Antibiotic-resistant Campylobacter: could efflux pump inhibitors control infection?

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1 Journal of Antimicrobial Chemotherapy Advance Access published November 20, 2006 Journal of Antimicrobial Chemotherapy doi: /jac/dkl470 Antibiotic-resistant Campylobacter: could efflux pump inhibitors control infection? Teresa Quinn 1, Jean-Michel Bolla 2, Jean-Marie Pagès 2 and Séamus Fanning 1 * Introduction 1 Centre for Food Safety, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland; 2 EA 2197, IFR 48, Faculté de Médecine, Université de la Méditerranée, 27 Boulevard Jean Moulin 13385, Marseille Cedex 05, France Campylobacter is the most common cause of bacterial gastroenteritis in the world. Poultry is the main reservoir of human infections. The widespread use of antibiotics in agriculture and veterinary medicine has resulted in the emergence of an increasing number of antibiotic-resistant Campylobacter strains that can be transmitted to humans through the food chain. Of particular concern to public health is the prevalence of resistance to macrolides and fluoroquinolones that are used in the treatment of life-threatening campylobacteriosis. The CmeABC efflux system has been shown to contribute to the intrinsic and acquired resistance to these antibiotics. In addition, by mediating resistance to bile, it is essential for colonization of the chicken gut in vivo. Inhibition of CmeABC may provide an effective means of reversing antibiotic resistance and decreasing the transmission of Campylobacter via the food chain. This would positively impact on public health by decreasing the morbidity, mortality and increased healthcare costs associated with the treatment of antibiotic-resistant Campylobacter. Keywords: fluoroquinolones, macrolides, colonization, resistance to bile, campylobacteriosis Since it was first isolated from faeces in the 1970s Campylobacter has risen from near obscurity to currently being heralded as one of the major causative agents of bacterial gastroenteritis in humans worldwide. 1,2 Greater than 90% of cases of Campylobacter gastroenteritis are caused by Campylobacter jejuni, with Campylobacter coli being responsible for most of the remaining infections. 3 Campylobacter is a zoonotic pathogen that colonizes a variety of wild and domestic animals. Commercial poultry are considered the main reservoir of human Campylobacter infections even though other risk factors for infection such as raw milk, water and the direct zoonotic transmission from contact with infected pets have been identified. 2,4,5 Since most sporadic cases of human campylobacteriosis have been associated with the consumption of, 6,7 or contact with raw or undercooked poultry, 8 reduction of pathogens at relevant points of the farm to fork continuum could potentially reduce the risks to human health. Unfortunately, the heavy bacterial burden in poultry flocks makes it virtually impossible to control Campylobacter during poultry processing. 9 Consequently, user-friendly and consumer acceptable intervention strategies are currently being sought that will reduce colonization of poultry with Campylobacter Although most cases of Campylobacter enteritis are selflimiting and require only supportive therapy such as maintenance of hydration and electrolyte balance, antibiotic therapy is warranted in immunocompromised individuals or in cases of persistent enteritis or extraintestinal infection. 2 Under these circumstances, macrolides or fluoroquinolones are the antibiotics of choice. 1,2 The prevalence of macrolide and fluoroquinolone resistance in Campylobacter raises a concern for human health as it could compromise treatment. Indeed, infection with antibioticresistant Campylobacter is associated with longer duration of illness, increased risk of death and invasive disease and escalating healthcare costs Multiple mechanisms for antibiotic resistance exist in Campylobacter. 18 Efflux was first postulated as a mechanism of multidrug resistance in Campylobacter in In 2002, a chromosomally encoded multidrug resistance-nodulation-cell division (RND) efflux system CmeABC, was identified and characterized in C. jejuni, 20,21 and later in C. coli. 22 It is constitutively expressed in wild-type Campylobacter strains and extrudes a broad range of antibiotics, dyes, bile salts and detergents. 20 Although the genetic mechanisms for fluoroquinolone and macrolide resistance have largely been attributed to target gene mutations in the quinolone resistance determining... *Corresponding author. Tel: ; Fax: ; sfanning@ucd.ie... Page 1 of 7 Ó The Author Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 region of the gyra 23 and in domain V of the 23S rrna, 23,24 there is considerable evidence that the CmeABC efflux pump contributes to the intrinsic and acquired resistance to these antibiotics. 20,21,25 29 In addition to its role in antibiotic resistance, by mediating resistance to bile in the intestinal tract CmeABC has been reported to be essential for the in vivo colonization of chickens by Campylobacter. 30 Here we review current trends in resistance to clinically relevant antibiotics in Campylobacter and the contribution of efflux pumps to antibiotic resistance, in particular to fluoroquinolones and macrolides. The potential for the use of efflux pump inhibitors as therapeutic agents for the treatment of human campylobacteriosis and for the prevention of colonization in animal reservoirs is discussed. Antibiotic resistance in Campylobacter trends and driving forces Antibiotic resistance in both medicine and agriculture has become a major public health concern in recent years. As Campylobacter may be transferred from animals to humans, the development of antibiotic resistance among Campylobacter spp., arising from the use of antibiotics in food animals as therapeutic agents or growth promoters, is a matter of concern. 31 Increasing numbers of resistant C. jejuni strains makes the clinical management of infection more difficult by prolonging illness and compromising the treatment of cases of bacteraemia. 32 Of particular concern in Campylobacter is resistance to fluoroquinolones and macrolides, which are the antibiotics of choice for the treatment of gastroenteritis. Susceptibility data indicate that ciprofloxacin resistance is emerging at a rapid pace. 2,17,33 Rates of fluoroquinolone resistance in Taiwan, Thailand and Spain as high as 90% have been reported, limiting the usefulness of fluoroquinolones in the treatment of community-acquired diarrhoea in these regions. Although lower frequencies of up to 45% have been reported in other countries, current trends show that resistance is increasing. 33,37 40 Emergence of fluoroquinolone resistance coincided with the introduction of fluoroquinolones in veterinary medicine. 14,41 It has been postulated that the increase in the numbers of fluoroquinolone-resistant Campylobacter isolated from human infections results from the emergence of resistant strains in poultry as a result of fluoroquinolone use. 14,41,42 Support for this comes from the findings of Smith et al. 14 who showed that the strain types were similar in domestically acquired human infections and local poultry flocks in Minnesota. Furthermore, fluoroquinolone treatment of Campylobacter-colonized broiler chickens has been reported to induce resistance under experimental conditions. 43 The strongest evidence to support the connection between Campylobacter infections in humans and fluoroquinolone use in poultry production systems comes from a study of human campylobacteriosis in Australia where fluoroquinolones are prohibited for use in poultry production. Despite regular use of fluoroquinolones in medicine in Australia there are few confirmed cases of domestically acquired fluoroquinoloneresistant campylobacteriosis. 44 Poultry producers in some countries have elected to voluntarily ban the use of fluoroquinolone drugs in commercial flocks. However, fluoroquinolone resistance has persisted even in the absence of selective pressure. 45 The rapid emergence of fluoroquinolone-resistant isolates may be attributable at least in part to an enhanced fitness. 46 When fluoroquinolone-resistant and -susceptible isolates are co-inoculated the former out-compete the latter, indicating that these isolates may be biologically fitter, conferring a selective advantage. Since the 1990s, a significant increase in the prevalence of resistance to macrolides amongst human Campylobacter isolates has been reported. 47 The widespread use of macrolides in veterinary medicine has accelerated this resistance trend. Macrolide resistance can also evolve during antibiotic treatment in humans, however it has been infrequently reported. 48 Although trends over time show stable and low rates of macrolide resistance in many countries including Japan, Sweden and Finland 47,49 and in parts of Canada, 50 a rise in macrolide resistance has been reported in human strains in several countries. 33,51,52 Fortunately, the prevalence of erythromycin resistance in C. jejuni has remained low, often well below 12% of isolates. 24,47 In contrast, a higher frequency of resistance is reported in C. coli (up to 70%) which causes <10% of human infections. 24,47 Similarly, in food animals, the prevalence of resistance to erythromycin is generally reported to be higher in C. coli than in C. jejuni In particular, high rates of macrolide resistance have been reported among C. coli isolates from swine, possibly as a consequence of the extensive veterinary use of macrolides in pig production. 56 Thus, pigs may represent an important source of infections with erythromycin-resistant C. coli. Globally, multiple antibiotic resistance (MAR; defined as resistance to three or more different classes of antibiotics) is emerging in Campylobacter clinical isolates. 50,52,57 Of particular concern is the emergence of co-resistance to macrolides and fluoroquinolones, 52,57,58 as these are the front-line drugs for treatment of campylobacteriosis. Tetracycline is considered an alternative therapeutic; however, the widespread prevalence of tetracycline resistance, 33,50,52,59 largely attributable to the use of tetracyclines in veterinary medicine for control of infection and growth promotion, 60 makes it a poor choice for treatment. Intravenous aminoglycoside therapy may be considered in more serious cases of infections, such as bacteraemia and other systemic infections. 1 Currently, gentamicin resistance remains negligible; however, the lack of an oral preparation may limit its widespread use. 33 Campylobacter spp. are also generally susceptible to chloramphenicol, clindamycin, nitrofurans and imipenem. 2 Role of active efflux in antibiotic resistance in Campylobacter In 2002, the RND efflux pump CmeABC was described in C. jejuni. 20,21 Insertional mutagenesis of the cmeb gene increased the susceptibility to multiple antibiotics (including fluoroquinolones, erythromycin, tetracycline, chloramphenicol and ampicillin), detergents and dyes, 20,21 defining an active role of the CmeABC multidrug efflux system in the intrinsic resistance of C. jejuni to antibiotics. To date, a number of studies have defined a contributory role of the CmeABC efflux system to acquired fluoroquinolone and macrolide resistance and to MAR 26,61,62 in Campylobacter. The CmeABC efflux system is composed of three proteins, CmeB, the energy-dependent efflux pump, CmeC, the outer Page 2 of 7

3 membrane channel forming protein and CmeA, the adaptor protein. 20 CmeB functions with CmeA and C as an energydependent efflux tripartite complex. Insertional mutagenesis of cmer, an open reading frame coding for a TetR-like regulator, proximal to the cmeabc operon, 21 resulted in overexpression of cmeabc. 63 Moreover, in vitro, recombinant CmeR bound specifically to an inverted repeat sequence located within the promoter region of cmeabc. 63 A single mutation in the inverted repeat region decreased the binding of CmeR in vitro, and resulted in an overexpression of cmeabc. 63 Taken together, these results indicate that CmeR functions as a local repressor for CmeABC. Although a number of other putative efflux pumps, including the RND efflux pump CmeDEF, have been identified by genome analysis, inactivation of the genes encoding these pumps revealed that they do not contribute to the intrinsic or acquired resistance to erythromycin, ciprofloxacin, chloramphenicol or tetracycline. 26,64 However, on the basis of recent evidence that interaction between CmeDEF and CmeABC confers intrinsic resistance to ciprofloxacin and tetracycline in some strains, 65 the possibility of interaction between other efflux pumps in the genesis of resistance cannot be ruled-out and remains to be evaluated. Role of efflux in fluoroquinolone resistance Studies investigating the contribution of efflux to acquired fluoroquinolone resistance in Campylobacter have been somewhat hampered by the unavailability of an efflux inhibitor capable of selectively inhibiting fluoroquinolone efflux. Although the efflux pump inhibitor L-phenylalanine-L-arginine-bnaphthylamine (PAbN) has been shown to increase susceptibility to fluoroquinolones in other bacteria, only one study to date has shown a significant decrease in the MICs of fluoroquinolones in Campylobacter in the presence of this inhibitor. 29 We 22,28,69,70 among others, 25,71 73 have refuted this finding by clearly demonstrating a lack of efficacy of PAbN in reversing fluoroquinolone resistance, thereby dismissing its future diagnostic utility in assessing the contribution of efflux to fluoroquinolone resistance in Campylobacter. Nevertheless, by use of a genetic approach involving inactivation of the cmeb gene, direct evidence for the contribution of the CmeABC efflux system to acquired fluoroquinolone resistance has been provided. Compared with wild-type isolates, all cmeb mutants showed a significant decrease in their resistance to fluoroquinolones Furthermore, several studies have shown that the CmeABC pump acts synergistically with gyra mutations to confer fluoroquinolone resistance. 26,27 In contrast to other Gram-negative bacteria where overexpression of efflux pump activity is largely associated with acquired fluoroquinolone resistance, fluoroquinolone resistance in Campylobacter does not appear to require overexpression of efflux pumps and may be mediated by single-step point mutations in gyra in the presence of the constitutively expressed CmeABC pump. 26,27 However, based on the findings of Pumbwe et al. 64 that (i) ciprofloxacin-resistant mutants were selected with the same frequency from separate cmeb and cmef knockout strains as from their parent strain and (ii) selected mutant phenotypes from each of the knockout strains showed decreased susceptibility to ciprofloxacin (in the absence of gyra and gyrb mutations), chloramphenicol, detergents and dyes and accumulated less ciprofloxacin and ethidium bromide than their respective parent strains, it would appear that a non-cmeb or -CmeF efflux system may contribute to the ciprofloxacin/multidrug resistance phenotype selected after exposure to a fluoroquinolone. This genetic locus remains to be identified. Role of efflux in macrolide resistance The first report documenting the possible contribution of active efflux to acquired macrolide resistance in Campylobacter was by Mamelli et al. 70 in They showed that susceptibility to erythromycin and clarithromycin was restored in three of four macrolide-resistant isolates when they were grown in the presence of PAbN. Subsequent studies have highlighted a significant role for the CmeABC efflux system in macrolide resistance. Several studies reported that PAbN 25,28,29 and cmeb inactivation 25,26,28 restored susceptibility in low-level erythromycin-resistant isolates (MICs 4 16 mg/l) carrying no mutated copies of 23S rrna genes. In addition, inactivation of cmeb in four high-level erythromycin-resistant strains (MICs >1024 mg/l) containing the A2075G transitional mutation in the 23S rrna genes decreased the MICs 128- to 512-fold, restoring susceptibility in three strains and returning the remaining strain to a low-level resistance phenotype. 25 cmeb inactivation also restored susceptibility in a high-level erythromycin-resistant isolate (MIC 512 mg/l) with no identified mutations in the 23S rrna genes. 26 In contrast to these findings, PAbN either had no effect, or caused 2- to 8-fold decrease in the MICs of high-level resistant isolates without restoring susceptibility. 25,28,29,79 Despite the limited competitive inhibition of PAbN in high-level resistant isolates, these data provide convincing evidence that the CmeABC efflux system is responsible for low-level erythromycin resistance and that it may act independently or in synergy with 23S rrna mutations to confer high-level erythromycin resistance. The CmeABC pump also contributes to resistance to the macrolides, azithromycin and tylosin 25 and the ketolide telithromycin. 25,28 However, the possibility of the involvement of another PAbN-sensitive efflux system in macrolide resistance cannot be ruled-out, as PAbN was still efficient against erythromycin, clarithromycin and telithromycin in cmeb mutants. 28 Role of efflux in MAR Overexpression of multidrug-resistant efflux pumps mediated by mutations in their regulators is usually associated with acquired resistance to multiple antibiotics In Campylobacter a number of studies have tentatively linked overexpression of cmeb with MAR. 29,62 High levels of expression of CmeB were documented in a number of C. coli MAR isolates from pigs. 29 In addition, Pumbwe et al. 62 reported that MAR was more associated with cmeb overexpression than cmef (9/32 MAR C. jejuni isolates from humans and poultry overexpressed cmeb alone and 3/32 overexpressed both cmeb and cmef). All cmeb overexpressing isolates accumulated less ciprofloxacin than wild-type strains and contained mutations in the CmeR transcriptional repressor protein. However, increased expression of CmeB, as has been demonstrated in a cmer mutant, may not be sufficient alone to generate clinically significant levels of antibiotic resistance. 63 Nevertheless, it may allow bacteria to survive under the pressure of high antibiotic concentrations and promote the emergence of mutants with target gene mutations that are highly resistant to antimicrobials. 82 Direct evidence for the contribution of CmeABC to MAR has been provided by inactivation of cmeb in several MAR-resistant isolates, which resulted in 4 to 256-fold decreases Page 3 of 7

4 in the MICs of ciprofloxacin, erythromycin and tetracycline. 26 However, overexpression of cmeb was not observed when comparing antibiotic-resistant to antibiotic-susceptible strains. 26 The exact contribution of CmeABC overexpression to MAR needs to be more clearly defined. Interestingly, Lin et al. 20 reported an 8-fold decrease in the MIC of tetracycline in a cmeb mutant that carried the teto gene, suggesting that cmeabc functions synergistically with teto to contribute to acquired tetracycline resistance. This warrants further investigation. A study by Pumbwe et al. 64 postulated the involvement of a non-cmeb or -CmeF efflux pump or reduced uptake in conferring MAR in mutants selected with ciprofloxacin from two efflux knockout strains, cmeb::kan r and cmef::kan r. However, critical evaluation of their data shows that a MAR phenotype was not selected after exposure to ciprofloxacin as only resistance to ciprofloxacin and chloramphenicol was observed. Nonetheless, the possibility of the interaction between different efflux pumps in the genesis of MAR remains to be explored. Role of efflux pumps in Campylobacter in bile resistance and avian gastrointestinal colonization Chickens represent a natural host and major reservoir for C. jejuni. Colonization occurs mainly in the lower intestine where the organism localizes in caecal and cloacal crypts. 84 Normally, colonization of chickens results in a harmless commensal relationship, 85 unlike in mammals where the pathogenesis of campylobacteriosis is associated with destruction of the colonic epithelium and invasion of the lamina propria. 86,87 In order to survive and multiply within the host, Campylobacter and other enterics need to resist multiple stresses in the gastrointestinal tract including the antimicrobial activity of bile and peptides, competition with other flora and attack by the immune system. Bile, which is produced by the liver, is composed primarily of bile salts, bile pigments and to a lesser extent phospholipids and cholesterol. In addition to being essential for the digestion and absorption of fats, bile salts function as antimicrobial agents by disaggregating the lipid bilayers of cellular membranes. 88 In Gram-negative bacteria, bile salts can pass directly across the outer membrane or through porins. 89 Thus, survival of enterics in the intestinal tract has necessitated the evolution of multiple selfdefence mechanisms against the bactericidal effects of bile salts, including use of efflux pumps, producing bile salt hydrolase or modulating synthesis of lipopolysaccharide and porins Of these, efflux of bile salts from the cytoplasm by multidrug efflux pumps is the best-characterized mechanism of bile resistance in Gram-negative bacteria. To date, the only documented mechanism of bile salt resistance in Campylobacter is active efflux. Inactivation of cmeabc significantly decreased the resistance to various bile salts in C. jejuni. 20,30 CmeABC is also essential for growth of Campylobacter in bile-containing media and in animal intestinal tracts as evidenced by the inability of a cmeb null mutant to colonize chickens. 30 More recently, bile salts have been shown to dramatically increase the expression of the cmeabc operon by inhibiting binding of the CmeR repressor protein to the promoter for cmeabc as well as by a CmeR-independent activation pathway. 93 Thus by mediating resistance to bile salts, CmeABC appears to play a significant role in the adaptation of Campylobacter to the intestinal environment in animal hosts. Although the natural function of multidrug efflux pumps is still largely undefined, 94,95 the broad distribution and expression of CmeABC in Campylobacter strains from different sources, 20 its inducibility by bile 93 and its demonstrated role in the export of and resistance to bile salts 20,30 is consistent with a natural role of this system in protecting the microbial cell from the action of these agents in the gut. Of the other identified efflux pumps in Campylobacter, only the contribution of the CmeDEF efflux system to bile resistance has been investigated. However, studies determining the effects of cmef inactivation on susceptibility to bile salts have yielded contradictory results. 64,65 In contrast, cmeb/cmef double mutants showed a significant decrease in resistance to detergents and bile salts compared with their corresponding cmeb mutants, 65 indicating that interaction between both CmeDEF and CmeABC efflux pumps may facilitate the adaptation of Campylobacter to various environments. Furthermore, it appears that CmeABC and CmeDEF interact in maintaining cell viability in Campylobacter and that at least one of them is required for optimal growth, 65 thus defining a role for efflux pumps in Campylobacter physiology. The future potential of efflux pump inhibitors to control Campylobacter infection The increasing recognition of the contribution of multidrug efflux pumps to antibiotic resistance in clinical bacterial isolates, 96 combined with the lack of new antibiotics in development, has resulted in the identification of the efflux machinery as bona fide pharmacological targets in the war against resistance. 97 Over the past decade, many efflux pump inhibitors have been identified by systematic screening of large collections of natural and synthetic compounds and by the testing of known inhibitors of mammalian multidrug efflux pumps and drugs used in human medicine for conditions other than the treatment of infectious diseases Many of these compounds have proved useful for studying the contribution and prevalence of efflux in acquired and intrinsic resistance to multiple antibiotics in bacteria. 97,100 However, to date, no efflux pump inhibitor has been licensed for use in the treatment of bacterial infections in human or veterinary medicine. As knowledge of the structures of efflux pump proteins and their substrate binding domains increase, there will be greater potential to rationally design new inhibitors or increase the efficacy of known ones. The pace at which our understanding of mechanisms of resistance in Campylobacter to important antimicrobial agents has developed has been very impressive. However, our understanding of many of the identified drug transporters in Campylobacter is still limited. The level of synergistic interaction between varying efflux pumps in relation to antibiotic resistance and adaptation to different environmental niches is currently unclear. Nonetheless, the well-documented contribution of the CmeABC efflux pump to antibiotic resistance and its critical role in the in vivo adaptation of Campylobacter in chickens 30 suggests that it may be a suitable target for control of Campylobacter infections. Efflux pump inhibitors capable of inhibiting CmeABC could have a potential clinical application in the treatment of antibioticresistant Campylobacter infections by reversing resistance to fluoroquinolones, macrolides and possibly tetracyclines. A further exciting possibility would be the use of inhibitors of CmeABC as Page 4 of 7

5 feed additives in poultry production systems to prevent/reduce intestinal colonization with Campylobacter. As a significant proportion of all Campylobacter infections have been attributed to the consumption of undercooked poultry or the handling of raw poultry, reduction of colonization in chickens would reduce carcass contamination during slaughter, thereby potentially reducing the incidence of C. jejuni infections in humans. Moreover, the use of efflux inhibitors in poultry production systems could possibly decrease the emergence of antibioticresistant strains that can be transmitted to humans through the food chain. Fluoroquinolone and multidrug resistance in Campylobacter is not clearly associated with overproduction of CmeABC, 26,27 suggesting that CmeABC might have unique regulatory features. To date, this pump has been shown to be controlled by CmeR, which functions as a transcriptional repressor; 63 however, it is unknown whether or not CmeR is controlled by a global regulator. Further elucidation of the regulatory mechanisms modulating the expression of CmeABC together with high-resolution structural information for the regulators may provide opportunities for the design of specific inhibitors that could potentially decrease the transcription of the transport genes. Finally, elucidation of the structures, function and regulation of other efflux pumps present in Campylobacter may in the future identify other novel targets for the therapeutic intervention of Campylobacter infection. Discussion Campylobacter can no longer be described as the unsung bug when it comes to food poisoning, as it is currently heralded as the world leader in foodborne diseases. Infection is largely associated with consumption of undercooked poultry and cross-contamination of other foods with drippings from raw poultry. Increasing prevalence of resistance to macrolides and fluoroquinolones has become a major public health concern as it can compromise treatment in cases of lifethreatening campylobacteriosis. Antibiotic resistance in Campylobacter is mainly a consequence of the use of antimicrobials in food-producing animals. However, even in the absence of antibiotic selective pressure, resistance can persist. The contribution of the CmeABC efflux pump to antimicrobial resistance in Campylobacter together with its role in the in vivo colonization of chickens mark it as a potential future target for the control of Campylobacter infection. The development of rationally designed efflux pump inhibitors that will specifically block its function may potentially reverse clinical antibiotic resistance and decrease the transmission of Campylobacter via the food chain. Other useful strategies, based on the modulation of the avian gut microbial content could also have a significant impact towards reducing the transmission of Campylobacter via the food chain. However, until new control systems are developed, the management of risks associated with the transmission of antibiotic-resistant isolates from food animals to humans should include the following: continuing improvements in food safety, promotion of prudent use of antibiotics in agriculture and veterinary medicine and the implementation and maintenance of global antimicrobial resistance surveillance studies to guide empirical prescribing of antimicrobial agents and to detect newly emerging resistances. Transparency declarations None to declare. References 1. Aarestrup FM, Engberg J. Antimicrobial resistance of thermophilic Campylobacter. Vet Res 2001; 32: Allos BM. Campylobacter jejuni Infections: update on emerging issues and trends. 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