JAC Advance Access originally published online on October 5, 2006
Journal of Antimicrobial Chemotherapy 2006 58(6):1154-1159; doi:10.1093/jac/dkl412
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Role of the CmeABC efflux pump in the emergence of fluoroquinolone-resistant Campylobacter under selection pressure
Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine Iowa State University, Ames, IA 50011, USA
*Corresponding author. Tel: +1-515-294-2038; Fax: +1-515-294-8500; E-mail: zhang123{at}iastate.edu
Received 23 June 2006; returned 7 August 2006; revised 14 September 2006; accepted 14 September 2006
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Abstract |
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Objectives: The objective of this study was to determine the contribution of the CmeABC efflux pump to the emergence of fluoroquinolone (FQ)-resistant mutants in Campylobacter jejuni under various levels of selection pressure.
Methods: The frequency of emergence of ciprofloxacin-resistant mutants was measured in wild-type C. jejuni NCTC 11168 and its isogenic cmeB mutant and cmeR mutant (overexpressing cmeABC) using plates containing various concentrations of ciprofloxacin. Representative ciprofloxacin-resistant mutants were selected for gyrA sequence analysis and MIC determination. Accumulation of ciprofloxacin in Campylobacter cells was measured using spectrofluorometry.
Results: Mutation of cmeB drastically reduced the frequency of emergence of FQ-resistant mutants at 10x and 32x the MIC of ciprofloxacin, while the cmeR mutant displayed an approximately 17-fold increase in the frequency of emergence of the mutants at 32x the MIC when compared with the wild-type strain. Various point mutations occurred in gyrA in the FQ-resistant mutants selected at 5x and 10x the MIC, while the Thr-86
Ile mutation was predominant in the mutants selected at 32x the MIC. The Thr-86Ile change conferred a high-level resistance to FQs, but other mutations only conferred an intermediate-level FQ resistance. In contrast, all types of gyrA mutations in the CmeABC-overexpressed background conferred high-level resistance to ciprofloxacin. Overexpression of cmeABC significantly reduced the amount of ciprofloxacin accumulated within bacterial cells.
Conclusions: CmeABC is not only important for maintaining high-level resistance to FQs but also contributes significantly to the emergence of FQ-resistant mutants. Inhibition of this efflux pump may prevent the emergence of clinically relevant FQ-resistant Campylobacter mutants.
Keywords: ciprofloxacin , gyrA mutations , efflux , antibiotic resistance
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Introduction |
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Campylobacter jejuni is a major enteric pathogen causing gastroenteritis in humans worldwide.1–3 Patients with campylobacteriosis often experience severe abdominal pain, fever, and watery/bloody diarrhoea. In addition, Campylobacter infections are also occasionally associated with Guillain-Barré syndrome (GBS), an autoimmune disorder of the peripheral nervous system characterized by muscle weakness.4,5 Fluoroquinolone (FQ) antimicrobials are often prescribed for clinical treatment of diarrhoea caused by enteric bacterial pathogens including Campylobacter.6 However, the effectiveness of treatment has been compromised due to the increasing prevalence of FQ-resistant Campylobacter commonly found in many countries, with some studies reporting a resistance rate up to 99%.7–10 In developed countries, most of the Campylobacter infections are food-borne, primarily from contaminated food, water or raw milk, while person-to-person transmission rarely happens.11
Campylobacter spp. are highly prevalent in food-producing animals, especially in poultry. Thus, many FQ-resistant Campylobacter strains in human patients are believed to be transferred from food producing animals, mostly from poultry.12 The usage of FQs in food-producing animals is considered a significant selective force for the development of FQ resistance in Campylobacter.3,13 Acquisition of FQ resistance also influences the fitness of Campylobacter in vivo, as was reported recently by Luo et al.14 who found that many FQ-resistant Campylobacter outcompete the FQ-susceptible Campylobacter in the intestinal tract of chickens. In addition, FQ resistance can also prolong the duration of the clinical illness caused by C. jejuni.15 Another recent report showed that patients infected with FQ-resistant Campylobacter strains had a more than 6-fold increase in the risk of having an adverse event compared with patients infected with FQ-susceptible Campylobacter strains.16 Therefore, FQ-resistant Campylobacter is a significant threat to public health, and an augmented effort is needed to mitigate the emergence of FQ-resistant Campylobacter strains.
The main targets of FQs are DNA gyrase and/or topoisomerase IV.17,18 In Campylobacter, spontaneous point mutations in the A subunit of DNA gyrase (GyrA) are mainly responsible for the resistance. The locations of the point mutations are clustered in the quinolone resistance-determining region (QRDR) of gyrA, and different gyrA mutations confer varied levels of resistance to FQs.19–21 Among different types of gyrA mutations found in Campylobacter, the Thr-86Ile change is the most frequently identified and can confer high-level resistance to FQs (MIC 
16 mg/L), while Asp-90Asn and Thr-86Lys can only achieve an intermediate-level resistance (4 mg/L < MIC < 16 mg/L).20,22–24 Another mechanism associated with FQ resistance in Campylobacter is mediated by active drug efflux systems.24,25 CmeABC is the best characterized drug efflux system in Campylobacter and contributes to its intrinsic and acquired resistance to a broad-spectrum of antibiotics including FQs.26–28 In our previous study using the cmeB mutant and the efflux pump inhibitor CCCP, we showed that CmeABC played a major role in the efflux of ciprofloxacin in C. jejuni.26 Constitutive expression of cmeABC occurs at a moderate level in wild-type Campylobacter strains; however, CmeABC is subject to regulation by a transcriptional repressor, named CmeR.29 CmeR binds to the inverted repeat in the promoter region of cmeABC and represses the transcription of the efflux operon. Insertional mutagenesis of the cmeR gene or mutation in the inverted repeat sequences results in overexpression of CmeABC, which enhances the resistance to multiple antibiotics including FQs. CmeABC and gyrA mutations work together to confer high-level FQ resistance in Campylobacter because inactivation of CmeABC resulted in a significant reduction in the resistance level of clinical isolates harbouring the resistance-associated mutations in gyrA.23
Spontaneous Campylobacter mutants resistant to FQ antimicrobials occur at a frequency of 
5 x 10–8 when selected in culture containing 4 mg/L ciprofloxacin.30 In addition, Campylobacter displays a highly mutable phenotype in vivo in response to FQ treatment because a large number of FQ-resistant Campylobacter were detected in chickens or human patients treated with FQ antimicrobials.23,24,31–33 Although the genetic mechanisms for FQ resistance have been well characterized, it is unclear what influences the frequency of emergence of FQ-resistant Campylobacter. In Streptococcus pneumoniae and Pseudomonas aeruginosa, it has been shown that efflux pumps play an important role in the emergence of FQ-resistant mutants.34,35 Since CmeABC contributes significantly to the intrinsic and acquired resistance in Campylobacter to FQs, we hypothesized that disruption of the CmeABC efflux system in Campylobacter would dramatically reduce the frequency of the emergence of FQ-resistant mutants, while overexpression of this efflux pump could increase the emergence of FQ-resistant mutants. To test this hypothesis, we compared the frequencies of emergence of FQ-resistant Campylobacter in various genetic backgrounds and determined the impact of various gyrA mutations and the expression status of CmeABC on the levels of FQ resistance.
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Bacterial strains and culture conditions
C. jejuni NCTC 11168 and its isogenic cmeB mutant (11168B) and cmeR mutant (11168R)36 were used in this study. Despite the repression by CmeR, CmeABC is constitutively expressed in 11168 and other wild-type Campylobacter strains at a moderate level.36–38 In 11168B, the cmeB gene was inactivated by insertion of a kanamycin resistance cassette (cmeB::kan), which abolished the expression of cmeBC and the function of CmeABC, resulting in increased sensitivity to various antibiotics.36 In 11168R, the cmeR gene was disrupted with a chloramphenicol resistance cassette, leading to the overexpression of CmeABC.29 These strains were routinely grown in Mueller–Hinton (MH) broth (Difco) or agar at 42°C under microaerobic conditions, which were generated using the Campypak Plus (Becton Dickinson) gas pack in an enclosed jar.
Detection of the frequencies of emergence of ciprofloxacin-resistant mutants
Strains 11168, 11168B and 11168R were grown in 10 mL of antibiotic-free MH broth for 24 h under microaerobic conditions. The cultures were collected by centrifugation and re-suspended in 1 mL of MH broth. The total cfu in the concentrated cultures were measured by serial dilutions and plating on MH plates. To estimate the frequencies of emergence of FQ-resistant mutants, each concentrated sample was plated onto duplicate antibiotic-free MH agar plates and ciprofloxacin-containing MH plates. This experiment was repeated three times. The concentrations of ciprofloxacin used for selection in MH agar were 0.625 mg/L, which is 5-fold (5x) the MIC for 11168; 1.25 mg/L, which is 10x the MIC, and 4 mg/L, which is 32x the MIC and the suggested breakpoint for ciprofloxacin resistance in Campylobacter.39 The frequency of emergence of FQ-resistant mutants was calculated as the ratio of the cfu on ciprofloxacin-containing MH agar plates to the cfu on ciprofloxacin-free MH agar plates after 2 days of incubation at 42°C under microaerobic conditions.
Sequence analysis of the QRDR of gyrA
Multiple FQ-resistant colonies were randomly selected from the ciprofloxacin-containing plates. The QRDR of gyrA was amplified by PCR using primer pair GyrAF1 (5'-CAACTGGTTCTAGCCTTTTG-3') and GyrAR1 (5'-AATTTCACTCATAGCCTCACG-3').20 PCR was carried out for 35 cycles, each consisting of a denaturation (45 s at 95°C), annealing (30 s at 52°C) and extension step (1 min at 72°C). The amplified PCR products were purified with the QIAquick PCR purification kit (Qiagen) prior to sequencing. DNA sequence analysis was carried out using an automated ABI Prism 377 sequencer (Applied Biosystems) and analysed by the Omiga 2.0 (Oxford Molecular Group) sequencing analysis software.
Antimicrobial susceptibility test
MICs for all the strains including the ciprofloxacin-resistant mutants were determined with the agar dilution method as recommended by the CLSI (formerly NCCLS).39 Ciprofloxacin (Pentex Products), erythromycin, doxycycline, gentamicin (Sigma Chemical) and meropenem (AstraZeneca) were used in the susceptibility tests.
Accumulation assay for ciprofloxacin
The accumulation of ciprofloxacin in C. jejuni cells was determined as previously described with some modifications.26 Strains were grown in MH broth for 24 h to the late log phase. The cells were harvested, washed once in 15 mM PBS (pH 7.2), resuspended in PBS to 1010–11 cfu/mL (10 mg wet bacterial pellet) and incubated at 37°C for 10 min. Then, ciprofloxacin was added to the culture to a final concentration of 10 mg/L. After the addition of ciprofloxacin, 0.5 mL of each sample was removed at different time points, immediately diluted in 2.5 mL of ice-cold PBS and then centrifuged for 5 min at 6000 g at 4°C. The pellets were washed once with 2 mL of ice-cold PBS, resuspended in 2 mL of 0.1 M glycine hydrochloride (pH 3.0) and shaken at room temperature for 16 h. The samples were then centrifuged at 6000 g for 15 min. The fluorescence of the supernatant was measured with a Perkin–Elmer (Norwalk, CT, USA) spectrofluorometer at excitation and emission wavelengths of 279 and 452 nm, respectively. The results were expressed as ng of ciprofloxacin per mg (wet weight) of bacteria. Three independent experiments were performed to measure the accumulation of ciprofloxacin.
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Frequency of emergence of FQ-resistant Campylobacter in different genetic backgrounds
To determine the influence of CmeABC on the emergence of FQ-resistant Campylobacter, three strains with different expression levels of cmeABC were used in this study, including 11168 (wild-type with a moderate level of CmeABC), 11168B (CmeB-negative) and 11168R (overexpressing cmeABC).29,36 The results are summarized in Table 1. When 0.625 mg/L ciprofloxacin was used for selection, all three strains showed similar frequencies of emergence of FQ-resistant mutants (10–6; P 0.05). At 1.25 mg/L ciprofloxacin, 11168 and 11168R showed similar frequencies of mutant emergence (10–6), while the frequency of mutant emergence in 11168B decreased dramatically to 10–9. The effect of the efflux pump on the emergence of FQ-resistant mutants was even more prominent at the higher selection pressure (4 mg/L ciprofloxacin), at which the frequency of emergence of ciprofloxacin-resistant mutants was about 17-fold higher (P < 0.05) in 11168R than that in 11168, while the rate of mutant emergence in 11168 was more than 20-fold greater (P < 0.05) than that in 11168B (Table 1). These results clearly indicated that inactivation of CmeABC decreased the frequencies of emergence of FQ-resistant mutants under selection pressure, especially at the higher antibiotic concentrations, while overexpression of CmeABC increased the frequency of emergence of FQ-resistant mutants.
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Different point mutations in gyrA were detected in the FQ-resistant mutants
Mutations in gyrA were found to be present in all the ciprofloxacin-resistant mutants examined in this study, and each of the mutants carried a single point mutation in the QRDR (Table 2). Four different types of point mutations were observed, including Thr-86Ile, Thr-86Lys, Asp-90Asn and Asp-90Tyr. The first three types of mutations have been reported previously,20,22,24,40 while the Asp-90Tyr change represents a new mutation identified in the FQ-resistant C. jejuni mutants. For the mutants selected on plates containing 0.625 and 1.25 mg/L ciprofloxacin, all four types of mutations were detected; however, the Thr-86Ile change was the only mutation detected in 11168 mutants selected on plates containing 4 mg/L ciprofloxacin, indicating that only this type of mutant could survive the high selection pressure. This finding is consistent with previous findings that the Thr-86Ile mutation can confer a high-level resistance to FQs, while other mutations confer an intermediate-level of resistance in Campylobacter.20,23,24 Notably, no mutants were detected in 11168B on plates containing 4 mg/L ciprofloxacin, indicating that CmeABC is essential for the survival of the gyrA mutants under high selection pressure. Another interesting finding was that the mutants of 11168R selected on 4 mg/L ciprofloxacin had two types of mutations including Thr-86Ile and Thr-86Lys, suggesting that Thr-86Lys was also able to confer high-level FQ resistance in the CmeABC-overexpressed background.
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Effects of different GyrA mutations and the expression level of CmeABC on ciprofloxacin MIC
The MIC of ciprofloxacin for the mutants selected from different genetic backgrounds and under different selection pressures was determined by the agar dilution method (Table 2). In the wild-type 11168 background, all mutants carrying the Thr-86Ile mutation had a ciprofloxacin MIC of 16 mg/L, while the mutants carrying other types of GyrA mutations had a ciprofloxacin MIC of 4 mg/L, confirming that the Thr-86Ile change confers a high level resistance to ciprofloxacin. All of the mutants selected from 11168B had ciprofloxacin MICs 
4 mg/L, regardless of the types of GyrA mutations, which suggests that without a functional CmeABC, none of the GyrA mutations can confer a high-level FQ resistance and explains why no mutants were selected from 11168B at 4 mg/L ciprofloxacin. In contrast, all of the mutants selected from 11168R had a ciprofloxacin MIC of 16 mg/L regardless of the types of GyrA mutations, indicating that in the CmeABC-overexpressed background, all types of GyrA mutations can confer a high level of resistance to ciprofloxacin. These findings clearly indicate that FQ resistance in C. jejuni mutants is affected not only by the point mutations in GyrA but also by the expression level of CmeABC. We also tested the MICs of several other antibiotics in the FQ-resistant mutants, none of which showed cross-resistance to the tested antibiotics including erythromycin, doxycycline, gentamicin and meropenem (data not shown).
Decreased accumulation of ciprofloxacin in 11168R
Previous studies have shown that CmeABC functions as an active efflux system and the cmeB mutants accumulate more ciprofloxacin than their wild-type strains.26,28 Since all of the 11168R mutants showed high-level resistance to ciprofloxacin, we suspected that this was due to the reduced accumulation of ciprofloxacin within the cells since cmeABC was overexpressed in 11168R. To test this possibility, we compared the accumulation of ciprofloxacin in 11168 and 11168R. Three independent experiments consistently showed that 11168R accumulated 40–50% less ciprofloxacin than the wild-type 11168. The differences are statistically significant (P < 0.05) and the result from a representative experiment is shown in Figure 1. This finding suggests that the increased efflux contributes to the high FQ resistance in the mutants selected from 11168R.
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This study demonstrated that the expression level of cmeABC affects the frequency of emergence of FQ-resistant mutants in Campylobacter. This conclusion was based on several observations. Firstly, the frequency of emergence of FQ-resistant mutants in the CmeB null mutant (11168B) was significantly lower than that of the wild-type strain at 10x and 32x the MIC (Table 1). Secondly, 11168R, in which cmeABC is overexpressed, had a significantly higher frequency of emergence of FQ-resistant mutants than the wild-type strain (Table 1). Thirdly, the same type of gyrA mutation conferred different levels of resistance to ciprofloxacin in different strains with varying levels of CmeABC expression (Table 2). Finally, 11168R accumulated significantly less ciprofloxacin than the wild-type strain (Figure 1). Together, these findings indicate that CmeABC facilitates the emergence and survival of FQ-resistant mutants by reducing the accumulation of FQ antimicrobials in Campylobacter cells.
The frequency of emergence of FQ-resistant mutants was similar (10–6) at 5x the MIC, regardless of the expression status of cmeABC (Table 1), suggesting that all of the resistance-associated gyrA mutations can confer a level of resistance to ciprofloxacin 0.625 mg/L. However, when ciprofloxacin was used at 10x the MIC (1.25 mg/mL) for selection, the frequency of emergence of FQ-resistant mutants in 11168B dropped about 1000-fold to 10–9, while the frequency of mutant emergence stayed at about the same level (10–6) for the wild-type strain and 11168R mutant. This finding suggests that without CmeABC, some of the gyrA mutants could not survive the selection by 1.25 mg/L ciprofloxacin. On the other hand, a moderate-level expression of CmeABC is sufficient and overexpression of this pump is not required for the gyrA mutants to survive the selection at 1.25 mg/L ciprofloxacin. This explanation is further supported by the MIC data shown in Table 2, in which all of the tested mutants had a ciprofloxacin MIC of 2 mg/L. At 32x the MIC (4 mg/L ciprofloxacin), which is the recommended breakpoint for Campylobacter resistance,39 the frequency of mutant emergence dropped about 100-fold to 10–8 for the wild-type strain and 10-fold to 10–7 for 11168R overexpressing CmeABC, resulting in a 17-fold difference between 11168 and 11168R (Table 1). In addition, no resistant colonies were detected from the 11168B mutant strain at 4 mg/L ciprofloxacin (Table 1). These results clearly indicated that without CmeABC, none of the gyrA mutants could survive the high selection pressure, while overexpression of CmeABC facilitated the emergence of FQ-resistant mutants at 4 mg/L ciprofloxacin. Since 4 mg/L is considered as the breakpoint for clinical resistance, inactivation of CmeABC may abolish the emergence of FQ-resistant Campylobacter mutants with clinical relevance. The observed differences between 11168 and 11168B in the frequencies of mutant emergence can be directly attributed to the loss of the CmeABC efflux function and are unlikely a non-specific effect of the cmeB knockout because disruption of CmeF, another RND-type drug transporter in Campylobacter,36 did not show any effects on the frequency of emergence of ciprofloxacin-resistant mutants (data not shown).
Several interesting observations were made with the types of GyrA mutations in different strains. In 11168, the Thr-86Ile mutation was not predominant in the mutants selected on plates containing 0.625 and 1.25 mg/L ciprofloxacin, but was the only one selected on plates containing 4 mg/L ciprofloxacin (Table 2). This finding suggests that a variety of point mutations spontaneously occur in the QRDR of GyrA, but the Thr-86Ile change is the sole one that can be selected by the high selection pressure in the background with a moderate-level expression of cmeABC. In 11168B, several types of GyrA mutations appeared in the mutants selected at the low selection pressure (0.625 and 1.25 mg/L ciprofloxacin), while no mutants were detected on plates containing 32x the MIC of ciprofloxacin, indicating that even the Thr-86Ile mutation cannot confer a high-level resistance without the function of CmeABC. In 11168R, more than one type of GyrA mutations occurred even in the mutants selected on the plates containing 32x the MIC of ciprofloxacin (Table 2). The observed mutation types in various genetic backgrounds (in terms of cmeABC expression) and at different levels of selection pressure can be explained by the MIC data of the examined mutants (Table 2). For example, no mutants were detected from 11168B at 4 mg/L ciprofloxacin because none of the mutants had a ciprofloxacin MIC > 4 mg/L regardless of the types of GyrA mutations. In contrast, all of the FQ-resistant mutants from the 11168R strain showed a ciprofloxacin MIC of 16 mg/L, which explains the fact that the mutants carrying a mutation other than the Thr-86Ile change can also be selected by 4 mg/L ciprofloxacin from 11168R.
To our knowledge, this is the first study showing the direct effect of CmeABC expression on the emergence and resistance levels of FQ-resistant mutants in C. jejuni, although similar observations have been described with efflux pumps in other bacteria.34,35 Even though the findings of this study are derived from in vitro experiments, they may have direct implications for the occurrence of FQ-resistant mutants in vivo. Previously we and others demonstrated that FQ-resistant mutants rapidly emerged in Campylobacter-infected chickens treated with enrofloxacin.23,31 We also showed that bile salts induce the expression of CmeABC in C. jejuni.37 Considering the ubiquitous presence of bile compounds in animal intestine, it is likely that the CmeABC efflux pump is up-regulated in the animal intestinal tract, which may facilitate the emergence of FQ-resistant mutants in vivo when treated with FQ antimicrobials. The new results from this study, along with the previous findings that CmeABC is an important player for multidrug resistance and in vivo colonization by mediating bile resistance,26,37 provide a strong rationale for targeting CmeABC to reduce the occurrence and spread of antibiotic-resistant Campylobacter. Since CmeABC is present in different C. jejuni strains,26,28,38 an inhibitor or blocker of CmeABC may have a broad anti-Campylobacter activity. This possibility remains to be investigated in future studies.
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None to declare.
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Present address. Department of Animal Science, University of Tennessee, Knoxville, TN 37996, USA 
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This study is supported by National Institute of Health grant DK063008 and USDA CSREES grant 2005-34211-15629.
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