Spontaneous ciprofloxacin-resistant mutants of Pseudomonas aeruginosa PAO2 were isolated on ML agar containing 0.5 ,g 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), PA0236 nalA2, PA04704 cfxA2, and PA04700 cfxAl cfxBl. 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 naL4).

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 ,ug of ciprofloxacin per ml. PA04701 cfxA2 contains a mutation in the gyrA gene, PA04742 cfxBS may represent a permeability mutant based on pleiotropic drug resistance, and PA04700 cfxAl cfxBl 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 ,ug/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.Ciprofloxacin is a highly active carboxy-quinolone antimicrobial agent that is therapeutically effective against infections caused by either gram-positive or gram-negative bacteria including Pseudomonas aeruginosa. The bactericidal activity of the drug has been attributed to the inhibition of DNA gyrase (21), an enzyme active in the replication, repair, and transcription of bacterial DNA (11). In gram-negative organisms, chromosomal mutations affecting DNA gyrase cause reduced susceptibility to quinolones (3,19,(21)(22)(23)35). However, quinolones must penetrate the cell envelope before they can act on their intracellular target. Recent investigations have suggested that the hydrophilic quinolones including ciprofloxacin permeate the outer membranes of gram-negative bacteria through water-filled channels created by porin proteins (21). The principal porin in P. aeruginosa, porin F, produces channel openings that are substantially smaller than those of other gram-negative bacteria (15,40). This confers intrinsically lower permeability to hydrophilic antibiotics, which may account for the relatively lower susceptibility of P. aeruginosa to quinolones. We and other investigators have observed that chromosomal mutations which are associated with pleiotropic drug resistance and altered permeability further enhance quinolone resistance in P. aeruginosa (8,19,35). One such mutation is cfxB, which recently was described by this laboratory (35).This study examines ciprofloxacin accumulation in P. aeruginosa and the nature of cfxB mutations in strain PAO2. We provide evidence that (i) ciprofloxacin accumulates in P. aeruginosa by diffusion; (ii) disruption of membrane potential or electron transport or both promotes drug accumulation; (iii) drug accumulation in cfxB mutants is less than in other ...

The Escherichia coli gyrase A gene was cloned in the broad-host-range cosmid vector pLA2917. The resulting plasmid, pNJR3-2, conferred quinolone susceptibility on a gyrA mutant of E. coli. To analyze the expression of this E. coli gene in Pseudomonas aeruginosa, pNJR3-2 or pLA2917 was mobilized via conjugation into P. aeruginosa PAO2 and several well-characterized quinolone-resistant mutants of this strain. The vector pLA2917 did not significantly affect the quinolone susceptibilities of any of the P. aeruginosa strains. However, pNJR3-2 conferred wild-type quinolone susceptibility on P. aeruginosa cfxA (gyrA) mutants and intermediate quinolone susceptibility on cfxA-cfxB double mutants of P. aeruginosa. The quinolone susceptibility of P. aeruginosa PAO2 gyrA + was unaffected by pNJR3-2. Also, pNJR3-2 had no significant effect on P. aeruginosa cfxB (permeability) mutants. These results demonstrate that the DNA gyrase A gene from E. coli is expressed in P. aeruginosa and confers dominant susceptibility on gyrA mutants. Thus, pNJR3-2 can be used to detect the quinolone resistance mutations that occur in the gyrase A gene of this organism. pNJR3-2 also appears to discriminate between mutations in gyrA and mutations which alter permeability. This gyrase A probe was used successfully in the analysis of quinolone resistance in clinical isolates of P. aeruginosa.The newer 4-quinolones are highly effective as therapeutic agents because of their extreme potencies and broad spectra of activity. These agents are indicated for the treatment of infections caused by gram-positive or gram-negative bacteria, including Pseudomonas aeruginosa. The bactericidal activities of 4-quinolones have been attributed to the inhibition of DNA gyrase (9, 14, 15, 24), a type II topoisomerase which is unique to bacteria. DNA gyrase, which is essential for the replication of bacterial DNA, is involved in DNA repair, transcription, and recombination (8). The gyrase holoenzyme is composed of two dimeric subunits. The A subunit, which is susceptible to quinolone inhibition, mediates breakage and reunion of double-stranded DNA (9, 29), while the B subunit, which is susceptible to novobiocin and coumermycin, is the site of ATP hydrolysis (28). The holoenzyme, using energy supplied by ATP, converts relaxed DNA to the supercoiled form (5).Analyses of quinolone-resistant mutants of P. aeruginosa by this laboratory (3, 24) and others (13,15,23) have identified three classes of mutation which reduce susceptibilities to 4-quinolones. cfxA (gyrA) mutations alter the A subunit of DNA gyrase and result in an 8-to 16-fold reduction in susceptibility to 4-quinolones. Two other classes of mutation appear to affect quinolone permeation. cfxB (3, 24) mutations reduce quinolone susceptibility by four-to eightfold and also reduce susceptibility to novobiocin, carbenicillin, chloramphenicol, and tetracycline. The nfxB mutation (13), which is distinct from cfxB, decreases quinolone susceptibility by 4-to 16-fold, has no effect on tetracycline or chloramphenicol susc...

Ciprofloxacin, one of the most active new quinolone antimicrobial agents, is bactericidal against a broad spectrum of gram-positive and gram-negative microorganisms, including Pseudomonas aeruginosa. Although P. aeruginosa is intrinsically less susceptible to most antibiotics than other clinically significant gram-negative organisms, 90% of P. aeruginosa strains are inhibited by ciprofloxacin at 0.5 ,ug/ml (7).There have been reports of decreased susceptibility to ciprofloxacin during therapy in clinical studies of P. aeruginosa infections (9, 10, 27, 35). These reports did not specify whether the decrease in susceptibility was due to the selection of naturally occurring resistant strains or the mutation of the original strain to a resistant variant.Kaatz and Seo (24), however, have described a P. aeruginosa strain whose identity was based on serotype, pyocin type, and plasmid profile. The MIC of ciprofloxacin for this strain increased from 0.57 to 5.42 ,ug/ml during parenteral therapy, with no significant change in susceptibility to other antibiotics. Resistance involved a decrease in the sensitivity of DNA synthesis to inhibition by ciprofloxacin, but the basis of this decreased sensitivity was not investigated further. Ogle et al. (31) analyzed 25 pairs of pre-and posttherapy isolates of P. aeruginosa from patients treated with imipenem, norfloxacin, or ciprofloxacin. Southern hybridization showed clonal identity between 23 of the 25 paired isolates, confirming the development of quinolone resistance rather than superinfection in these strains. The mechanism of this acquired resistance was not studied.Quinolones interfere with the activity of DNA gyrase (12,39), an essential bacterial topoisomerase that converts relaxed DNA to the supercoiled form. DNA gyrase has been isolated from a variety of bacteria (3,11,26,40) including P. aeruginosa (28). In vitro studies with P. aeruginosa PAO have shown that alterations in DNA gyrase (gyrA cfxA nalA mutations) or permeability (cfxB nfxB) can result in de-* Corresponding author. creased susceptibility to quinolones, including ciprofloxacin (32), norfloxacin (20), and nalidixic acid (22).To further understand the factors which mediate quinolone susceptibility in P. aeruginosa, we studied three clinical isolates of P. aeruginosa from a patient with a complicated infection. The MIC of ciprofloxacin increased from 0.5 ,ug/ml at the initiation of parenteral ciprofloxacin therapy to 16 ,ug/ml during therapy. All isolates were verified as the same strain by Southern hybridization with a strainspecific P. aeruginosa DNA probe (John Ogle, University of Colorado School of Medicine, Denver). To characterize the mechanisms involved in the acquisition of resistance to ciprofloxacin in vivo, we examined the outer membrane proteins (OMPs), lipopolysaccharide (LPS) content, antimicrobial susceptibilities, accumulation of ciprofloxacin, and DNA gyrase of these isolates. We provide evidence that the acquired resistance to quinolones in this clinical strain is primarily due to an altera...

Bacteriophage CP-51, a generalized transducing phage for Bacillus anthracis, B. cereus, and B. thuringiensis, mediates transduction of plasmid DNA. B. cereus GP7 harbors the 2.8-megadalton multicopy tetracycline resistance plasmid, pBC16. B. thuringiensis 4D11A carries pC194, the 1.8-megadalton multicopy chloramphenicol resistance plasmid. When phage CP-51 was propagated on these strains, it transferred the plasmid-encoded antibiotic resistances to the nonvirulent Weybridge (Sterne) strain of B. anthracis, to B. cereus 569, and to strains of several B. thuringiensis subspecies. The frequency of transfer was as high as 10-5 transductants per PFU. Tetracycline-resistant and chloramphenicol-resistant transductants contained newly acquired plasmid DNA having the same molecular weight as that contained in the donor strain. Antibiotic-resistant transductants derived from any of the three species were effective donors of plasmids to recipients from all three species.