JAC Advance Access originally published online on April 10, 2006
Journal of Antimicrobial Chemotherapy 2006 57(6):1223-1226; doi:10.1093/jac/dkl139
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Production of the cryptic EefABC efflux pump in Enterobacter aerogenes chloramphenicol-resistant mutants
Enveloppe Bactérienne, Perméabilité et Antibiotiques, EA2197, IFR48, Faculté de Médecine, Université de la Méditerranée 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
*Corresponding author. Tel: +33-4-91-32-45-87; Fax: +33-4-91-32-46-06; E-mail: Jean-Marie.Pages{at}medecine.univ-mrs.fr
Received 14 December 2005; returned 21 January 2006; revised 17 February 2006; accepted 21 March 2006
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Abstract |
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Objectives: AcrAB-TolC is the major tripartite multidrug efflux pump in Enterobacter aerogenes while EefABC is a cryptic efflux system. This study was conducted to identify and characterize E. aerogenes mutants producing the EefABC efflux pump.
Methods: Four spontaneous chloramphenicol-resistant (CMR) mutants were isolated. The expression level of the eefABC promoter and the production of the EefA and B proteins were analysed in the mutants. Antibiotic susceptibilities were compared for wild-type and mutant strains. Efflux activity was investigated using an efflux pump inhibitor.
Results: The activation of the eefABC promoter was detected in four CMR mutants. These mutants showed increased resistance to erythromycin and ticarcillin, but not to fluoroquinolones, ketolides and detergents. Two additional efflux proteins were detected in the mutants. The CMR mutants bear no mutation in hns, which encodes a repressor of eefABC. No alteration of porin expression, a phenotype observed in marA or ramA multidrug-resistant mutants, was detected in the mutants.
Conclusions: These observations suggest that eefABC activation can occur in vitro independently of the H-NS, MarA or RamA global regulators.
Keywords: drug efflux pumps , efflux inhibitors , multidrug resistance
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Introduction |
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Enterobacter aerogenes emerged as an important hospital pathogen in the 1990s. This Gram-negative bacterium is now the third most common pathogen recovered from the respiratory tract and is often isolated in the urine and gastrointestinal tracts of patients.1,2 E. aerogenes strains isolated from hospitalized patients generally exhibit high resistance levels to a wide variety of structurally unrelated antibiotics.1–5 Production of a chromosomal cephalosporinase and a plasmid-borne extended-spectrum ß-lactamase (ESBL) confers resistance both to ß-lactams and combinations of ß-lactams and ß-lactamase inhibitors. An epidemic clone of E. aerogenes producing TEM-24 ESBL has spread to nearly all French university hospitals.3,5 Moreover, decrease in porin synthesis and induction of active drug efflux have been reported in several clinical isolates.5,6 To date, two tripartite multidrug resistance (MDR) efflux systems, the AcrAB-TolC and EefABC pumps, have been identified and characterized in E. aerogenes.7,8 The contribution of antibiotic exposure in the development of MDR due to efflux pumps expression has been demonstrated in vitro and in vivo.9–11 AcrAB-TolC expression is constitutive in vitro and is enhanced in the presence of antimicrobial agents in the growth medium; furthermore the overexpression of this pump has been observed during imipenem therapy of E. aerogenes-infected patients.9 In contrast, eefABC expression has never been detected under any laboratory growth conditions, except in hns (H-NS, histone-like nucleoid-structuring protein) mutants.7 We report here the isolation and characterization of E. aerogenes mutants producing the EefABC pump in the absence of an hns mutation or a pleiotropic MarA activation.
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Materials and methods |
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Bacterial strains, growth media and antibiotic susceptibility tests
Bacteria were grown at 37°C in Luria–Bertani (LB) broth. E. aerogenes BW16627 is an rpsL derivative of ATCC 15038, and EAEP295 is a spontaneous nalidixic acid-resistant mutant of BW16627.7 EA27, a multidrug-resistant isolate, was described previously.5,8
Susceptibility to chloramphenicol, tetracycline, norfloxacin, ciprofloxacin, erythromycin, telithromycin, ticarcillin, amikacin, acriflavine, deoxycholate (DOC) and sodium dodecyl sulphate (SDS) was measured by the broth dilution method, as described previously,5,8 according to the standard 2-fold dilution method of the French Society for Microbiology. Approximately 106 cells were inoculated into 1 mL of Mueller–Hinton broth containing 2-fold serial dilutions of each antibiotic. Results were read after 18 h at 37°C and are expressed as MICs in mg/L. The efflux pump inhibitor phenylalanine-arginine ß-naphthylamide (PAßN) was used as described previously.6,10
DNA techniques
Plasmid DNA was isolated using the Wizard® Plus SV Minipreps DNA Purification System kit (Promega, Madison, WI, USA). E. aerogenes genomic DNA was isolated using the Wizard® genomic DNA purification kit (Promega). DNA fragments were gel-extracted using a QIAquick® gel extraction kit (Qiagen, Courtaboeuf, France). DNA sequencing was carried out using universal or custom synthesized primers at the Eurogentec Sequencing Department, Seraing, Belgium.
Construction of the reporter plasmids
The eefABC promoter region (Peef) was extracted from pEP1377 as a 0.6 kb BamHI DNA fragment and cloned into pBBR1-KGFP12 upstream of the green fluorescent protein (GFP) reporter gene to generate pEP90 (Peef::gfp, Kmr). The same Peef DNA fragment had been cloned upstream of the ß-galactosidase gene to construct pMM38 (Peef::lacZ, Kmr) as described previously.7
ß-Galactosidase assay
ß-Galactosidase was assayed on cells cultured overnight according to the method of Miller.13 The data are expressed as the means of a minimum of three experiments. Standard deviations are indicated.
Preparation of total cell extracts and immunodetection
After centrifugation of bacterial suspension from exponential growth phase cultures (14 000 g, 15 min, 4°C), pellets were resuspended in 100 µL of sample buffer and heated for 5 min at 96°C. Proteins were separated by 10% SDS–PAGE and electrotransfered onto a nitrocellulose membrane (Schleicher & Schuell BioSience Inc., Keene, NH, USA). Membranes were probed with antibodies raised against E. coli AcrA or AcrB, and immunoreactive proteins were visualized with alkaline phosphatase-conjugated secondary antibodies.
Detection of chloramphenicol acetyltransferase
A chemical chloramphenicol acetyltransferase assay was performed using the method described previously.10 Acetyl coenzyme A and 5,5'-di-thio-bis (2-nitrobenzoic acid) (DNTB) were used as reagents. A positive reaction was indicated by the development of a deep yellow colour.
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Results and discussion |
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Isolation of chloramphenicol-resistant (CMR) mutant
The eefABC operon is silent in E. aerogenes grown in the laboratory, but its expression is derepressed in an hns mutant.7 To identify other regulators of the Peef promoter, we mutagenized E. aerogenes EAEP295,7 bearing a Peef::gfp reporter plasmid (pEP90, GFP– phenotype) with the mini-Tn5Cm transposon.7 Clones were selected on LB agar supplemented with chloramphenicol (30 mg/L) and kanamycin (30 mg/L), and Cmr Kmr colonies were examined under fluorescent light to screen for GFP+ clones. Four fluorescent Cmr Kmr colonies were isolated from four different plates and purified. Genomic DNA of the four mutants was prepared and analysed by Southern hybridization with a mini-Tn5Cm probe. None of the mutants contained the transposon. Thus, they were spontaneous Cmr mutants. Plasmids were prepared from each mutant and reintroduced into EAEP295. All Kmr transformants were GFP–, indicating that the GFP+ phenotype of the mutants was not due to a mutation in the pEP90 reporter plasmid. Each of the four Cmr GFP+ mutants was grown in LB in the absence of Km, and a GFP– Kms plasmid-cured derivative was isolated from each mutant to obtain clones CMR-1 to CMR-4. The reintroduction of pEP90 in these four derivatives restored the GFP+ phenotype, which showed that the original Peef expression had been conserved.
Characterization of CMR mutants
The Peef expression level in the four CMR mutants was estimated using the Peef::lacZ reporter plasmid pMM38.7 Peef::lacZ expression level was 200- to 300-fold higher in the CMR mutants transformed with pMM38 than in the BW16627 strain bearing the same plasmid (Figure 1a); the same low-level expression was also observed with the parental strain EAEP295 (data not shown).
The MICs of various antibiotics were determined for the four CMR mutants. Although chloramphenicol was the selective agent, the MICs of chemically unrelated antibiotics were also increased in the mutants compared with the parental strain (Table 1). All were clearly less susceptible to erythromycin and ticarcillin. In contrast, no noticeable modifications in the susceptibilities to fluoroquinolones (norfloxacin and ciprofloxacin), amikacin, tetracycline, telithromycin, acriflavine and detergents (DOC and SDS) were observed (Table 1). This phenotype is quite different from that of the AcrAB-overexpressing MDR clinical isolate EA27 that exhibits a more general drug resistance profile.5 These results confirm previous reports showing that AcrAB-TolC is involved in the efflux of various drugs including fluoroquinolone and tetracycline families, when EefABC exhibited a more restricted substrate spectrum.7,8 Several studies reported that bacterial MDR arises as a consequence of the expression of cryptic efflux pumps.11 The drug selectivity of the pumps varies, although the majority of them may transport a broad range of substrates.11
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The presence of new efflux components was investigated in the CMR mutants. EefA has been shown previously to cross-react with anti-AcrA antibodies and to migrate like a 38 kDa protein in SDS–PAGE.7 Proteins from whole-cell extracts of E. aerogenes BW16627 and CMR-1 to CMR-4 were separated by SDS–PAGE, transferred to a nitrocellulose membrane and analysed by western immunoblotting with polyclonal antibodies raised against the E. coli AcrA or AcrB pump components. AcrA and AcrB were immunodetected in BW16627 (Figure 1) and in EAEP295 with the same intensity (data not shown). In addition to the E. aerogenes AcrA and AcrB proteins, products of
38 and 110 kDa were detected in the four CMR mutants, but not in the parental strain (Figure 1b). These observations suggest that the CMR mutants produce the EefABC pump.
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To further confirm the presence of an active efflux of chloramphenicol and other antibiotics in the mutants, we compared the MICs in the absence or in the presence of PAßN, the previously described efflux pump inhibitor that is active in E. aerogenes strains expressing efflux pumps.10 This inhibitor increased the susceptibilities to the three structurally unrelated antibiotics––chloramphenicol, erythromycin and ticarcillin (Table 1). The recovery of drug susceptibilities indicates that an active PAßN-sensitive efflux is involved in the resistance mechanism to these antibiotics. In addition, no significant chloramphenicol acetyltransferase activity was detected in the four CMR mutants, indicating the absence of an enzymic barrier for chloramphenicol activity (data not shown).
MDR phenotypes can be the consequence of mutations in a local or a global regulatory gene.11,14 Expression of the eefABC operon has been previously shown to be repressed by the nucleoid binding protein H-NS.7 The hns gene of E. aerogenes BW16627 and that of the four CMR mutants was PCR-amplified using the primers hns1 (5'-TTACATTCCCCCCTATTG-3') and hns2 (5'-ATGACTGCGGTAATAAGC-3') and sequenced. Comparison of the nucleotide sequences indicated the absence of hns mutation in the eefABC-derepressed mutants.
In conclusion, the overexpression of the global regulators MarA, RamA, SoxS or Rob generates an MDR phenotype due to a marked increase in AcrAB production and a strong reduction of porin synthesis in E. aerogenes and other Gram-negative bacteria.6,10,11,14 It is noteworthy that we did not observe any modification in the production of the Omp36 major porin in the resulting CMR mutants (data not shown). The present study demonstrates the capacity of chloramphenicol to select the expression of the cryptic EefABC efflux pump in E. aerogenes via a MarA and RamA independent regulatory pathway that remains to be deciphered.
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Nothing to declare.
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Footnotes |
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Present address. School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA 
Present address. Inserm U801, IBL, Institut Pasteur de Lille, BP447, 59021 Lille Cedex, France
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Acknowledgements |
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We thank V. Koronakis for generously providing E. coli anti-AcrA and anti-AcrB antibodies. We are indebted to J.-M. Bolla and A. Davin-Régli for the helpful advice and discussions. This work was supported by the Université de la Méditerranée and by an Astra-Zeneca–ESCMID Grant (J.-M. P.).
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References |
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