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Antimicrobial Agents and Chemotherapy, April 2009, p. 1652-1656, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.00822-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Occurrence of Efflux Mechanism and Cephalosporinase Variant in a Population of Enterobacter aerogenes and Klebsiella pneumoniae Isolates Producing Extended-Spectrum β-Lactamases{triangledown}

Que-Tien Tran,1,3 Myrielle Dupont,1 Jean-Philippe Lavigne,1 Jacqueline Chevalier,1 Jean-Marie Pagès,1 Albert Sotto,2 and Anne Davin-Regli1*

Transporteurs Membranaires, Chimioresistance et Drug-Design, UMR-MD1, Facultés de Médecine et Pharmacie, Université de la Méditerranée, 13385 Marseille,1 INSERM, Espri 26, UFR Medecine, Universite Montpellier-1, 30029 Nîmes, France,2 Jacobs University Bremen, Campus Ring 1, School of Engineering and Science, D-28759 Bremen, Germany3

Received 23 June 2008/ Returned for modification 22 July 2008/ Accepted 15 January 2009


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ABSTRACT
 
We investigated the occurrence of multidrug resistance in 44 Enterobacter aerogenes and Klebsiella pneumoniae clinical isolates. Efflux was involved in resistance in E. aerogenes isolates more frequently than in K. pneumoniae isolates (100 versus 38% of isolates) and was associated with the expression of phenylalanine arginine β-naphthylamide-susceptible active efflux. AcrA-TolC overproduction in E. aerogenes isolates was noted. An analysis of four E. aerogenes isolates for which cefepime MICs were high revealed no modification in porin expression but a new specific mutation in the AmpC β-lactamase.


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INTRODUCTION
 
Enterobacter aerogenes and Klebsiella pneumoniae, two of the most prevalent nosocomial enterobacterial species, can frequently express a multidrug resistance (MDR) phenotype by the acquisition or high-level production of β-lactamases in combination with the overproduction of efflux pumps, associated or not with porin alterations in the outer membrane (1, 3, 7, 9, 13). Such modifications of envelope permeability, including efflux and influx (porin), in 28 Enterobacter aerogenes and 16 Klebsiella pneumoniae clinical isolates from Nîmes University Hospital, previously studied for extended-spectrum β-lactamase (ESBL) profiling and clonal identification, were investigated (11). Moreover, we ascertained the mechanism involved in cefepime resistance in four E. aerogenes isolates showing high-level resistance (MIC = 128 mg/liter).

Characterization of the MDR phenotype was carried out by determining the MICs of various structurally unrelated antibiotics selected for their capacities to be expelled and/or to enter cells via porins (Table 1). We used the twofold broth dilution method with Mueller-Hinton broth according to the CLSI guidelines: the tests were carried out with or without the broad-spectrum efflux pump inhibitor (EPI) phenylalanine arginine β-naphthylamide (PAβN). This diamine compound, used at a low concentration (26.3 mg/liter), can increase the activities of chloramphenicol, tetracycline, norfloxacin, and sparfloxacin against various enterobacterial isolates which overproduce efflux pumps such AcrAB-TolC (4, 9, 16). The test results confirmed the existence of a PAβN-susceptible efflux mechanism in all E. aerogenes and many K. pneumoniae strains, with marked decreases in the MICs of fluoroquinolones and chloramphenicol in the presence of PAβN (Table 1). Efflux pump activity was identified when the PAβN addition induced a threefold decrease in the MIC of an antibiotic molecule (4, 9). For the two fluoroquinolones, the PAβN addition had better efficiency in decreasing the MICs of sparfloxacin than those of norfloxacin. The observed difference in PAβN efflux inhibition between the two fluoroquinolones may reflect the respective locations of ligands inside the AcrB cavity and the flux competition (20). Yu et al. demonstrated, during previous cocrystalization analyses, the presence of different affinity sites located inside the AcrB pump and suggested that the benefit generated by the conjoint use of an EPI and an antibiotic is due to a variable synergistic effect (23). In addition, it is important that PAβN, a well-described EPI, can restore a high intracellular ATP concentration but cannot bypass other resistance mechanisms, such as a target mutation or altered permeability, existing in clinical isolates (5). Moreover, some isolates may also overproduce other efflux pumps less susceptible to PAβN (12).


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  		TABLE 1. Susceptibility data for E. aerogenes and K. pneumoniae clinical isolates and E. aerogenes and E. coli transformantsa

All strains were susceptible to imipenem, with MICs for E. aerogenes strains slightly higher than those for K. pneumoniae strains and MICs for all strains ranging from 0.25 to 2 mg/liter. High MICs of ceftazidime and cefoxitin were associated with the expression of an ESBL and the AmpC cephalosporinase, respectively. Two representative groups of isolates were selected to investigate (i) active drug efflux by studying the expression of the AcrAB pump element and measuring the accumulation of radiolabeled chloramphenicol and (ii) porin expression by using immunodetection. Eight isolates (E. aerogenes EA6, EA13, EA17, and EA20 and K. pneumoniae KP2, KP12, KP13, and KP14 [designations beginning with EA indicate E. aerogenes isolates, and those beginning with KP indicate K. pneumoniae isolates]) showing significant responses to PAβN, with some MICs for these strains decreasing, were selected for the examination of the efflux mechanism. Strains with susceptibility to imipenem corresponding to a MIC of 2 mg/liter (EA5, EA7, EA13, EA19, EA21, and KP3) were chosen for permeability studies. For the chloramphenicol accumulation test, the isolates picked out were EA6, EA13, EA17, KP13, and KP14. The method of measuring the uptake of radiolabeled chloramphenicol into fresh cells was adapted from previous studies in order to monitor the involvement of an active efflux mechanism in the intracellular drug concentration (8). Inhibition assays were performed by adding the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), which collapses the membrane transporter activity, to evaluate the energy involvement in the active efflux for three exposure times (13). In comparison with the data for reference strains, the results displayed the inhibitory effects of CCCP on the efflux pumps of clinical isolates, with increases in the intracellular chloramphenicol concentrations (by two to five times) (Fig. 1). The antibiotic uptake by the reference strains in the presence or absence of CCCP indicated no active efflux. The PAβN showed an effect similar to that of CCCP on chloramphenicol accumulation when added during accumulation in selected isolates (data not shown).


Figure 1
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  		FIG. 1. Effect of the EPI on the intracellular accumulation of [14C]chloramphenicol. The assays were carried out in the presence or absence of CCCP. Values (expressed as counts per minute per unit of optical density [OD]) were obtained from duplicate independent experiments.

It was reported previously that the overproduction of AcrA and TolC pump elements is involved in the resistance levels of clinical isolates (4, 8, 9). The membrane proteins of selected isolates were extracted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection with polyclonal antibodies directed against AcrA and TolC components (9, 18). Immunoblots were stained by colorimetric detection with alkaline phosphatase-conjugated secondary antibodies. Three reference strains, EAEP289 (a Kans derivative of EA27, which is a clinical MDR strain), EAEP294 (EAEP289 acrA::Kanr), and EAEP298 (EAEP289 tolC::Kanr), were used as internal controls (18). Concerning K. pneumoniae, similar AcrA and TolC profiles were obtained with the ATCC reference strain and isolates. The selected E. aerogenes strains exhibited the overproduction of the tested efflux components, in comparison with the production of these components in the reference strain (Fig. 2C and D). Regarding K. pneumoniae isolates, an efflux mechanism was detected but the AcrAB-TolC pumps in these strains seemed to be less involved in resistance than those in E. aerogenes strains.


Figure 2
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  		FIG. 2. (A and B) Immunodetection of porins (A) and internal loop 3 (B) in E. aerogenes and K. pneumoniae strains. (C and D) Immunoblots of whole-cell extracts with polyclonal antibodies against AcrA (C) or TolC (D). Only the relevant parts of the gels are shown. MW, molecular weight marker; SM, size marker.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting methods were carried out as described previously using polyclonal antibodies directed against denatured porins and a polyclonal antiserum (F4) prepared against the internal loop 3 peptide sequence (7, 13). The results showed that all tested isolates expressed the major nonspecific porin (Fig. 2A). For E. aerogenes isolates, we obtained identical results with F4 antibodies, indicating a conserved sequence for the internal loop 3 porin epitope. However, F4 failed to recognize the peptides belonging to the loop 3 epitopes of porins from the tested K. pneumoniae strains.

In this clinical population of enterobacteria, the drug resistance was associated with the expression of PAβN-susceptible efflux and no alteration of porin synthesis was observed. It has been reported that these two mechanisms, influx (via porins) and efflux (via pumps), are controlled by the same complex regulation cascade (5, 10). However, they can be independently expressed (7, 8, 9, 10, 13). Regarding K. pneumoniae isolates, the signals corresponding to AcrAB-TolC components in the tested MDR strains did not show significant overstaining while a noticeable reversing effect of PAβN on these strains was obtained. These observations suggest that another efflux pump may be involved or, alternatively, that some changes occurring in antigenic sites may alter the immunodetection of the K. pneumoniae isolate efflux components.

Strikingly, the level of cefepime resistance in four E. aerogenes strains (EA12, EA21, EA24, and EA27) was especially high (MIC = 128 mg/liter). Three of the four strains were epidemiologically unrelated, and all were isolated after the use of broad-spectrum cephalosporin therapy (11). High levels of resistance to cephalosporins (up to céphèmes) are associated with the production of ESBLs or the hyperproduction of wild-type AmpC, but MICs have not exceeded 4 mg/liter (17). Thiolas et al. described a specific resistance phenotype of E. aerogenes due to a mutation in loop 3 of the Omp36 major porin that alters the pore properties (21). This substitution alters the antigenic site of the F4 antibody, and no signal was revealed by immunodetection with F4 (21). Positive F4 signals were obtained for the four E. aerogenes isolates tested here, indicating that the loop 3 epitope was preserved (data not shown). It was demonstrated in previous studies that cefepime is not efficiently removed by efflux and that PAßN has no effect on the MIC of cefepime for E. aerogenes, similar to its lack of effect for K. pneumoniae (7, 9).

Mutations in the AmpC cephalosporinase were recently reported to be the source of cefepime resistance in Escherichia coli, Enterobacter cloacae, and E. aerogenes (2, 6, 14, 15, 22). To evaluate the relationship between AmpC and cefepime resistance, the four E. aerogenes strains were transformed with plasmid pNH5 bearing an ampD gene (2). The MICs of cefepime for the transformants decreased by a factor of 128, confirming the involvement of ampC in the resistance. The four E. aerogenes isolates were singled out for the amplification and sequencing of the ampC to ampR genes with primers E1 (5'-TGCGTGTCATAACATTATCCG-3') and E2 (5'-AACCCGTAGCCCAGGTAAAC-3') as described previously (2). After the amplification of the genes and the sequencing of the PCR products, the sequences were compared to the E. aerogenes 97B sequence deposited in GenBank (accession no. AF211348). Three substitutions corresponding to the replacements Q-90-H, W-101-C, and L-108-Y, located on the H3 helix of the enzyme, were observed in the four strains. The final impact of these mutations on the functional structure of the enzyme is difficult to assess, but the H3 helix seems to be a region involved in cefepime resistance, as reported previously for the H10 helix (2). However, to confirm the involvement of mutations in the level of resistance to cefepime, PCR products were cloned with primers E1 and E2 into the pDrive PCR cloning vector (Qiagen) and recombinant plasmids were used to transform E. coli DH5{alpha}, E. coli ATCC 35218, and E. aerogenes ATCC 15038. For a control, pACM204, a plasmid bearing the wild-type ampC gene, was used to transform E. coli DH5{alpha} and E. aerogenes 15038 (19). The MICs for the transformants are presented in Table 1. The altered AmpC induced increases in cefepime resistance (raising the MICs by 32- to 258-fold compared to those for susceptible strains) and ceftazidime resistance (raising the MICs by 16- to 256-fold). No modification of the MIC of cefepime was observed with the wild-type AmpC, demonstrating the involvement of the AmpC variant in the high resistance levels. These mutations seem to induce key modifications in the structure of the AmpC β-lactamase, leading to changes in the catalytic properties that result in an extension of the enzymatic spectrum to include cefepime.

In the United States, 20% of K. pneumoniae infections and 31% of Enterobacter infections in intensive care units involve strains resistant to cephalosporins (17). Moreover, MICs of cefepime for E. aerogenes strains possessing ESBLs and overproducing AmpC are about 4 to 8 mg/liter (17). This work sheds new light on the occurrence of an active efflux mechanism and the identification of a new AmpC variant involved in high cefepime MICs for E. aerogenes. These two emerging mechanisms of resistance may become more frequent as broad-spectrum antibiotics are used. This is the first description of the association of these two resistance mechanisms in E. aerogenes. Such resistance to cefepime requires further attention to define the best options for detection and treatment.


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ACKNOWLEDGMENTS
 
This work was supported by EU grant MRTN-CT-2005-O19335 (Translocation), the COST Action BM0701 (Atens), the Université de la Méditerannée, and the Service de Santé des Armées.


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FOOTNOTES
 
* Corresponding author. Mailing address: UMR-MD1, Faculté de Médecine, 27 Blvd. Jean-Moulin, 13385 Marseille Cedex 05, France. Phone: (33) 4 91 32 45 29. Fax: (33) 4 91 32 46 06. E-mail: Anne.Regli{at}medecine.univ-mrs.fr Back

{triangledown} Published ahead of print on 21 January 2009. Back


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Antimicrobial Agents and Chemotherapy, April 2009, p. 1652-1656, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.00822-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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