e a
Institut National de la Recherche Agronomique, UR086 BioAgresseurs, Sante´, Environnement, 37380 Nouzilly, France b EA 2197, IFR48, Faculte´ de Me´decine, 27 Bd Jean Moulin, F-13385 Marseille Cedex, France c Centre for Food Safety, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland d INSERM ERI 10, Centre National de Re´fe´rence des Campylobacters, Universite´ Victor Segalen, Bordeaux 2, 33076 Bordeaux, France e Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50011, USA Available online 29 March 2006
Abstract The incidence of human Campylobacter infections is increasing worldwide, as well as the proportion of isolates resistant to fluoroquinolones and/or macrolides, the drugs of choice to treat campylobacteriosis. In this review, we report recent developments in the understanding of the resistance mechanisms to fluoroquinolones and macrolides. In addition, we will discuss the recent findings on multidrug resistance in Campylobacter spp. Ó 2006 Elsevier SAS. All rights reserved. Keywords: Campylobacter; Antibiotic resistance mechanisms; Efflux pumps; Fluoroquinolones; Macrolides
1. Introduction
2. Mechanisms of fluoroquinolone resistance
Campylobacter jejuni and C. coli are recognized as major causes of human gastroenteritis worldwide [1,2]. Campylobacteriosis is usually a self-limiting disease, and antimicrobial therapy is required only for severe, prolonged or systemic infections or to control infection in high-risk groups. In these cases, macrolides and fluoroquinolones are the drugs of choice for treatment [2]. However, resistance to these antibiotics, especially to fluoroquinolones, is on the rise in many countries, and this could compromise future treatment (see accompanying review ‘‘The epidemiology of antibiotic resistance in Campylobacter’’ [3]). In this article, we will review the mechanisms of antimicrobial resistance developed by Campylobacter, focusing on fluoroquinolone and macrolide resistance as well as multidrug resistance.
Quinolones available for clinical use are classified into four generations, according to their spectrum of activity [4]. The second generation of quinolones, fluoroquinolones (including ciprofloxacin and levofloxacin), derived from the first compounds by a fluorine substitution at position 6, thereby increasing their activity against Gram-negative bacteria. A substantial improvement in activity against Gram-positive bacteria occurred with the introduction of third-generation molecules (including moxifloxacin) containing new substitutions at positions 7 and 8 [4]. Three mechanisms providing resistance to quinolones have been described in Gram-negative bacteria. These include target mutations, reduced antibiotic intracellular accumulation (by lowering outer membrane permeability or increasing efflux activity), and target protection mediated by the Qnr protein [5]. The plasmid-borne qnr gene, confers a low level of resistance to fluoroquinolones in other bacterial species, but has not been described in Campylobacter.
* Corresponding author. Tel.: þ33 2 47 42 79 88; fax: þ33 2 47 42 77 74. E-mail address: [email protected] (S. Payot). 1286-4579/$ - see front matter Ó 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2005.12.032
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2.1. Target mutations
2.2. Decrease in antibiotic intracellular concentration
In Gram-negative bacteria, DNA gyrase (also known as topoisomerase II) and DNA topoisomerase IV are the primary and secondary targets of fluoroquinolones [4]. The corresponding gene products are large enzymatic quaternary structures consisting of two pairs of subunits, named GyrA and GyrB (DNA gyrase), and ParC and ParE (topoisomerase IV), respectively. Resistance to this class of antimicrobial agents arises from amino acid(s) substitution(s) in a target region of the corresponding topoisomerase termed the ‘‘quinolone resistance determining region’’ (QRDR). The latter is located within the DNA-binding domain on the surface of these enzymes. In Escherichia coli and Salmonella spp., common mutations have been reported located at positions 83 and 87 in the QRDR region of the GyrA subunit. Once a first-step mutation has occurred, the organism exhibits a phenotype consistent with a reduction in susceptibility to these agents. Additional mutations in gyrA and in gyrB or parC can further increase the level of resistance [5]. In Campylobacter, there is increasing evidence that the secondary target (topoisomerase IV) is absent. Only one report of a parC homologue exists in the literature [6], and in this case the sequence was highly similar to the corresponding gene in E. coli (95% nucleotide identity) with a percentage of GC higher than expected for Campylobacter (52% GC compared to a mean percentage of GC from 29.6% to 34.5% for the current Campylobacter genomes [7,8]). Furthermore, several laboratories have reported experimental attempts to amplify parC; all were unsuccessful [9e13] even when using the same C. jejuni strain and primers previously described in the parC report (Payot et al., Corcoran et al., unpublished data). In addition, searches of the annotated genomes failed to identify a parC homologue, suggesting that this gene is absent from the sequenced genomes of C. jejuni NCTC 11168 [8], and RM1221 [7] and C. coli RM2228 [7] [a tBLASTn analysis on these genomes using amino acid sequence of the reported ParC sequence of C. jejuni or ParC of E. coli found no homologue (Payot, unpublished data)]. The absence of a secondary target for fluoroquinolones in Campylobacter leads to a situation where a unique modification in the GyrA subunit (Thr86Ile, corresponding to position 83 in GyrA of E. coli or Salmonella) is sufficient to confer a resistant phenotype to fluoroquinolones in C. jejuni and C. coli [9e14]. Other modifications of the GyrA subunit have also been reported to be associated with quinolone resistance. These include Asp90Asn [9,11e14] and Thr86Lys [13] associated with moderate resistance (MIC ¼ 8e16 mg/ml) and the less frequent Thr86Ala (associated with a MIC of 2 mg/ml for ciprofloxacin) [9], Thr86Val [12] and Asp90Tyr [9]. Double mutations resulting in two amino acid modifications were also reported: Thr86Ile-Pro104Ser [12] and Thr86Ile-Asp90Asn [11,15]). This latter conferred a higher resistance level to moxifloxacin, whereas a single Thr86Ile mutant was still susceptible to this third-generation molecule [9,15]. No mutations in the GyrB subunit have been described in Campylobacter at the time of writing [9e12].
Modification of the outer membrane permeability as a mechanism of resistance to antibiotics has not yet been described in Campylobacter. Two porins have been characterized in C. jejuni: the major outer membrane protein (MOMP) [16] and a minor one (Omp50) [17]. This latter appears to be absent in C. coli [18]. No modification in expression nor porin sequence has been found to be associated with antibiotic resistance in Campylobacter [11,13,19]. Lin et al. [20] and Pumbwe and Piddock [21] both described the first efflux system in C. jejuni (CmeABC), conferring intrinsic resistance to multiple antibiotics (including fluoroquinolones, erythromycin, ampicillin, tetracycline, chloramphenicol), detergents and dyes (including ethidium bromide). The CmeB protein is structurally and functionally similar to members of the resistance nodulation and cell division (RND) superfamily of transporters described in other Gram-negative bacterial pathogens [22]. The cmeABC operon is widely distributed in different Campylobacter strains, including C. coli [23,24] and is constitutively expressed in wild-type strains [11,20,25]. Luo et al. showed that this pump functions synergistically with the gyrA mutation to confer a high level of resistance to fluoroquinolones in in vivo selected isolates of C. jejuni [13]. Lin et al. [26] characterized the CmeR transcriptional regulator of the CmeABC efflux pump which is a TetR-like repressor that is highly conserved in nature. An insertional cmeR mutant of the 81e176 C. jejuni strain exhibited a higher resistance level to ciprofloxacin, and this correlated with the overexpression of the CmeABC pump components [26]. A similar correlation was observed in a multidrug-resistant mutant of the same strain, obtained after three rounds of selection on increasing amounts of ciprofloxacin [26]. This mutant contained a single nucleotide deletion in the binding region of CmeR located in the promoter region of the cmeABC operon [26]. Pumbwe et al. [19] measured the expression of the cmeB gene in multidrug-resistant isolates of C. jejuni and found that one third of the isolates overexpressed the cmeB gene. These isolates accumulated less ciprofloxacin [19], suggesting that efflux may be involved in the multidrug resistance. These isolates contained a Gly86Ala modification in the CmeR repressor. Additional mutations have been described in the cmeR gene (Gln9Pro [19], A22G and L25F [27]) but their functional significance is unknown at present. A second efflux pump, CmeDEF, was described in C. jejuni [26,28]. The cmeF gene encoding the RND-type transporter of this efflux system was uniformly distributed in multiple strains of C. jejuni examined [26]. Although the cmeDEF genes were transcribed, the expression level was substantially lower than that of cmeABC. Insertional mutagenesis of cmeF in the NCTC 11168 strain only resulted in modest changes in the susceptibility to a few antimicrobials (excluding ciprofloxacin and erythromycin) [28], while the cmeF mutation in C. jejuni 81e176 did not change its susceptibility to ciprofloxacin, erythromycin, tetracycline, and chloramphenicol [26,29]. It is likely that cmeDEF is regulated by a transcriptional factor,
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but the identity of the regulator and the impact of overexpression of this system with respect to resistance to antimicrobials in Campylobacter remain to be determined. Pumbwe et al. [19] investigated the expression of the CmeDEF efflux pump in multidrug-resistant isolates of C. jejuni. These authors showed that 3 out of 32 isolates overexpressed both the cmeB and cmeF genes, but no isolates overexpressed the cmeF gene alone [19]. Further work is thus needed to determine the precise role of CmeDEF in antimicrobial resistance of Campylobacter. Pumbwe et al. exposed cmeB and cmeF insertional mutants to increasing concentrations of ciprofloxacin [28]. In vitro multidrug-resistant mutants (resistant to ciprofloxacin and also to chloramphenicol and tetracycline) were obtained that expressed the cmeB and cmeF genes at the same levels compared to original genetic backgrounds used for selection [28]. This observation suggested the involvement of another efflux pump or a reduced uptake of these antibiotics. Many other genes encoding putative efflux systems of the different families of transporters (MFS, SMR, ABC and MATE) are present in the sequenced genomes of C. jejuni NCTC 11168 [8] and RM1221 [7] and C. coli RM 2228 [7]. Recently, Ge et al. [29] inactivated genes encoding 8 putative efflux pumps (4, 2 and 2 homologues of the MFS, MATE and DMT families of transporters, respectively) in addition to the cmeB and cmeF genes. They found that, except for the cmeB gene, inactivation of the genes encoding these putative efflux pumps has no effect on susceptibility to chloramphenicol, ciprofloxacin, erythromycin and tetracycline [29]. The role of these putative efflux systems in antimicrobial resistance of Campylobacter needs further investigation. 3. Mechanisms of macrolide resistance Macrolide compounds inhibit bacterial growth by binding to bacterial ribosomes and interfering with protein synthesis. Erythromycin, a 14C member of the macrolide family, binds at a site on the ribosome that includes 23S rRNA and ribosomal proteins and causes early release of peptidyl-tRNA by physical blockage of the tunnel entrance of the ribosome [30]. Three mechanisms of macrolide resistance have been described in bacteria: (i) modification of the antibiotic, (ii) modification of the antibiotic target by methylation or mutation, and (iii) efflux of the antibiotic from the bacterial cell [31]. Modification of antibiotics through the activity of esterases and/or phosphotransferases has only been reported in staphylococci [31]. The two remaining mechanisms have been described in Campylobacter [26,32]. 3.1. Modification of the ribosomal target Modification of the ribosomal target of macrolides can occur by enzyme-mediated methylation. This mechanism gives rise to cross-resistance to macrolides, lincosamides and streptogramins B (MLSB phenotype). This is mediated by methylases encoded by erm (erythromycin ribosome methylase) genes. Methylation occurs at the A2058 (E. coli numbering) position
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of the 23S rRNA. This modification probably sterically hinders the proper positioning of the desosamine sugar of the macrolides and disrupts the alignment of the hydrogen bonding between the A2058 nucleotide and the sugar moiety of macrolide [30]. In Campylobacter, mobile erm determinants have been described only in C. rectus (previously Wolinella recta) [33]. Modification of the ribosomal target of macrolides is the more common mechanism, and this occurs by mutation. The two 23S rRNA nucleotides known to be targeted by this mechanism are in close proximity to the desosamine sugar of macrolides, and these mutations probably reduce the interaction between the tunnel wall of the ribosome and the macrocyclic ring of the macrolide [30]. Point mutation at position 2075 (A2075G, corresponding to position 2059 in E. coli) of the 23S rRNA was found to be associated with a high level of erythromycin-resistance phenotype (MIC > 128 mg/ml) in clinical strains of C. jejuni and C. coli [32,34e40]. In most of the cases, the three copies of 23S rRNA gene were all mutated [35e37,39], but a genetic mosaic of wild-type and mutated copies has also been described [37e39]. Vacher et al. described transversion mutations at position 2074 (A2074C or A2074T, equivalent to position 2058 in E. coli) of the 23S rRNA along with a double mutation A2074C/A2075G in one isolate [34,35]. The A2074T conferred a lower level of resistance and was not present in all copies of the 23S rRNA gene [35]. Recently, Gibreel et al. [39] described a A2074G mutation also in a clinical isolate of C. jejuni. The latter was associated with a very high level of resistance to erythromycin (MIC ¼ 512 mg/ml) but appeared to be less stable after successive subculturing than the A2075G mutation [39]. In order to circumvent macrolide resistance in Grampositive bacteria, new molecules of the macrolide family, ketolides, were developed. Telithromycin, for example, displayed greater efficiency against macrolide-resistant streptococci [41]. However, as recently reported by Mamelli et al. [42] and Cagliero et al. [23], Campylobacter strains that were resistant to erythromycin were also affected in their susceptibility to telithromycin (with MIC of 32 to >128 mg/ml for strains exhibiting mutations in 23S rRNA). The affinity of telithromycin was lower in mutated ribosomes than for the wild-type sensitive control [42]. Mutations affecting macrolide binding were also identified in the ribosomal proteins L4 and L22. Both are thought to form portions of the polypeptide exit tunnel within 70S bacterial ribosomes [30]. These mutations have been described in several bacterial species including Streptococci and Haemophilus influenzae [30]. Although no mutations in either the L4 or L22 proteins in Campylobacter were detected in a recent study [39], Corcoran et al. [40] described 13 Campylobacter isolates (6 C. jejuni and 7 C. coli) all possessing the characteristic A2075G polymorphism along with one or more amino acid substitutions in the L4 protein together with two or more substitutions in L22. In some cases these substitutions were associated with in-frame insertions or deletions of six consecutive amino acids. Interestingly a unique A103V substitution was identified in the L22 protein in each of two high-level erythromycin-resistant C. jejuni and C. coli. This
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substitution may be linked to the resistant phenotype in these isolates. Other substitutions in L4 and L22 described [40] do not appear to influence resistance to erythromycin, as these occur in both resistant and susceptible isolates. However, their role (if any) requires further investigation. 3.2. Efflux of macrolides The CmeABC pump that was proved to be involved in the intrinsic and acquired resistance to fluoroquinolones also includes erythromycin in its panel of expelled antimicrobials [20,21]. This pump could thus also be involved in macrolide resistance in Campylobacter. This hypothesis was examined by Mamelli et al. [43] by testing an efflux pump inhibitor (EPI) specific for RND transporters (Phe-Arg-b-naphthylamide, PAbN) on four macrolideresistant isolates of Campylobacter (2 C. jejuni isolates and 2 C. coli isolates) in association with erythromycin and clarithromycin. The EPI restored the susceptibility to erythromycin of three of the four isolates tested, suggesting that efflux may contribute to macrolide resistance in C. jejuni and C. coli. The contribution of efflux to macrolide resistance was also examined by Payot et al. [37] in 38 animal isolates of C. coli resistant to both fluoroquinolones and erythromycin. The EPI PAbN, tested in association with erythromycin, restored the susceptibility to erythromycin of the 26 isolates with a low level of resistance to erythromycin (MIC ¼ 8e16 mg/ ml). By contrast, it was not efficient to restore the susceptibility of the 12 isolates with a high level of resistance to erythromycin (MIC 512 mg/ml) that had mutated copies of the 23S rRNA gene. These results suggested that efflux played a role not only in intrinsic resistance but also in acquired low level of resistance to erythromycin in Campylobacter [37]. The cmeB gene, encoding the transporter component of the CmeABC efflux pump, was inactivated in strains of C. coli from various sources (human, poultry and pig), with different patterns of resistance to erythromycin [23]. The inactivation of the CmeABC efflux pump led to the restoration of the susceptibility of the intermediate- and low-level resistant strains (context of absence of mutations in the 23S rRNA genes). The same finding was also observed by other laboratories ([42]; Zhang, unpublished study). In the highly resistant strains (context of presence of mutations in the 23S rRNA genes), the erythromycin MIC decreased 128 to 256 fold upon interruption of the cmeB gene (with MIC 8 mg/ml) [23]. The CmeABC efflux pump thus acts synergically with 23S rRNA mutations to confer a high level of resistance to erythromycin in C. coli [23] as previously shown for the synergy between the Thr86Ile modification in the GyrA subunit and efflux by the CmeABC efflux pump leading to fluoroquinolone resistance [25]. Macrolide efflux mechanism in Campylobacter was shown to be also efficient on the ketolide telithromycin [23,42]. By contrast, azithromycin appears to be less affected by efflux [23]. Mamelli et al. [42] found that PAbN was still efficient in the cmeB mutants (also observed by another laboratory, Payot, unpublished results). This suggested either that another
PAbN-sensitive efflux pump is active in these cmeB mutants or that these strain derivatives are susceptible to PAbN due to the toxicity of this compound that could be substrate of the CmeABC efflux pump. The cmeR mutant and the mutant selected in vitro on ciprofloxacin by Lin et al. [26] exhibited a multidrug-resistance pattern that included erythromycin resistance. This was also the case for the human and poultry multidrug isolates examined by Pumbwe et al. [19]. These results indicated that cmeB overexpression may be associated with erythromycin resistance and more largely with multidrug resistance in C. jejuni. 4. Conclusions In C. jejuni and C. coli, the most frequently reported mechanism of resistance to fluoroquinolones and macrolides is target mutation in the gyrA gene or in the 23S rRNA gene, respectively. However, efflux mediated by the CmeABC system is also required and acts in synergy with these mutations to confer a high level of resistance to fluoroquinolones or macrolides. Overexpression of this efflux system can confer a multidrug pattern to Campylobacter, and mutations associated with this overexpression have been described in the repressor and the operating region of this repressor. In addition, other target mutations and efflux systems likely play a role in Campylobacter resistance to antimicrobials, which remains to be examined in future studies. Understanding of the mechanisms responsible for antimicrobial resistance will facilitate the design of strategies to control antibiotic-resistant Campylobacter. Acknowledgements Except for the corresponding author, who played a major and leading role in preparing the manuscript, all other authors are listed in alphabetical order as each of them played an equal role in the preparation of the manuscript. References [1] C.R. Friedman, J. Neimann, H.C. Wegener, R.V. Tauxe, Epidemiology of Campylobacter jejuni infections in the United States and other industrialized nations, in: I. Nachamkin, M.J. Blaser (Eds.), Campylobacter, American Society for Microbiology, Washington, 2000, pp. 121e138. [2] J.E. Moore, D. Corcoran, J.S.G. Dooley, S. Fanning, B. Lucey, M. Matsuda, D.A. McDowell, F. Me´graud, B.C. Millar, R. O’Mahony, L. O’Riordan, M. O’Rourke, J.R. Rao, P.J. Rooney, A. Sails, P. Whyte, Campylobacter, Vet. Res. 36 (2005) 351e382. [3] J.E. Moore, M.D. Barton, I.S. Blair, D. Corcoran, J.S.G Dooley, S. Fanning, I. Kempf, A.J. Lastovica, C.J. Lowery, M. Matsuda, D.A. McDowell, A. McMahon, B.C. Millar, J.R. Rao, P.J. Rooney, B.S. Seal, W.J. Snelling, O. Tolba, The epidemiology of antibiotic resistance in Campylobacter, Microb. Infect. in press. [4] F. Van Bambeke, J.M. Michot, J. Van Eldere, P.M. Tulkens, Quinolones in 2005: an update, Clin. Microbiol. Infect. 11 (2005) 256e280. [5] G.A. Jacoby, Mechanisms of resistance to quinolones, Clin. Infect. Dis. 41 (Suppl.2) (2005) S120eS126. [6] A. Gibreel, E. Sjogren, B. Kaijser, B. Wretlind, O. Skold, Rapid emergence of high-level resistance to quinolones in Campylobacter jejuni associated with mutational changes in gyrA and parC, Antimicrob. Agents Chemother. 42 (1998) 3276e3278.
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