The Effect of Ofloxacin on Production of Exoenzymes by Pseudomonas aeruginosa

Abstract

Exoenzymes produced by Pseudomonas aeruginosa are known for their important virulence factors. Some studies have suggested pathogenic roles for P. aeruginosa exoenzymes in animal infection models. The effects of sub inhibitory concentrations of antibiotics on P. aeruginosa exoenzyme expression have been examined. Tetracycline inhibits protease production at subinhibitory concentrations (Shibl & AI-Sowaygh 1980). When present at sub-MICs, ciprofloxacin, tobramycin and ceftazidime are all able to suppress production of exoenzymes in vitro (Grimwood et al. 1989). Growth of P. aeruginosa in broth is not significantly atTected during 24-hour culture with subMICs of erythromycin, although the production of elastase, protease and leucocidin is inhibited by subMICs of this agent (Kita et al. 1991). In this study, the etTect of ofloxacin on the production of exoenzymes by P. aeruginosa was evaluated and compared with the etTect of minocycline, gentamicin and piperacillin. The P. aeruginosa strain used in this study was the B16 strain isolated from human blood, and the MICs of ofloxacin, minocycline, gentamicin and piperacillin against this strain were 1, 16, 4 and 4 mg/L, respectively. After 24-hour culture of P. aeruginosa with each concentration (1 MIC, 1/5 MIC, 1/10 MIC, 1/20 MIC) of antibiotics, the activities of exoenzymes were assayed and the growth of variable cells was examined in each culture broth. For assay of total protease activity, P. aeruginosa was grown in brain heart infusion broth containing antibiotics at 37°C for 24 hours. The culture supernatant was added to hide powder azure substrate and the mixture was incubated at 37"C for I hour. Undissolved substrate was removed by centrifugation. Total protease activity was determined by comparing the A595 values of the supernatants with those of a standard curve prepared by using purified P. aeruginosa protease. The method for determining elastase activity was the same as that for total protease, except that elastin Congo red substrate was used, incubation time was 2 hours, and the absorbance value was A495. Values were compared with those of a standard curve prepared by using purified P. aeruginosa elastase (Grimwood et al. 1989). For assay of phospholipase C activity, P. aeruginosa was grown in tryptose minimal medium containing antibiotics at 32°C for 24 hours. Decolourising carbon was added to the culture supernatant. After centrifugation, the supernatant was added to a solution containing Tris butTer, glycerol, ZnCl2 and p-nitrophenylphosphorylcholine in individual wells ofa 96-well microdilution tray. These were incubated at 37"C for I hour and phospho-