JAC Advance Access originally published online on November 22, 2005
Journal of Antimicrobial Chemotherapy 2006 57(1):52-60; doi:10.1093/jac/dki419
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Interaction of CmeABC and CmeDEF in conferring antimicrobial resistance and maintaining cell viability in Campylobacter jejuni
Department of Microbiology and Preventive 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 15 August 2005; returned 21 September 2005; revised 11 October 2005; accepted 21 October 2005
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Abstract |
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Objectives: To determine the role of CmeDEF in conferring antimicrobial resistance in Campylobacter jejuni and examine the interaction of CmeABC and CmeDEF in mediating antimicrobial resistance and maintaining cell viability.
Methods: Single and double mutants of cmeF and cmeB were generated in multiple strains using insertional mutagenesis. The mutants were compared with their wild-type strains for antimicrobial susceptibility and growth characteristics. Transcription fusion was used to quantify the expression of cmeDEF and cmeABC. Ethidium bromide (EB) accumulation assay was used to measure the efflux function.
Results: Insertional mutagenesis of the cmeF gene in C. jejuni NCTC 11168 resulted in a 2-fold decrease in the resistance to ampicillin, polymyxin B and EB, whereas the same mutation in C. jejuni 81-176 and 21190 led to a 2–4-fold increase in the resistance to multiple antimicrobials and toxic compounds. The increased resistance in the cmeF mutants of 81-176 and 21190 was associated with the elevated efflux in the mutants. Compared with the cmeB mutant, the cmeF/cmeB double mutants of 81-176 and 21190 showed further decrease in the resistance to various antimicrobials and toxic compounds. Transcription fusion assay indicated that the expression level of cmeF was substantially lower than that of cmeB. Notably, the cmeB/cmeF double mutation, not the single mutations, impaired cell viability in Campylobacter.
Conclusions: CmeDEF interacts with CmeABC in conferring antimicrobial resistance and maintaining cell viability in C. jejuni. CmeABC is the predominant efflux pump in C. jejuni, whereas CmeDEF plays a secondary role in conferring intrinsic resistance to antimicrobials.
Keywords: antibiotics , efflux , mutagenesis
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Introduction |
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Campylobacter jejuni is one of the most common food-borne bacterial pathogens of humans in many industrialized countries.1 This pathogenic bacterium causes abdominal pain, watery diarrhoea and/or haemorrhagic colitis, and is also associated with Guillain–Barré syndrome, an acute flaccid paralysis that may lead to respiratory muscle compromise and death.2 Most of the sporadic cases of human campylobacteriosis are associated with consumption of, or contact with, raw or undercooked poultry meat.1 As a zoonotic pathogen, C. jejuni colonizes a variety of wild and domestic animals, but commercial poultry is considered the major reservoir of human C. jejuni infections.1,3 Owing to the extensive use of antibiotics in human medicine and animal agriculture, the incidence of antibiotic resistance in Campylobacter has dramatically increased over the last decade.4 Campylobacter resistance to macrolides and fluoroquinolones is considered a major threat to public health as these two classes of antibiotics are the drugs of choice for treating gastroenteritis caused by Campylobacter spp.4
Campylobacter has evolved multiple mechanisms for antibiotic resistance.5,6 Some of the mechanisms, such as gyrA mutation and ß-lactamase production, are associated with Campylobacter resistance to specific classes of antibiotics, whereas other mechanisms (e.g. efflux systems) are involved in the resistance to structurally diverse antimicrobials. Recently, a multidrug efflux system named CmeABC was identified in C. jejuni.7,8 This system is encoded by a three-gene operon on the Campylobacter chromosome and is composed of a transporter protein (CmeB) belonging to the resistance-nodulation-cell division (RND) family, a periplasmic membrane fusion protein (CmeA) and an outer membrane factor (CmeC). CmeABC is constitutively expressed in wild-type Campylobacter strains and extrudes a broad range of antibiotics (including fluoroquinolones), dyes, heavy metals, bile salts and detergents.7–9 This efflux system is a key player for intrinsic antibiotic resistance and is also required for the acquired resistance to fluoroquinolones.10 In addition, CmeABC is essential for Campylobacter colonization in vivo by mediating resistance to bile, which is normally present in the intestinal tract of animals.11 CmeABC is controlled by CmeR,12 a transcriptional repressor encoded by a gene located immediately upstream of cmeABC. CmeR binds to the inverted repeat in the promoter region of cmeABC and inhibits the expression of the efflux operon.
The genomic sequence of C. jejuni NCTC 1116813 revealed the presence of multiple putative efflux pumps in Campylobacter.5 However, the functions of the majority of these putative efflux systems are unknown. In addition to CmeABC, C. jejuni also possesses another RND-type efflux pump named CmeDEF,5,14 in which CmeD, CmeE and CmeF are predicted to be an outer membrane channel protein, periplasmic fusion protein and inner membrane transporter, respectively. Although two recent studies have tried to characterize the function of CmeDEF,14,15 its role in antimicrobial resistance is still controversial owing to conflicting findings and possible masking of CmeDEF by CmeABC. In this study, we generated single and double mutants of cmeF and cmeB in different C. jejuni strains, determined the contribution of CmeDEF to antimicrobial resistance in relation to CmeABC and measured the effect of the mutations on Campylobacter growth. It was found that although CmeDEF is expressed at a low level, it acts interactively with CmeABC in conferring resistance to antimicrobials and toxic compounds. In addition, CmeDEF and CmeABC are important for maintaining cell viability in C. jejuni. Interestingly, inactivation of cmeF alone in several C. jejuni strains elevated the efflux machinery in the mutants, suggesting that the cmeF mutation up-regulated other efflux transporters. These findings provide new insights into the biological functions of efflux pumps and their complex interaction in mediating antibiotic resistance in Campylobacter.
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Materials and methods |
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Bacterial plasmids, strains and culture conditions
The key bacterial plasmids and strains used in this study are listed in Table 1. Other C. jejuni strains used in this work include human clinical isolates E46972, F26747 [GenBank] , H30769 [GenBank] , H49024 [GenBank] , S9801, S13530 [GenBank] , S20237, T37597 [GenBank] , X77136 [GenBank] , M402 and W11805 [GenBank] ; chicken isolate S2b; and ovine isolates 19084751 and 19094451. Escherichia coli strains were grown in Luria–Bertani (LB) broth or on LB agar with or without ampicillin (100 mg/L), kanamycin (50 mg/L) or chloramphenicol (50 mg/L) at 37°C. C. jejuni strains were grown in Mueller–Hinton (MH) broth or on MH agar at 42°C under microaerobic conditions, which were generated using the CampyGen (Oxoid, Basingstoke, England) gas packs in enclosed jars. All media were purchased from Difco (Detroit, MI, USA).
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PCR
PCR primers used in this study and the expected sizes of the products are listed in Table 2. PCR was performed in a volume of 50 µL containing 200 µM of each deoxynucleoside triphosphate, 200 nM of primers, 2.5 mM of MgSO4, 50 ng of template DNA and 5 U of Taq DNA polymerase (Promega) or Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA, USA). Cycling conditions varied according to the estimated annealing temperatures of primers and the expected sizes of products. PCR products were purified by QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) when needed for cloning or sequence analysis. For reverse transcriptase PCR (RT–PCR), total RNA was isolated from C. jejuni strains using RNeasy Kit (Qiagen). RNA samples were treated with RNase-free DNase (Epicentre, Madison, WI, USA) at 37°C for 30 min, followed by heat inactivation at 75°C for 5 min. RT–PCR was conducted using the MasterAmp Kit (Epicentre). Cycling conditions for the RT–PCR included an initial incubation at 60°C for 20 min, followed by 35 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 30 s. A RT–PCR mixture lacking the RT was included as a negative control.
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Insertional mutation of cmeF
An isogenic cmeF mutant of NCTC 11168 was constructed by insertional mutagenesis. Primers FF1 and FR1 were used to amplify a 1874 bp cmeF fragment (Table 2) with a unique BsrBRI site in the middle region of the fragment. The PCR product was cloned into the pGEMT-Easy vector (Promega), resulting in the construction of pCMEF. Primers CHLF and CHLR were used to amplify the cat gene encoding chloramphenicol resistance from pUOA1816 using the Pfu Turbo DNA polymerase (Stratagene). The cat PCR product was ligated to the BsrBRI-digested pCMEF to obtain plasmid construct pCMEFC, which was then transformed into E. coli JM109. pCMEFC, which served as a suicide vector in Campylobacter, was introduced into C. jejuni NCTC 11168 using an electroporator (Gene Pulser Xcell System; Bio-Rad Laboratories, Richmond, CA, USA). Transformants were selected on MH agar containing chloramphenicol 4 mg/L. PCR analysis of the cmeF mutant (named 11168F) confirmed that the cat gene was inserted into the cmeF gene in the same orientation as the cmeF gene. To create the insertional cmeF mutant in other Campylobacter strains, the genomic DNA of 11168F was used to transform strains 81-176 and 21190 using natural transformation as described previously.17 The cmeF mutation in 81-176 and 21190 was again confirmed by PCR. The cmeF mutants of 81-176 and 21190 were named 81-176F and 21190F, respectively (Table 1).
Construction of cmeB/cmeF double mutants
The cmeB gene in 81-176 was inactivated by the EZ::TN <Kan-2> transposon (Epicentre; containing a kanamycin resistance cassette) to generate 81-176B (previously named 9B6) in a previous study.7 The cmeB mutation in 81-176B was introduced by natural transformation to NCTC 11168 and 21190 to create 11168B and 21190B (Table 1), respectively. To generate double mutants, the cmeB mutation in 81-176B was transferred into 81-176F by natural transformation. Transformants were selected on MH agar containing kanamycin 30 mg/L and chloramphenicol 4 mg/L. The cmeB mutation in the double mutant was confirmed by PCR using a transposon-specific primer (Epicentre) and a cmeB-specific primer as described previously.7 The cmeB/cmeF double mutant of 81-176 was named 81-176B/F (Table 1). To generate the cmeB/cmeF double mutant of 21190, the genomic DNA of 21190B (cmeB::KanR) was used to transform 21190F (cmeF::cmR) and the transformant was selected on MH plates containing kanamycin 30 mg/L and chloramphenicol 4 mg/L. The double mutant of 21190 was named 21190B/F (Table 1). We also tried to generate a double mutant in strain 11168 by introducing the cmeB mutation into 11168F or the cmeF mutation into 11168B. Both natural transformation and electroporation were used for the transformation. Despite multiple trials, we were unable to generate a cmeB/cmeF double mutant in 11168.
Production of recombinant proteins and generation of polyclonal antisera
The histidine (His)-tagged recombinant peptide of CmeF was produced in E. coli by using the pQE-30 vector of the QIAexpress expression system (Qiagen). Based on the predicted antigenic profiles of CmeF (Cj1033), an 873 bp fragment spanning the predicted loop region of CmeF was amplified using primers FF2 and FR2 (Table 2). After digestion with BamHI and HindIII, this fragment encoding 278 amino acids (nt 91–924 of cmeF) was cloned into the pQE30 vector. Transformation and screening for positive recombinant clones were performed according to the manufacturer's instruction (Qiagen). The plasmid in the E. coli clone expressing the recombinant CmeF peptide was sequenced and no mutation occurred in the coding sequence of the amplified cmeF fragment (data not shown). Purification of the His-tagged recombinant CmeF under native conditions was performed as described previously.18 Rabbit anti-CmeF sera were prepared by immunizing two New Zealand White rabbits with the His-tagged recombinant CmeF as described previously.7 The rabbit anti-CmeB antibody prepared in previous work7 was also used in this study.
SDS–PAGE and immunoblotting
Cell envelopes of Campylobacter strains were prepared as described previously.19 Membrane fractions (10 µg) were loaded in each lane and separated by SDS–PAGE with a 9% (w/v) polyacrylamide separating gel.20 Immunoblotting using rabbit anti-CmeF or anti-CmeB antibodies was conducted as described previously.7
Construction of transcriptional fusions and ß-galactosidase assay
Primers BSF1 and AR2 were used to amplify a 650 bp fragment containing the putative cmeABC promoter, and primers PROF and PROR were used to amplify a 676 bp fragment containing the putative cmeDEF promoter (Table 2). These fragments were digested with BamHI and XbaI, and then cloned into the pMW10 shuttle vector, which contained a promoterless lacZ gene.21 The names of the promoter–lacZ fusion plasmids and their relevant characteristics are listed in Table 1. The promoter fusion plasmids in E. coli DH5
were mobilized into various C. jejuni strains by triparental mating using DH5/pRK2013 as the helper strain as described by Miller et al.22 The pMW10 plasmid was also introduced into C. jejuni as a background control for LacZ activity. ß-Galactosidase activity in Campylobacter was measured according to the method of Miller.23 Statistical analysis was performed using Student's t-test on data from three independent experiments done in triplicate.
Ethidium bromide accumulation assay and antimicrobial susceptibility tests
Accumulation of ethidium bromide (EB) in C. jejuni was measured as described in a previous study.7 The MICs of various antimicrobials were determined using the standard microtitre broth dilution method in MH broth with an inoculum of 106 bacteria/mL as described previously.7 Bacterial growth was assessed after the microtitre plates were incubated for 48 h at 42°C under microaerobic conditions. With each mutant or strain tested in this study, the MIC experiment was repeated at least three times with each of various antimicrobials.
Growth rates of 81-176 and its mutants
To compare the growth kinetics of various mutants of 81-176 with that of the wild-type, the cultures were separately inoculated into MH broth with an initial cell density of 
3x104 cfu/mL. The cultures were incubated at 42°C under microaerobic conditions. Aliquots of the cultures were collected at different time points, serially diluted and plated onto MH plates for enumeration of bacterial colonies. Three independent experiments were conducted using the same strains and conditions. For Experiment 1, samples were collected at 0, 6, 10, 24, 48 and 72 h post-inoculation, whereas samples were collected at 0, 6, 12, 24, 48 and 72 h post-inoculation for Experiments 2 and 3.
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Effect of the cmeF mutation on antimicrobial susceptibility
To examine the role of CmeDEF in antimicrobial resistance, we inactivated the cmeF (encoding the inner membrane transporter) gene of the cmeDEF operon in three different strains of C. jejuni and compared the susceptibilities of the wild-type strains and their corresponding cmeF mutants with structurally unrelated antimicrobials and toxic compounds (Table 3). The cmeF mutant of 11168 (11168F) showed a 2-fold decrease in the resistance to ampicillin, polymyxin B and EB. No changes were observed in the susceptibility to other tested antimicrobials. The increased susceptibility of 11168F to ampicillin and EB was in agreement with the study by Pumbwe et al. who found that inactivation of cmeF in 11168 increased the susceptibility to several antimicrobials. In contrast to their findings, we did not observe any changes in the susceptibility of 11168F to bile salts. Surprisingly, inactivation of cmeF in strains 81-176 and 21190 resulted in a 2–4-fold increase in the resistance to multiple antimicrobials and toxic compounds (Table 3). The MIC differences between the cmeF mutant and its corresponding wild-type strain were reproducible in at least three experiments. This same trend of increased resistance associated with the cmeF mutation was also observed with strain 164 (M. Akiba, unpublished results).
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The increased antimicrobial resistance in the cmeF mutants of 81-176 and 21190 suggested that the efflux machinery in the mutants was elevated. To examine this possibility, the EB accumulation assay was conducted with the cmeF mutants. In comparison with their wild-type strains, the cmeF mutants of 81-176 and 21190 showed
20–30% reduction in the accumulation of EB, whereas the cmeF mutant of 11168 showed 30% increase in the accumulation of EB (Table 4). These results are consistent with the MIC data for EB (Table 3) and indicated that the cmeF mutation up-regulated the efflux machinery in 81-176 and 21190.
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Effect of double mutations in cmeABC and cmeDEF on antimicrobial resistance
To examine if the constitutively expressed CmeABC pump potentially masked the effect of the cmeF mutation on antimicrobial resistance, we constructed cmeB/cmeF double mutants and compared the MICs of various antimicrobials between the cmeB mutant and the cmeB/cmeF double mutant (Table 5). The double mutant was successfully obtained from strains 81-176 and 21190. In 81-176, comparing with the cmeB mutant, the cmeB/cmeF double mutant showed a 2-fold increase in the susceptibility to ciprofloxacin, tetracycline, fusidic acid and acriflavine, and a 2–16-fold increase in the susceptibility to various detergents and bile salts (Table 5). In comparison with 21190B, the double mutant 21190B/F showed a 2-fold increase in the susceptibility to several antimicrobials (fusidic acid, acriflavine, polymyxin B and novobiocin) and showed a 2–128-fold increase in the susceptibility to detergents and bile salts (Table 5). In both 81-176 and 21190, the impact of the cmeF mutation in the cmeB-defective background on antimicrobial susceptibility was greater with detergents and bile salts than with antibiotics. Nevertheless, the results from the double mutants clearly indicate that CmeDEF also contributes to the intrinsic resistance of Campylobacter to antimicrobials and toxic compounds, although the contribution is not at the same scale as that of CmeABC.
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Notably, a double mutant was not obtained with strain 11168 despite the fact that single mutants of cmeB or cmeF were successfully generated in the strain. Using natural transformation and electroporation, we tried to introduce the cmeF mutation to the cmeB mutant or the cmeB mutation to the cmeF mutant. All failed to generate a double mutant in 11168. Similar work conducted by Pumbwe et al.14 was also unable to generate a cmeB/cmeF double mutant in 11168. Together, these results strongly suggest that the double mutation is lethal to strain NCTC 11168. This observation indicates the essential role of CmeABC and CmeDEF in maintaining cell viability in this particular strain. However, the cmeB/cmeF double mutation is dispensable for the survival of 81-176 and 21190, suggesting that a compensatory mechanism may exist in the two strains, which compensates for the loss of function of both CmeB and CmeF.
Expression of cmeDEF
The relatively moderate contribution of CmeDEF to the intrinsic antimicrobial resistance may be related to its low expression level. To measure the transcription of cmeDEF, RT–PCR was performed using primer pairs (Table 2) specific for cmeD (F1 and R1), cmeE (F2 and R2), cmeF (F3 and R3) and Cj1034c (F4 and R4) with RNA samples extracted from wild-type strains (81-176 or NCTC 11168) or their cmeF mutants (81-176F or 11168F). cmeDEF-specific mRNA sequences were detected in both 81-176 and NCTC 11168 (partially shown in Figure 1), indicating that the cmeDEF operon is transcribed in wild-type Campylobacter strains. In the cmeF mutants, the RT–PCR result was positive with cmeD and cmeE, but negative with cmeF. This finding is consistent with the fact that the cmeF gene was inactivated by a cat cassette in the cmeF mutants. The cat insertion in cmeF did not cause a polar effect on the transcription of the downstream gene (Cj1034c), because the transcript of Cj1034c was detected in both wild-type strains and their cmeF mutants (Figure 1). The control RT–PCR reactions with no reverse transcriptase yielded negative results from the RNA samples, indicating that there was no DNA contamination in the RNA template.
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We further determined the expression of cmeDEF with immunoblotting using anti-CmeF antibodies. As shown in Figure 1, the anti-CmeF antibody reacted strongly with the recombinant CmeF peptide, indicating the high quality of the antibody to CmeF. In wild-type 81-176 and its CmeB mutant, the anti-CmeF antibody detected a faint protein band of 99 kDa (Figure 1), which is slightly smaller than the deduced molecular mass of CmeF (108 kDa). This band was absent in the CmeF mutant, and was only detectable in the wild-type strain and the cmeB mutant when excess amounts of total membrane proteins were loaded in the SDS–PAGE gel (Figure 1). With this high concentration of membrane proteins loaded in each lane, the CmeB band as detected by the anti-CmeB antibody was so strong that it formed a dark smear on the immunoblot (Figure 1). The same results shown in Figure 1 were also obtained when membrane preparations of strains 11168 and 21190 were used in the immunoblotting (data not shown). The difference in protein production between CmeB and CmeF indicated that the two efflux transporters are expressed at different levels.
To further quantify the expression of cmeDEF in comparison with that of cmeABC, promoter–lacZ fusions were used to measure the promoter activities of cmeDEF and cmeABC in different genetic backgrounds. According to the measured activity (Miller units) of LacZ, the transcription of cmeDEF was 8- and 12-fold lower than that of cmeABC in strains 11168 and 81-176, respectively (Table 6). The findings from the transcription fusion were consistent with the result from immunoblotting, which indicated that cmeDEF is expressed at a greatly lower level than cmeABC. We also examined if inactivation of cmeF affected the expression of cmeABC. Interestingly, the promoter activity of cmeABC in the cmeF mutants (81-176F or 11168F) was increased compared with that in the wild-type background (81-176 and NCTC 11168; Table 6). Although the increase was of a small scale (9–15%), the difference was experimentally reproducible and statistically significant (P < 0.01). This finding suggests that the two efflux pumps have intrinsic links in function and inactivating cmeDEF would lead to increased expression of cmeABC.
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cmeDEF is not regulated by CmeR
In previous studies, we showed that CmeABC is regulated by CmeR,12 a member of the TetR family of regulators. To examine if CmeDEF is subject to CmeR regulation, the promoter activity of cmeDEF was measured in wild-type 81-176 and its CmeR mutant (JL107)12 using pDEF81 (Table 1). There was no difference in the LacZ activity between 81-176 and its cmeR mutant (data not shown), indicating that CmeR does not modulate the expression of cmeDEF.
Effect of cmeB and cmeF mutations on Campylobacter growth in MH broth
The inability to generate a cmeB/cmeF double mutant in 11168 suggested that the function of CmeABC or CmeDEF is essential for the viability of this particular strain. To further examine the role of CmeABC and CmeDEF in maintaining Campylobacter growth and viability, we compared the growth rates of the single mutants (81-176B and 81-176F) and the double mutant (81-176B/F) of 81-176 in MH broth. When cultured in MH broth without antibiotics, the growth rates of the mutants and the wild-type were similar in the exponential phase (Figure 2). In the stationary phase, the single mutants and the wild-type showed similar growth kinetics, whereas the viable cell count of the double mutant (81-176B/F) was consistently about 1 log unit lower than those of the wild-type and the single mutants (Figure 2). This result was reproducible in three independent experiments and indicated that a functional CmeABC or CmeDEF is required for maintaining the normal growth/survival in the stationary phase of 81-176. Together, these findings with 11168 and 81-176 indicate that CmeABC and CmeDEF play an important role in maintaining cell viability in Campylobacter.
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Distribution of cmeDEF among C. jejuni isolates
To determine whether cmeDEF is present in different C. jejuni strains, cmeF-specific primers (FF2 and FR2) were used in PCR to amplify an 873 bp sequence of cmeF from various strains including ATCC33291, E46972, F26747 [GenBank] , H30769 [GenBank] , H49024 [GenBank] , S9801, S13530 [GenBank] , S20237, T37597 [GenBank] , X77136 [GenBank] , M402, W11805 [GenBank] , 19084751, 19094451, 21190 and S2b. The cmeF-specific sequence was amplified from every isolate examined in this study (data not shown), indicating cmeF is present in different C. jejuni strains isolated from different animal species. RT–PCR using primer F3 and R3 also showed a positive product from all of the strains examined (data not shown), suggesting that cmeF is expressed in the clinical isolates. The broad distribution and expression of cmeDEF in different strains further support its role in intrinsic antimicrobial resistance and maintaining cell viability.
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Discussion |
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In this study, we clearly demonstrated using single and double mutants that CmeDEF is involved in the intrinsic resistance of Campylobacter to antimicrobials and toxic compounds (Tables 3 and 5). Compared with CmeABC, the level of resistance conferred by CmeDEF is relatively moderate and is masked by the function of CmeABC in wild-type Campylobacter strains. The moderate contribution of CmeDEF to antimicrobial resistance is consistent with its low-level expression in wild-type Campylobacter as determined by immunoblotting (Figure 1) and transcription fusion (Table 6). Thus, the constitutively expressed CmeABC appear to be the predominant efflux pump in C. jejuni, whereas CmeDEF probably functions as a secondary efflux mechanism and may be primarily responsive to certain conditions. Despite the differences in their expression and contribution to the intrinsic antimicrobial resistance, CmeABC and CmeDEF appear to function interactively because inactivation of CmeF increased the transcription of cmeABC and retention of either efflux pump is required for optimal cell viability.
Our finding with 11168B is consistent with the report by Pumbwe et al.14 in that inactivation of cmeF decreases the resistance of NCTC 11168 to several antimicrobials, but differs from their study in the specific antimicrobials that are affected by the cmeF mutation. In both studies, the cmeF mutant showed decreased resistance to ampicillin and EB. Different from their study, our work did not find any differences between 11168B and its wild-type in the susceptibility to bile salts and other detergents. In fact, we noticed that the wild-type 11168 strain (P270) used in their study was quite susceptible to bile salts compared with the 11168 culture used in our laboratory. This discrepancy may be due to differences in phenotypes associated with the 11168 cultures maintained in different research laboratories. In another work by Ge et al.,15 it was found that inactivation of cmeF in 81-176 had no effect on the susceptibility to ciprofloxacin, erythromycin, tetracycline and chloramphenicol, which is consistent with the finding in this study (Table 3). However, the effect of the cmeF mutation on the susceptibility to other antimicrobials or toxic compounds was not investigated in their report.15 In our study, we found that the cmeF mutation in 81-176 slightly enhanced resistance to several antimicrobials including cefotaxime, novobiocin, fusidic acid, EB and acriflavine (Table 3). Furthermore, we successfully generated double cmeB/cmeF double mutants in different strains and determined the expression level of CmeABC and CmeDEF and their relative contributions to antimicrobial resistance in Campylobacter.
One of the interesting findings from this study is that the single CmeF mutants of 81-176 and 21190 showed enhanced resistance to antimicrobials and toxic compounds (Table 3). The same observation also extended to the cmeF mutant in strain 164 (M. Akiba, unpublished results). The enhanced resistance associated with the single cmeF mutants are probably the consequence of increased efflux because 81-176B and 21190B accumulated less EB as compared with their corresponding wild-type strains (Table 4). It is tempting to speculate that CmeDEF has a functional link with other drug transporters and inactivation of cmeF up-regulates the other transporters to compensate for the loss of CmeF. We indeed noticed that expression of cmeABC is increased in the cmeF mutant of 11168 and 81-176 (Table 6). Although the increased expression of cmeABC is at a moderate scale, the difference is statistically significant (P < 0.01) and can be consistently demonstrated in independent experiments with different strains. The elevated transcription of cmeABC alone cannot explain the MIC difference between the single cmeF mutants and their wild-type strains because 11168F showed a 2-fold decrease in resistance to several antimicrobials (Table 3), even though cmeABC expression was elevated in this mutant (Table 6). It is likely that other uncharacterized efflux mechanisms were also activated in the cmeF mutants of 21190 and 81-176, which collectively contributed to the enhanced resistance in 81-176B and 21190B. Although it is known that NCTC 11168 contains 13 putative drug efflux transporters of different superfamilies,5 it is unclear how many efflux transporters exist in strains 21190 and 81-176 and if there are any efflux pumps that are unique to the two strains, which could explain the difference in the resistance phenotype of their cmeF mutants from that of 11168.
The increased antimicrobial resistance associated with the cmeF mutation in 81-176 and 21190 was completely abolished when cmeABC was inactivated by a null mutation. In fact, the cmeB/cmeF double mutants showed a moderate decrease in the resistance to antibiotics and a significant reduction in the resistance to detergents and bile salts compared with their corresponding cmeB mutants (Table 5), which clearly showed the role of CmeDEF in mediating antimicrobial resistance in C. jejuni. The greatest MIC differences between the double mutants and the cmeB mutants were observed with SDS, Triton X-100, Tween 20, Empigen and bile salts (Table 5). Since surfactants are present in the natural habitat (e.g. animal intestine and food-producing environments) of C. jejuni, the high-level resistance to detergents conferred by CmeABC and CmeDEF indicate that these two efflux systems act interactively and play an important role in facilitating the adaptation of Campylobacter to various environments.
It was found that CmeR, a transcriptional repressor for cmeABC,12 is not involved in the regulation of cmeDEF. At present, it is unknown which regulator controls the expression of cmeDEF and what conditions can trigger the overexpression of cmeDEF. We tried to overexpress cmeDEF in Campylobacter or E. coli by cloning the operon into a shuttle vector. Despite extensive efforts in the cloning experiments, we were unable to clone the entire cmeDEF operon including its promoter region (data not shown), and were only successful in obtaining clones with truncations in the 5' region (including the promoter and part of cmeD coding sequence) of the operon, suggesting that cmeDEF is tightly controlled in wild-type strains and artificial expression of this system without the inducing condition may cause a deleterious effect on the growth of C. jejuni.
Another important finding of this work is that CmeABC and CmeDEF are involved in maintaining cell viability in C. jejuni. A single mutation of either cmeB or cmeF did not affect the viability or growth characteristics of Campylobacter in conventional culture media. However, the cmeB/cmeF double mutation appears to be lethal to NCTC 11168 because our work and another independent study by Pumbwe et al.14 were unable to generate the cmeB/cmeF double mutant in this strain. Although the cmeB/cmeF double mutation was not lethal to 81-176, the double mutant showed decreased viability in the stationary phase (Figure 2). These observations indicate that CmeABC and CmeDEF are involved in the regulation of cell homeostasis and at least one of the two efflux pumps is required for maintaining the physiological processes in Campylobacter cells. At this stage it is unknown how these two efflux pumps contribute to the homeostatic process in Campylobacter. One possibility is that the efflux pumps are involved in the extrusion of endogenous toxic wastes. This hypothesis needs to be examined in future studies. Nevertheless, findings from this work strongly suggest that CmeABC and CmeDEF act interactively in maintaining cell viability in Campylobacter and at least one of them is needed for optimal growth. Thus, the efflux pumps are not only important for antimicrobial resistance, but also play significant roles in Campylobacter physiology.
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None to declare.
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Footnotes |
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Present address. Department of Safety Research, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan 
Present address. Department of Animal Science, The University of Tennessee, 2505 River Drive, Knoxville, TN 37996-4574, USA
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Acknowledgements |
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We thank Drs W. G. Miller and R. Mandrell for providing plasmid pMW10 used in this study. This study was supported by National Institute of Health grant DK063008.
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