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Antimicrob Agents Chemother. Jan 2003; 47(1): 390–394.
PMCID: PMC148968

In Vivo Selection of Campylobacter Isolates with High Levels of Fluoroquinolone Resistance Associated with gyrA Mutations and the Function of the CmeABC Efflux Pump

Abstract

Enrofloxacin treatment of chickens infected with fluoroquinolone(FQ)-sensitive Campylobacter 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)-resistant Campylobacter 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 FQ-resistant 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 Table1.1. 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 = 0.125 μg/ml) and treatment with enrofloxacin (Baytril; Bayer Corporation) are detailed in Table Table1.1. Cloacal swabs were collected periodically, resuspended in MH broth, and plated onto MH plates containing Campylobacter-specific growth supplements (Oxoid Ltd., Basingstoke, England) for the enumeration of Campylobacter cells. The number of FQ-resistant colonies in each sample of experiment B was also determined by using MH plates supplemented with 4 μg of ciprofloxacin/ml. From each group of chickens, 10 to 15 Campylobacter colonies were selected at each sampling time from the regular MH agar plates (no ciprofloxacin) for MIC testing. MICs were determined using Etest strips (AB Biodisk, Solna, Sweden) following the manufacturer's instructions. The sensitive S3B isolate with a ciprofloxacin MIC of 0.125 μg/ml was used as an internal control for testing the isolates obtained from the chickens. Since there are presently no NCCLS-approved standards for C. jejuni (13), “resistant” refers to those isolates with ciprofloxacin MICs of ≥4 μg/ml and “susceptible” refers to those isolates with ciprofloxacin MICs of ≤1.0 μg/ml.

TABLE 1.
Experimental design of the chicken studies (experiments A and B)

Changes in Campylobacter colonization in response to enrofloxacin treatment.

The chickens inoculated with S3B were quickly colonized and started to shed C. jejuni at the level of 106 CFU/g of feces 2 days after inoculation. As shown in Fig. Fig.1,1, the number of colonized chickens in the enrofloxacin-treated groups dropped substantially after initiation of the enrofloxacin treatment and then increased steadily, even with continuation of the treatment, and reached the pretreatment level 3 days after the cessation of treatment. In the few chickens that remained colonized at day 1 after initiation of quinolone treatment, the numbers of C. jejuni cells in feces also decreased considerably (data not shown). In parallel to the steady recovery of the percentage of colonized chickens, the shedding levels of C. jejuni increased in the colonized chickens and reached the levels of nontreated groups after the cessation of treatment (data not shown). The chickens inoculated with S3B but not receiving FQ treatment remained colonized during the entire experimental period (Fig. (Fig.1).1). The control chickens without inoculation of S3B were Campylobacter free throughout the experiment. These results indicate that FQ treatment reduced Campylobacter colonization transiently but did not eliminate the organism from the infected chickens. Instead, Campylobacter colonization recovered steadily even with the continuation of FQ treatment.

FIG. 1.
Percentage of chickens colonized with C. jejuni in experiments A and B as determined by direct plating. A chicken was considered noncolonized if the level of colonization was below the detection limit (102 organisms/g of feces). Each bar represents the ...

Emerging dynamics of FQ-resistant C. jejuni in enrofloxacin-treated chickens.

Enrofloxacin and ciprofloxacin are structurally related FQ antibiotics. Ciprofloxacin is one of the key antibiotics used for treating human campylobacteriosis (6), while enrofloxacin is licensed exclusively for veterinary use. To determine if the therapeutic use of enrofloxacin in chickens selects for ciprofloxacin-resistant Campylobacter, 10 to 15 Campylobacter isolates were selected from each group at each sampling time and subjected to Etest with ciprofloxacin strips. As shown in Fig. Fig.2,2, Campylobacter isolates from chickens in the nontreated groups remained sensitive (ciprofloxacin MICs = 0.064 to 0.125 μg/ml) to FQ throughout the entire experiment. In the treated groups, FQ-resistant C. jejuni was not detected before initiation of the treatment. However, resistant C. jejuni organisms were isolated from the treated chickens as early as 1 day after the initiation of enrofloxacin treatment (Fig. (Fig.2B).2B). Once FQ-resistant C. jejuni cells appeared in the chickens, the majority of the recovered isolates became highly resistant (MICs > 32 μg/ml) to ciprofloxacin. Only a few resistant isolates showed intermediate levels of resistance (ciprofloxacin MICs = 6 to 16 μg/ml). The ciprofloxacin-resistant isolates were also cross-resistant to nalidixic acid and enrofloxacin (Table (Table22).

FIG. 2.
Dynamics of emergence of FQ-resistant Campylobacter in chickens of experiments A and B. (A and B1) Resistance rates of randomly selected Campylobacter isolates as determined by Etest. (B2) Change of resistance rates in Campylobacter populations in the ...
TABLE 2.
Effect of cmeB mutation on the susceptibility to ciprofloxacin, nalidixic acid, and enrofloxacin

The above observations revealed the occurrence of FQ-resistant Campylobacter in enrofloxacin-treated chickens, which was consistent with the results reported by other investigators (10, 12). In addition to demonstrating the development of resistance, we further monitored the dynamic changes of Campylobacter populations in individual chickens by differential plating, in which the resistant colonies and the total Campylobacter colonies in each sample were simultaneously enumerated by using two different types of plates (Table (Table1)1) (experiment B). This unique plating method revealed that, once the treatment was initiated, the FQ-sensitive Campylobacter populations rapidly diminished in the treated chickens, with the concurrent occurrence of resistant organisms in the birds (Fig. (Fig.2).2). Only about 20% of the chickens in each treated group remained colonized and showed resistant Campylobacter initially. Then, the number of chickens colonized by the resistant organisms steadily increased during the course of treatment, and eventually all chickens in the treated groups were colonized by large numbers (up to 108 CFU/g of feces) of resistant Campylobacter. Together, these observations suggest the possibility that resistant Campylobacter initially occurred in only a small number of chickens and subsequently spread to other penmate chickens probably via horizontal transmission. It was also noticed that, due to FQ treatment, the background bacterial counts in the fecal samples were also drastically reduced. Thus, the antibiotic treatment diminished numbers of not only FQ-sensitive Campylobacter but also other competing bacterial flora in the gut, resulting in a favorable environment for the rapid propagation and transmission of FQ-resistant Campylobacter mutants in the experimental chickens.

Representative Campylobacter isolates obtained at different sampling times were analyzed by pulsed-field gel electrophoresis (PFGE). Both FQ-sensitive and FQ-resistant C. jejuni isolates were identical to each other as well as to the original S3B strain in terms of PFGE patterns (data not shown). This result indicates that the FQ-resistant isolates were not derived from environmental contamination but rather had evolved from the original FQ-sensitive C. jejuni populations inoculated into the experimental chickens. Interestingly, in the 25-ppm group of experiment B, sensitive Campylobacter populations reemerged after the cessation of FQ treatment (Fig. (Fig.2B).2B). The recovered sensitive organisms had the same PFGE patterns as the original S3B inoculum and a ciprofloxacin MIC of 0.125 μg/ml, suggesting that they most likely represented the reestablishment of the original sensitive organisms that survived the FQ treatment because of inadequate water intake in some birds or other unknown reasons.

Association of gyrA mutations with the acquired resistance.

To determine the mechanisms responsible for the acquired FQ resistance, primers P1 and P2, reported by Wang et al. (21), were used to amplify the quinolone resistance-determining region of the gyrA gene in Campylobacter. Four FQ-sensitive and 11 FQ-resistant isolates were used for the PCR. Sequence analysis of the PCR products revealed no mutations in the QRDR of gyrA in the FQ-sensitive isolates. However, the Thr-86-Ile change in gyrA was observed in the isolates that were highly resistant to ciprofloxacin (MIC > 32 μg/ml), while the Asp-90-Asn and Thr-86-Lys mutations were detected in the isolates with intermediate levels of resistance (MIC = 6 to 16 μg/ml). These types of gyrA mutations were also observed in the clinical human isolates reported in previous studies (8, 16, 21). This finding provides direct evidence that FQ-resistant Campylobacter isolates from the human and chicken share the same types of gyrA mutations.

To determine if mutations in parC were involved in the acquired FQ resistance, multiple pairs of primers were designed from the published parC sequence (8) and were used to amplify the gene sequence from the Campylobacter isolates used in this study. Despite extensive efforts involving multiple experiments, we were unable to detect any parC-related sequences in the Campylobacter isolates although the same pairs of primers consistently amplified parC-specific products from Escherichia coli. Similar findings were recently reported by Bachoual et al. (2), who, using various parC-specific primers, failed to identify parC in the Campylobacter strains used in their study. In addition, the genomic sequence of C. jejuni NCTC11168 did not reveal a parC homolog (14). Based on these facts, we conclude that C. jejuni lacks parC. The absence of parC in the Campylobacter isolates ruled out its contribution to the acquired FQ resistance.

Contribution of CmeABC to the acquired FQ resistance.

CmeABC is a newly characterized multidrug-resistant pump encoded by a three-gene operon (cmeA, cmeB, and cmeC) located on a Campylobacter chromosome (11). It was shown previously that CmeABC functions as an energy-dependent efflux system and contributes to the intrinsic resistance of C. jejuni to FQ (11). However, the role of CmeABC in the acquired FQ resistance of C. jejuni has not been examined. To determine the contribution of CmeABC to the acquired FQ resistance, this pump was inactivated in six FQ-resistant isolates (with different gyrA mutations) and one FQ-sensitive isolate by inserting the EZ::TN <KAN-2> transposon (Epicentre) into the cmeB gene (Table (Table2).2). Compared with the wild-type resistant isolates, the CmeB mutants showed a marked decrease in their resistance to ciprofloxacin and enrofloxacin (Table (Table2).2). The residual resistance in the CmeB mutants of the FQ-resistant isolates was still above the level of the wild-type sensitive isolates but below the level of clinical significance (Table (Table2).2). The cmeB-specific mutation had a less drastic but still substantial effect on the resistance to nalidixic acid (Table (Table2).2). Together, these results suggest that CmeABC is required to maintain high-level FQ resistance in the in vivo-selected clinical isolates.

To examine if CmeABC was overexpressed in the FQ-resistant isolates, the cell envelope proteins of representative sensitive and resistant isolates grown in MH broth (with no FQ antibiotics) were analyzed by immunoblotting with antibodies specific for CmeB and CmeC as described previously (11). In all, 20 sensitive isolates (ciprofloxacin MICs = 0.064 to 0.125 μg/ml) and 20 resistant isolates (ciprofloxacin MICs > 32 μg/ml) recovered from the chickens were examined in this study. No difference was observed between the sensitive and resistant isolates regarding the expression levels of CmeB and CmeC (data not shown). Similar amounts of the major outer membrane protein, which is encoded by the cmp gene (23) and functions as a porin in Campylobacter (3, 9), were observed in the sensitive and resistant isolates (data not shown). In addition, there were no changes in the gene sequences encoding the porin (data not shown), suggesting that change of membrane permeability was unlikely associated with the acquired FQ resistance. To determine if the in vivo-selected FQ-resistant isolates were cross-resistant to other antibiotics, 29 highly FQ-resistant (ciprofloxacin MIC > 32 μg/ml) isolates were tested with erythromycin, ampicillin, cefotaxime, and chloramphenicol by the microtiter broth dilution method in MH broth (11). None of the isolates displayed a resistance phenotype to the antibiotics. These observations contradict the findings with other gram-negative bacteria that acquired FQ resistance is often associated with the overexpression of efflux pumps (15). However, this study only examined the expression of CmeABC in MH broth with no FQ antibiotics. It is unknown if the production of CmeABC can be enhanced in the presence of ciprofloxacin. Future studies are needed to examine the possible induction of this efflux pump by FQ antibiotics.

In summary, results from this study demonstrate that C. jejuni displays a hypermutable phenotype in infected chickens under the selection pressure of FQ antibiotics, highlighting the need for the prudent use of FQ antibiotics in farm settings. The findings also open new avenues for further investigation into reducing or eliminating FQ resistance in C. jejuni. As proposed by Zhao and Drlica (24), development of resistant bacterial mutants may be avoided by the use of antibiotic concentrations beyond the mutant selection window. It would be interesting to know if the administration of enrofloxacin can be modified to achieve the mutant prevention concentrations in vivo so that the emergence of FQ-resistant Campylobacter can be eliminated. In addition, the CmeABC efflux system may be targeted in vivo to prevent the occurrence of resistant Campylobacter in clinical settings or in animal reservoirs.

Acknowledgments

We thank Jerrel C. Meitzler for technical help.

This work was supported in part by USDA CSREE competitive grant 00-51110-9741.

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