Imipenem and expression of multidrug efflux pump in Enterobacter aerogenes

BBRC Biochemical and Biophysical Research Communications 301 (2003) 985–990 www.elsevier.com/locate/ybbrc

Imipenem and expression of multidrug efflux pump in Enterobacter aerogenes Charl
eric Bornet, Renaud Chollet, Monique Mall
ea, Jacqueline Chevalier, Anne Davin-Regli, Jean-Marie Pages,* and Claude Bollet Enveloppe Bact
erienne, Perm
eabilit
e et Antibiotiques, EA2197, IFR48, Facult
e de M
edecine, Universit
e de la M
editerran
ee, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France Received 13 January 2003

Abstract Imipenem is often used to treat intensive care unit patients infected by Enterobacter aerogenes, but it is leading to an increasing number of antibiotic resistant strains. Clinical isolates and imipenem resistant variants presented a high level of resistance to b-lactam antibiotic group and to chemically unrelated drugs. We report here that imipenem selects strains which contain active efflux pumps ejecting various unrelated antibiotics including quinolones, tetracycline, and chloramphenicol. An increase of AcrA, an efflux pump component, was observed in the imipenem resistant variants. The overexpression of marA, involved in the genetic control of membrane permeability via porin and efflux pump expression, indicated the activation of the resistance genetic cascade in imipenem resistant variants. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Efflux pump; Enterobacter aerogenes; Imipenem; Efflux pump inhibitor; Multidrug resistance

Enterobacter aerogenes has emerged as an important hospital pathogen since the 1990s [1–4]. E. aerogenes strains isolated from hospitalized patients generally exhibit high resistance to a broad spectrum of antibiotics, including third generation cephalosporins [5]. In addition, several studies indicated that a reduction in porin synthesis decreases sensitivity to the most recently developed cephalosporins, including cefepime and cefpirome [2,6]. As regards carbapenems, imipenem has been successfully used for a decade to treat multiresistant organisms involved in nosocomial infections. Recently, analysis of clinical strains collected in several patients showed that the use of imipenem to treat E. aerogenes infections could lead to resistance to this antibiotic [2,7– 9]. This resistance has been associated with a lack of porin or an alteration of the lipopolysaccharide [2,10,11]. Imipenem has been shown to be a natural in*

Corresponding author. Fax: +33-4-91-32-46-06. E-mail address: [email protected] (J.-M. Pages).

ducer of the chromosomal cephalosporinase, but this enzyme is not directly involved, or only slightly involved, in imipenem resistance of E. aerogenes [12]. To investigate imipenem as a potent selector for multidrug resistance phenotype, we obtain resistant strains under increasing imipenem concentrations in vitro. The resulting variants exhibit resistance not only to b-lactam antibiotics but also to other chemically unrelated antibiotics. The genetic and biochemical characteristics of these strains were defined to determine the involvement of imipenem in the emergence of multidrug resistance.

Materials and methods Bacterial strains, growth media, and selection of imipenem resistant strains. Bacteria were grown at 37 °C in Luria–Bertani (LB) broth (Difco Laboratories, Detroit, MI, USA). E. aerogenes clinical strains EA3, 11,668 (no imipenem treatment), 12,515 (5 days imipenem treatment), and 13,165 (10 days imipenem treatment) have been previously described [2,13]. E. aerogenes ATCC 13048-type strain was used as the control strain. Strains I-1, I-2, I-3, I-4, I-5, I-6, and I-32

0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00074-3

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C. Bornet et al. / Biochemical and Biophysical Research Communications 301 (2003) 985–990 5 lM. At various intervals, 100 ll of the suspension was removed and immediately filtered through GF/C filters (Whatman, Maidstone, Kent, UK). After three washes with 5 ml of cold buffer (50 mM sodium phosphate buffer containing 0.1 M lithium chloride), filters were dried and radioactivity was measured in a Packard scintillation counter. To de-energize the bacteria, 50 lM carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added 10 min before the radiolabelled antibacterial agent. Analyses of marA expression. Total RNA was extracted using the RNeasy mini kit (Qiagen SA, France). The quality of samples was checked electrophoretically and quantification was done spectrophotometrically. Quantification of marA RNA transcripts was carried out by reverse transcription with the Access RT PCR System (Promega WI, USA). Expression of the constitutively expressed housekeeping gene gyrA (encoding gyrase) from the same RNA preparation was used as an internal standard [18]. The resulting RT-PCR products were separated on a 2% agarose gel and analysed densitometrically after ethidium bromide staining (Image Master 1D, Amersham Pharmacia Biotech, NJ, USA). Experiments were two time reproduced and each sample was duplicated for analyses. Fig. 3 presents the mean from two independent experiments carried out under these conditions. Results were normalized from the gyrA band density and standard deviations were calculated.

were obtained sequentially from ATCC 13048 following growth in the presence of imipenem at concentrations of 0.5, 1, 1.5, 2, 2.5, 3, and 16 lg/ml, respectively. For each step, about 108 cells cultured at a given imipenem concentration were subsequently cultured for 12 h at 37 °C in 100 ml LB, containing the next increased concentration of imipenem. Imipenem (Tienam), cefpirome, norfloxacin, ciprofloxacin, chloramphenicol, gentamicin, and tetracycline were obtained from Aventis Hoescht Marrion Roussel (Swindon, UK and Paris, France), Bristol-Myers Squibb (Paris, France), Astra-Zeneca (Cheshire, UK), Merck Sharp Dohme and Chibret (Paris, France), and Sigma Chemical (MO, USA). Antibiotic susceptibility tests. Susceptibilities to antibiotics were measured by the broth dilution method, as previously described [14]. 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 minimal inhibitory concentrations (MICs) in lg=ml. The efflux pump inhibitor phenylalanine–arginine b-naphthylamide, ðPAbNÞ (Sigma Chemical, MO, USA), was used as previously described [15]. SDS–polyacrylamide gel electrophoresis and immunodetection of AcrA. Exponential phase bacterial cells grown in LB broth were collected. Total membrane proteins were separated by SDS–PAGE. Electrotransfer to nitrocellulose was performed as previously described [14]. We systematically evaluate the amount of proteins included in nitrocellulose sheet by using ponceau red and silver staining after electrotransfer to check quantitative protein-transfer and use nitrocellulose exhibiting similar protein concentration to carry out immunodetection analyses. After an initial saturating step, the nitrocellulose sheet was incubated with polyclonal antibodies directed against AcrA [16]. The antigen–antibody complexes were detected with alkaline phosphatase-conjugated affinitiPure goat anti-rabbit immunoglobulin G antibodies (Jackson ImmunoResearch, West Grove PA, USA). Membrane protein. Bacterial cell membrane proteins were isolated as described previously [17]. Briefly, E. aerogenes cells grown in LB broth were harvested by centrifugation and resuspended. The suspensions were sonicated with 7  30 s pulses on ice. Total cell envelopes were pelleted by ultracentrifugation of sonicate at 48,000g for 45 min. The pellet containing the membrane proteins was solubilized in loading buffer at 96 °C and protein samples were analysed by SDS–PAGE, as previously described [14]. The gels were stained with Coomassie blue. Chloramphenicol uptake. The uptake of ½14 Cchloramphenicol by intact cells has been described previously [13]. Briefly, exponentialphase bacteria grown in LB broth were pelleted, washed once, and suspended to a density of 1010 CFU/ml in 50 mM sodium phosphate buffer, pH 7, containing 5 mM magnesium chloride. The ½14 Cchloramphenicol was a gift from Aventis Hoescht Marrion Roussel (Romainville, France). ½14 CChloramphenicol (specific radioactivity, 59.46 mCi/mmol) was added to 500 ll of cell suspension at 37 °C in a shaking water bath, yielding a final chloramphenicol concentration of

Results In an attempt to characterize general resistance mechanisms selected by imipenem, other than specific enzyme production, we focused the analysis on structurally unrelated antibiotics such as quinolones, chloramphenicol, and tetracycline. Antibiotic resistance and membrane protein expression of selected variants Imipenem resistant derivatives were obtained from E. aerogenes ATCC 13048-type strain cultured in the presence of imipenem (0.5–16 lg/ml). Significant increase in resistance to norfloxacin, chloramphenicol, polymyxin, and tetracycline was observed for I-1, I-2, and subsequent variants (Table 1). The diminution of polymyxin and imipenem susceptibilities suggested modifications of lipopolysaccharide structure occurring in I-1 and subsequent variants as reported in clinical isolates [9].

Table 1 Antibiotic susceptibility of the E. aerogenes imipenem resistant derivatives E. aerogenes strains

ATCC 13048 I-1 I-2 I-4 I-5 I-6 I-32

MIC ðlg=mlÞ IMI

FEP

CPO

NOR

CM

TC

PB

GM

0.5 4 4 8 16 16 32

0.5 1 0.5 8 8 16 32

1 1 1 16 16 16 64

0.5 16 16 16 16 16 16

8 16 32 32 nd 64 64

1 8 8 8 8 8 16

2 16 16 16 16 16 32

2 1 1 1 nd 1 1

Antimicrobial agent abbreviations: CPO, cefpirome; FEP, cefepime; IMI, imipenem; NOR, norfloxacin; CM, chloramphenicol; TC, tetracycline; PB, polymyxin B; GM, gentamicin; nd, not done. Values are means of three independent determinations.

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increase was observed with cefepime and cefpirome. In contrast, the gentamicin susceptibility was unchanged in all tested variants. Analyses of membrane protein profiles indicated that porins were not observed in I-4 and in successive isolates (Fig. 1A) reflecting the general decrease of b-lactam susceptibilities. We also observed increased expression of a band migrating with an approximate molecular weight of 45 kDa in variants. Conjointly, an overexpressed product was immunorecognized by an antiAcrA antiserum (Fig. 1B), suggesting the increased expression of the efflux complex in the corresponding variants. These results clearly show that imipenem selects for independent resistant mechanisms acting on chemically unrelated drugs. Evidence for an active drug efflux in the resistant variants

Fig. 1. Analyses of membrane proteins of imipenem variants. (A) Membrane proteins stained with Coomassie blue. E. aerogenes ATCC 13048 presented a porin. The variants presented a lack of porin and an additional band at 42 kDa. Arrows indicate the postulated porin migration and molecular weight standards are indicated in kilodaltons. Only the relevant part of the gel is shown. (B) Immunodetection of E. aerogenes membrane proteins was performed with polyclonal antibodies directed against AcrA. Strains E. aerogenes I-1 to I-32 presented a positive signal while a weak intensity was observed with ATCC 13048.

In addition, concerning cephalosporins, MICs for I-4 and subsequent strains were noticeably higher than those of the parent strain (Table 1): about 32-fold

To assess the presence of an active efflux of fluoroquinolones and other antibiotics, we compared the MICs of four imipenem resistant variants in the presence or in the absence of the pump inhibitor, PAbN [15], (Table 2). The inhibitor increased the susceptibilities to the three structurally unrelated antibiotics norfloxacin, chloramphenicol, and tetracycline, while the MICs for imipenem were unchanged. Clearly, the complete recovery of drug susceptibilities indicates that active efflux determines resistance to these antibiotics. In addition, no mutations were detected in the quinolone resistance determining region (QRDR) [19], the gyrA sequences from ATCC 13048, I-6, and I-32 were identical (data not shown). To define the efflux activity, we analysed the accumulation of intracellular ½14 Cchloramphenicol in the I-6 variant. When the assays were carried out in the absence of energy uncoupler, the drug intracellular accumulation was similar to that obtained with the isolate EA3 (Fig. 2), a strain previously characterized as expelling chloramphenicol and norfloxacin by active efflux [13,14]. In the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), which collapses the energy component of the drug efflux system, the accumulation of

Table 2 Effect of efflux pump inhibitor phenylalanine–arginine b-naphthylamide ðPAbNÞ, on various antibiotic susceptibilities E. aerogenes strains

MIC ðlg=mlÞ IMI

ATCC 13048 I-1 I-4 I-6 I-32

NOR

CM

TC

)

þPAbN

)

þPAbN

)

þPAbN

–

þPAbN

0.5 4 8 16 32

0.5 4 8 32 32

0.5 16 16 16 nd

0.5 1 1 1 nd

8 16 16 64 64

2 2 2 2 2

1 8 8 8 16

2 2 2 2 2

Antimicrobial agent abbreviations: IPM, imipenem; NOR, norfloxacin; TC, tetracycline; CM, chloramphenicol; nd, not done. Values are means of three independent determinations.

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C. Bornet et al. / Biochemical and Biophysical Research Communications 301 (2003) 985–990

Fig. 2. Uptake of ½14 Cchloramphenicol by E. aerogenes strains. Accumulation of ½14 Cchloramphenicol was measured in E. aerogenes I-6 in the absence ðÞ and presence ðjÞ of CCCP, in E. aerogenes isolate EA3 in the absence ðsÞ and presence ðdÞ of CCCP, and in E. aerogenes ATCC 13048 in the absence (+) of CCCP. Each point is the mean of four independent experiments.

chloramphenicol showed a 2.3-fold increase (Fig. 2). This intracellular level was similar to that obtained in isolate EA3 with CCCP and in the wild type ATCC 13048 strain in the absence of CCCP. These concentration changes reflect an active energy-dependent efflux system in I-6. Similar results were obtained with strain I32 (data not shown). Genetic analyses of imipenem variants To investigate the possible modulation of MDR regulation which supports the emergence of efflux pump, the transcriptional level of marA was analysed in the imipenem resistant variants, using gyrA transcript as an internal standard [18]. We observed that the level of marA transcript was 6-fold increased in derivatives I-1 and I-2 compared to ATCC 13048. A 15-fold increase was observed in strains I-4 and I-6 (Fig. 3). In addition to the increase observed for acrA, these results suggest that imipenem may select for a high level of marA expression. We research possible mutations in genes marR, soxR, emrR, acrR, and ramA, described as playing a key role in the multidrug resistance in Enterobacteriacae [20–24]. The sequence comparison of the genes in the various mutants with the ATCC 13048 strain indicated no mutation in these regulators. It is especially interesting to note that no mutations were found in the two major regulatory genes marR and soxR involved in the genetic control of MDR in Escherichia coli. Similarly, no mutations were detected in the marO regions containing the two major regulator binding sites of the mar operon [20].

Fig. 3. marA expression in the E. aerogenes imipenem resistant variants. The level of marA transcript (presented as percent to gyrA trancript) was determined in each strain. Each point (marA/gyrA transcript ratio in %) is the mean of independent four measurements and standard deviations were indicated.

Emergence of an efflux phenotype during imipenem treatment To examine the effect of imipenem therapy on the emergence of efflux mechanism, E. aerogenes imipenem resistant isolates previously recovered during imipenem treatment of colonized patient [2] are analysed. Their high level of resistance to structurally unrelated drugs including quinolones, chloramphenicol, and tetracycline suggested the presence of an efflux pump in these isolates (Table 3) and the susceptibilities were tested in the presence of efflux pump inhibitor PAbN. The respective decrease of MICs for ciprofloxacin and chloramphenicol obtained with PAbN (Table 3) indicated the presence of an active pump, expelling these drugs in the E. aerogenes isolates collected during imipenem treatment. The partial recovery of antibiotic susceptibility suggests the presence of additional resistance mechanisms in these isolates [2,13].

Discussion Enterobacter aerogenes is a commensal Gram negative bacterium which adapts easily to antibiotic therapy during the treatment of nosocomical infections [2]. Imipenem remains one of the most powerful antibiotics used today, thus it is important to decipher its selecting activity on sensitive E. aerogenes and its role in the emergence of resistant strains during therapy. In this work, we showed that imipenem selects the activation of MDR in E. aerogenes. The imipenem resistant variants are resistant to structurally unrelated drugs such as norfloxacin, chloramphenicol, and tetracycline and

C. Bornet et al. / Biochemical and Biophysical Research Communications 301 (2003) 985–990

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Table 3 Identification of a drug efflux in imipenem resistant E. aerogenes isolates collected during antibiotherapy E. aerogenes isolates

11,668 12,515 13,165

Patient treatment

0 Imipenem (5 days) Imipenem (10 days)

MIC ðlg=mlÞ GM

IMI

CIP

CM

)

þPAbN

)

þPAbN

)

þPAbN

2 2

1 64

1 64

1 64

1 16

8 256

8 32

2

64

64

64

16/32

256

32

Antimicrobial agent abbreviations: GM, gentamicin; IMI, imipenem; CIP, ciprofloxacin; CM, chloramphenicol; PAbN, phenylalanine–arginine b-naphthylamide. Values are means of three independant determinations.

possess an efflux pump. Interestingly, in the various imipenem resistant variants, we observed the preservation of gentamicin susceptibility, a similar phenotype also being detected in clinical strains. We provide molecular evidence for a CCCP sensitive energy-dependent chloramphenicol efflux, in strains I-6 and I-32. The efflux mechanism is detected in the first selected variants and concerns various antibiotic families structurally unrelated to imipenem. The use of ðPAbNÞ, an efflux pump inhibitor [13,15], clearly indicates the activity of a multidrug efflux pump in all variants in contrast to strain ATCC 13048. Protein analysis of the membranes in the resistant variants indicated both a decrease of porin expression and the presence of an additional product recognized by an antiAcrA antiserum. AcrA protein, involved in the complex which expels antibiotics, has been observed in previous studies [16]. Moreover, the absence of mutations located in the QRDR region of gyrA, described in quinolone resistant strains [19], indicates a role for active efflux in fluoroquinolone resistance in the selected variants. Studies have shown that mutations in regulators can modulate antibiotic resistance levels [20,21,23,24]. Consequently we analysed the major known bacterial regulator genes, marR, soxR, acrR, ramA, and emrR. No mutations were detected in their respective sequence nor in the marO region reported in the E. aerogenes mar operon [25]. However, we identified overexpression of marA transcript, which is a key regulator of the MDR cascade [20], in the first imipenem variants. This increase is correlated (i) with the efflux pump activity detected by using efflux inhibitor and (ii) with the increased synthesis of AcrA, the periplasmic fusion protein of efflux pump. This study demonstrates the capacity of imipenem to influence the expression of an active efflux mechanism in Enterobacteriacae. This multidrug efflux mechanism cannot directly participate in imipenem resistance. This leads us to propose that this drug is not only an inducer of enzymatic response, but is also involved in the selection of E. aerogenes strains expressing multidrug efflux which expels various antibiotic families structurally unrelated to imipenem.

Acknowledgments We thank Aventis Hoescht Marrion Roussel (Romainville, France) for its generous gift of radiolabelled chloramphenicol. We are indebted to Dr. E. Pradel and Dr. P. Nordmann for helpful advice and discussions. This work was supported by the Assistance Publique de Marseille (Recherche Clinique) and the Universit
e de la M
editerran
ee.

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