Unrelated Antimicrobial Agents Research Articles

Multidrug resistant Klebsiella pneumoniae isolated from respiratory tract infection

An increasing antimicrobial resistance among Klebsiella pneumoniae (K. pneumoniae) has been rapidly growing. The cross-sectional, analytical and descriptive study was conducted to investigate the multidrug resistance (MDR) of K. pneumoniae isolated from respiratory tract infection. One hundred and fifty-one K. pneumoniae isolates were recovered from 330 sputum specimens (45.75%). The isolates showed high levels of resistance to the most of antibiotics and intermediate resistance prevalence against imipenem and meropenem. Random and misuse of antibiotics lead to the emergence of superbugs. Prospective studies must focus on new strategies for facing of the tsunami of drug resistance.K. pneumoniae is a Gram-negative bacterium, belongs to family Enterobacteriaceae. K. pneumoniae can be stated as the major cause of different types of lower respiratory infections followed by septicemia. The majority of the infections were diagnosed in hospitalized patients, it indicates that K. pneumoniae is a potent nosocomial pathogen in our hospitals(Kramer et al., 2006).Treatment of infections caused by K. pneumoniae is hindered by emergence of bacterial resistance to antimicrobial agents. K. pneumoniae is capable of employing a multitude of mechanisms by which to confer resistance to most available antibiotics, efflux pumps play a major role. Bacterial efflux pumps are membrane proteins that decrease intracellular concentration of antimicrobial agents through pumping them outside the cells. Importantly, bacterial efflux pumps are non-specific in action and therefore could mediate bacterial resistance to structurally unrelated antimicrobial agents (Nahar and Rashid, 2017).

Xylose Improves Antibiotic Activity of Chloramphenicol and Tetracycline against K. pneumoniae and A. baumannii in a Murine Model of Skin Infection.

Increased resistance to antimicrobials in clinically important bacteria has been widely reported. The major mechanism causing multidrug resistance (MDR) is mediated by efflux pumps, proteins located in the cytoplasmic membrane to exclude antimicrobial drug. Some efflux pumps recognize and expel a variety of unrelated antimicrobial agents, while other efflux pumps can expel only one specific class of antibiotics. Previously, we have reported that xylose decreases the efflux-mediated antimicrobial resistance in Salmonella typhimurium, Pseudomonas aeruginosa, and Acinetobacter baumannii in vitro. In this work, we assessed the effectiveness of combining xylose with antibiotics to kill resistant Acinetobacter baumannii and Klebsiella pneumoniae in a murine model of skin infection. Skin infections were established by seeding 109 bacteria onto eroded skin of mice. Mice treated with the antibiotic alone or with a mixture of glucose and antibiotics or xylose and antibiotics were compared to a control group that was infected but received no further treatment. We observed that the mixtures xylose-tetracycline and xylose-chloramphenicol produced a decrease of at least 10 times viable Acinetobacter baumannii and Klebsiella pneumoniae recovered from infected skin, compared with mice treated with the antibiotic alone. Our results show that xylose improves the antibiotic activity of tetracycline and chloramphenicol against efflux-mediated resistance Acinetobacter baumannii and Klebsiella pneumoniae, in a murine model of skin infection. We envision these combined formulations as an efficient treatment of skin infections with bacteria presenting efflux-mediated resistance, in both humans and animals.

Pseudomonas fluorescens tolerance to benzyldimethyldodecyl ammonium chloride: Altered phenotype and cross-resistance.

Benzyldimethyldodecyl ammonium chloride (BDMDAC) is a quaternary ammonium compound (QAC) with bactericidal action that is used as an active molecule in detergent formulations. Pseudomonas fluorescens is a Gram-negative bacterium with versatile metabolism that is frequently present in biofilms on industrial surfaces. This work reports P. fluorescens adaptation to BDMDAC and subsequent concurrent reduced susceptibility to the QAC benzalkonium chloride (BAC) and the antimicrobial ciprofloxacin (CIP). Stepwise adaptation to increasing concentrations of BDMDAC was easily achieved and caused changes in the bacterial phenotype of P. fluorescens. Adaptation was evaluated through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination and was subsequently confirmed by time-kill curves. Biofilm phenotype (biomass and number of cells) was characterised for the adapted and reference strains after treatment with BDMDAC, BAC and CIP. Susceptibility to BAC and CIP was reduced in adapted P. fluorescens. Biofilms developed by the adapted strain had 20% more mass and a higher number of bacteria (2 log). This study revealed that exposure to sublethal concentrations of BDMDAC may select tolerant strains to that product as well as to related products and unrelated antimicrobial agents.

Prevalence of Antibiotic-resistance Enterobacteriaceae strains Isolated from Chicken Meat at Traditional Markets in Surabaya, Indonesia

Antibiotic resistance in bacteria from the family Enterobacteriaceae is an important indicator of the emergence of resistant bacterial strains in the community. This study investigated the prevalence of antibiotic-resistant Enterobacteriaceae isolated from chicken meat sold at traditional markets in Surabaya Indonesia. In all, 203 isolates (43 Salmonella spp., 53 Escherichia coli, 16 Shigella spp., 22 Citrobacter spp., 13 Klebsiella spp, 24 Proteus spp., 15 Yersinia spp., 7 Enterobacter spp., 6 Serratia spp., 3 Edwardsiella spp. were resistant to tetracycline (69.95 %), nalidixid acid (54.19 %), sulfamethoxazole/sulfamethizole (42.36 %), chloramphenicol (12.81%), cefoxitin (6.40 %), gentamicin (5.91 %). Tetracycline was the antimicrobial that showed the highest frequency of resistance among Salmonella, E. coli, Citrobacter, Proteus and Erdwardsiella isolates, and nalidixid acid was second frequency of resistance. Overall, 124 (61.08 %) out of 203 isolates demonstrated multidrug resistance to at least two unrelated antimicrobial agents. The high rate of antimicrobial resistance in bacterial isolates from chicken meat may have major implications for human and animal health with adverse economic implications.

Exposure to Multiple and Unrelated Antimicrobial Agents Is Associated With Bloodstream Infection Due to Antibiotic Resistant Bacteria Among Hospitalized Children

Exposure to Multiple and Unrelated Antimicrobial Agents Is Associated With Bloodstream Infection Due to Antibiotic Resistant Bacteria Among Hospitalized Children

Development and analysis of furazolidone-resistant Escherichia coli mutants.

Furazolidone-resistant mutants were obtained from four clinical isolates of diarrhoeagenic Escherichia coli. The stability of the resistance and the frequency of mutation were established. The minimal inhibitory concentration of furazolidone, nitrofurantoin, nalidixic acid, ampicillin, chloramphenicol and tetracycline was established both in the presence and absence of the efflux pump inhibitor Phe-Arg-β-Naphtylamyde. The presence of mutations in the nitroreductase genes nfsA and nfsB was analysed by PCR; sequencing and their enzymatic activity was assessed by a spectrophotometric assay. Alterations in outer membrane proteins were studied by SDS-PAGE. The frequency of mutation ranged from <9.6×10(-10) to 9.59×10(-7) . Neither an effect on efflux pumps inhibited by Phe-Arg-β-Naphtylamyde nor cross-resistance with the antibiotics studied was observed. Nineteen mutants (52.94%) presented mutations in the nitroreductase-encoding genes: 17 in the nfsA gene (15 mutants with an internal stop codon, 2 with amino acid changes), 2 in the nfsB (all amino acid changes). Alterations in the outer membrane proteins OmpA and OmpW were also observed. Although more studies are necessary to find other resistance mechanisms, present data showed the low potential of selecting furazolidone-resistant mutants, together with the lack of cross-resistance with unrelated antimicrobial agents.

Prevalence of Extended Spectrum β-Lactamase inKlebsiella pneumoniaIsolates in a Teaching Hospital of Zahedan City, Iran

Background: Extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumonia has become widespread in hospitals and is increasing in community settings. Most of K. pneumonia that harbor these enzymes, display resistance to other unrelated antimicrobial agents and thus, often pose a therapeutic dilemma. Objectives: This study was conducted to determine the prevalence of ESBL-producing K. pneumonia in a major university hospital in Zahedan, Iran. Materials and Methods: The susceptibility of 83 K. pneumonia isolates to 12 antibiotics was assessed using Kirby-Bauer disc diffusion method. For the ESBL phenotypic test, double-disk diffusion (DD) method was used. Results: The highest resistance rates of the isolates were seen against cefixime (82%), cefotaxime (81%), ceftriaxone (73%), ceftazidime (72%), and azithromycin (60%), consecutively. The lowest resistance rates were observed against gentamicin (58%), tetracycline (59%), nalidixic acid (59%), and amikacin (63%), consecutively. ESBLs were found in 65% of K. pneumonia isolates. Conclusions: We found that 65% of K. pneumonia isolates produced ESBL. Therapeutic strategies to control infections should be carefully formulated in teaching hospitals. The high percentage of drug resistance in ESBL-producing K. pneumonia suggests that routine detection of ESBL by reliable laboratory methods is required.

Crystal structure of the transcriptional regulator Rv1219c of Mycobacterium tuberculosis

The Rv1217c-Rv1218c multidrug efflux system, which belongs to the ATP-binding cassette superfamily, recognizes and actively extrudes a variety of structurally unrelated toxic chemicals and mediates the intrinsic resistance to these antimicrobials in Mycobacterium tuberculosis. The expression of Rv1217c-Rv1218c is controlled by the TetR-like transcriptional regulator Rv1219c, which is encoded by a gene immediately upstream of rv1218c. To elucidate the structural basis of Rv1219c regulation, we have determined the crystal structure of Rv1219c, which reveals a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. The N-terminal domains of the Rv1219c dimer are separated by a large center-to-center distance of 64 Å. The C-terminal domain of each protomer possesses a large cavity. Docking of small compounds to Rv1219c suggests that this large cavity forms a multidrug binding pocket, which can accommodate a variety of structurally unrelated antimicrobial agents. The internal wall of the multidrug binding site is surrounded by seven aromatic residues, indicating that drug binding may be governed by aromatic stacking interactions. In addition, fluorescence polarization reveals that Rv1219c binds drugs in the micromolar range.

Antimicrobial Drug Resistance and Disinfectants Susceptibility of Pseudomonas aeruginosa Isolates from Clinical and Environmental Samples in Jimma University Specialized Hospital, Southwest Ethiopia

P. aeruginosa is one of the most important pathogen that causes nosocomial infections often acquired from hospital environment and contaminated medical devices. The infections caused by this bacterium are particularly problematic because it is inherently resistant to several unrelated antimicrobial agents and antiseptics. Hence, the aim of this study was to determine drug resistance and disinfectants susceptibility of P. aeruginosa isolated from clinical samples and hospital environments. A laboratory based cross sectional study was conducted from May to September, 2012 on a total of 81 P. aeruginosa isolates. A standard bacteriological technique (conventional biochemical tests and pigment production) was used to identify the bacterium. Drug resistance and disinfectant susceptibility tests were determined by Kirby-Bauer disc-diffusion and through the classic method of successive dilutions respectively. In this study a total of 305 (160 clinical and 145 environmental) samples were investigated for P. aeruginosa and 81 isolates were obtained. This gives an overall P. aeruginosa isolation rate of 26.5% (81/305). About 47.5% of the clinical and 34.2% environmental isolates were detected from wounds and ward sinks respectively. Of these P. aeruginosa isolates, 95.1% were resistance to Trimethoprim-sulphametoxazole, 62% to Gentamicin, and 58% to Ceftriaxone. But, only 4.9% of isolates were resistance to Amikacin. Moreover, disinfectant susceptibility test revealed that hydrogen peroxide and sodium hypochlorite had a higher (92.6% & 91.4%) bactericidal activity compared to ethanol and savlon at the recommended user dilution. In general, our results indicated that P. aeruginosa was significantly resistance to commonly prescribed antimicrobial drugs, a situation that demands a more rational and appropriate use of antibiotics. Hydrogen peroxide and sodium hypochlorite were relatively more effective when used in recommended dilution. However, all tested disinfectants or antiseptics showed reduced bactericidal activities in higher dilutions. Therefore, strict adherence to the recommended dilution is important for better activity. The correct use of them also has to be considered appropriately as part of infection control practices.

Gene Cloning and Characterization of MdeA, a Novel Multidrug Efflux Pump in Streptococcus mutans

Multidrug resistance, especially multidrug efflux mechanisms that extrude structurally unrelated cytotoxic compounds from the cell by multidrug transporters, is a serious problem and one of the main reasons for the failure of therapeutic treatment of infections by pathogenic microorganisms as well as of cancer cells. Streptococcus mutans is considered one of the primary causative agents of dental caries and periodontal disease, which comprise the most common oral diseases. A fragment of chromosomal DNA from S. mutans KCTC3065 was cloned using Escherichia coli KAM32 as host cells lacking major multidrug efflux pumps. Although E. coli KAM32 cells were very sensitive to many antimicrobial agents, the transformed cells harboring a recombinant plasmid became resistant to several structurally unrelated antimicrobial agents such as tetracycline, kanamycin, rhodamin 6G, ampicillin, acriflavine, ethidium bromide, and tetraphenylphosphonium chloride. This suggested that the cloned DNA fragment carries a gene encoding a multidrug efflux pump. Among 49 of the multidrug-resistant transformants, we report the functional gene cloning and characterization of the function of one multidrug efflux pump, namely MdeA from S. mutans, which was expressed in E. coli KAM32. Judging from the structural and biochemical properties, we concluded that MdeA is the first cloned and characterized multidrug efflux pump using the proton motive force as the energy for efflux drugs.

Fecal microbiota of calves in the clinical setting: Effect of penicillin treatment

The effect of parenteral penicillin treatment on the intestinal microbiota was determined by monitoring the phenotypic antimicrobial resistance among Escherichia coli in 19 calves (15 calves received treatment and four calves were healthy controls) and by examining changes in the fecal microbial community structure using molecular fingerprinting techniques in a subset of eight calves (five treated calves and three control calves). After five days of penicillin treatment an increased resistance to multiple unrelated antimicrobial agents, including non-β-lactams, was seen in E. coli from treated calves, and this was not seen in the controls. Automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphism (TRFLP) revealed that penicillin treatment causes a significant variation in the microbial structure within an individual calf. The study shows that parenteral administration of penicillin has an impact on the composition of the fecal microbiota in calves, and on the antimicrobial resistance pattern of their fecal E. coli.

Fecal microbiota of horses in the clinical setting: Potential effects of penicillin and general anesthesia

Antimicrobial treatment is associated with the spread of antimicrobial resistance and disturbances in the ecological balance of intestinal microbiota. In horses, the main adverse effect of antimicrobial treatment is colitis. We used culture and 16S rRNA gene based molecular methods to monitor the prevalence of antimicrobial resistance and changes in predominant fecal populations during penicillin treatment and general anesthesia of horses in the clinical setting. After 5 days of parenteral administration of penicillin, fecal Escherichia coli were resistant to multiple unrelated antimicrobial agents when compared to the pre-exposure situation. Denaturing gradient gel electrophoresis (DGGE) profiles indicated that horses have an extremely diverse fecal microbiota, with marked differences between individual horses. Most of the variation in DGGE profiles could be attributed to horse-specific factors, and penicillin, general anesthesia or both could not explain the remaining variation. Within-animal variation remained less than between-animal variation despite treatment. However, real-time PCR quantification (qPCR) indicated subclinical changes in selected bacterial groups of the penicillin treated horses.

Determination of the Efflux Pump-Mediated Resistance Prevalence in Pseudomonas aeruginosa, Using an Efflux Pump Inhibitor

In gram negative bacteria, fluoroquinolone resistance is acquired by target mutations in topoisomerase genes or by reducing the permeation of drugs due to the increase in expression of endogenous multidrug efflux pumps that expel structurally unrelated antimicrobial agents. An ongoing challenge is searching for new inhibitory substances in order to block efflux pumps and restore the antibiotic drugs susceptibility. In this research, we sought to investigate the interplay between ciprofloxacin and an efflux pump inhibitor (EPI), phenyl alanine arginyl beta-naphtylamide (PAbetaN), to determine the prevalence of efflux pump overexpression in clinical isolates of Pseudomonas aeruginosa. Ciprofloxacin was tested at different concentrations (256-0.25 microg/ml) with a fixed concentration of PAbetaN (50 microg/ml). The isolates susceptibility profiles were analyzed by disc diffusion and agar dilution methods using 10 antibiotic discs and 4 powders. It was found that in the presence of PAbetaN, resistance to ciprofloxacin was inhibited obviously and MIC values were decreased. The comparison between subgroups of P. aeruginosa isolates with different resistance profiles indicates that efflux pump overexpression (EPO) is present in 35% of ciprofloxacin resistant isolates with no cross resistance and in variable frequencies among isolates showing cross resistance to other tested antibiotics: gentamicin (31%), ceftazidime (29%), and imipenem (18%). Altogether, these results imply that PAbetaN maybe effective to restore the fluoroquinolone drugs susceptibility in clinical treatment procedures. Results also show that increased use of a fluoroquinolone drug such as ciprofloxacin can affect the susceptibility of P. aeruginosa to other different antipseudomonal agents.

Associated antimicrobial resistance in Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes

Associated resistance to four to six related and unrelated antimicrobial agents was investigated in consecutive non-duplicate isolates of Escherichia coli (n = 39 425), Pseudomonas aeruginosa (n = 1070), Staphylococcus aureus (n = 7489), Streptococcus pneumoniae (n = 1604) and Streptococcus pyogenes (n = 2531). In all species, high proportions (76.5–88.9%) of isolates were susceptible to all the drugs investigated. Irrespective of species, isolates resistant to one drug were more likely to be resistant to any of the other drugs than were susceptible isolates. Thus, trimethoprim resistance in E. coli was 38.4% among ampicillin-resistant vs. 3.9% among ampicillin-susceptible isolates, and erythromycin resistance in Strep. pneumoniae was 41% among doxycycline-resistant vs. 1% among doxycycline-susceptible isolates. In all five species investigated, there was also significant associated resistance among unrelated drugs, highlighting the fact that resistance development occurs primarily among bacteria already resistant to one or more antimicrobial agents. For the clinician, pronounced resistance associations mean that when empirical therapy fails because of resistance, there is a reduced chance of choosing an alternative successful empirical agent. For the epidemiologist, who uses routine clinical susceptibility data to describe resistance development, resistance associations mean that if the dataset contains results for isolates selected on the basis of their susceptibility to another drug, structurally related or not, a bias of false resistance is introduced.

Functional Gene Cloning and Characterization of the SsmE Multidrug Efflux Pump from Serratia marcescens

We cloned a gene responsible for multidrug resistance from chromosomal DNA of Serratia marcescens, and determined the nucleotide sequence. We designated the gene as ssmE. The deduced amino acid sequence of SsmE showed high similarity with the small multidrug resistance (SMR)-type multidrug efflux pumps. Cells of Escherichia coli KAM32, a drug hyper-susceptible mutant, transformed with a plasmid pESM437 carrying the ssmE gene showed elevated minimum inhibitory concentrations of structurally unrelated antimicrobial agents. E. coli KAM32/pESM437 showed elevated energy dependent efflux of ethidium. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that ssmE was expressed in cells of S. marcescens.

SmdAB, a Heterodimeric ABC-Type Multidrug Efflux Pump, in Serratia marcescens

We cloned genes, designated smdAB, that encode a multidrug efflux pump from the chromosomal DNA of clinically isolated Serratia marcescens NUSM8906. For cells of the drug-hypersensitive strain Escherichia coli KAM32 harboring a recombinant plasmid carrying smdAB, structurally unrelated antimicrobial agents such as norfloxacin, tetracycline, 4',6-diamidino-2-phenylindole (DAPI), and Hoechst 33342 showed elevated MICs. The deduced amino acid sequences of both SmdA and SmdB exhibited similarities to the sequences of ATP-binding cassette (ABC)-type multidrug efflux pumps. The efflux of DAPI and Hoechst 33342 from E. coli cells expressing SmdAB was observed, and the efflux activities were inhibited by sodium o-vanadate, which is a well-known ATPase inhibitor. The introduction of smdA or smdB alone into E. coli KAM32 did not elevate the MIC of DAPI; thus, both SmdA and SmdB were required for function. These results indicate that SmdAB is probably a heterodimeric multidrug efflux pump of the ABC family in S. marcescens.

Bile salts enhance bacterial co-aggregation, bacterial-intestinal epithelial cell adhesion, biofilm formation and antimicrobial resistance of Bacteroides fragilis

Bacteroides fragilis is the most common anaerobic bacterium isolated from human intestinal tract infections. Before B. fragilis interacts with the intestinal epithelial cells, it is exposed to bile salts at physiological concentrations of 0.1–1.3%. The aim of this study was to determine how pre-treatment with bile salts affected B. fragilis cells and their interaction with intestinal epithelial cells. B. fragilis NCTC9343 was treated with conjugated bile salts (BSC) or non-conjugated bile salts (BSM). Cellular ultrastructure was assessed by electron microscopy, gene expression was quantified by comparative quantitative real-time RT-PCR. Adhesion to the HT-29 human intestinal cell line and to PVC microtitre plates (biofilm formation) was determined. Exposure to 0.15% BSC or BSM resulted in overproduction of fimbria-like appendages and outer membrane vesicles, and increased expression of genes encoding RND-type efflux pumps and the major outer membrane protein, OmpA. Bile salt-treated bacteria had increased resistance to structurally unrelated antimicrobial agents and showed a significant increase in bacterial co-aggregation, adhesion to intestinal epithelial cells and biofilm formation. These data suggest that bile salts could enhance intestinal colonization by B. fragilis via several mechanisms, and could therefore be significant to host–pathogen interactions.

Porins, efflux pumps and multidrug resistance in Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic pathogen, causing infections mainly in patients in intensive care units where the extensive use of antimicrobial agents can select for the emergence of multiresistant strains. In fact, since strains resistant to all antimicrobial agents have been reported, A. baumannii is considered the paradigm of multiresistant bacteria. Both acquired and intrinsic resistance can contribute to multiresistance. The ability to acquire multidrug resistance can be due to either the acquisition of genetic elements carrying multiple resistant determinants or mutations affecting the expression of porins and/or efflux pump(s), which can affect unrelated antimicrobial agents. Meanwhile, intrinsic resistance can be generated by the interplay of decreased permeability and constitutive expression of active efflux systems and it too can affect unrelated antimicrobial agents. This review is focused on the current knowledge of porins and efflux pump(s) in this microorganism.

Effector-Repressor Interactions, Binding of a Single Effector Molecule to the Operator-bound TtgR Homodimer Mediates Derepression

The RND family transporter TtgABC and its cognate repressor TtgR from Pseudomonas putida DOT-T1E were both shown to possess multidrug recognition properties. Structurally unrelated molecules such as chloramphenicol, butyl paraben, 1,3-dihydroxynaphthalene, and several flavonoids are substrates of TtgABC and activate pump expression by binding to the TtgR-operator complex. Isothermal titration calorimetry was employed to determine the thermodynamic parameters for the binding of these molecules to TtgR. Dissociation constants were in the range from 1 to 150 microm, the binding stoichiometry was one effector molecule per dimer of TtgR, and the process was driven by favorable enthalpy changes. Although TtgR exhibits a large multidrug binding profile, the plant-derived compounds phloretin and quercetin were shown to bind with the highest affinity (K(D) of around 1 microm), in contrast to other effectors (chloramphenicol and aromatic solvents) for which exhibited a more reduced affinity. Structure-function studies of effectors indicate that the presence of aromatic rings as well as hydroxyl groups are determinants for TtgR binding. The binding of TtgR to its operator DNA does not alter the protein effector profile nor the effector binding stoichiometry. Moreover, we demonstrate here for the first time that the binding of a single effector molecule to the DNA-bound TtgR homodimer induces the dissociation of the repressor-operator complex. This provides important insight into the molecular mechanism of effector-mediated derepression.

Functional Gene Cloning and Characterization of MdeA, a Multidrug Efflux Pump from Staphylococcus aureus

A DNA fragment conferring drug resistance was cloned from the chromosomal DNA of Staphylococcus aureus N315 using a drug hypersensitive Escherichia coli KAM32 as the host. Although E. coli KAM32 cells were sensitive to many antimicrobial agents, transformed cells harboring a recombinant plasmid carrying the DNA region became resistant to several structurally unrelated antimicrobial agents, such as tetraphenylphosphonium chloride, Hoechst 33342 and norfloxacin. These results suggest that the cloned DNA fragment carries a gene(s) encoding a multidrug efflux pump. We partially determined the nucleotide sequence of the cloned DNA and found the mdeA gene within it. The E. coli cells transformed with the mdeA gene showed efflux activity of Hoechst 33342. On the other hand, S. aureus cells transformed with mdeA showed elevated resistance to doxorubicin, daunorubicin, tetraphenylphosphonium chloride, Hoechst 33342, ethidium bromide and rhodamine 6G. Elevated energy-dependent efflux of ethidium was observed with transformed S. aureus. We found that the mdeA gene was expressed under normal growth conditions in S. aureus N315.