JAC Advance Access originally published online on February 8, 2007
Journal of Antimicrobial Chemotherapy 2007 59(3):378-386; doi:10.1093/jac/dkl504
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A combined phenotypic and genotypic method for the detection of Mex efflux pumps in Pseudomonas aeruginosa
1 Unité de Pharmacologie cellulaire et moléculaire, Brussels, Université catholique de Louvain, UCL 7370 avenue E. Mounier 73, B-1200 Bruxelles, Belgium 2 Laboratoire de Microbiologie, Cliniques universitaires UCL de Mont-Godinne, avenue G. Therasse 1, B-5530 Yvoir, Belgium
* Corresponding author. Tel: +32-2-7622136 / 7647371; Fax: +32-2-7647373; E-mail: tulkens{at}facm.ucl.ac.be
Received 10 August 2006; returned 29 September 2006; revised 10 November 2006; accepted 14 November 2006
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Abstract |
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Background: Mex efflux pumps contribute to multidrug resistance in Pseudomonas aeruginosa. Evidencing their expression in clinical isolates would help in rationalizing antibiotic selection.
Methods: We have developed a combined phenotypic and genotypic approach for the differential diagnosis of resistance mediated by four major transporters (MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM). The methodology was validated using reference strains harbouring only one specific transporter and its applicability evaluated towards seven selected clinical isolates, the resistance mechanisms of which could not be assigned by conventional techniques. Phenotypic detection used MIC measurements with reporter antibiotics [carbenicillin (MexAB-OprM); erythromycin (MexCD-OprJ); norfloxacin and imipenem (MexEF-OprN); gentamicin (MexXY-OprM)] with and without Phe-Arg-ß-naphthylamide. Genotypic detection was made by semi-quantitative reverse transcription PCR (RT-PCR) for mexC and mexE, and by quantitative competitive RT-PCR and real-time PCR for mexA and mexX (correlation between both methods: > 88 % ; overexpression levels ranging between 4.8 and 8.1).
Results: Convergence between phenotypic and genotypic methods was observed in control strains for all pumps. For clinical isolates, convergence was obtained in 6 of 7 strains for MexXY-OprM and MexEF-OprM, and in 5 of 7 for MexAB-OprM and MexCD-OprJ, mostly due to hard to interpret phenotypic data.
Conclusions: The data plead for combining phenotypic and genotypic approaches in the diagnosis of efflux-mediated resistance in P. aeruginosa.
Keywords: multidrug resistance , Phe-Arg-ß-naphthylamide , diagnostic , efflux pump , Pseudomonas aeruginosa
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Introduction |
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The emergence of multidrug resistance phenotypes1 makes epidemiological surveillance of Pseudomonas aeruginosa susceptibility more and more essential for the selection of successful empirical regimens. In this respect, resistance by efflux is of particular interest. Even though efflux usually confers a moderate level of resistance, its impact in the clinics could be important, because it may make antibiotics inefficient in infected sites where antibiotic concentrations are less than optimal,2 and it confers cross-resistance to unrelated antibiotic classes in Gram-negative bacteria,3,4 which may be difficult to anticipate based on standard, routine susceptibility testing. The Mex efflux pumps of P. aeruginosa are of particular interest in this context because of their exceptionally broad substrate specificity. While 12 potential efflux systems of this family have been identified in the P. aeruginosa genome,5 4 of them (MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM) are best characterized as antibiotic transporters.6
Diagnosis of efflux-mediated resistance based only on phenotypic methods is often ill-informed due (i) to the frequent concomitant presence of other mechanisms of resistance affecting partly or totally the same drugs, (ii) to the variable expression of resistance among different strains, and (iii) to the difficulty of detecting mechanisms conferring low to moderate levels of resistance.2,7 The specific detection of efflux pumps has long relied on western blot8–11 or northern blot analysis,12 but these methods cannot easily be implemented in the clinical laboratory. As recently presented in a review paper,13 nucleic acid-based diagnostics are gradually replacing or complementing culture-based biochemical and immunological assays in routine microbiology laboratories, because of their speed and potentially high level of specificity. They can now be used not only for detection and identification of microbial pathogens, but also for genotyping as applied to the determination of antibiotic resistance or to microbial fingerprinting. Accordingly, semi-quantitative reverse-transcription PCR (RT-PCR),14–17 or quantitative real-time PCR,18–21 which directly identify the presence or the expression level of genes of interest, have been successfully used for detecting the expression of efflux pumps in P. aeruginosa. However, they do not provide direct information on the level of production and on the functionality of the corresponding proteins. We, therefore, have developed a combined phenotypic and genotypic approach for the detection of resistance by Mex-mediated efflux in P. aeruginosa, concentrating on MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM. Validation was sought with references strains, and potential applicability in the clinical setting assessed by testing a limited number of clinical isolates.
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Materials and methods |
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Strains and growth conditions
All strains are described in Table 1. Reference strains were chosen as carrying only one specific efflux mechanism; clinical isolates were selected as ‘resistance mechanism(s) difficult to assign’ based on conventional interpretative analysis of resistance phenotypes by disc diffusion method. All bacteria were grown overnight in Muller–Hinton broth (Becton Dickinson & Co., Franklin Lakes, NJ, USA) at 37°C, with constant shaking at 100 rpm and under aerobic conditions.
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Phenotypic analysis
The following reporter antibiotics were used as phenotypic markers of the Mex efflux pumps of interest: carbenicillin (MexAB-OprM), erythromycin (MexCD-OprJ), gentamicin (MexXY-OprM) and norfloxacin (MexEF-OprN).3,22–25 We also tested imipenem as an indirect indicator of MexEF-OprN since it induces the expression of this pump,26 and because its MIC is increased in MexEF-OprN overproducers (probably through a decreased expression of OprD).10,27 MICs were determined by broth microdilution28 in the absence or presence of a broad-spectrum inhibitor of Mex pumps, namely Phe-Arg-ß-naphthylamide (PaßN), also known as MC-207,11029 at a concentration of 50 mg/L, which fully restored the susceptibility of reference strains to the reporter antibiotics but did not impair the bacterial growth. (Lower concentrations were shown to be insufficient to reverse resistance to all substrates of a given pump;29 large concentrations have also been used by others for clinical isolates able to express several efflux mechanisms.30,31) Routine assays were made with log2 dilutions. To detect minimal changes, however, arithmetic dilutions were occasionally used for carbenicillin (4 mg/L increments in the 8–32 mg/L range) and gentamicin (0.1 mg/L increments in the 0.25–1 mg/L range).
Extraction of RNA and synthesis of cDNA. Pseudomonas aeruginosa cultures were harvested at the late log-phase of growth, based on the following considerations: (i) the expression of MexAB-OprM is phase-regulated, being minimally expressed in lag phase and increasing in log to late-log phase,32 suggesting that collecting material at this stage of growth may improve the sensitivity of the assay; (ii) this metabolic state is reached by overnight growth, which may facilitate the routine implementation of the procedure as well as the correlation with MIC data of reporter antibiotics, which are also measured on bacterial cultures having grown overnight. Total RNA was isolated and purified as described previously.16 Absence of residual genomic DNA was checked by PCR control reactions using RNA as template. Generation of cDNAs was carried out by reverse transcription using 5 µg of DNA-free RNA as template (Promega Reverse Transcription System, Promega Co, Madison, WI, USA). All PCR amplifications were performed using a Bio-Rad Thermocycler model 100, with the primers pairs presented in Table 2. The specificity of all these primers was checked in silico for absence of potential cross-hybridization [using the Primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi)], and experimentally by performing (i) PCR with each of the primer pairs and demonstrating the presence of a single band at the expected size, and (ii) real-time PCR in the absence of cDNA template and observing the absence of amplification signal.
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Detection of mexA and mexX genes by real-time PCR. We used the primers mexA3 and mexA4, and mexX3 and mexX4, respectively, with amplification reactions performed in the presence of Sybr Green iQTM Supermix (Bio-Rad) using a iCycler iQ® Single-Color Real-Time-PCR Detection System (MyiQTM real time PCR software; Bio-Rad) with the following conditions: 10 min denaturation at 95°C; 40 cycles (15 s, 95°C/1 min, 60°C); melting curve analysis 60–95°C with continuous fluorescence readings; rpsL used as housekeeping gene.18,19,33 The threshold cycle of samples was always within the range of standards (R2 > 0.995).
Detection of mexA and mexX genes by quantitative competitive RT-PCR (QC-RT-PCR)34. Standard techniques were used with the following specific conditions. Internal competitor DNAs35 were synthesized by PCR using 0.6 µM primers (mexA-F and mexA-40mer-R or mexX-40mer-F and mexX-R), 10 ng template DNA, 1.5 M betaine (Roche Molecular Biochemicals, Mannheim, Germany), 1.25 mM mixed deoxynucleoside triphosphates (Fermentas GmbH, St Leon-Rot, Germany) and 2 U of Taq DNA Polymerase in the corresponding buffer (1 x dilution; Biotools, B & M Labs S.A., Madrid, Spain). The PCR conditions consisted of (i) 10 min denaturation at 95°C, (ii) 30 cycles of 30 s at 95°C, 30 s at 60°C and 30 s at 72°C for mexA amplification, and 30 cycles of 1 min at 95°C, 1 min at 60°C and 1 min at 72°C for mexX amplification, and (iii) a final elongation at 72°C for 10 min. For quantification of the target genes expression level, different amounts of internal competitor (see Supplementary Material) were co-amplified with a constant amount of target cDNA. Amplifications were carried out as described for the synthesis of the internal competitors, but using the primer pairs mexA-F and mexA-R (252 bp) or mexX-F and mexX-R (451 bp). Band intensities of each product were analysed optically [Gel-Doc 1000 camera system operated with the QuantityOne® image analysis software (Bio-Rad, Richmond, CA, USA)]. Raw data were corrected for the size of the amplified fragments.36
Detection of mexC and mexE expression by semi-quantitative RT-PCR. We used a published method17 with the primers listed in Table 2. For a positive control, mexA (expressed at a basal level in all strains)37 was amplified in parallel.
Assessment of reproducibility and sensitivity of the quantitative methods
MICs were determined in triplicate using independent cultures (differences observed are shown in the Results) and cDNA preparation was done in triplicate from independent bacterial cultures. The PCR quantification reactions were routinely performed in triplicate for each of the cDNA preparations, generating a total of nine data sets for each strain. The intra-day (n = 3) and inter-day (n = 9) coefficients of variations ( % ) were 16.1 and 7.9 for mexA, and 10.3 and 8.3 for mexX in QC-RT-PCR; and 12.3 and 7.3 for mexA, and 9.2 and 9.6 for mexX in real-time PCR. The lowest limit of detection (i.e. lowest concentration of the standard giving a highly reproducible signal) was 102 copies/sample by real time PCR and 3.5 x 102 copies/sample by QC-RT-PCR.
Definition of convergence/divergence between the phenotypic and the genotypic methods
In the phenotypic analysis, efflux was considered likely if the MIC of a given strain was at least 2 log2 dilutions higher than in the wild-type strain, and if the addition of PAßN lowered its value to that which was measured in the wild-type strain ( ± 1 dilution). If the MIC was increased by 1 dilution only or if the MIC remained elevated upon addition of PAßN (compared with the wild strain), efflux was considered as possible. In the genotypic analysis: (i) MexAB-OprM- and MexXY-OprM-based efflux was considered likely for a 
3-fold mexA and mexX overexpression by QC-RT-PCR or real-time PCR compared with the wild strain; (ii) MexCD-OprJ- and MexEF-OprN-based efflux was considered likely if mexC and mexE were detected. Convergence between the phenotypic and genotypic methods was considered as complete if both methods showed the likely presence (or absence) of the corresponding efflux mechanism, and partial if efflux was scored only as possible by the phenotypic approach, while being scored as likely by the genotypic approach. All other situations were scored as denoting divergence.
Carbenicillin and norfloxacin were purchased from Sigma-Aldrich Co, St Louis, MO, USA. Gentamicin, imipenem and erythromycin were obtained as the corresponding branded products distributed for clinical usage in Belgium by Schering-Plough, Merck Sharp and Dohme, and Abbott, respectively. PAßN was obtained from Sigma-Aldrich and, upon its discontinuation from their product list, received from Mpex Pharmaceuticals Inc., San Diego, CA, USA. Unless stated otherwise, all other reagents were obtained from E. Merck AG, Darmstadt, Germany, or Sigma-Aldrich.
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Results |
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Phenotypic characterization of resistance mediated by Mex efflux pumps
Table 1 shows the MIC values measured in the absence and in the presence of 50 mg/L of the broad-spectrum efflux inhibitor PAßN. For reference strains, there was a 2 log2 dilutions increase of MICs for each reporter antibiotic according to their established resistance mechanism, and the addition of PAßN brought these values to, or close to (1 dilution), those of the wild-type strain. Addition of PAßN in PAO1 caused a 33 and 23 % reduction of the MICs of carbenicillin and gentamicin, respectively (based on arithmetic dilutions measurements), denoting a low, basal expression of MexAB-OprM and MexXY-OprM.9,38 The MIC of erythromycin was markedly reduced in PAO1 by addition of PAßN, suggesting the presence of another PAßN-inhibitable efflux transporter in this strain (erythromycin has been described so far as a potential substrate of MexJK and MexVW39). Considering the 7 clinical isolates, MICs 2 to 3 log2 dilutions higher than in the wild-type strain PAO1 were observed in 6 strains for carbenicillin, in 3 strains for erythromycin, in 6 strains for norfloxacin, in 4 strains for imipenem, and in 3 strains for gentamicin. The pattern of restoration of activity by PAßN led us to suspect that MexAB-OprM was active in strains 324, 411, 699, 785 and 905; MexCD-OprJ in strains 324, 699 and 762; MexEF-OprN in strains 324, 376, 411 and 762, and MexXY-OprM in strains 376, 785 and 905. Isolate 376 showed a non-significant increase of MIC compared with the wild-type strain PAO1 but PAßN decreased this value by 2 dilutions.
Genotypic characterization of resistance mediated by Mex efflux pumps
Three techniques were used for this analysis. For mexA and mexX, methods quantifying the amount of mRNA were considered essential because these genes are constitutively expressed at a basal level in wild-type strains (contributing to intrinsic resistance), and overexpressed to variable levels in resistant strains.37,40 We therefore designed a real-time PCR assay, which was validated by a parallel examination of the samples using QC-RT-PCR assay [see Supplementary Data—available at JAC Online (http://jac.oxfordjournals.org/)]. For mexC and mexE, a semi-quantitative method was considered sufficient because the expression of these genes is strictly down-regulated in wild-type strains but expressed to high levels in resistant strains.10,17,37,41 Thus, we selected a semi-quantitative PCR assay, which allowed us to easily detect the appearance of an amplification product in resistant strains only [see Supplementary Data—available at JAC Online (http://jac.oxfordjournals.org/)].
Determination of the overexpression of mexA and mexX. Table 3 shows that mexA and mexX were overexpressed by 4 to 7 times in reference strains. Similar overexpression levels were detected in 6 and 3 clinical isolates for mexA and mexX, respectively. A good agreement was observed between the overexpression levels detected by real-time PCR or by QC-RT-PCR.
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Determination of mexC and mexE expression by semi-quantitative RT-PCR. In reference strains, mexC and mexE were specifically amplified in EryR and JFL28, and in PAO7H and JFL05, respectively, the other strains showing only the amplification of mexA used as the internal control. This technique was then applied to the clinical isolates included in the study, and the results (expression of the corresponding genes or not) are shown in Table 4.
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Applicability of phenotypic and genotypic methods to the diagnosis of Mex-mediated resistance in clinical isolates
Table 4 summarizes and compares the results of the phenotypic and the genotypic methods as applied to the reference strains and the 7 clinical isolates. For reference strains, an excellent correlation was observed between the two methods. For the clinical strains, the correlation was excellent for MexEF-OprN and MexXY-OprM (6 strains with complete convergence; 1 strain with partial convergence; no divergence), good for MexAB-OprM (5 strains with complete convergence; 2 strains with partial convergence; no divergence), and fair for MexCD-OprJ (5 strains with complete convergence; 1 strain with partial convergence; 1 strain with divergence; for the latter strain, the phenotypic method yielded a positive or likely positive result for the presence of an efflux-mediated resistance, but the semi-quantitative genotypic method was negative).
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Discussion |
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The present study proposes a combined phenotypic and genotypic approach for the specific diagnostic of resistance mediated by four Mex efflux pumps in P. aeruginosa. MexAB-OprM and MexXY-OprM are expressed at a basal level in wild-type strains, contributing to the low intrinsic susceptibility of P. aeruginosa to the corresponding antibiotics, but are overproduced in resistant strains.9,23,38,42 MexCD-OprJ and MexEF-OprN are not detected in standard conditions of culture for wild-type bacteria, but their expression is up-regulated in resistant bacteria.8,10,16 As opposed to diagnostic methods based solely on MIC determinations towards a single antibiotic,43 or using a non-specific inhibitor such as CCCP,15 the phenotypic approach devised here provides a first level of differentiation among these four Mex efflux pumps. However, and as already noted by others,7 interpretation of phenotypic data remains difficult with clinical strains, probably because of the co-expression of resistance mechanisms other than efflux. While interpretation could be made easier by increasing the number of reporter antibiotics, this would considerably weigh down the interest of the method. Using more specific inhibitors44 faces the problem of lack of commercial supply of the corresponding products.
Specific methods to directly evidence the genes encoding the proteins responsible for efflux appear, therefore, desirable. For detecting overexpression of constitutively expressed pumps, quantitative assays are necessary. We show here that two methods can be used interchangeably, namely real-time PCR (rapid but expensive) or QC-RT-PCR (laborious but less expensive). This conclusion, and the levels of overexpression observed, are in accordance with what has been demonstrated in other applications,45,46 as well as in the present application with different amplicons and other clinical strains.18,19,33,47 Of the two, real-time PCR technology is more likely to become the standard method in routine, based on a series of advantages which include excellent sensitivity and specificity, low contamination risk, and speed,48 and on the development of high throughput systems, allowing for the concomitant run of several reactions in parallel. However, we did not apply this expensive method for the detection of the genes of inducible pumps (MexCD-OprJ and MexEF-OprN), since all previous studies consistently show that the expression of mexC or mexE is strictly down-regulated in wild-type strains,10,41 but is large (100–1000 x the wild-type value) in nfxB or nfxC resistant mutants.14,18 Inclusion of the detection of mexA, a constitutively expressed gene, as control decreases the risk of false negatives.
Genotypic diagnostic methods do not necessarily provide information on the final expression of the gene product and its functionality.19 We addressed this issue by examining the correlation between the genotypic and the phenotypic methods developed here (Figure 1). It clearly appears that the use of combined thresholds—namely 3 for gene overexpression (close to what was proposed in an independent recent study21), and a 1.5 log2 dilution reduction of MIC—allows differentiation between low, probably meaningless, changes from those that may require reconsideration of the susceptibility pattern of the corresponding clinical isolate.
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Besides the technical and potential clinical interest of the diagnostic methodology presented here, some biological observations made during our study also deserve attention. First, we have shown that the basal expression level of mexX is much lower than that of mexA but that both efflux pumps are overexpressed 4 to 8 times in resistant strains, suggesting that a lower quantity of MexXY-OprM than MexAB-OprM protein may be needed for effective transport of the corresponding substrates. Second, and as in another study,8 overexpression of mexX in clinical isolates is systematically associated with that of mexA. This may be related to the fact that MexXY uses OprM as a porin,9 the expression of which is under the control of the regulator of the MexAB-OprM operon.49 Third, PAßN was found to reduce the MIC of imipenem in MexEF-OprN reference strains (PAO7H and JFL05), as if it were a substrate of this pump. Potential reasons may include a partial transport of imipenem by MexEF-OprN, or by a still uncharacterized Mex pump inhibitable by PAßN, or an action of PAßN on the common regulator, causing a reduction in the pump expression and an increased expression of the OprD porin. Fourth, clinical isolates were found to express several efflux pumps at the same time, causing particularly complex multidrug-resistant phenotypes. Even though these conclusions still need to be considered with caution due to the small number of strains included in the present study, our data plead for a larger implementation of diagnosis of efflux, through the combined use of appropriate phenotypic and genotypic methods in routine clinical microbiology, for the correct identification of resistance mechanisms in these organisms. This knowledge may be helpful, at the level of the individual patient, for rationalizing the antibiotic choice and dosing and, at the level of the hospital, for defining antibiotic policies, based on epidemiological surveys evidencing the most prevailing resistance mechanisms.
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None to declare.
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Supplementary data |
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Supplementary data are available at JAC Online (http://jac.oxfordjournals.org/).
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Acknowledgements |
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N. M. was recipient of a ‘Prospective Research for Brussels’ fellowship, and L. A. of a post-doctoral fellowship ‘Research in Brussels’ from the Région de Bruxelles-capitale / Brussels Hoofdstedelijk Gewest; N. E. C. is Post-doctoral Fellow, and F. V. B. Maître de Recherches of the Belgian Fonds National de la Recherche Scientifique. We thank: P. Plesiat, T. Kohler, and J. Martinez for providing us with reference strains of P. aeruginosa; O. Lomovskaya for the gift of PAßN (MC-207,110); and J. C. Pechère for useful comments and suggestions in the preparation of this paper. This work was supported by the Région de Bruxelles-capitale / Brussels Hoofdstedelijk Gewest, the Belgian Fonds National de la Recherche Scientifique Médicale (grants no. 1.5.230.05 [EC] F, 3.4.549.00 [EC] F and 3.4.639.04 [EC] F), and the Belgian Federal Science Policy Office (Research project P5/33; research action P5).
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References |
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