Mode of action of the dual-action cephalosporin Ro 23-9424.

Ro 23-9424 is a broad-spectrum antibacterial agent composed of a cephalosporin and a quinolone moiety. Its biological properties were compared with those of its two components and structurally related cephalosporins and quinolones. Like ceftriaxone and cefotaxime but unlike its decomposition product, desacetyl cefotaxime, Ro 23-9424 bound at less than or equal to 2 micrograms/ml to the essential penicillin-binding proteins 1b and 3 of Escherichia coli and 1, 2, and 3 of Staphylococcus aureus. In E. coli, Ro 23-9424 produced filaments exclusively and decreased cell growth; cefotaxime produced both filaments and lysis. Like its decomposition product fleroxacin but unlike quinolone esters, Ro 23-9424 also inhibited replicative DNA biosynthesis in E. coli. In an E. coli strain lacking OmpF, growth continued after addition of Ro 23-9424, decreased after addition of cefotaxime, and stopped immediately after addition of fleroxacin. The results, together with the chemical stability of Ro 23-9424 (half-life, approximately 3 h at pH 7.4 and 37 degrees C), suggest that in E. coli the compound acts initially as a cephalosporin with intrinsic activity comparable to that of cefotaxime but with poorer penetration. Subsequent to the decomposition of Ro 23-9424 to fleroxacin and desacetyl cefotaxime, quinolone activity appears. The in vitro antibacterial activity reflects both mechanisms of action.

Radiochemical method for evaluating the effect of antibiotics on Escherichia coli biofilms.

A simple radiochemical method for evaluating the action of antibiotics on Escherichia coli cells in biofilms is reported. After growth, biofilms of E. coli ATCC 25922 on disks of urinary catheter material were suspended in fresh medium containing or lacking an antibiotic, incubated for 4 h at 37 degrees C, and pulse-labeled with [3H]leucine for 5 min. Radioactivity in trichloracetic acid-precipitable material in the biofilm and in the surrounding medium (planktonic E. coli) was then measured. Antibiotic-induced inhibition of incorporation of [3H]leucine into the cells in the biofilm was far less pronounced than incorporation into planktonic cells and, furthermore, correlated well with loss in viable counts. The method is simple, inexpensive, and extremely timesaving.

Method of evaluating effects of antibiotics on bacterial biofilm.

Antibiotics are generally not effective against organisms in exopolysaccharide biofilms. A simple method of studying the effect of antibiotics on bacteria in established biofilms is reported. Escherichia coli ATCC 25922 cells grown overnight at 37 degrees C on Mueller-Hinton agar were suspended in buffer and dispensed on 0.5-cm2 catheter disks. The disks were incubated for 1 h at 37 degrees C, washed, transferred to petri dishes containing 20 ml of broth, and incubated at 37 degrees C for 20 to 22 h, at which time thick biofilms were established. Disks were washed, placed in broth or broth containing antibiotic, and incubated at 37 degrees C for 4 h. The disks were removed, and viable counts were determined. This process was repeated at other selected time intervals (e.g., 8 and 24 h). Viable bacterial counts decreased from 10(3) to 10(4) CFU/cm2 in 24 h with 400 micrograms of amdinocillin or cefamandole per ml. A combination containing 400 micrograms of each antibiotic per ml decreased the viable counts to an undetectable level (less than 100 CFU/cm2) in 24 h. Other antibiotics and organisms were also examined in this system.

Monocyclic and tricyclic analogs of quinolones: mechanism of action.

The mode of action of Ro 13-5478 and Ro 14-9578, monocyclic and tricyclic quinolone analogs, respectively, was examined for Escherichia coli and Staphylococcus aureus. The compounds showed antibacterial activity and effects on cell morphology, replicative DNA biosynthesis, and gyrase-catalyzed DNA supercoiling that were comparable to those shown by nalidixic acid and by oxolinic acid compounds. The results suggest that their site of action is DNA gyrase and that a bicyclic quinolone nucleus is not essential for activity.

Effect of antifungal agents on lipid biosynthesis and membrane integrity in Candida albicans.

Eight antifungal agents were examined for effects on lipid biosynthesis and membrane integrity in Candida albicans. Lipids were labeled in vivo or in vitro with [14C]acetate and analyzed by thin-layer and gas chromatography. Membrane integrity was measured by a recently developed [14C]aminoisobutyric acid radiolabel release assay. The imidazole antifungal agents miconazole, econazole, clotrimazole, and ketoconazole, at concentrations inhibiting ergosterol biosynthesis (0.1 microM), decreased the ratio of unsaturated to saturated fatty acids in vivo but not in vitro. Similarly, naftifine, tolnaftate, and the azasterol A25822B, at concentrations inhibiting ergosterol biosynthesis (10, 100, and 1 microM, respectively), decreased the ratio of unsaturated to saturated fatty acids in vivo only. This suggests that the effect on fatty acids observed with ergosterol biosynthesis inhibitors may be secondary to the effect on ergosterol. With imidazoles, oleic acid antagonized inhibition of cell growth but not inhibition of ergosterol. This suggests that, with the C-14 demethylase inhibitors, decreased unsaturated fatty acids, rather than decreased ergosterol, are responsible for growth inhibition. Cerulenin, previously reported to be a potent inhibitor of both fatty acid and ergosterol biosynthesis, was found in the present study to inhibit the former (at 5 microM) but not the latter (up to 100 microM). Of the antifungal agents tested, econazole and miconazole (at 100 microM) produced complete release of [14C]aminoisobutyric acid, which is consistent with membrane damage.

Possible physiological functions of penicillin-binding proteins in Staphylococcus aureus.

There are four penicillin-binding proteins (PBPs) in Staphylococcus aureus, of which PBPs 2 and 3 are essential. Cefotaxime binds selectively to PBP 2, and cephalexin binds to PBP 3, each at its respective MIC. The morphology of S. aureus strains grown in the presence of the two antibiotics was examined by phase-contrast and scanning electron microscopy. Exposure of the cells to cefotaxime at concentrations at which it bound selectively to PBP 2 resulted in the extrusion of cytoplasm and cell lysis, whereas exposure to cephalexin at concentrations at which it bound exclusively to PBP 3 resulted in cell enlargement and the cessation of septation. The latter morphological response was very similar to that produced by norfloxacin. The results suggest that in S. aureus, PBP 2 may be the primary peptidoglycan transpeptidase, and PBP 3 may be involved in septation.

A modified radiometric assay for 3-hydroxy-3-methylglutaryl coenzyme A reductase.

A radiometric assay for measuring the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is described. The assay is based on the separation of the mevalonate product from HMG-CoA by high-voltage electrophoresis. This method is more sensitive and more specific than the NADPH-based spectrophotometric assay, and less tedious than available radiometric assays. It has been used to measure HMG-CoA reductase activity in crude extracts of Saccharomyces cerevisiae and in human skin fibroblasts.

Brain monoamine oxidase and replacement of its coenzyme flavin in rats.

The influence of 7-ethyl-8-methylflavin and 7-methyl-8-ethylflavin, vitamin-like homologues of riboflavin, on rat brain mitochondrial monoamine oxidase (MAO) was studied using tyramine as substrate. While riboflavin deficiency caused the enzyme activity to fall to 80% of norma, when 7-ethyl-8-methylflavin replaced riboflavin as the precursor of its coenzyme, it caused essentially complete loss of the enzyme activity. We showed that while 7-ethyl-8-methylflavin can serve as a coenzyme for MAO, 7-methyl-8-ethylflavin cannot serve as coenzyme for this enzyme.