Immunoblotting with antibody against AcrA, an obligatory component of the AcrAB multidrug efflux system, showed that this protein was overexpressed by >170% in 9 of 10 clinical isolates of Esherichia coli with high-level ciprofloxacin resistance (MICs, >32 g/ml) but not in any of the 15 isolates for which the MIC was <1 g/ml.It is well known that resistance to fluoroquinolones usually requires alteration in the genes that code for the targets of these drugs, gyrA and parC (4)(5)(6)(7)16). On the other hand, it is not clear whether these mutations alone could produce very high levels of resistance, and one of the major factors that produce such resistance is sometimes thought to be an increased drug efflux. This hypothesis has been examined in several laboratories. Thus, Piddock and associates (2) examined 36 high-level ciprofloxacin-resistant isolates of Escherichia coli and found that 22 strains accumulated lower levels of ciprofloxacin than the wild type, in addition to the gyrA mutations found in all of them. Levy and associates (12) found that 21 of 57 high-level fluoroquinolone-resistant clinical isolates of E. coli showed tolerance to cyclohexane, suggesting an elevated broad-spectrum efflux activity. Furthermore, some of the regulatory factors that may cause the overproduction of AcrAB, the major, constitutively expressed multidrug efflux pump of E. coli, were examined: in a 1996 study dealing with 23 fluoroquinolone-resistant E. coli isolates (9), 3 were shown to produce MarA, a positive regulator of acrAB transcription (1), constitutively at high levels, and 8 were shown to produce MarA to higher levels than the wild type upon "induction" with tetracycline or salicylate; and in a 1999 study of 25 fluoroquinolone-resistant, cyclohexane-tolerant strains (13), 9 were shown to have mutations in marR, the repressor for MarA expression, or in soxR, the repressor for the expression of SoxS, a close homolog of MarA. All these studies, however, were somewhat indirect in view of our knowledge that the AcrAB pump alone contributes in a decisive manner to the MarA-induced multidrug resistance of E. coli (14). Thus, the identity of the efflux pump remained unknown in the direct efflux study (2) or in the solvent resistance study (12), and the examination of mar and sox systems (9, 13) left open the question of whether other regulatory pathways were altered. We have therefore examined directly the level of expression of AcrA, one of the essential components of the AcrAB-TolC multidrug efflux system (10), by immunoblotting.The 25 clinical isolates of E. coli, randomly chosen from the isolates collected during the period of October 1995 through December 1998, were obtained from the Clinical Microbiology Laboratory of the University of Verona Hospital. Each strain was from a different patient. Their ciprofloxacin susceptibilities were tested by the broth microdilution method in Luria-Bertani broth at 37°C with a standard inoculum of 10 4 cells ml Ϫ1 . Ciprofloxacin was obtained from Sigma (St. Louis, Mo.). In terms of...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.