Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and norfloxacin

Abstract

Most strains of Pseudomonas aeruginosa are significantly more resistant, even in the absence of R plasmids, to many antimicrobial agents, including P-lactams, tetracycline, chloramphenicol, and fluoroquinolones, than most other gram-negative rods. This broad-range resistance has so far been assumed to be mainly due to the low permeability of the P. aeruginosa outer membrane. The intrinsic-resistance phenotype becomes further enhanced in "intrinsically carbenicillin-resistant" isolates, which were often assumed to produce outer membranes of even lower permeability. It has been shown, however, that this hypothesis cannot explain the f-lactam resistance of these isolates (D. M. Livermore and K. W. M. Davy, Antimicrob. Agents Chemother. 35:916-921, 1991). In this study, we examined the uptake of tetracycline, chloramphenicol, and norfloxacin by intact cells using strains showing widely different levels of intrinsic resistance. Their accumulation and the response to the addition of a proton conductor showed that even relatively susceptible strains of P. aeruginosa actively pump out these compounds from the cell and that the efflux activity becomes much stronger in strains showing higher levels of intrinsic resistance. We conclude that the efilux mechanism(s) are likely to contribute significantly to the intrinsic resistance of P. aeruginosa isolates to tetracycline, chloramphenicol, and fluoroquinolones, as does the low permeability of the outer membrane. This conclusion is supported by the observation that the hypersusceptibility to various agents of the mutant K799/61 (W. Zimmermann, Antimicrob. Agents Chemother. 18:94-100, 1980) was apparently caused by the lack of active eftlux. Although the hypersusceptibility of this mutant has hitherto been assumed to be solely due to its higher outer membrane permeability, its outer membrane was shown to have a coefficient of permeability to cephaloridine that was not significantly different from that of the parent, resistant strain K799/WT. The strains with elevated intrinsic resistance overproduced two cytoplasmic membrane proteins and one outer membrane protein; at least two of these proteins appeared different from the proteins overproduced in the recently described mutant with a derepressed multidrug efilux system, MexA-MexB-OprK (K. Poole, K. Krebes, C. McNally, and S. Neshat, J. Bacteriol. 175:7363-7372, 1993).It is well known that most strains of Pseudomonas aeruginosa show significant degrees of intrinsic resistance to a wide variety of antimicrobial agents, including most ,-lactams, tetracyclines, chloramphenicol, and fluoroquinolones. The outer membrane of this species shows a very low nonspecific permeability to small, hydrophilic molecules (1, 46), and this has generally been thought to be the main cause of the resistance of this organism.Nevertheless, the low outer membrane permeability cannot be the entire explanation of the intrinsic resistance. First, theoretical analysis shows that even the low-permeability outer membrane of this species should allow a suf...

Silver-resistant mutants of Escherichia coli display active efflux of Ag+ and are deficient in porins

“…Other heavy metal salts (analytical grade) were from commercial sources. The sources of antibiotics were as previously described (8,9). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), disodium ATP, and sodium sulfadiazine were purchased from Sigma Chemical Co. (St. Louis, Mo.).…”

“…The OMs and cytoplasmic membranes (CMs) were prepared by sonic disruption followed by differential extraction with Sarkosyl (Sigma) (8). The protein composition was analyzed by sodium dodecyl sulfate-slab polyacrylamide (13%) gel electrophoresis (8).…”

“…When an energy inhibitor such as CCCP (prepared as a 10 mM stock solution in 90% ethanol), arsenate, or vanadate was used, the cells were pretreated with the inhibitor for 5 min at 37°C before the addition of radioactive Ag. Because Ag ϩ tends to adsorb to various material, including membrane filters, we used a silicone oil centrifugation assay (8). Duplicate 0.1-ml samples were removed at the indicated times and placed on top of a silicone oil mixture (0.25 ml; a 1:1 [vol/vol] mixture of Dow Corning silicone oils 510 [50cs] and 550) in a narrow plastic centrifuge tube (0.4 ml; VWR Scientific, West Chester, Pa.).…”

Triclocarban Influences Antibiotic Resistance and Alters Anaerobic Digester Microbial Community Structure

“…(36,37) The MexAB system is able to pump antibiotics, organic dyes, detergents, and organic solvents from within a cell and can decrease bacterial susceptibility to several classes of antibiotics and TCS. (37,38) The genera Pseudomonas and Cupriavidus, along with other bacteria, are known to carry the mexB gene. (39,40) Results from Zhang et al, suggest that mexB is found on plasmids as well and may be mobile in the environment.…”

Characterization of the MSMEG_2631 Gene ( mmp ) Encoding a Multidrug and Toxic Compound Extrusion (MATE) Family Protein in Mycobacterium smegmatis and Exploration of Its Polyspecific Nature Using Biolog Phenotype MicroArray

“…Initially, intrinsic drug resistance was characterized as a passive mechanism associated with the absence of the drug target or lack of bacterial permeability (15,16), but recent studies have established that it can depend on drug efflux (46)(47)(48). Efflux pumps can contribute to acquired drug resistance due to overexpression (24,49) and to MDR (24,50,51).…”