Cloning, Nucleotide Sequencing, and Analysis of the AcrAB-TolC Efflux Pump of Enterobacter cloacae and Determination of Its Involvement in Antibiotic Resistance in a Clinical Isolate

Abstract

Enterobacter cloacae is an emerging clinical pathogen that may be responsible for nosocomial infections. Management of these infections is often difficult, owing to the high frequency of strains that are resistant to disinfectants and antimicrobial agents in the clinical setting. Multidrug efflux pumps, especially those belonging to the resistance-nodulation-division family, play a major role as a mechanism of antimicrobial resistance in gram-negative pathogens. In the present study, we cloned and sequenced the genes encoding an AcrAcBTolC-like efflux pump from an E. cloacae clinical isolate (isolate EcDC64) showing a broad antibiotic resistance profile. Sequence analysis showed that the acrR, acrA, acrB, and tolC genes encode proteins that display 79.8%, 84%, 88%, and 82% amino acid identities with the respective homologues of Enterobacter aerogenes and are arranged in a similar pattern. Deletion of the acrA gene to yield an AcrA-deficient EcDC64 mutant (Ec⌬acrA) showed the involvement of AcrAB-TolC in multidrug resistance in E. cloacae. However, experiments with an efflux pump inhibitor suggested that additional efflux systems also play a role in antibiotic resistance. Investigation of several unrelated isolates of E. cloacae by PCR analysis revealed that the AcrAB system is apparently ubiquitous in this species.Multidrug efflux pumps, especially those belonging to the resistance-nodulation-division family, play a major role in establishing the "intrinsic or acquired" resistance of gram-negative bacteria to a wide range of toxic compounds, including antibiotics (20).A well-studied example is the AcrAB-TolC multidrug resistance (MDR) tripartite pump system of Escherichia coli, which confers resistance to a wide variety of lipophilic and amphiphilic compounds, including dyes, detergents, and antimicrobial agents such as ethidium bromide, crystal violet, sodium dodecyl sulfate, bile acids, tetracycline, chloramphenicol, fluoroquinolones, ␤-lactams, erythromycin, and fusidic acid (17, 18). The presence of similar systems has been reported for other members of Enterobacteriaceae (2,9,11,12,14,15,16,21,28).The species of Enterobacter that most commonly cause nosocomial infections are E. cloacae and E. aerogenes (24). The existence of an acrB-like gene (11) has previously been identified in E. cloacae, although none of the components of the efflux pump have been cloned.The objectives of the present study were (i) to characterize the genes encoding the AcrAB-TolC-like efflux pump of E. cloacae and (ii) to determine the involvement of this efflux pump in MDR in E. cloacae. The clinical strain used in the study was an MDR strain of E. cloacae (EcDC64) which overexpressed the AcrAB system and also overexpressed the chromosomal ampC gene and exhibited a drastic reduction of ompC gene expression.(These results were presented in part at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy [2a].) MATERIALS AND METHODSBacterial strains and growth media. The bacterial strains used in the study are listed i...

Landscape of Resistance-Nodulation-Cell Division (RND)-Type Efflux Pumps in Enterobacter cloacae Complex

“…AcrABTolC is a well-characterized multidrug efflux pump of the RND family in E. cloacae (20,26,27). Based on the sequence of the protein AcrB (designated ECL_01233 in ECL13047), the inner membrane transporter protein of the system, we searched for the presence of other members of this family of transporters.…”

“…Sequence similarities to AcrB varied from 41% to 89%, while amino acid identities were between 23% and 80% ( Table 2). To date, two transporters of a tripartite MDR efflux system have been identified (AcrB and EefB [ECL_00054]) in the genome of the ATCC reference strain (20,28), whereas two others were annotated as belonging to the RND family (ECL_02125 and ECL_02244) (21). ClustalW alignments of all RND-type transporters revealed that three of the five residues corresponding to the amino acids essential for the proton transport within the AcrB identified in E. coli (D407, R970, and T977) were present in all sequences (29,30).…”

“…All ECC strains naturally possess the cephalosporinaseencoding ampC gene, the expression of which can be induced by some ␤-lactams, and it is responsible for the intrinsic resistance to ampicillin, amoxicillin-clavulanate, and first-and second-generation cephalosporins (19). The second chromosomal mechanism implicated in antimicrobial resistance in ECC is associated with the presence of the AcrAB-TolC efflux pump system (20).…”

Antimicrobial Resistance and Virulence: a Successful or Deleterious Association in the Bacterial World?

“…Expression of efflux pumps is usually associated with increased MICs of the clinically used aminoglycosides (amikacin, gentamicin, or tobramycin). Examples include efflux pumps belonging to the RND family, such as AcrAB-TolC of E. cloacae (109), AdeABC of A. baumannii (110), or MexXY of P. aeruginosa (105). The impact of efflux pump expression on virulence is described above in the section on ␤-lactams and resistance conferred by efflux pumps.…”

Caracterização molecular dos mecanismos de resistência aos carbapenêmicos de isolados clínicos de Enterobacter aerogenes e Enterobacter cloacae

“…Quando analisaram todos os genes da proteinas da bomba de efluxo acrR, acrA, acrB e tolC, observaram que estas proteinas eram homologas a isolados de E. aerogenes (151,152) .…”