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 ...

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...

A total of 19 strains of oral Cytophaga sp. obtained from subgingival plaque deposits were tested for their ability to coaggregate with strains of Actinlc)nyes israelii, A. viscosus, A. naesllundii, Streptococclus sanguis, S. miltans, S. salivarills, and S. mitis. Coaggregation was observed only with A. israelii. Based on their coaggregation patterns with eight A. israelii strains, the Cvtophaga strains were distributed among three distinct groups: those that coaggregated with A. israelii PK16 but not with A. israelii W1011 (ATCC 29322), those that coaggregated with A. israelii ATCC 29322 but not with A. israelii PK16, and those that coaggregated with none of the eight A. isr-aelii strains. In each of the coaggregations, prior heat treatment (85°C, 30 min) of the Cytophaga cells prevented coaggregation, whereas identical treatment of the A. israelii cells had no effect. The ability of A. isr-aelii PK16 to form adherent plaque on a tooth surface previously coated with Cytophaga plaque was tested with one of the coaggregating Cytophaiga strains. White patches of A. israelii plaque were found covering both the amber-colored Cytophaga plaque on the cementum surface as well as the enamel surface to which Cytophaga strains do not adhere. Electron micrographs of thin-sectioned mixed-plaque material revealed both cell types in close proximity. In addition, electron micrographs of negatively stained coaggregated cells showed interbacterial adherence between surface fimbrae on A. israelii and outer membrane blebs on the gram-negative Cytophaga sp. The kinetics of binding of A. i.sraelii to spheroidal hydroxyapatite and to root powder were indicative of a high-affinity binding system with comparatively large numbers of available binding sites on both substrata. These results indicate the highly specific nature of Cvtophaga sp.-A. israelii recognition. The contribution of such recognition toward the mechanisms that are responsible for the indigenous nature of these oral bacteria is discussed.

By using in vitro assays, a group of related, filamentous gram-negative bacteria isolated from subgingival plaque deposits of patients with periodontal disease were found to colonize intact teeth. Tentatively identified as members of the genus Cytophaga, these isolates exhibited a preference for colonizing the cementumr surface of the root. Examination of intact teeth after several weeks of colonization revealed that the root substructure had been extensively demineralized.

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