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In this study, a chromosomally encoded putative drug efflux pump of the SMR family, named AbeS, from a multidrug-resistant strain of Acinetobacter baumannii was characterized to elucidate its role in antimicrobial resistance. Expression of the cloned abeS gene in hypersensitive Escherichia coli host KAM32 resulted in decreased susceptibility to various classes of antimicrobial agents, detergents, and dyes. Deletion of the abeS gene in A. baumannii confirmed its role in conferring resistance to these compounds.
Acinetobacter baumannii is an important nosocomial pathogen frequently reported to be associated with a variety of infections, including respiratory tract infections, urinary tract infections, bacteremia, and skin and skin structure infections (3). Reports of the increased isolation of multidrug-resistant A. baumannii clinical isolates from different regions of the United States are appearing at a startling rate (1, 4, 10, 25, 30, 33).
Antibiotic resistance in A. baumannii has been attributed to either intrinsic or acquired mechanisms (21). The resistance mechanisms in A. baumannii are diverse and include enzymatic modification of antibiotics, target gene mutation, altered outer membrane permeability, and upregulated multidrug efflux pump activity (20).
Efflux systems involve transport proteins that function to reduce the concentration of drugs or toxic substances by transporting them across the inner and outer membranes into the external medium (24). These multidrug efflux systems are classified into five different families: ATP-binding cassette (ABC), major facilitator super family (MFS), resistance/nodulation/cell division (RND), multidrug and toxic-compound extrusion (MATE), and the small multidrug resistance (SMR) family of bacterial integral membrane proteins (22). ABC transporters are ATP-driven efflux pumps; MFS, RND, and SMR are proton driven; and MATE transporters consist of an Na+/H+ drug antiporter system (22, 23). Genome sequence analyses reveal that, on average, efflux pumps constitute at least 10% of the transporters in bacterial species, and they usually are capable of extruding a broad range of structurally unrelated compounds (18).
Multidrug efflux pumps of the SMR type are made of a transport protein located in the inner membrane (19). The polypeptide chains of SMR efflux pumps, found in the inner membranes of gram-negative bacteria, are 110 amino acid residues in length and contain four transmembrane helices (29). Reports show that 52% of currently sequenced genomes of eubacteria and archaea contain at least one SMR homologue (2). Well-studied examples of SMR efflux pumps include EmrE of Pseudomonas aeruginosa, EbrAB of Bacillus subtilis, SsmE of Serratia marcescens, and EmrE of Escherichia coli, which are involved in resistance to a variety of antimicrobial agents and quaternary ammonium compounds (14, 16, 17, 34).
The 3.9-Mb genome of A. baumannii AYE is reported to harbor 46 open reading frames (ORFs) encoding putative efflux pumps of different families (8). The efflux systems functionally characterized so far include AdeABC and AdeIJK (RND type), AbeM (MATE type), and CraA (MFS type) (20, 27). Albeit comparative genomics clearly reveal the existence of several chromosomally borne putative efflux transporters (8, 12), apparently the role of the Acinetobacter SMR efflux pump was never examined. Therefore, the objective of the present study was to investigate the function of one putative SMR efflux pump from a clinical isolate, A. baumannii AC0037.
(Part of this work was presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2008 [8a].)
A. baumannii AC0037 used in this study (isolated from the respiratory system of an infected patient in The Ohio State University Medical Center) is a multidrug clinical strain (31) with the following MICs for antibiotics: amikacin, >128 μg/ml; ceftazidime, 32 μg/ml; cefepime, >16 μg/ml; ciprofloxacin, >72 μg/ml; imipenem, 4 μg/ml; and meropenem, 7.5 μg/ml. The MICs were determined using the broth dilution method, and interpretation was done per the criteria approved by the Clinical and Laboratory Standards Institute (CLSI) (7). The drug-hypersusceptible E. coli KAM32 (generously provided by Tomofusa Tsuchiya) that lacks the major multidrug efflux pumps AcrAB and YdhE was used as a host (5).
The protein encoded by ORF ABAYE1181 in the AYE genome is annotated as a putative multidrug efflux protein. It exhibits 51.8% identity (E value of 1e−25 and score of 108) to E. coli EmrE (GenBank accession number NC_013364), a well-characterized member of the SMR family (34). Thus, the ABAYE1181 ORF was selected as the putative SMR-type-pump gene to be investigated in this study. The genomic DNA of A. baumannii was extracted using the DNeasy tissue kit (Qiagen, Valencia, CA). The putative efflux gene from AC0037 was amplified by PCR as described previously (28), with minor modifications. The primers used were P1 and P2 (5′-TAGAGAATTCATGTCTTATCTTTATTTAGC-3′ [EcoRI sequence underlined] and 5′-CGCTCTGCAGTTATAGATGGGTGTTTTTAG-3′ [PstI sequence underlined]). The PCR product was purified using the QiaQuick PCR purification kit (Qiagen, Valencia, CA). The amplicon was digested with EcoRI and PstI and subsequently ligated with vector pUC18 (New England Biolabs, MA) to obtain recombinant plasmid pVBS1. At least two independently generated recombinant plasmids were sequenced bidirectionally using the CEQ 8000 capillary electrophoresis system (Beckman Coulter Instruments, Inc., Palo Alto, CA) to rule out mutations introduced during PCR. Analysis revealed the presence of one ORF that was designated abeS (A. baumannii efflux pump of SMR family). Further analysis revealed that abeS was a 330-bp gene encoding a 109-amino-acid protein with a calculated mass of 11.14 kDa. AbeS exhibited 100% identity to the product of ORF ABAYE1181 found in the A. baumannii AYE genome (8). AbeS exhibited different degrees of identity with other SMR transporters from various gram-negative and gram-positive species (Fig. (Fig.11).
To evaluate the role of abeS in antimicrobial resistance, the MICs of various antibiotics, detergents, and dyes (Sigma, St. Louis, MO) were determined in triplicate in Mueller-Hinton broth (Difco, Sparks, MD) by the broth dilution method, according to the guidelines of CLSI (7).
The SMR efflux pump-expressing cells exhibited a higher MIC for erythromycin (sixfold) as well as novobiocin (fivefold). Minor increases (twofold) were observed with MICs of aminoglycosides (amikacin), quinolones (ciprofloxacin, norfloxacin), tetracycline, and trimethoprim. No significant differences in MICs were found for chloramphenicol, nalidixic acid, and rifampin (rifampicin) (Table (Table1).1). KAM32/pVBS1 also showed increased levels of resistance to detergents, such as deoxycholate (25-fold) and sodium dodecyl sulfate (16-fold), and dyes, such as acridine orange (fivefold), acriflavine (eightfold), and benzalkonium chloride (sixfold). A nearly fourfold increase in resistance was observed for 4′,6-diamidino-2-phenylindole (DAPI), ethidium bromide (EtBr), and rhodamine 123. A marginal decrease in susceptibility toward chlorhexidine, pyronin Y, and tetraphenylphosphonium chloride was also noted (Table (Table11).
Overall, results demonstrated that AbeS could decrease susceptibility to some antibiotics, disinfectants, dyes, and detergents. The broad substrate specificity displayed by AbeS was very similar to those of other SMR transporters reported previously (6, 14, 16, 17, 34).
To confirm the role of AbeS as an efflux pump, the MICs of substrates with strong specificity were monitored in the presence of 25 μg/ml efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP; an uncoupler of oxidative phosphorylation which disrupts the proton gradient on the membrane) (31). Results were consistent with the notion that the abeS gene product conferred resistance to various antimicrobial compounds through an efflux mechanism (Table (Table11).
EtBr is highly fluorescent when bound to DNA. Upon being extruded from the cell through an efflux pump, the fluorescence decreases, and this difference can be measured with a spectrofluorometer (13, 17). To confirm the role of AbeS as an efflux pump, we compared the rates of EtBr accumulation in E. coli KAM32 cells carrying either pUC18 or recombinant plasmid pVBS1. We found that the intracellular level of EtBr in E. coli KAM32/pVBS1 (carrying abeS) was significantly lower than that of the control strain E. coli KAM32/pUC18 under energized conditions (in the absence of CCCP) (Fig. (Fig.2A).2A). After the addition of CCCP, at 25 μg/ml, there was a rise in the intracellular accumulation level of EtBr in E. coli KAM32/pVBS1, which eventually reached a plateau equal to that of the control strain E. coli KAM32/pUC18 (Fig. (Fig.2A).2A). These results showed that AbeS is an energy-dependent active efflux pump. Subsequently, we tested the influence of monovalent cations, such as Na+ and Li+, in the efflux process because the activities of several multidrug efflux pumps are coupled with monovalent cations (9). The addition of either NaCl or LiCl caused no difference in the efflux activity from that of the control strain (data not shown). Thus, results demonstrated that AbeS is an efflux pump and the active extrusion is proton (H+) driven.
To evaluate the role of AbeS in A. baumannii AC0037, an abeS deletion mutant was generated. The vector pUC18, not capable of replicating in A. baumannii, was used as a suicide vector (15). To construct the abeS deletion mutant, the 548-bp upstream region (amplicon A) and 542-bp downstream region (amplicon B) of the efflux gene were amplified using specific primers. The primers for amplicon A were P3 (5′-TGATTCTAGATGTGATATGTGCTTACCAGAATGC-3′, with an XbaI linker) and P4 (5′-TCATCTGCAGAGTAAAACCTTGGATGCTTT-3′, with a PstI linker). The primers for amplicon B were P5 (5′-GATACTGCAGTGCATTGGTTTAGCTTTA-3′, with a PstI linker) and P6 (5′-ACAGGGATCCTCTGAGGCGGAAGAACGTGCAC-3′, with a BamHI linker) (enzyme sequences are underlined). PCR products were generated from the genome of AC0037. The digested fragments were ligated with the PstI-kanamycin resistance cassette (K) obtained with the pUC-4K vector (Pharmacia). The generated AKD fragment was inserted into the XbaI- and BamHI-digested plasmid pUC18, resulting in the pabeS-Kan plasmid. It was bidirectionally sequenced to ensure the presence of the kanamycin resistance cassette and to confirm authenticity. The plasmid pabeS-Kan was then introduced into A. baumannii AC0037 for the double homologous recombination event to occur by electroporation as described previously (15).
Selection of transformants was made on 50 μg/ml kanamycin- and 80 μg/ml ticarcillin-containing plates. Inactivation of the abeS gene by insertion of pabeS-Kan was confirmed by PCR amplification and DNA sequencing, and the null mutant was designated AC0037ΔabeS.
The primers used for confirmation were the gene-specific primers P1 and P2, as well as the flanking chromosomal region primers P11 (5′-GCTTATTCAGCGAGTTAAATATG-3′) and P12 (5′-CACATGACAGTACTGGAAAATGT-3′).
Following the confirmation of the abeS deletion mutant, the role of abeS in A. baumannii was evaluated. Susceptibility testing data showed that deletion of abeS resulted in increased susceptibility to various antimicrobial compounds (Table (Table11).
The EtBr accumulation experiments demonstrated that the efflux was more efficient in the wild-type AC0037 strain, whereas the efflux was significantly less efficient (increased accumulation of >3-fold for EtBr) in the AC0037ΔabeS deletion mutant. Addition of efflux inhibitor CCCP increased the intracellular accumulation of EtBr in AC0037ΔabeS (Fig. (Fig.2B2B).
The role of the A. baumannii abeS gene was confirmed by gene complementation experiments. Briefly, a DNA fragment containing a functional aadA1 gene was amplified from an AC0019 clinical isolate (GenBank accession number EU977568.1) (26, 31) by PCR using primers P7 (5′-TACAGATATCATGAGGGAAGCGGTGATC-3′ [EcoRV linker underlined]) and P8 (5′-TACGGTCGACTTATTTGCCTACTACCTTGGTGA-3′ [SalI linker underlined]). The cassette was ligated into the E. coli-Acinetobacter shuttle vector pWH1266 (11). The resulting plasmid, pWH-Spc, was modified by cloning a PCR-amplified wild-type abeS gene with the primer pair P1/P2 into the PstI site of the plasmid, yielding a recombinant plasmid, pWH-abeS. Electroporation of the recombinant plasmid pWH-abeS into the A. baumannii AC0037ΔabeS mutant resulted in strain AC0037ΔabeSΩabeS. Selection of the AC0037ΔabeSΩabeS mutant was made on 200 μg/ml streptomycin, 50 μg/ml kanamycin, and 80 μg/ml of ticarcillin-containing plates. Complementing the mutant with the wild-type abeS gene nearly restored reduced susceptibility to antimicrobial agents, detergents, and dyes, similar to that of the parental strain AC0037 (Table (Table1).1). These findings confirmed the role of abeS in mediating resistance also in A. baumannii.
In conclusion, this study demonstrated for the first time the role of the SMR efflux pump AbeS in mediating resistance to some antibiotics, hydrophobic compounds, detergents, and disinfectants in A. baumannii clinical strain AC0037.
The nucleotide sequence data reported in this paper have been deposited in the GenBank nucleotide sequence database under the accession number FJ843079.
This study was funded by The Ohio State University intramural funding to W.A.G.
We thank the members of the Infectious Diseases Molecular Epidemiology Laboratory (IDMEL) team for technical assistance. We also thank Tomofusa Tsuchiya, Craig Altier, Preeti Pancholi, Kurt Stevenson, and Mario Marcon for providing the E. coli host strain, plasmids, and A. baumannii clinical isolates for this study.
Published ahead of print on 21 September 2009.