Differences in susceptibility to quinolones of outer membrane mutants of Salmonella typhimurium and Escherichia coli

Abstract

The mechanism of penetration of quinolones through the bacterial outer membrane was studied with lipopolysaccharide-deficient and porin-deficient mutants. The data indicated that the lipopolysaccharide layer might form a permeability barrier for hydrophobic quinolones such as nalidixic acid but not for hydrophilic quinolones such as norfloxacin and ciprofloxacin. The results also showed that quinolones with a low relative hydrophobicity appeared to permeate through OmpF porin, whereas quinolones with a high relative hydrophobicity appeared to permeate through both OmpF porin and phospholipid bilayers.New quinolone derivatives which have broader and more potent antibacterial activity against gram-negative and grampositive bacteria than old quinolones, such as nalidixic acid, have recently been developed. They also have greater antibacterial activity against nalidixic acid-resistant bacteria (5,7,9,21,24,25).We previously reported that the greater antibacterial activity of norfloxacin, one of these new quinolones, might be due to its greater ability to permeate bacterial cells and its potent inhibitory action on in vivo DNA synthesis (5).The ability of drugs to pass through the bacterial outer membrane is a very important factor in their antibacterial activity and spectrum (2,10,13,14,22,23,26). There are many reports on the penetration into bacterial cells of various antimicrobial agents, especially beta-lactam antibiotics (4,8,15,22,23,26), but the penetration mechanisms of quinolones have not been studied in detail.We report here on the mechanisms of penetration of quinolones through the bacterial outer membrane with lipopolysaccharide ( ofloxacin were from Daiichi Pharmaceutical Co. Ltd.; pipemidic acid, piromidic acid, and enoxacin were from Dainippon Pharmaceutical Co. Ltd.; and ciprofloxacin was from Bayer Yakuhin Co. Ltd. The partition coefficients of the quinolones were determined by the modified method of Nikaido (13). Solutions (10 ,ug/ml) of quinolones were made in 0.1 M phosphate buffer (pH 7.2). After shaking with an equal volume of n-octanol at 25°C for 48 h and centrifuging at 1,870 x g for phase separation, the concentrations of quinolones in the aqueous phase were determined with spectrophotometric assay by measuring the A272 for enoxacin, norfloxacin, and pefloxacin, the A286 for ciprofloxacin, the A264 for pipemidic acid, the A288 for cinoxacin, the A292 for AM-833 and piromidic acid, the A294 for ofloxacin, the A264 for miloxacin, the A290 for oxolinic acid, the A258 for nalidixic acid, the A285 for rosoxacin, and the A254 for flumequine. The partition coefficients were expressed as the ratio of the amount of compound in the n-octanol phase to that in the aqueous phase.Susceptibility to quinolones was measured by the agar dilution method with Mueller-Hinton agar and an inoculum of 106 CFU per spot as described previously (7). The data were expressed as MICs.We tested the antibacterial activity of quinolones against LPS-deficient (rfa) mutants of S. typhimurium LT2 (19) ( Table 1). The suscepti...

Influence of growth media on Escherichia coli cell composition and ceftazidime susceptibility

“…The increased susceptibility of E. coli to nalidixic acid in 4% phosphate was not as obvious (a twofold reduction of the MIC), but could be seen by using the MS-2 Abbott Research System (data not shown). These data indicated that the hydrophobic antibiotic, nalidixic acid, may be able to penetrate bacteria by passing through the hydrophobic domain of the outer membrane, as was suggested in a study with an LPS-deficient mutant (13).…”

“…The hope was to change E. coli surface properties (hydrophobicity and phospholipid content) to investigate a possible nonspecific lipophilic diffusion pathway for ceftazidime. Cephaloridine, a dipolar ionic ,-lactam that penetrates readily through the water-filled hydrophilic pores of the outer membrane of gram-negative bacteria (34), and the more hydrophobic antibiotic nalidixic acid, which may diffuse through the LPS-phospholipid bilayer in a nonspecific manner (a nonporin pathway) (13), served as controls in this study.…”

Mutations producing resistance to norfloxacin in Pseudomonas aeruginosa

“…These results suggest that the nfxB gene might be a novel gene in P. aeruginosa PAO; however, further genetic studies are in progress. We found previously that the outer membrane permeability to new quinolones such as norfloxacin in E. coli and Salmonella typhimurium differed from that to nalidixic acid (12). The incomplete cross-resistance among quinolones in E. coli is known (18,41), and this phenomenon was also observed in P. aeruginosa (32).…”

“…The uptake of norfloxacin by P. aeruginosa cells was measured by the method described previously (12,15). Bacterial cells were grown at 37°C in antibiotic medium 3 (Difco), and norfloxacin was added to the bacterial culture (A570 = 0.7) to a final concentration of 10 ,ug/ml.…”

To Study the Formulation of Niosome of Ofloxacin and Its Evaluation for Efficacy of Anti-Microbial Activity

“…Ofloxacin is freely soluble in acetic acid, slightly soluble in water, methanol, ethanol or acetone. The octanol/water partition coefficient for ofloxacin was reported to be -0.48 [1,2] Ofloxacin which is the focus of this review is a second generation fluoroquinolone with a 6-fluoro substituent and a 7-piperazinyl substituent on the quinolone ring structure [11] Entrapped niosomes were prepared by hand shaking and ether injection process with different ratios of (1:1, 1:2 and 1:3) cholesterol and Span-80 (Non-ionic surfactant). The niosomes prepared were in the size of 0.5-2.5 microns in case of hand shaking process and 0.5-2.5 microns in case of Ether injection process.…”