Functional cloning and characterization of a multidrug efflux pump,mexHI-opmD,from a Pseudomonas aeruginosa mutant

We isolated mutant YM644, which showed elevated resistance to norfloxacin, ethidium bromide, acriflavine, and rhodamine 6G, from Pseudomonas aeruginosa YM64, a strain that lacks four major multidrug efflux pumps. The genes responsible for the resistance were mexHI-opmD. Elevated ethidium extrusion was observed with cells of YM644 and YM64 harboring a plasmid carrying the genes. Disruption of the genes in the chromosomal DNA of YM644 made the cells sensitive to the drugs.     

An RND-type multidrug efflux pump SdeXY from Serratia marcescens

OBJECTIVES: Serratia marcescens, an important cause of nosocomial infections, shows intrinsic resistance to a wide variety of antimicrobial agents (multidrug resistance). Multidrug efflux pumps are often involved in the multidrug resistance in many bacteria. A study was undertaken to characterize the multidrug efflux pumps in S. marcescens. METHODS: The genes responsible for the multidrug resistance phenotype in S. marcescens were cloned into Escherichia coli KAM32, a drug-hypersusceptible strain, for further analysis. RESULTS: We cloned sdeXY genes and determined the nucleotide sequence. Clones that carried the sdeXY genes displayed reduced susceptibility to several antimicrobial agents including erythromycin, tetracycline, norfloxacin, benzalkonium chloride, ethidium bromide, acriflavine and rhodamine 6G. A protein similarity search using GenBank revealed that SdeY is a member of the resistance nodulation cell-division (RND) family of multidrug efflux proteins and SdeX is a member of the membrane fusion proteins. Introduction of sdeXY into E. coli cells possessing tolC, but not in cells lacking tolC, resulted in multidrug resistance. We observed energy-dependent ethidium efflux in cells of E. coli KAM32 possessing sdeXY and tolC. CONCLUSIONS: SdeXY is the first RND-type multidrug efflux pump to be characterized in multidrug-resistant S. marcescens.     

A multidrug efflux phenotype mutant of Streptococcus pyogenes

We describe a mutant of Streptococcus pyogenes NCTC 8198 with a multidrug efflux phenotype. A mutant selected with ethidium bromide showed a four-fold rise in MIC of norfloxacin, a 16-fold rise in MIC of ethidium bromide and an eight-fold rise in MIC of acriflavine when compared with the parent strain. The MICs were unaffected by the efflux pump inhibitors reserpine, rescinnamine and verapamil. The mutant's ethidium bromide MIC was reduced two-fold by norfloxacin. Ethidium bromide accumulation after 10 min was 58% lower in the mutant compared with the parent. This difference was not affected by carbonyl cyanide m-chlorophenylhydrazone.     

Prevalence of a Putative Efflux Mechanism among Fluoroquinolone-Resistant Clinical Isolates of Streptococcus pneumoniae

Twenty-three norfloxacin-selected first-step mutants of Streptococcus pneumoniae showed low-level fluoroquinolone resistance. Their susceptibility to norfloxacin in the presence or absence of reserpine and known efflux pump substrates was determined by an agar dilution method. Five mutants showed four- to eightfold increases in their susceptibility to norfloxacin in the presence of reserpine and four- to eightfold decreases in their susceptibility to acriflavine and ethidium bromide. This phenotype is suggestive of an efflux mechanism of resistance. A representative of these mutants, 1N27, accumulated significantly less ethidium bromide than the parent strain; reserpine abolished these differences. No changes in the quinolone resistance-determining regions of parC, parE, gyrA, or gyrB were found in this mutant. By our validated agar dilution method, the efflux phenotype was sought in clinical isolates of S. pneumoniae. Of 1,037 clinical isolates examined from the United Kingdom, 273 showed reduced susceptibility to norfloxacin or ciprofloxacin. Of these, 45.4% showed the efflux phenotype. Our findings suggest that an efflux mechanism may be a frequent cause of clinically significant fluoroquinolone resistance in pneumococci.     

A new member of the tripartite multidrug efflux pumps,MexVW-OprM,in Pseudomonas aeruginosa

OBJECTIVES: Multidrug efflux pumps are thought to be involved in mediating multidrug resistance in Pseudomonas aeruginosa. Here we aim to characterize hitherto uncharacterized multidrug efflux pumps from P. aeruginosa. MATERIALS AND METHODS: We isolated a mutant, YM442, which showed elevated resistance to several antimicrobial agents from P. aeruginosa YM44 lacking four major multidrug efflux pumps, MexAB, MexCD-OprJ, MexEF-OprN and MexXY. We cloned genes responsible for the resistance from chromosomal DNA of YM442 using YM44 as host. RESULTS: We designated the genes mexVW. Introduction of a recombinant plasmid pTAJ2 carrying the mexVW into YM44 cells conferred resistance to fluoroquinolones, tetracycline, chloramphenicol, erythromycin, ethidium bromide and acriflavine. Elevated ethidium bromide extrusion was observed with cells of YM442 and of YM44/pTAJ2. An outer membrane protein OprM was able to cooperate with MexVW. Elevated expression of the mexV gene was observed with YM442 compared with YM44. CONCLUSIONS: MexV (membrane fusion protein)-MexW (RND-type membrane protein)-OprM is a tripartite multidrug efflux pump. It is suggested that other outer membrane component(s) could cooperate with MexVW.     

Rv2686c-Rv2687c-Rv2688c, an ABC fluoroquinolone efflux pump in Mycobacterium tuberculosis

The Mycobacterium tuberculosis Rv2686c-Rv2687c-Rv2688c operon, encoding an ABC transporter, conferred resistance to ciprofloxacin and, to a lesser extent, norfloxacin, moxifloxacin, and sparfloxacin to Mycobacterium smegmatis. The resistance level decreased in the presence of the efflux pump inhibitors reserpine, carbonyl cyanide m-chlorophenylhydrazone, and verapamil. Energy-dependent efflux of ciprofloxacin from M. smegmatis cells containing the Rv2686c-Rv2687c-Rv2688c operon was observed.     

Role of the Mmr Efflux Pump in Drug Resistance in Mycobacterium tuberculosis

Efflux pumps are membrane proteins capable of actively transporting a broad range of substrates from the cytoplasm to the exterior of the cell. Increased efflux activity in response to drug treatment may be the first step in the development of bacterial drug resistance. Previous studies showed that the efflux pump Mmr was significantly overexpressed in strains exposed to isoniazid. In the work to be described, we constructed mutants lacking or overexpressing Mmr in order to clarify the role of this efflux pump in the development of resistance to isoniazid and other drugs in M. tuberculosis. The mmr knockout mutant showed an increased susceptibility to ethidium bromide, tetraphenylphosphonium, and cetyltrimethylammonium bromide (CTAB). Overexpression of mmr caused a decreased susceptibility to ethidium bromide, acriflavine, and safranin O that was obliterated in the presence of the efflux inhibitors verapamil and carbonyl cyanide m-chlorophenylhydrazone. Isoniazid susceptibility was not affected by the absence or overexpression of mmr. The fluorometric method allowed the detection of a decreased efflux of ethidium bromide in the knockout mutant, whereas the overexpressed strain showed increased efflux of this dye. This increased efflux activity was inhibited in the presence of efflux inhibitors. Under our experimental conditions, we have found that efflux pump Mmr is mainly involved in the susceptibility to quaternary compounds such as ethidium bromide and disinfectants such as CTAB. The contribution of this efflux pump to isoniazid resistance in Mycobacterium tuberculosis still needs to be further elucidated.     

Functional Analysis of Pneumococcal Drug Efflux Pumps Associates the MATE DinF Transporter with Quinolone Susceptibility

The pneumococcal chromosome encodes about 140 transporters, many of which are predicted to be involved in efflux. In order to critically evaluate pneumococcal efflux, a series of transporter mutants were constructed, and their phenotypes were assayed by disk diffusion, microdilution drug susceptibility testing (MIC testing), growth of cultures at sub-MIC concentrations, and phenotype microarray analysis. Mutants with mutations in seven ATP binding cassette (ABC) transporters, three multiantimicrobial extrusion (MATE) family efflux pumps, and one major facilitator superfamily (MFS) transporter were obtained in Streptococcus pneumoniae strain DP1004. The susceptibility of these 11 mutants to over 250 different substances was compared to that of the parent strain. Of the tested transporters, only the ABC transporter PatAB (SP2073-5) presented a clear multidrug resistance (MDR) profile, as the mutant showed significantly increased susceptibility to ethidium bromide, acriflavine, and berberine. Among the other transporters analyzed, the mutants devoid of the MATE efflux pump SP2065 exhibited reduced susceptibility to novobiocin, and those with mutations of the MATE family DinF transport system (SP1939) exhibited increased susceptibility to moxifloxacin, ciprofloxacin, and levofloxacin. This change in quinolone MIC was found to be independent from the competence-mediated effect of quinolones on the cinA-recA-dinF operon. Furthermore, the dinF mutant, in contrast to the parental strain, allowed selection for quinolone-resistant mutants when exposed to moxifloxacin. These data confirm the clear MDR profile of the PatAB ABC transporter and suggest for the MATE DinF a phenotype associated with quinolone susceptibility, particularly for moxifloxacin.     

Prevalence of multiple antibiotic resistance in 443 Campylobacter spp. isolated from humans and animals

AIMS: In view of recent findings that a multidrug efflux pump CmeABC exists in Campylobacter jejuni, 391 C. jejuni and 52 Campylobacter coli of human and animal origin were examined for a multidrug resistance phenotype. MATERIALS AND METHODS: The MICs of ampicillin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, tetracycline, cetrimide, triclosan, acridine orange, paraquat and ethidium bromide were determined. Resistance to organic solvents and the effect of salicylate (known inducer of the marRAB operon in Escherichia coli and Salmonella) were also examined. RESULTS: Two C. coli and 13 C. jejuni isolates, mainly from pigs or poultry, were resistant to three or more antibiotics and 12 of these strains had reduced susceptibility to acridine orange and/or ethidium bromide. Strains (n = 20) that were less susceptible to acridine orange, ethidium bromide and triclosan were significantly more resistant (P < 0.05) to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, nalidixic acid and tetracycline, with two- to four-fold increases in MIC values compared with strains (n = 20) most susceptible to acridine orange, ethidium bromide and triclosan. Growth of strains with 1 mM salicylate caused a small (up to two-fold) but statistically significant (P < or = 0.005) increase in the MICs of chloramphenicol, ciprofloxacin, erythromycin and tetracycline. CONCLUSIONS: These data indicate that multiple antibiotic resistant (MAR)-like Campylobacter strains occur and it may be postulated that these may overexpress cmeABC or another efflux system.     

Efflux pump Lde is associated with fluoroquinolone resistance in Listeria monocytogenes

Five Listeria monocytogenes isolates (CLIP 21369, CLIP 73298, CLIP 74811, CLIP 75679, and CLIP 79372) were found to be resistant to fluoroquinolones during the screening for antibiotic resistance of 488 L. monocytogenes isolates from human cases of listeriosis in France. On the basis of a fourfold or greater decrease in the ciprofloxacin MIC in the presence of reserpine, fluoroquinolone resistance was attributed to active efflux of the drugs. The lde gene (Listeria drug efflux; formerly lmo2741) encodes a 12-transmembrane-segment putative efflux pump belonging to the major facilitator superfamily of secondary transporters that displayed 44% identity with PmrA from Streptococcus pneumoniae. Insertional inactivation of the lde gene in CLIP 21369 indicated that the corresponding protein was responsible for fluoroquinolone resistance and was involved in the level of susceptibility to dyes such as ethidium bromide and acridine orange.     

Characterization of P55, a multidrug efflux pump in Mycobacterium bovis and Mycobacterium tuberculosis

The Mycobacterium bovis P55 gene, located downstream from the gene that encodes the immunogenic lipoprotein P27, has been characterized. The gene was identical to the open reading frame of the Rv1410c gene in the genome of Mycobacterium tuberculosis H37Rv, annotated as a probable drug efflux protein. Genes similar to P55 were present in all species of the M. tuberculosis complex and other mycobacteria such as Mycobacterium leprae and Mycobacterium avium. By Western blotting, P55 was located in the membrane fraction of M. bovis. When transformed into Mycobacterium smegmatis after cloning, P55 conferred aminoglycoside and tetracycline resistance. The levels of resistance to streptomycin and tetracycline conferred by P55 were decreased in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the pump inhibitors verapamil and reserpine. M. smegmatis cells expressing the plasmid-encoded P55 accumulated less tetracycline than the control cells. We conclude that P55 is a membrane protein implicated in aminoglycoside and tetracycline efflux in mycobacteria.     

Evidence of active efflux in resistance to ciprofloxacin and to ethidium bromide by Mycoplasma hominis

The uptake of fluoroquinolones was characterized for the fluoroquinolone-susceptible strain PG21 of Mycoplasma hominis. Accumulation of fluoroquinolones appeared to occur by passive diffusion. Addition of arginine as the energizer significantly reduced the uptake of fluoroquinolones, suggesting the presence of an energy-dependent efflux process. Reserpine and orthovanadate, two multidrug pump inhibitors, increased significantly the ciprofloxacin (CIP) uptake. In contrast, such a strong effect was not observed for moxifloxacin and pefloxacin uptakes. Two ethidium bromide (EtBr)-resistant strains, selected in vitro, showed a resistance profile compatible with a multidrug-resistant phenotype, with increased MICs for the hydrophilic fluoroquinolones, CIP and norfloxacin, EtBr, and acriflavine. Taking the EtBr-resistant strain RB1La as a model, a significant decrease of the CIP and EtBr uptakes was observed compared to the reference strain PG21. In the presence of reserpine and orthovanadate, both inhibitors of ATP-dependent efflux pumps, the CIP uptake increased significantly, reaching approximately the same level as that of the susceptible strain. Similar results were obtained with EtBr uptake and efflux experiments. Our data suggest the presence of an active efflux system, possibly an ABC-type efflux pump, implicated in the resistance to CIP and unrelated compounds like EtBr in the human mycoplasma M. hominis.     

Acriflavine-Resistant Mutant of Streptococcus cremoris

Selection for resistance to acriflavine in Streptococcus cremoris resulted in cross-resistance to the drugs neomycin, streptomycin, ethidium bromide, mitomycin C, and proflavine. Furthermore, the mutants showed resistance to lytic bacteriophages to which the parental strain was sensitive, and, unlike the parent, the mutants grew well at higher temperatures (40°C). Revertants selected independently either for temperature sensitivity or for acriflavine sensitivity lost resistance to all the drugs and dyes but retained the bacteriophage resistance phenotype. The acriflavine-resistant mutation resulted in an increase in resistance by the bacterial cells to sodium dodecyl sulfate, a potent solvent of lipopolysaccharide and lipoprotein. It is suggested that the acriflavine resistance mutation determines the synthesis of a membrane substance resistant to higher temperatures.     

Expression of Escherichia coli TehA gives resistance to antiseptics and disinfectants similar to that conferred by multidrug resistance efflux pumps.

The genes tehAB located at 32.3 min on the Escherichia coli chromosome were initially identified by their ability to mediate resistance to potassium tellurite (128 micrograms of K2TeO3 per ml) when overexpressed with a high-copy-number plasmid. The genes encode an integral membrane protein (TehA) of 36 kDa with 10 putative transmembrane segments and a second protein (TehB) of 23 kDa. Overexpression of TehAB results in hypersensitivity to dequalinium CI and methyl viologen (paraquat). Expression of TehA alone gives similar hypersensitivity. Overexpression of TehA gave resistance to tetraphenylarsonium CI, ethidium bromide, crystal violet and proflavin. The efflux of ethidium, measured by fluorescence quenching, revealed that TehA transported ethidium at twice the control rate and 10% of the rate of the highly resistant efflux transporter Emr Eco. Addition of tellurite had no effect on ethidium transport. In addition to the ethidium transport assay, a proflavin fluorescence assay which was approximately 200-fold more sensitive was also used. TehA was also found to have proflavin efflux activity. The addition of TeO32- to the proflavin transport assay on TehA caused a 20% increase in transport rate. Both ethidium and proflavin transport were found to be energy dependent.