Ciprofloxacin-resistant Mutants Research Articles

Escherichia coli cells resistant to the DNA gyrase inhibitor, ciprofloxacin, overproduce a 60 kD protein homologous to GroEL

Using a variety of mutagenic methods, we have generated a series of ciprofloxacin-resistant mutants derived from Escherichia coli strains which overproduce the DNA gyrase A protein. Many of these mutants are found to overexpress a 60 kD protein which is shown to be highly homologous in terms of N-terminal amino acid sequence to the E. coli heat-shock protein, GroEL. Other evidence confirms that the 60 kD protein is unrelated to DNA gyrase and is similar, but not identical, to GroEL.

In vitro emergence of quinolone-resistant mutants of Escherichia coli, Enterobacter cloacae, and Serratia marcescens

Norfloxacin- and ciprofloxacin-resistant mutants of several Enterobacter cloacae and Serratia marcescens isolates occurred at frequencies of greater than or equal to 10(-7)/CFU, which were higher than those of Escherichia coli isolates, in accordance with the increasing emergence of less-susceptible or resistant strains in clinical isolates of E. cloacae and S. marcescens.

Factors influencing the accumulation of ciprofloxacin in Pseudomonas aeruginosa

Ciprofloxacin accumulation in Pseudomonas aeruginosa was measured by a bioassay. Drug accumulation in strain PAO2 was compared with that of three spontaneous ciprofloxacin-resistant mutants selected with 0.5 micrograms of ciprofloxacin per ml. PAO4701 cfxA2 contains a mutation in the gyrA gene, PAO4742 cfxB5 may represent a permeability mutant based on pleiotropic drug resistance, and PAO4700 cfxA1 cfxB1 contains both types of mutations. In all strains, drug accumulation was similar, reaching steady state during the first minute of exposure. Drug accumulation was unsaturable over a range of 5 to 80 micrograms/ml, suggesting that ciprofloxacin accumulates by diffusion in P. aeruginosa. Although all four strains accumulated two- to sevenfold more ciprofloxacin in the presence of the inhibitor carbonyl cyanide m-chlorophenylhydrazone, the cfxB mutants accumulated two- to fourfold less drug than either PAO2 or the cfxA2 mutant. Polyacrylamide gel analysis revealed a protein common to cfxB mutants only, while all strains had similar lipopolysaccharide profiles. The results suggest that ciprofloxacin accumulation in P. aeruginosa is a complex phenomenon that may be affected by both an energy-dependent drug efflux process and outer envelope composition.

Genetic and physiological characterization of ciprofloxacin resistance in Pseudomonas aeruginosa PAO.

Spontaneous ciprofloxacin-resistant mutants of Pseudomonas aeruginosa PAO2 were isolated on ML agar containing 0.5 microgram of ciprofloxacin per ml (2 times the MIC). The mutants were 8- to 64-fold more resistant to ciprofloxacin and showed complete cross resistance to nalidixic acid, ofloxacin, enoxacin, and norfloxacin. Two chromosomal resistance genes, cfxA and cfxB, were mapped between eda-9001 and phe-2 and near pyrB52 distal to proC130, respectively. The cfxB mutation was identical to a nalB mutation and conferred cross resistance to novobiocin, tetracycline, carbenicillin, and chloramphenicol, suggesting that there is an effect on permeability. DNA gyrase A and B subunits were purified from strain PAO2 (wild type), PAO236 nalA2, PAO4704 cfxA2, and PAO4700 cfxA1 cfxB1. Inhibition of gyrase-mediated DNA supercoiling by ciprofloxacin or nalidixic acid was greatly reduced in preparations derived from each of the mutants. Inhibition studies on reconstituted heterologous gyrase subunits showed that decreased inhibition was dependent on the mutant gyrase A subunit. We conclude that ciprofloxacin resistance in P. aeruginosa PAO2 can occur by mutation in the nalB gene or the gene for DNA gyrase A (formerly nalA).