JAC Advance Access originally published online on September 7, 2006
Journal of Antimicrobial Chemotherapy 2006 58(5):966-972; doi:10.1093/jac/dkl374
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Effect of efflux pump inhibitors on bile resistance and in vivo colonization of Campylobacter jejuni
Department of Animal Science, The University of Tennessee 2505 River Drive, Knoxville, TN 37996, USA
*Corresponding author. Tel: +1-865-974-5598; Fax: +1-865-974-7297; E-mail: jlin6{at}utk.edu
Received 19 July 2006; returned 31 July 2006; revised 5 August 2006; accepted 15 August 2006
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Abstract |
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Objectives: The multidrug efflux pump CmeABC is essential for Campylobacter colonization in animal intestine by mediating bile resistance. The objective of this study is to examine the effect of inhibition of the CmeABC pump by efflux pump inhibitor (EPI) on the susceptibility of Campylobacter to bile salts and to evaluate the in vivo efficacy of two EPIs on the colonization of Campylobacter in a host.
Methods: Two wild-type Campylobacter jejuni strains and their isogenic cmeB mutants were used to determine the susceptibilities of the strains to various bile salts in the presence of EPI MC-207,110 or MC-04,124. The in vivo effect of the EPIs on the colonization of C. jejuni in a host was evaluated using a chicken model system.
Results: The presence of EPIs resulted in a 16- to 512-fold reduction in the MICs of bile salts in both C. jejuni strains. Compared with wild-type strains, cmeB mutants displayed much smaller magnitudes of reduction in the MICs of bile salts, indicating that the in vitro effect of the EPI is primarily mediated by the CmeABC efflux pump. Investigation of 21 Campylobacter isolates from various origins further showed that the EPI MC-207,110 decreased bile resistance in all isolates. Single oral administration of EPI (MC-207,110 or MC-04,124) at two different doses reduced colonization of C. jejuni in chickens at 2–4 days post-inoculation only. Oral administration of MC-207,110 for three consecutive days following inoculation of C. jejuni did not result in a more significant reduction in the level of Campylobacter colonization in chickens.
Conclusions: Inhibition of Campylobacter efflux pumps by EPIs is a potential means for therapeutic intervention to reduce colonization of C. jejuni in humans and animal reservoirs.
Keywords: antimicrobial , pathogenesis , therapeutic intervention
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Introduction |
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Campylobacter jejuni is the leading bacterial cause of human enteritis in many industrialized countries.1,2 Campylobacter infections in humans vary from mild diarrhoea to severe cramping and abdominal pain.3 This pathogenic organism is also associated with Guillain–Barre syndrome, an autoimmune disease that may lead to respiratory muscle compromise and death.4 The majority of human C. jejuni infections are epidemiologically linked to ingestion of contaminated poultry meat.2,5 In parallel to its increased prevalence, C. jejuni has become increasingly resistant to antibiotics, greatly compromising the effectiveness of antibiotic treatments and posing a serious threat to public health.6 Therefore, development of effective strategies to prevent or eliminate Campylobacter infections is urgently needed. To achieve this goal, it is essential to develop an understanding of host–pathogen interaction, such as the mechanisms utilized by Campylobacter to adapt in the intestinal environment in the presence of various antimicrobial agents (e.g. bile salts). Understanding the in vivo adaptation mechanisms may facilitate the development of effective means to prevent and control Campylobacter infections in humans and animal reservoirs.
The CmeABC is characterized as a resistance-nodulation-division (RND)-type multidrug efflux system in Campylobacter.7,8 A unique feature of this efflux pump is that CmeABC not only contributes to multidrug resistance but also plays an essential role in in vivo colonization of C. jejuni by mediating bile resistance, as evidenced by the inability of a cmeB null mutant to colonize chickens.7,9 Notably, the CmeABC efflux pump can be dramatically induced by bile salts present in the intestine, which may facilitate rapid adaptation of Campylobacter to the stresses encountered in the intestine.10 This notion is further supported by a recent study in which the expression of cmeABC was found to be highly up-regulated (up to 300-fold using real-time RT–PCR) in rabbit ileal loops.11 Together, these findings demonstrate the significance of CmeABC in Campylobacter adaptation to the intestinal environment in the host and support the notion that bile resistance is a natural function of CmeABC. Thus, the CmeABC efflux system is an attractive target for the development of intervention strategies against Campylobacter infections in humans and animal reservoirs.
It has been proposed that inhibition of multidrug efflux systems by efflux pump inhibitors (EPIs) is a novel approach to enhance drug accumulation inside the bacterial cell, thereby increasing bacterial susceptibility to antimicrobials.12,13 Recently, promising EPI lead compounds have been discovered and demonstrated to prevent and control antibiotic resistance in bacteria, based on extensive in vitro but very limited in vivo investigations.13–15 Given the essential role of the CmeABC efflux pump in bile resistance and in vivo colonization, we speculate that inhibitors targeting the CmeABC efflux pump may not only control antibiotic resistance but also increase the susceptibility of C. jejuni to intestinal bile salts, consequently decreasing the colonization level of Campylobacter in the host. Such pump inhibitors could be directly used as novel antimicrobials for therapeutic intervention of Campylobacter infection. In this study, we demonstrate that EPIs inhibit the function of the CmeABC efflux pump and dramatically increase the susceptibility of C. jejuni to various bile salts. Oral administration of EPIs also partly reduces the colonization of C. jejuni in animal intestinal tracts. Inhibition of Campylobacter efflux pumps by EPIs is a potential means for therapeutic intervention to reduce colonization of C. jejuni in human and animal reservoirs.
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Materials and methods |
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Bacterial strains and culture conditions
C. jejuni strains 81–176 (a human isolate) and S3B (a chicken isolate) have been described in previous studies.16,17 Both C. jejuni 81–176 and S3B have been used for the characterization of the CmeABC efflux system in previous studies.7,9,10,17,18 C. jejuni 81–176 cmeB isogenic mutant was originally created using EZ::TN <KAN-2> Tnp Transposome.7 The cmeB insertional mutation in 81–176 was introduced to S3B by standard biphasic natural transformation.19 Twenty-one C. jejuni isolates from various origins (human, chicken and bovine) that have been described in a previous study were also used in this study for susceptibility assay.20 All strains were routinely grown in Mueller–Hinton (MH) broth or agar at 42°C under microaerophilic conditions, which were generated using a Campypak gas pack (Oxoid) in an enclosed jar. When needed, MH media was supplemented with 30 mg/L kanamycin. All media were purchased from Difco.
EPIs
EPI MC-207,110 (Phe-Arg ß-naphthyl-amide dihydrochloride) was purchased from Sigma. MC-207,110 is a lead compound inhibiting RND-type efflux pumps and was successfully identified by screening large synthetic compounds and natural product libraries.21 EPI MC-04,124 is an analogue of MC-207,110 and is less toxic in vivo.22MC-04,124 was a gift from Olga Lomovskaya (MPex Pharmaceuticals, CA, USA). Stock solutions of both EPIs were made using de-ionized distilled H2O and were sterilized by membrane filtration.
Susceptibility tests
MIC tests were performed to determine the susceptibility of Campylobacter to detergent SDS and various bile salts with or without a specific EPI. A standard microtitre broth dilution method was used to determine MICs in MH broth with an inoculum of 106 bacteria/mL.23 Microtitre plates were incubated for 2 days under microaerophilic conditions at 42°C. All antimicrobials used in this study were purchased from Sigma.
Chequerboard titration assay
To determine whether the effect of EPI MC-207,110 on the susceptibility of C. jejuni to bile salts is dose-dependent, chequerboard assays were performed using 96-well plates as described previously.21 Representative bile cholate was tested at 11 concentrations ranging from 0.03 to 32 g/L. The EPI MC-207,110 was tested at seven concentrations ranging from 0.25 to 16 mg/L. The microtitre plates were inoculated with one of the C. jejuni strains (81–176 or S3B) and were incubated for 2 days under microaerophilic conditions at 42°C. Bacterial growth in each well was recorded and MICs were determined.
Effect of EPIs on the colonization of C. jejuni in chickens
Two animal challenge experiments were conducted in this study and the experimental design is detailed in Table 1. C. jejuni strain S3B was chosen for the challenge studies because S3B colonizes chickens effectively and has been used in our previous chicken studies.17,18 For both trials, 1-day-old broiler chickens were obtained from Hubbard Hatchery, a commercial hatchery company in Pikeville, Tennessee. The chickens were randomly assigned into different treatment groups. Each group was maintained in a sanitized wire-floored cage and provided with unlimited access to feed and water. Before use, these chickens were screened for Campylobacter by culturing cloacal swabs, which were plated onto MH agar plates containing Campylobacter-specific growth supplements (SR084E and SR117E; Oxoid). All of the birds were negative for Campylobacter. For both experiments, 5-day-old chickens were weighed and all chicks were inoculated via oral gavage one time with C. jejuni S3B at a dose of 105 cfu/chick. In the first experiment, specific EPIs were administered into each chick 30 min post-inoculation at appropriate doses (Table 1). In the second experiment, a wider dose range was used for MC-207,110 (3–75 mg/kg) and an additional two EPI treatments were given at 24 and 48 h post-inoculation. Faecal samples were collected using cloacal swabs, which were taken at 2, 4, 7 and 9 days post-inoculation. The swabs were diluted in MH broth and spread onto MH plates containing Campylobacter-specific selective supplements. The plates were then incubated for 2 days at 42°C under microaerophilic conditions and the number of colonies was counted. Some of the selected colonies were tested by PCR to ensure that the output Campylobacter populations were the same as the inoculum and that there was no contamination of the chickens by other sources. The percentage of chickens colonized by C. jejuni and the shedding level of Campylobacter in chickens colonized by C. jejuni after inoculation and treatment were determined. Fisher's exact test was used to measure the significant differences in the percentage of colonized chickens at each sampling point between groups (SAS 9.1). One-way analysis of variance followed by a least-significant difference was used to calculate the significant differences in shedding level (log transformed). A P value 
0.05 was considered significant. All animal studies were conducted following a standard protocol approved by the Institutional Animal Care and Use Committee at The University of Tennessee (IACUC protocol number: 1428).
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EPI increased the susceptibility of C. jejuni to bile salts
The MICs of MC-207,110 and MC-04,124 in MH broth were 20 and 66 mg/L, respectively. Since sublethal concentrations of MC-207,110 (10 mg/L) and MC-04,124 (33 mg/L) fully supported Campylobacter growth in MH broth, we routinely added sublethal concentrations of specific EPIs to MH broth for MIC testing in this study. As shown in Table 2, the MICs of the detergent SDS and four bile salts were dramatically decreased in the presence of EPI MC-207,110. When C. jejuni 81–176 was grown in MH broth with MC-207,110, the MICs of bile salts were decreased from 16-fold (cholate) to 512-fold (taurocholate). Similar MIC reductions were also observed in C. jejuni S3B in response to MC-207,110 and the MIC reduction ranged from 32- to 128-fold (Table 2).
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We also determined the effect of another EPI, MC-04,124, on the susceptibilities of C. jejuni to the four bile salts. Addition of EPI MC-04,124 to MH broth to a final concentration of 33 mg/L resulted in comparable MIC reductions for all antimicrobials to those for MC-207,110 described in Table 2 (data not shown). Since higher concentrations of MC-04,124 (33 mg/L) were used in the MIC test to achieve similar MIC reductions for bile salts when compared with MC-207,110 (10 mg/L), the in vitro activity of MC-04,124 is not as potent as that of MC-207,110 to inhibit the efflux pump contributing to bile resistance in C. jejuni.
Effect of MC-207,110 on the susceptibility of cmeB mutants to bile salts
Isogenic cmeB mutants were constructed for MIC testing to determine whether the effect of MC-207,110 is primarily mediated by the CmeABC efflux pump. As shown in Table 2, insertional mutation in cmeB still resulted in significant MIC reductions for all bile salts and detergent in two cmeB mutants. However, the magnitude of MIC reduction was smaller than that in wild-type strains. For example, the presence of MC-207,110 in broth led to 512- and 128-fold reductions in the MIC of taurocholate in 81–176 and S3B, respectively. When cmeB was knocked out in 81–176 and S3B, MC-207,110 still resulted in marked reductions in the MIC of taurocholate, but the magnitude of MIC reduction (16- to 32-fold) was much smaller than that in the wild-type parent strains.
Effect of MC-207,110 on the susceptibility of C. jejuni to bile salts is dose-dependent
To determine whether the effect of EPI MC-207,110 on reducing MICs of bile salts observed above was dose-dependent, standard chequerboard assays were performed using cholate as representative of bile salts. The highest concentration of MC-207,110 (16 mg/L) displayed bactericidal effect against both strains. However, the sublethal concentration of MC-207,110 (8 mg/L) fully supported Campylobacter growth and led to the most dramatic MIC reduction in both strains (64-fold) compared with the MICs tested in MH broth. With a decrease in MC-207,110 concentration in MH broth, the MIC reduction also declined. As low as 1 mg/L of the EPI still resulted in slight but reproducible MIC reductions (2-fold) in 81–176 and S3B.
MC-207,110 increased susceptibilities of various Campylobacter isolates to cholate
To determine whether MC-207,110 not only increases the susceptibilities of clinical strains C. jejuni 81–176 and S3B but also reduces the MICs of bile salts for other Campylobacter isolates from various sources, MICs of cholate were measured for 21 C. jejuni isolates of different origins. All 21 C. jejuni isolates grew normally in MH broth containing 10 mg/L MC-207,110 alone. However, the presence of the EPI dramatically increased the susceptibilities of all isolates to cholate (Figure 1). In response to MC-207,110, the majority of the isolates (76%) displayed 8- to 16-fold reductions in the MIC of cholate while two isolates (9.5%) showed 32-fold MIC reductions (Figure 1).
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EPI reduced colonization of C. jejuni in chickens
As shown in Figure 2, colonization levels of C. jejuni in EPI treatment groups were lower than that in the control group. Specifically, the control group without EPI treatment had a 60% colonization rate 2 days post-inoculation. However, there was no colonization in any chicken in the groups treated one time with a low dose of MC-207,110 or a high dose of MC-04,124 at 2 days post-inoculation (Figure 2). A high dose of MC-207,110 or a low dose of EPI MC-04,124 resulted in lower colonization levels compared with the control group at 2 days post-inoculation (Figure 2). Surprisingly, MC-207,110 at high dose (25 mg/kg) has less effect than MC-207,110 at low dose (5 mg/kg) in experiment A (Figure 2). As the study continued, the differences between treatment and control groups lessened (Figure 2), most probably owing to horizontal transmission of C. jejuni among chickens within a group and the single administration of EPI at day 0. There was no significant difference in colonization between control and EPI-treated chickens at 7 and 9 days post-inoculation.
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To further define the dose effect of MC-207,110 and examine the effect of multiple EPI treatments, we conducted another chicken experiment using three different EPI doses and administered EPI MC-207,110 three times for each dosage. The second chicken study (Figure 3a) also showed that MC-207,110 reduced the percentage of chickens colonized by C. jejuni S3B when compared with the control group (90%). Similar to the unexpected dose–response with MC-207,110 in the first chicken study, the highest dose of MC-207,110 resulted in the highest colonization rate (70%) among the three EPI treatment groups at 2 days post-inoculation (Figure 3a). Although MC-207,110 was administered for three consecutive days, inhibition of C. jejuni colonization by MC-207,110 lessened throughout the study and by day 9 post-inoculation C. jejuni S3B colonized 80–90% of chickens for all groups. Shedding levels of chickens colonized with Campylobacter were also evaluated and no significant difference was observed among four groups (Figure 3b).
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To confirm that the isolates recovered from the experimental chickens were derived from the inoculated S3B, the cmp gene encoding the major outer membrane protein was PCR-amplified with representative Campylobacter isolates obtained from the chickens. A recent study indicated that cmp-based typing is a simple, yet highly discriminatory, approach for molecular differentiation of C. jejuni strains.20 The sequence data showed that the cmp sequences were identical to that of S3B, indicating that the output Campylobacter population was the same as the inoculum and there was no contamination of chickens by other sources.
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Discussion |
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The results of this study demonstrate the feasibility of developing a CmeABC efflux pump-based intervention strategy against Campylobacter colonization. This conclusion is supported by the following evidence. First, EPI MC-207,110 and MC-04,124 greatly potentiated the bactericidal effect of bile salts against C. jejuni in vitro, primarily mediated by inhibition of the CmeABC efflux pump in a dose-dependent manner (Table 2). Secondly, EPI MC-207,110 also greatly increased susceptibility of the Campylobacter isolates from various origins to bile salt cholate (Figure 1). Finally, oral administration of EPI MC-207,110 and MC-04,124 reduced C. jejuni colonization at 2–4 days post-inoculation only in the host using a chicken challenge model system (Figures 2 and 3). Together, these findings strongly suggest that inhibiting the C. jejuni CmeABC efflux pump by EPIs may be a novel approach to directly prevent and control Campylobacter infection in humans and animal reservoirs.
Although EPIs have been demonstrated to be effective to potentiate clinical antibiotics against a variety of Gram-negative bacteria,21,24–28 this is the first report showing that EPIs also dramatically increase the susceptibility of Campylobacter to intestinal bile salts that form a barrier to limit enteric pathogen infections. Consistent with the in vitro observation, the in vivo studies using a chicken model system also showed a reduced colonization rate following oral administration of EPIs. Despite significant dilution of the EPIs in the long digestive tract in chickens upon administration, we cannot rule out the possibility that the reduced colonization is also attributed to the direct killing effect of the EPIs on Campylobacter, which makes it difficult to conclusively interpret our in vivo results. However, given the high concentrations of bile salts in the intestine, increased susceptibility to bile via CmeABC inhibition by EPIs is probably a major reason for the reduced colonization observed in this study. Furthermore, from the standpoint of developing antimicrobials for clinical use, the antimicrobial activity of EPIs may be desired because such EPIs can inhibit both bacterial growth and the functions of bacterial efflux systems. It is very likely that antibacterial activity of the EPIs is not linked to the inhibition of multidrug efflux pumps because multidrug efflux systems are dispensable in many cases and the identified efflux systems in C. jejuni are not essential for growth.7,29–31 Together, our findings clearly support the feasibility of using EPIs directly as therapeutic agents against Campylobacter.
In Escherichia and Salmonella, the AcrAB-TolC efflux pump (a homologue of the CmeABC pump) also contributes to bile resistance and is inducible by bile salts.32,33 Thus, it is likely that inhibiting bacterial efflux of bile salts with EPIs may be a general approach for developing therapeutic measures for enteric pathogens. However, the effect of EPIs in other enteric pathogens may not be as significant as that observed for C. jejuni in this study because expression of the AcrAB-TolC pump in E. coli and Salmonella was only moderately induced by bile salts and the magnitude of MIC reduction of bile due to the mutation in the pump is much smaller in E. coli and Salmonella than that in Campylobacter.32,33
Previous studies indicated that CmeABC is a primary multidrug efflux pump contributing to antibiotic and bile resistance in C. jejuni.30,31,34 According to the whole genome analysis,34 there are 13 putative multidrug efflux systems including only two RND-type efflux pumps (CmeABC and CmeDEF) in C. jejuni. Recent studies showed that CmeDEF did not contribute to Campylobacter resistance to bile salts.30,35 However, in this study, the EPIs still resulted in marked MIC reductions in isogenic cmeB mutants for all bile salts, which suggested that, in addition to RND-type CmeABC and CmeDEF efflux systems, other unknown efflux system(s) also contributed to Campylobacter resistance to bile salts. It is likely that the EPI MC-207,110 also inhibits efflux pumps belonging to other superfamilies in Campylobacter, such as the major facilitator superfamily. This possibility remains to be examined in future studies.
Development of clinically useful EPIs must address several key issues, such as toxicity, stability and bioavailability.14,15 MC-207,110 and its derivatives such as MC-04,124 have been evaluated for in vivo toxicity using a mouse model system.22 When administered via intravenous bolus injection, MC-207,110 displayed appreciable toxic effects (MLD <25 mg/kg) but MC-04,124 was less toxic in rodents (MLD >150 mg/kg).22 It has been observed that MC-207,110 was not stable in serum but MC-04,124 displayed high stability in serum (Dr Olga Lomovskaya, Mpex Pharmaceuticals, Inc., San Diego, CA, USA, personal communication). In this study, MC-207,110 also reduced the level of Campylobacter colonization at 2 days post-inoculation in chickens, indicating that this EPI still displays a certain level of stability in the intestinal tract although the compound is not stable in serum. However, multiple treatments of chickens with MC-207,110 did not result in further reduced colonization, suggesting that MC-207,110 may be not stable enough to maintain its efficacy after it passes through the long small intestine and finally reaches the caecum, the predominant site for Campylobacter colonization in chickens. In this study, none of the chickens experienced any specific EPI-induced health problems and none of the chickens died upon EPI treatment even when a high dosage of MC-207,110 (75 mg/kg) was used, suggesting that in vivo toxicity of EPI depends on administration route. It is possible that physiological factors of the gastrointestinal tract are responsible for the chickens being able to tolerate high doses of EPIs. However, the EPIs such as MC-207,110 probably have asymptomatic toxic effects on the chicken. We consistently observed the unexpected inverse dose–response with MC-207,110 in this study, which may be related to a side effect of the EPI on the host. The high dose of MC-207,110 may affect the physiology of the chicken intestine and such physiological changes probably facilitate Campylobacter to colonize in the intestine, consequently overshadowing the inhibitory effect of MC-207,110 on the CmeABC efflux pump. For example, given the pump-inhibitory nature of MC-207,110, this EPI may also target eukaryotic transporter(s) including those in chicken intestinal epithelial cells. Thus, the presence of MC-207,110 in the intestine may change the structure and composition of intestinal mucus through alteration of the function of epithelial cells. The intimate interaction of Campylobacter with intestinal mucus is important for Campylobacter to colonize chicken intestinal mucosa. Therefore, it is likely that the changes in intestinal mucus due to the EPI treatment may promote Campylobacter colonization in the intestine. This hypothesis remains to be examined in future studies. Overall, our chicken study indicated that inhibition of Campylobacter efflux pumps by appropriate EPIs is a potential means for therapeutic intervention to reduce colonization of C. jejuni in human and animal reservoirs. To develop clinically useful EPI compounds, more studies using new lead series in conjunction with pharmacokinetic/pharmacodynamic analysis are needed in the future.
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None to declare.
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Present address. Health Sciences Center, University of Louisville, 570 S. Preston Street, Louisville, KY 40202-1760, USA 
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Acknowledgements |
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We are grateful to Olga Lomovskaya for providing EPI MC-004,124 and insightful comments. This study is supported by the University of Tennessee Professional Development Awards and by the Tennessee Agricultural Experiment Station.
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 P. Malik-Kale, C. T. Parker, and M. E. Konkel Culture of Campylobacter jejuni with Sodium Deoxycholate Induces Virulence Gene Expression J. Bacteriol., April 1, 2008; 190(7): 2286 - 2297. [Abstract] [Full Text] [PDF]
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