Campylobacter jejuni, a major foodborne human pathogen, has become increasingly resistant to fluoroquinolone (FQ) antimicrobials. By using clonally related isolates and genetically defined mutants, we determined the fitness of FQ-resistant Campylobacter in chicken (a natural host and a major reservoir for C. jejuni) in the absence of antibiotic selection pressure. When monoinoculated into the host, FQ-resistant and FQ-susceptible Campylobacter displayed similar levels of colonization and persistence in the absence of FQ antimicrobials. The prolonged colonization in chickens did not result in loss of the FQ resistance and the resistance-conferring point mutation (C257 3 T) in the gyrA gene. Strikingly, when coinoculated into chickens, the FQ-resistant Campylobacter isolates outcompeted the majority of the FQ-susceptible strains, indicating that the resistant Campylobacter was biologically fit in the chicken host. The fitness advantage was not due to compensatory mutations in the genes targeted by FQ and was linked directly to the single point mutation in gyrA, which confers on Campylobacter a high-level resistance to FQ antimicrobials. In certain genetic backgrounds, the same point mutation entailed a biological cost on Campylobacter, as evidenced by its inability to compete with the FQ-susceptible Campylobacter. These findings provide a previously undescribed demonstration of the profound effect of a resistance-conferring point mutation in gyrA on the fitness of a major foodborne pathogen in its natural host and suggest that the rapid emergence of FQ-resistant Campylobacter on a worldwide scale may be attributable partly to the enhanced fitness of the FQ-resistant isolates.colonization ͉ gyrA mutation ͉ poultry A ntimicrobial resistance in bacterial pathogens has become a serious threat to public health. There is a general notion that the acquisition of drug resistance, particularly the resistance mediated by chromosomal mutations, entails a biological cost for pathogens, resulting in reduced fitness in the absence of antibiotic selection pressure (1, 2). However, evidence has accumulated that in vivo-selected or clinically derived isolates may develop compensatory mutations that reduce the fitness cost associated with antimicrobial resistance (3-7). Different environments (in vitro vs. in vivo) may select for different compensatory mutations and varied levels of restoration of fitness (8). Even without compensatory mutations, drug-resistant mutants may show little or no fitness cost (9). One important finding revealed by previous studies is that clinically derived drugresistant isolates display diverse fitness changes, with some showing a biological cost and others showing no cost or even enhanced fitness (4, 10). The retention of ecological fitness in resistant pathogens creates a significant barrier for the elimination of resistant organisms by natural selection.Campylobacter jejuni, a Gram-negative microaerobic bacterium, is a common causative agent of human enterocolitis (11). For antibiotic treatment of ca...

CmeABC functions as a multidrug efflux pump contributing to the resistance of Campylobacter to a broad range of antimicrobials. In this study, we examined the role of CmeABC in bile resistance and its contribution to the adaptation of Campylobacter jejuni in the intestinal tract of the chicken, a natural host and a major reservoir for Campylobacter. Inactivation of cmeABC drastically decreased the resistance of Campylobacter to various bile salts. Addition of choleate (2 mM) in culture medium impaired the in vitro growth of the cmeABC mutants but had no effect on the growth of the wild-type strain. Bile concentration varied in the duodenum, jejunum, and cecum of chicken intestine, and the inhibitory effect of the intestinal extracts on the in vitro growth of Campylobacter was well correlated with the total bile concentration in the individual sections of chicken intestine. When inoculated into chickens, the wild-type strain colonized the birds as early as day 2 postinoculation with a density as high as 10 7 CFU/g of feces. In contrast, the cmeABC mutants failed to colonize any of the inoculated chickens throughout the study. The minimum infective dose for the cmeABC mutant was at least 2.6 ؋ 10 4 -fold higher than that of the wild-type strain. Complementation of the cmeABC mutants with a wild-type cmeABC allele in trans fully restored the in vitro growth in bile-containing media and the in vivo colonization to the levels of the wild-type strain. Immunoblotting analysis indicated that CmeABC is expressed and immunogenic in chickens experimentally infected with C. jejuni. Together, these findings provide compelling evidence that CmeABC, by mediating resistance to bile salts in the intestinal tract, is required for successful colonization of C. jejuni in chickens. Inhibition of CmeABC function may not only control antibiotic resistance but also prevent the in vivo colonization of pathogenic Campylobacter.

CmeABC, a resistance-nodulation-division (RND) type of efflux pump, contributes to Campylobacter resistance to a broad spectrum of antimicrobial agents and is also essential for Campylobacter colonization of the animal intestinal tract by mediation of bile resistance. As one of the main systems for Campylobacter adaptation to different environments, CmeABC is likely subject to control by regulatory elements. We describe the identification of a transcriptional repressor for CmeABC. Insertional mutagenesis of cmeR, an open reading frame immediately upstream of the cmeABC operon, resulted in overexpression of cmeABC, as determined by transcriptional fusion (P cmeABC-lacZ ) and immunoblotting with CmeABC-specific antibodies. Overexpression of the efflux pump was correlated with a moderate increase in the level of resistance of the cmeR mutant to several antimicrobials. In vitro, recombinant CmeR bound specifically to the promoter region of cmeABC, precisely, to the inverted repeat sequences in the cmeABC promoter. A single nucleotide deletion between the two half sites of the inverted repeat reduced the level of CmeR binding to the promoter sequence and resulted in overexpression of cmeABC. Together, these findings indicate that cmeR encodes a transcriptional repressor that directly interacts with the cmeABC promoter and modulates the expression of cmeABC. Mutation either in CmeR or in the inverted repeat impedes the repression and leads to enhanced production of the MDR efflux pump.

Enrofloxacin treatment of chickens infected with fluoroquinolone(FQ)-sensitiveCampylobacter promoted the emergence of FQ-resistant Campylobacter mutants which propagated in the intestinal tract and recolonized the chickens. The recovered isolates were highly resistant to quinolone antibiotics but remained susceptible to non-FQ antimicrobial agents. Specific single-point mutations in the gyrA gene and the function of the CmeABC efflux pump were linked to the acquired FQ resistance. These results reveal that Campylobacter is hypermutable in vivo under the selection pressure of FQ and highlight the need for the prudent use of FQ antibiotics. Fluoroquinolone (FQ)-resistantCampylobacter jejuni strains are rapidly increasing throughout the world, which has posed a serious threat to public health (19,20). Although FQ resistance in Campylobacter can occur following the treatment of humans with the antibiotic (4, 17, 22), poultry are considered a significant source for FQ-resistant Campylobacter (1,5,7,18). Laboratory studies have demonstrated the emergence of FQ-resistant Campylobacter in experimental chickens treated with FQ antibiotics (10, 12). However, the previously published works revealed little information on the in vivo dynamics of the emergence of FQ-resistant Campylobacter in chickens. Specifically, it is unknown how Campylobacter populations shift in individual birds in response to the antibiotic treatment and how extensively the infected chickens are colonized by the resistant organisms. In addition, the genetic mechanisms responsible for the in vivo-acquired resistance to FQ in Campylobacter strains are not known. Answering these questions will greatly improve our understanding of the development and mechanisms of FQ resistance in Campylobacter and may facilitate the design of means to prevent the occurrence of FQresistant Campylobacter in vivo.In this study, we examined the dynamics of changes of Campylobacter populations in chickens treated with enrofloxacin and determined the molecular mechanisms associated with the acquired FQ resistance in the in vivo-selected resistant isolates. C. jejuni strain S3B was originally isolated from chicken feces in our laboratory. Bacterial cultures were routinely grown in Mueller-Hinton (MH) broth or plates (Becton Dickinson and Company, Sparks, Md.) at 42°C under microaerophilic conditions generated by the CampyPak Microaerophilic System (BBL). Day-old broiler chickens were obtained from a commercial hatchery. Prior to use, the birds were tested negative for Campylobacter by conventional culture methods. Two independent experiments (A and B) were conducted whose designs are detailed in Table 1. Each group of chickens was maintained in a sanitized wire-floored cage. Feed and water were provided ad libitum. Infection of the chickens with C. jejuni strain S3B (ciprofloxacin MIC ϭ 0.125 g/ml) and treatment with enrofloxacin (Baytril; Bayer Corporation) are detailed in Table 1. Cloacal swabs were collected periodically, resuspended in MH broth, and plated onto MH plates con...

Campylobacter infection is one of the major causes of ovine abortions worldwide. Historically, Campylobacter fetus subsp. fetus was the major cause of Campylobacter-associated abortion in sheep; however, Campylobacter jejuni is increasingly associated with sheep abortions. We examined the species distribution, genotypes, and antimicrobial susceptibilities of abortion-associated Campylobacter isolates obtained from multiple lambing seasons on different farms in Iowa, Idaho, South Dakota, and California. We found that C. jejuni has replaced C. fetus as the predominant Campylobacter species causing sheep abortion in the United States. Most strikingly, the vast majority (66 of 71) of the C. jejuni isolates associated with sheep abortion belong to a single genetic clone, as determined by pulsed-field gel electrophoresis, multilocus sequence typing, and cmp gene (encoding the major outer membrane protein) sequence typing. The in vitro antimicrobial susceptibilities of these isolates to the antibiotics that are routinely used in food animal production were determined using the agar dilution test. All of the 74 isolates were susceptible to tilmicosin, florfenicol, tulathromycin, and enrofloxacin, and 97% were sensitive to tylosin. However, all were resistant to tetracyclines, the only antibiotics currently approved in the United States for the treatment of Campylobacter abortion in sheep. This finding suggests that feeding tetracycline for the prevention of Campylobacter abortions is ineffective and that other antibiotics should be used for the treatment of sheep abortions in the United States. Together, these results indicate that a single tetracycline-resistant C. jejuni clone has emerged as the major cause of Campylobacter-associated sheep abortion in the United States.