Molecular basis of macrolide resistance in Campylobacter: role of efflux pumps and target mutations

Laurent Mamelli1, Valérie Prouzet-Mauléon2, Jean-Marie Pagès1, Francis Mégraud2 and Jean-Michel Bolla1,*

1 Université de la Méditerranée, Enveloppe Bactérienne, Perméabilité et Antibiotiques, EA2197, IFR48, Faculté de Médecine, 27 Bd Jean Moulin 13385, Marseille Cedex 05, France; 2 Université Victor Segalen Bordeaux 2, Laboratoire de Bactériologie, Centre National de Référence des Campylobacters, Bordeaux, France


* Corresponding author. Tel: +33-4-91-32-44-40; Fax: +33-4-91-32-46-06; E-mail: Jean-Michel.Bolla{at}medecine.univ-mrs.fr

Received 18 March 2005; returned 9 May 2005; revised 24 May 2005; accepted 23 June 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background: Erythromycin is the drug of choice to treat human campylobacteriosis. Campylobacter isolates exhibit two different phenotypes with regard to erythromycin resistance: high-level resistant strains (HLR) and low-level resistant strains (LLR).

Objectives: To study the mechanisms of resistance of Campylobacter to erythromycin, its 6-O-methyl derivative clarithromycin and the ketolide telithromycin.

Results: We observed a cross-resistance against these three molecules but in contrast, no cross-resistance to quinolones. Analyses of LLR showed no mutation on the 23S rDNA and the presence of a drug transport system, which can be inhibited by phenylalanine arginine ß-naphthylamide (PAßN), an efflux-pump inhibitor. In contrast, no PAßN-sensitive drug transport was identified in HLR but we found mutations in the rDNA, which were responsible for decreased binding of telithromycin to purified ribosomes. We further showed that the CmeB efflux pump already described in Campylobacter is not involved in the PAßN-sensitive transport of telithromycin.

Conclusions: Mutations in the ribosome confer high-level macrolide/ketolide resistance. Low-level resistance was mediated by an efflux mechanism which is sensitive to PAßN. This efflux pump was selective to macrolides/ketolide and was different from the previously described Campylobacter efflux pump.

Keywords: macrolides , ketolides , efflux pumps , efflux pump inhibitors , ribosome binding


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Campylobacter jejuni is the most common bacterium isolated in stool samples of humans suffering from gastrointestinal disease worldwide. Antibiotic therapy is needed when symptoms persist or when a complication occurs, and in such cases, ciprofloxacin and erythromycin are the drugs of choice.1 However, similarly to other Gram-negative bacteria, Campylobacter has developed resistance to these antibiotics by target mutations. Mutations in the quinolone resistance determining region (QRDR) region of the DNA gyrase for ciprofloxacin and in the 23S rDNA for erythromycin have been widely described.27 In addition to these mechanisms, many Gram-negative pathogens are developing resistance to fluoroquinolones and macrolides by efflux mechanisms.8 These efflux mechanisms can either be selective for one antibiotic family or they can account for resistance to a large panel of structurally unrelated drugs.9,10

The first report of an efflux mechanism in Campylobacter was made by Charvalos et al.11 but genes involved were not characterized. The complete genomic sequence released in 2000 allowed screening for Gram-negative efflux-pump homologues.12 These analyses indicated that several putative efflux pumps are present in Campylobacter (www.sanger.ac.uk/Projects/C_jejuni/functional_classes/5.G.shtml). Since then, reports by Lin et al.13 and by Pumbwe and Piddock14 have described the cloning and characterization of CmeABC, a tripartite efflux system. They showed that CmeABC was able to extrude from the cell a wide variety of compounds, from dyes (Acridine Orange, ethidium bromide) and detergents (SDS, cholic acid and chenodeoxycholic acid for example) to antibiotics of various families (ß-lactams, ciprofloxacin, chloramphenicol and tetracycline for example). A CmeB deficient mutant exhibited an increased drug susceptibility compared with the parental strain, which demonstrated the activity of the pump. The CmeABC pump thus appeared as a functional resistance mechanism with a basal level activity in wild-type Campylobacter. Moreover, while there is no direct correlation between the CmeABC expression level and resistance, this efflux system has been proposed to contribute to resistance in clinical isolates.15 Another efflux pump, CmeDEF, has recently been studied. However, at this time, it cannot be concluded unequivocally that CmeDEF is an antibiotic resistance mechanism.16

Phenylalanine arginine ß-naphthylamide (PAßN) first described as an inhibitor of the Pseudomonas aeruginosa efflux pumps,17 has been successfully used in several other cases in vitro.1722 We recently observed that PAßN was able to restore an erythromycin susceptibility to Campylobacter strains exhibiting a low-level resistance (LLR).21

Erythromycin and its derivative clarithromycin belong to the 14-member-ring macrolides.23 The subsequent spread of resistant strains prompted a search for newer macrolide derivatives. Among the new molecules, the ketolides exhibit an improved profile and, more importantly, show a significant activity against some macrolide-resistant isolates.19,24,25

In this study, we sought to determine whether the ketolide telithromycin was able to circumvent erythromycin resistance in high-level resistant strains (HLR) and, on the other hand, if the mechanism of transport evidenced in LLR strains was efficient for telithromycin. We further constructed an LLR cmeB mutant, in order to determine if CmeABC was involved in the PAßN-sensitive macrolide transport.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and growth conditions

Strains used in this study are listed in Table 1. Identification at the species level was performed by biochemical tests with the ApiCampy system (bioMérieux, Marcy l'Étoile, France). Campylobacter strains were routinely grown at 37°C either on Columbia agar supplemented with Campylosel (bioMérieux) as a selective medium, or on Mueller–Hinton agar (MH) or MH supplemented with 5% sheep blood if indicated, and under microaerobic conditions obtained with GenerBag (bioMérieux).


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Table 1. Origins, biotype and antimicrobial susceptibility of strains used in this study

 
Chemicals

Telithromycin (14C-labelled, standard susceptibility disc, and water free telithromycin) was kindly provided by Aventis-Pharma SA (Romainville, France). All others drugs (kanamycin, erythromycin, clarithromycin and ciprofloxacin) and PAßN were purchased from Sigma–Aldrich (Saint Quentin Fallavier, France). Standard susceptibility discs were purchased from Sanofi-Pasteur (Bio-Rad, Marnes la Coquette, France). Antibiotic-free discs were purchased from bioMérieux. All chemical products (HEPES, NH4Cl, MgCl2, 2-mercaptoethanol, lysozyme and deoxycholic acid) were purchased from Sigma–Aldrich.

Antibiotic susceptibility

MIC determination was performed on MH agar plates by the standard agar dilution method in accordance with the recommendations of the Comité de l'Antibiogramme de la Société Française de Microbiologie (CA-SFM)26 and confirmed by Etest for erythromycin (AB-Biodisk, Bio-Rad). For pump inhibitor assays in agar plate tests, the same procedure was used to inoculate MH plates supplemented with or without PAßN. Briefly, a strain was grown for 24 h on selective medium. A 3 mL suspension, containing 107 bacteria, was spread on 12 cm x 12 cm MH plates supplemented or not with 10 mg/L of PAßN as indicated. Antibiotic-free discs were loaded with 20 µL of an antibiotic dilution corresponding to 5 µg for erythromycin, clarithromycin and telithromycin, and 0.2 µg for ciprofloxacin, air dried, and placed on inoculated agar plates. The plates were incubated at 37°C under microaerobic conditions and zones of inhibition around each disc were measured after 36 h of incubation. According to the recommendations of the CA-SFM,26 strains with MICs ≤1 mg/L and >4 mg/L were susceptible and resistant strains, respectively. Strains with an MIC between >1 mg/L and ≤4 mg/L were intermediate. From a technical point of view, the strains that repeatedly presented an MIC of erythromycin of 4 mg/L (belonging to the borderline) were termed low-level resistant (LLR) strains.

[14C]Telithromycin uptake

Measurement of [14C]telithromycin uptake by intact cells was adapted from previous studies.8,19,20,27 Exponential-phase bacteria grown in D-MEM broth (Sigma–Aldrich) supplemented with 5 mM MgCl217,18 were separated into aliquots of 1.2 mL and supplemented with 5 µM [14C]telithromycin (specific radioactivity, 55 mCi/mmol) and incubated at 37°C under microaerobic conditions. At the indicated intervals, two 500 µL aliquots of the suspension were removed and immediately loaded on 1 mL of 1 M sucrose solution and further centrifuged for 10 min at 10 000 g. The pellet was suspended in 5 mL of liquid scintillation cocktail (Ready Flow III, Beckman-Coulter, Roissy, France). Radioactivity was measured using a Packard Tri-Carb 2100TR liquid scintillation counter (PerkinElmer, Courtaboeuf, France). Efflux-inhibition assays were performed in the presence of PAßN at a final concentration of 300 µM.

23S rDNA analysis

The presence of mutations in the 23S rDNA of each isolate was determined as described by Vacher et al.7

Isolation of 70S ribosomes

Ribosomes from NCTC 11168 (wild-type ribosomes) and from 99T403 (mutant ribosomes) were isolated by a modified version of a previously described method.28 The procedure was performed at 4°C, and sterilized glassware and tubes were used. Overnight culture (250 OD600 units) was harvested (15 min at 12 000 g) and resuspended in 30 mL of ribosome buffer (20 mM HEPES, 30 mM NH4Cl, 6 mM MgCl2, 4 mM 2-mercaptoethanol) supplemented with 5 mg/L of lysozyme. The cells were slowly shaken for 1 h. Then 30 mL of ribosome buffer supplemented with 0.5% deoxycholic acid was added and slowly shaken again for 1 h to obtain a bacterial lysate. The cell paste was subjected to two low-speed centrifugation steps (20 min at 15 000 g to remove entire cells followed by 60 min at 30 000 g to remove the cell debris). The resulting supernatant was centrifuged for 17 h at 110 000 g in a Beckman-Coulter 70Ti rotor. The crude ribosomal pellet was rinsed with ribosome buffer then resuspended in the same buffer with continuous shaking for about 1 h. The resuspended crude ribosomes were clarified (5 min at 10 000 g) and their concentration was determined (OD260). The crude ribosomes in ribosome buffer were loaded on a discontinuous sucrose gradient (10–30% sucrose, approximately two OD260 units per bucket) in ribosome buffer and centrifuged for 16 h at 48 000 g in a Beckman-Coulter SW55Ti rotor. After centrifugation, the tightly coupled 70S ribosomes were separated from subunits and other membrane fractions. The gradient was fractioned and the fractions harbouring an OD260/OD280 ratio of 1.8 were pooled and considered to be the 70S particles described by Blaha et al.28

[14C]Telithromycin binding to ribosomes

The 70S ribosomes (3 pmol) from macrolide susceptible and resistant strains (NCTC 11168 and 99T403, respectively) were incubated in 1.5 mL of ribosome buffer containing 0.1 nmol of [14C]telithromycin for 30 min at room temperature. The ribosomes (1 mL of reaction mixture) were applied to prewashed Supor Membrane Disc filters (0.22 µm pore size; PALL Life Science, Saint Germain en Laye, France) and washed three times with 5 mL of ice-cold ribosome buffer. The filters were dried for 30 min at 65°C, and the radioactivity associated with the filters was determined using liquid scintillation. Reaction mixtures without ribosomes were treated identically, collected on filters and the radioactivity associated with the filters was determined for each measurement to obtain the background count. To obtain the relative binding efficiency, the radioactivity of an aliquot (100 µL) of each reaction mixture was measured directly.

Construction of cmeB mutants

For generating the cmeB mutant, the genomic DNA of the 81176 cmeB mutant kindly provided by Q. Zhang13 was back-crossed to Campylobacter strains according to the standard biphasic method for natural transformation.29 Transformants were plated on MH agar containing 40 mg/L of kanamycin. The mutation was confirmed to be in cmeB by PCR using cmeB specific primers.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Campylobacter resistance to erythromycin is specific

The strains were tested with the agar dilution method and the disc diffusion assay. A representative set of strains, with regard to antibiotic resistance, is presented in Table 1. No correlation between erythromycin and quinolone (nalidixic acid and ciprofloxacin) susceptibility could be drawn. For example, the NCTC 11168 strain was susceptible to nalidixic acid, ciprofloxacin and erythromycin while strain 00251 was resistant to both quinolones and susceptible to erythromycin (Table 1). Moreover, strain 89503 was susceptible to nalidixic acid and ciprofloxacin and resistant to erythromycin (Table 1). These results showed that for macrolide and quinolone resistance, different mechanisms were involved in these strains, as previously described in Campylobacter.4,7,30

Campylobacter exhibited two levels of resistance to erythromycin

We performed a detailed analysis in order to differentiate resistant strains by the range of their MICs. With regard to erythromycin, three groups of strains were observed (Table 1): the first group was classified as susceptible, with MICs from 0.5 to 1 mg/L, the second group was low-level resistant (LLR) with MICs of 4 mg/L, and the last group with MICs over 128 mg/L contained strains with high-level resistance (HLR).

Erythromycin derivatives did not circumvent resistance

The 6-O-methyl-erythromycin clarithromycin showed the same range of activity on the LLR and the HLR strains (Table 2). The ketolide telithromycin had been efficiently used against erythromycin-resistant Staphylococci,24,25 we thus tried this new molecule against Campylobacter. Surprisingly, we obtained the same results as with the two other macrolides (Table 1). This suggested that Campylobacter developed macrolide resistance mechanisms, equally efficient to at least three members of this antibiotic family.


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Table 2. Effect of PAßN in the agar plate test and strains' corresponding 23S rDNA

 
Only the LLR strains were susceptible to the efflux pump inhibitor (EPI)

PAßN was first described as an efficient EPI in Pseudomonas17,18 and subsequently in other organisms.1921,27 We recently observed that PAßN could restore erythromycin- and clarithromycin-susceptibility to resistant Campylobacter. We show here that the HLR strains were not affected by PAßN, while the LLR strains tested showed their resistance inhibited by PAßN (Table 2). These results indicated that two different mechanisms were present in these two groups.

HLR strains exhibited mutations in their 23S rDNA

According to previous results showing that macrolide-resistant strains contained mutations in their 23S rDNA,7 we performed a combined PCR restriction length polymorphism analysis of the 23S rDNA region from the strains analysed here. As shown in Table 2, all the strains belonging to the HLR group exhibited a mutation at position 2075; an A2075G transition was shown in the majority of cases, with the exceptions of strain 89503 showing an A2075T transition and the double mutant 00039 which exhibited an A2074C transition in addition to A2075G. Conversely, no mutations were identified, either at nucleotide position 2074 or at nucleotide position 2075, in the LLR strains.

These mutations were described in other microorganisms at the equivalent position of their 23S rDNA, to be involved in macrolide resistance by a decreased affinity of the antibiotic to its target.23,31 To check that mutations were responsible for resistance in Campylobacter at the target level, ribosomes from 99T403 and NCTC 11168 used as control strain were purified following the method of Blaha et al.28 According to the equivalence rule of 1 OD260 70S ribosomes = 24 pmol 70S,28 the preparations were standardized to the same concentration before the binding assay was performed. The binding studies demonstrated that under identical conditions, 70S ribosomes from macrolide-resistant strain 99T403 bound significantly less [14C]telithromycin than did those from the macrolide-susceptible control strain NCTC 11168 (Figure 1). Therefore, these results indicated that the drug affinity site of 70S particles of the macrolide-resistant strain harbouring A2075G was modified and consequently the binding of telithromycin was altered.



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Figure 1. Relative binding efficiency of [14C]telithromycin to wild-type (NCTC 11168) and mutant (99T403) 70S ribosomes. Results were obtained with 0.1 nmol of [14C]telithromycin and 3 pmol of ribosomes. Grey bar, wild-type ribosomes; black bar, mutant ribosomes.

 
Evidence for a PAßN-sensitive telithromycin efflux

We show here that PAßN can restore susceptibility to the macrolides erythromycin and clarithromycin and the ketolide telithromycin of the LLR strains. This suggested that an efflux mechanism was active in these strains. To define the efflux pump activity, we measured the intracellular accumulation of [14C]telithromycin in the presence and absence of the EPI. The PAßN addition significantly modified the ketolide accumulation in the strain NCTC 11168. At the steady state, the EPI induced a noticeable increase in the amount of cell-associated antibiotic reaching about 200% that of the control (Figure 2a). These results indicated that intrinsic resistance to ketolides depends on an active efflux mechanism which is able to impair antibiotic accumulation in the bacteria and that this mechanism is PAßN-sensitive. The same experiments were performed on a low-level resistant representative. In the absence of the EPI, labelled telithromycin accumulated in strain 96C208 at a lower level than in NCTC 11168 and the EPI restored a high intracellular concentration of ketolide, which demonstrated that an efflux pump susceptible to PAßN is active in 96C208 (Figure 2b). In the case of the highly-resistant strain 99T403, no significant difference was observed in the accumulation of telithromycin in the presence or absence of the EPI (data not shown).



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Figure 2. Accumulation of [14C]telithromycin without (filled symbols) and with (open symbols) 300 µM PAßN. The values were obtained from three independent experiments carried out in duplicate. (a) Control; (b) 96C208; (c) 96C208 cmeB.

 
Role of CmeABC in telithromycin efflux

The CmeABC system is an efflux mechanism previously described in Campylobacter.13,14 This prompted us to study the role of CmeABC in Campylobacter macrolide/ketolide resistance. We first compared the antibiotic susceptibility of strain 81176 with its cmeB derivative. The strain 81176 was susceptible to antibiotics, and its cmeB derivative exhibited an increased susceptibility (Table 2) as previously described by Lin et al.13 PAßN induced an increased susceptibility to erythromycin, clarithromycin and telithromycin but not to fluoroquinolone in the strain 81176, in agreement with the results obtained with strain NCTC 11168 (Table 2). Interestingly, the EPI was still efficient on the 81176 cmeB derivative (Table 2). This suggested that, in addition to the CmeABC complex, a PAßN-sensitive efflux system participated in the intrinsic resistance to macrolides/ketolide. To determine the role of CmeABC in macrolide transport in LLR strains, we constructed a 96C208 cmeB derivative. The susceptibility to macrolides of the parental and the recombinant derivatives was tested in the presence or absence of the EPI. The EPI caused an increase in macrolide susceptibility in the cmeB mutant within a range which was approximately the same as that of the parental strain (Table 2). Moreover, the antibiotic accumulation assays showed that the intracellular concentration of [14C]telithromycin in the 96C208 cmeB derivative was not significantly modified compared with the level measured with the parental strain (compare Figure 2b with Figure 2c). This suggested that the mutant maintained its ability to overcome the intracellular concentration of macrolide. In addition, the intracellular amount was still dependent on the presence of PAßN (Figure 2). These results indicated that another efflux pump is fully active in this mutant, independent of CmeABC.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to decipher the mechanisms of erythromycin resistance in Campylobacter and to determine if other macrolides were able to circumvent this resistance.

Erythromycin resistance in Campylobacter has been widely reported and the principal mechanism already described is mutation within 23S rDNA.2,5,7,32,33 No rRNA modifying enzyme has been described to date in Campylobacter,34 while several groups reported that efflux mechanisms could be involved in this process.21,22 Moreover, the CmeABC efflux system described by Lin et al.13 and Pumbwe and Piddock14 suggested that resistance by an efflux process could work efficiently in Campylobacter.

Here we have shown that erythromycin-resistant isolates can be classified in two levels, a low level and a high level as also described by Payot et al.22 for C. coli. Our results indicated that target mutations are responsible for high-level telithromycin resistance and confirm previous reports.7,22 Interestingly, the low-level resistant strains exhibited no mutation in the target rRNA gene. In a previous study,21 we had shown that PAßN restored erythromycin susceptibility in some isolates. Here we have shown that PAßN was only active on LLR strains. Our results are slightly divergent from those of Payot et al.:22 we did not observe any effect on the HLR strains under our conditions. However, the high PAßN concentration (40 mg/L) used by these authors inhibited the growth of HLR strains assayed in the work presented here (data not shown). Moreover, one must also note that the effect was smaller on HLR (2- to 4-fold increase in macrolide susceptibility) than on LLR (16- to 64-fold).22

Our results support our previous conclusions that the PAßN effect suggested an active efflux mechanism, in susceptible and in resistant isolates. CmeB was described as a general efflux pump extruding a wide variety of compounds from the cell, and particularly antibiotics from different families (quinolones, fluoroquinolones, ß-lactams and macrolides for example). Here we have shown that PAßN was only active on the macrolide efflux pump. This may suggest that CmeB has different binding sites for macrolides and other molecules as previously mentioned for AcrB35 and that PAßN selectively inhibits the interaction of macrolides with the pump. Alternatively, another efflux mechanism could be concomitantly expressed with CmeB. Because in a cmeB genetic background, PAßN was still efficient, we concluded that another pump is active. The expression of the CmeDEF system was recently studied by Pumbwe et al.16 but no correlation with macrolide and quinolone resistance was drawn. Thus, it is possible that a third system is active against macrolides.

While erythromycin is recommended to treat Campylobacter infections, the increasing number of erythromycin-resistant human pathogens led to the research effort to overcome this resistance. Among new macrolides, the erythromycin derivative clarithromycin is used with strong efficiency against Helicobacter pylori, a species closely related to Campylobacter. However, we have shown here that the strains resistant to erythromycin were also resistant to clarithromycin, at the same level. Another family of macrolides, the ketolides, was reported to have activity against macrolide-resistant Gram-positive pathogens.24,25 Here we tested the ketolide telithromycin (HMR 3647) and we observed similar resistance patterns as for classical macrolides. In the case of HLR Campylobacter, these results are consistent with previous observations that the 23S rRNA mutations impair the binding of erythromycin.34 We also showed here that efflux mechanisms in Campylobacter are efficient on telithromycin. CmeB was not tested previously on ketolide and we showed here that a cmeB deficient mutant is more sensitive than the wild-type and that in a cmeB background PAßN is able to increase telithromycin susceptibility.

To conclude, we have shown here that an additional efflux mechanism distinct from CmeABC is active in Campylobacter. This mechanism is selective to macrolides and is sensitive to PAßN, which suggests a common binding site for these two molecules. Many putative efflux systems exist in Campylobacter37 and the efflux pump responsible for the resistance has still to be identified.


    Acknowledgements
 
We are greatly indebted to J. Chevalier for her involvement in antibiotic accumulation assays. We gratefully acknowledge Q. Zhang for providing us with the DNA from the cmeB strain and for helpful advice. We thank Dr A. Bryskier for his faithful advice. We thank Dr G. Ermel (AFSSA, Ploufragan, France), Dr P. Brisou (Hôpital d'Instruction des Armées Sainte-Anne, Toulon, France), Dr J. M. Vialle (Bastia, France) and Dr C. Bollet (Hôpital de la Timone, Marseille, France; deceased) for providing us with Campylobacter isolates. We thank L. Dedieu for fruitful discussions. L. M. is a scholarship recipient of the Collectivité Territoriale de Corse, France. This work was supported by the Université de la Méditerranée, Marseille, France.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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