Innate Aminoglycoside Resistance of Achromobacter xylosoxidans Is Due to AxyXY-OprZ,an RND-Type Multidrug Efflux Pump

Achromobacter xylosoxidans is an innately multidrug-resistant pathogen which is emerging in cystic fibrosis (CF) patients. We characterized a new resistance-nodulation-cell division (RND)-type multidrug efflux pump, AxyXY-OprZ. This system is responsible for the intrinsic high-level resistance of A. xylosoxidans to aminoglycosides (tobramycin, amikacin, and gentamicin). Furthermore, it can extrude cefepime, carbapenems, some fluoroquinolones, tetracyclines, and erythromycin. Some of the AxyXY-OprZ substrates are major components widely used to treat pulmonary infections in CF patients.     

KpnEF,a New Member of the Klebsiella pneumoniae Cell Envelope Stress Response Regulon,Is an SMR-Type Efflux Pump Involved in Broad-Spectrum Antimicrobial Resistance

Klebsiella pneumoniae has been frequently associated with nosocomial infections. Efflux systems are ubiquitous transporters that also function in drug resistance. Genome analysis of K. pneumoniae strain NTUH-K2044 revealed the presence of ∼15 putative drug efflux systems. We discuss here for the first time the characterization of a putative SMR-type efflux pump, an ebrAB homolog (denoted here as kpnEF) with respect to Klebsiella physiology and the multidrug-resistant phenotype. Analysis of hypermucoviscosity revealed direct involvement of kpnEF in capsule synthesis. The ΔkpnEF mutant displayed higher sensitivity to hyperosmotic (∼2.8-fold) and high bile (∼4.0-fold) concentrations. Mutation in kpnEF resulted in increased susceptibility to cefepime, ceftriaxone, colistin, erythromycin, rifampin, tetracycline, and streptomycin; mutated strains changed from being resistant to being susceptible, and the resistance was restored upon complementation. The ΔkpnEF mutant displayed enhanced sensitivity toward structurally related compounds such as sodium dodecyl sulfate, deoxycholate, and dyes, including clinically relevant disinfectants such as benzalkonium chloride, chlorhexidine, and triclosan. The prevalence of kpnEF in clinical strains broadens the diversity of antibiotic resistance in K. pneumoniae. Experimental evidence of CpxR binding to the efflux pump promoter and quantification of its expression in a cpxAR mutant background demonstrated kpnEF to be a member of the Cpx regulon. This study helps to elucidate the unprecedented biological functions of the SMR-type efflux pump in Klebsiella spp.     

Drug Efflux and parC Mutations Are Involved in Fluoroquinolone Resistance in Viridans Group Streptococci

Nine ciprofloxacin-resistant viridans group streptococci isolated from asymptomatic carriers were analyzed. Identification to the species level by using three different commercial systems and a PCR-based approach was inconsistent. The nucleotide sequences of fragments of the parC, parE, gyrA, and gyrB genes showed considerable intra- and interspecies variations, and these variations mainly involved silent mutations. Three isolates had changes in Ser-79 of ParC (to Phe or Tyr). Phenotypic characterization indicated that eight of the nine isolates had a putative efflux mechanism that would confer low-level resistance to ciprofloxacin.     

Resistance-Nodulation-Cell Division-Type Efflux Pump Involved in Aminoglycoside Resistance in Acinetobacter baumannii Strain BM4454

Multidrug-resistant strain Acinetobacter baumannii BM4454 was isolated from a patient with a urinary tract infection. The adeB gene, which encodes a resistance-nodulation-cell division (RND) protein, was detected in this strain by PCR with two degenerate oligodeoxynucleotides. Insertional inactivation of adeB in BM4454, which generated BM4454-1, showed that the corresponding protein was responsible for aminoglycoside resistance and was involved in the level of susceptibility to other drugs including fluoroquinolones, tetracyclines, chloramphenicol, erythromycin, trimethoprim, and ethidium bromide. Study of ethidium bromide accumulation in BM4454 and BM4454-1, in the presence or in the absence of carbonyl cyanide m-chlorophenylhydrazone, demonstrated that AdeB was responsible for the decrease in intracellular ethidium bromide levels in a proton motive force-dependent manner. The adeB gene was part of a cluster that included adeA and adeC which encodes proteins homologous to membrane fusion and outer membrane proteins of RND-type three-component efflux systems, respectively. The products of two upstream open reading frames encoding a putative two-component regulatory system might be involved in the regulation of expression of the adeABC gene cluster.     

Characterization of emeA, a norA Homolog and Multidrug Resistance Efflux Pump, in Enterococcus faecalis

We hypothesized that multidrug resistance efflux pumps (MDRs) may be contributing to the drug resistance of enterococci. We recently identified potential MDR-encoding genes in the Enterococcus faecalis V583 genome. Among the putative MDRs, we found a gene that encodes a NorA homolog and have characterized this enterococcal MDR in the present study. A mutant from which the enterococcal NorA homolog has been deleted has reduced resistance to several NorA substrates. Complementation of the deletion mutant with the wild-type gene verified the involvement of this enterococcal gene in resistance to ethidium bromide (EtBr) and norfloxacin. Known MDR inhibitors (reserpine, lansoprazole, and verapamil) inhibit the efflux of EtBr and norfloxacin in wild-type strain OG1RF. A fluorescence assay with EtBr allowed us to quantitate the efflux capability of the enterococcal NorA pump. On the basis of these results, we have named this enterococcal gene emeA (enterococcal multidrug resistance efflux).     

Involvement of an Active Efflux System in the Natural Resistance of Pseudomonas aeruginosa to Aminoglycosides

A mutant, named 11B, hypersusceptible to aminoglycosides, tetracycline, and erythromycin was isolated after Tn501 insertion mutagenesis of Pseudomonas aeruginosa PAO1. Cloning and sequencing experiments showed that 11B was deficient in an, at that time, unknown active efflux system that contains homologs of MexAB. This locus also contained a putative regulatory gene, mexZ, transcribed divergently from the efflux operon. Introduction of a recombinant plasmid that carries the genes of the efflux system restored the resistance of 11B to parental levels, whereas overexpression of these genes strongly increased the MICs of substrate antibiotics for the PAO1 host. Antibiotic accumulation studies confirmed that this new system is an energy-dependent active efflux system that pumps out aminoglycosides. Furthermore, this system appeared to function with an outer membrane protein, OprM. While the present paper was being written and reviewed, genes with a sequence identical to our pump genes, mexXY of P. aeruginosa, have been reported to increase resistance to erythromycin, fluoroquinolones, and organic cations in Escherichia coli hosts, although efflux of aminoglycosides was not examined (Mine et al., Antimicrob. Agents Chemother. 43:415-417, 1999). Our study thus shows that the MexXY system plays an important role in the intrinsic resistance of P. aeruginosa to aminoglycosides. Although overexpression of MexXY increased the level of resistance to fluoroquinolones, disruption of the mexXY operon in P. aeruginosa had no detectable effect on susceptibility to these agents.     

Importance of Multidrug Efflux Pumps in the Antimicrobial Resistance Property of Clinical Multidrug-Resistant Isolates of Neisseria gonorrhoeae

The contribution of drug efflux pumps in clinical isolates of Neisseria gonorrhoeae that express extensively drug-resistant or multidrug-resistant phenotypes has heretofore not been examined. Accordingly, we assessed the effect on antimicrobial resistance of loss of the three gonococcal efflux pumps associated with a known capacity to export antimicrobials (MtrC-MtrD-MtrE, MacA-MacB, and NorM) in such clinical isolates. We report that the MIC of several antimicrobials, including seven previously and currently recommended for treatment was significantly impacted.     

Role of AbeS,a Novel Efflux Pump of the SMR Family of Transporters,in Resistance to Antimicrobial Agents in Acinetobacter baumannii

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].)     

RND-Type Efflux Pumps in Multidrug-Resistant Clinical Isolates of Acinetobacter baumannii: Major Role for AdeABC Overexpression and AdeRS Mutations

Increased expression of chromosomal genes for resistance-nodulation-cell division (RND)-type efflux systems plays a major role in the multidrug resistance (MDR) of Acinetobacter baumannii. However, the relative contributions of the three most prevalent pumps, AdeABC, AdeFGH, and AdeIJK, have not been evaluated in clinical settings. We have screened 14 MDR clinical isolates shown to be distinct on the basis of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) for the presence and overexpression of the three Ade efflux systems and analyzed the sequences of the regulators AdeRS, a two-component system, for AdeABC and AdeL, a LysR-type regulator, for AdeFGH. Gene adeB was detected in 13 of 14 isolates, and adeG and the intrinsic adeJ gene were detected in all strains. Significant overexpression of adeB was observed in 10 strains, whereas only 7 had moderately increased levels of expression of AdeFGH, and none overexpressed AdeIJK. Thirteen strains had reduced susceptibility to tigecycline, but there was no correlation between tigecycline MICs and the levels of AdeABC expression, suggesting the presence of other mechanisms for tigecycline resistance. No mutations were found in the highly conserved LysR regulator of the nine strains expressing AdeFGH. In contrast, functional mutations were found in conserved domains of AdeRS in all the strains that overexpressed AdeABC with two mutational hot spots, one in AdeS near histidine 149 suggesting convergent evolution and the other in the DNA binding domain of AdeR compatible with horizontal gene transfer. This report outlines the high incidence of AdeABC efflux pump overexpression in MDR A. baumannii as a result of a variety of single mutations in the corresponding two-component regulatory system.     

AcrAB Efflux System: Expression and Contribution to Fluoroquinolone Resistance in Klebsiella spp

Seven Klebsiella pneumoniae and four Klebsiella oxytoca clinical isolates with different levels of resistance to ciprofloxacin were studied. Mutations in the topoisomerase genes were found in almost all strains, but the contribution of a multidrug efflux system homologous to AcrAB in Escherichia coli was also observed. Overexpression of this efflux system was demonstrated by immunoblotting with antibodies against E. coli AcrA.     

Evolution and Dissemination of OqxAB-Like Efflux Pumps,an Emerging Quinolone Resistance Determinant among Members of Enterobacteriaceae

The OqxAB efflux pump, a plasmid-mediated quinolone resistance (PMQR) determinant, has become increasingly prevalent among members of Enterobacteriaceae over the past decade. To investigate the evolution and dissemination routes of the oqxAB operon, we assessed the prevalence of oqxAB-like elements among various Gram-negative bacterial species and analyzed the genotypic and phenotypic characteristics of organisms harboring such elements. With a comprehensive genotyping approach, a chromosome-based oqxAB operon was detectable in all Klebsiella pneumoniae strains tested, including organisms isolated before the year 1984. Sequence and phylogenetic analyses confirmed that the oqxAB operon in K. pneumoniae isolates was genetically closest to their plasmid-borne counterparts recoverable only from Escherichia coli and Salmonella isolates collected from the year 2003 onward. Chromosomal elements with much lower sequence homology were also found among the Enterobacter spp. but not other Gram-negative species. Contrary to the quinolone resistance phenotypes which were consistently observable among organisms with oqxAB-harboring plasmids, chromosomal oqxAB elements generally did not confer quinolone resistance, except for K. pneumoniae strains, which exhibited a typical oqxAB-mediated phenotype characterized by cross-resistance to olaquindox, chloramphenicol, and the quinolones. Gene expression analysis illustrated that such phenotypes were due to elevated expression of the chromosomal oqxAB operon. Furthermore, transposition of the oqxAB operon from the bacterial chromosome to plasmids was found to result in a >80-fold increase in the level of expression of the OqxAB pump, confirming its status as the first constitutively expressed efflux system located in bacterial mobile elements.     

Antimicrobial resistance in community-acquired urinary tract infections: results from the Korean Antimicrobial Resistance Monitoring System

The Korean Antimicrobial Resistance Monitoring System was established and the first nationwide surveillance of bacterial uropathogens was conducted during the period from January 2008 to June 2009. With the cooperation of 34 medical centers throughout South Korea, a total of 1994 strains belonging to clinically relevant bacterial uropathogens were collected from patients with community-acquired urinary tract infections (UTIs). To compare with past data, understand a trend of antimicrobial resistance, and ultimately determine new regimens for empirical treatment of uncomplicated cystitis, the antimicrobial susceptibilities against Escherichia coli in uncomplicated cystitis to commonly prescribed drugs were investigated. In uncomplicated cystitis, the most prevalent causative organism was E. coli (72.7%), followed by Enterococcus faecalis (10.7%) and Klebsiella pneumoniae (3.5%). Among E. coli isolates from acute uncomplicated cystitis, 38.5% were susceptible to ampicillin, 80.7% to amoxicillin/clavulanate, 67.3% to trimethoprim/sulfamethoxazole, 74.6% to ciprofloxacin, 77.5% to levofloxacin, 86.0% to cefazolin, 86.1% to cefuroxime, 93.6% to cefpodoxime, 94.7% to ceftriaxone, 99.5% to amikacin, 80.9% to tobramycin, and 76.6% to gentamicin. An increasing tendency of resistance to ciprofloxacin (24.8%) has been observed compared with the similar studies in 2006 (23.4%) and 2002 (15.2%) from patients with uncomplicated cystitis. These data provide much needed information on the prevalence of antimicrobial resistance in community-acquired UTIs in South Korea and will be a useful reference for future periodic surveillance studies.