Antibiotic inhibition of protease production by Pseudomonas aeruginosa

Abstract

P S E U D O M O N A S AERUGZNOSAis an unusual gram-negative bacillus in that many of its pathogenic effects are believed to be caused by extracellular products (haemolysin, lecithinase, toxin and protease) and not by the so-called endotoxin moiety (Liu, Abe and Bates, 1961;Liu, 1974).Previous results (Meinke et al., 1970;Homma et al., 1975;Gray and Kreger, 1979) indicate that P. aeruginosa protease may be important in the pathogenesis of pseudomonas pneumonia. Homma et al. (1975) observed that the sera Of patients contained elevated titres of antibody to pseudomonas protease. Meinke et al. (1970) reported that intranasal administration of P. aeruginosa protease to mice produced focal and confluent haemorrhagic lung lesions, macroscopically identical to those seen in human patients (Shimizu et al., 1974). Gray and Kreger (1979) considered that protease was at least partially responsible for the lung damage in pseudomonas pneumonia.Because of the possible pathogenic importance of pseudomonas protease we investigated the in-vitro ability of antibiotics, in concentrations too low to affect greatly the growth and survival of P. aeruginosa, to suppress extracellular protease production. MATERIALS AND METHODSBacterial strain. P. aeruginosa strain SAR was isolated from a human pneumonia specimen at King Abdul Aziz University Hospital. It produced pyocyanin and was identified by the method of Buchanan and Gibbons (1974). This strain was the best producer of extracellular protease among 20 strains tested by the method described below.Culture methods and sampling. The kinetics of growth and protease production were followed for 24 h in 250-ml cultures in trypticase soy broth (TSB). The antibiotic solutions were added at the beginning of the experiment or at various times during incubation. The flasks were seeded with an overnight TSB culture grown at 37°C to give a final concentration of approximately lo5 viable organisms/ml, and were incubated at 37°C on an orbital shaker (Gallenkamp) at 140 r.p.m. Optical densities were obtained by removing aseptically 2-ml volumes of culture and reading the optical density (OD) at 490 nm in a Beckman spectrophotometer 20. Culture supernates were obtained from these samples by centrifugation at 10 000 g for 20 min. The supernates were sterilised by filtration and then used for protease assay.Assay ofprotease was by the method of Brown and Foster (1970) with agar containing 15% (v/v) sterile skimmed milk. In preliminary experiments, wells 4,9 and 12 mm in diameter were filled with serial twofold dilutions of supernate from a 24-h culture. The plates weremaintained at room temperature for 48 h. The wells 9 and 12 mm in diameter were equally satisfactory for detecting the smallest concentrations of protease. Wells 9 mm in diameter were used in all subsequent experiments. The titre was the highest dilution that produced clearing around the wells.Test for DNA. The method of Burton (1956) was used. Extraction of intracellularprotease. Ethylenediamine tetraacetate-lysozyme spheroplasts of P....

Subinhibitory Clindamycin Differentially Inhibits Transcription of Exoprotein Genes in Staphylococcus aureus

“…The clindamycin effect has been observed for extracellular proteins in a variety of organisms, including streptolysin S and M protein production by Streptococcus pyogenes (9,37) and extracellular lipase production by Propionibacterium acnes and P. granulosum (39). Similar effects have been observed for tetracycline and aminoglycosides, which down-regulate protease production by Pseudomonas aeruginosa (36) and hemolysin production by Escherichia coli (38). In some cases, disparate results have been reported by different investigators, which may well represent interstrain differences.…”

Inhibition of Pseudomonas aeruginosa exoenzyme expression by subinhibitory antibiotic concentrations

“…These results support and extend the observations of other workers who have reported the inhibition of exoenzyme function in the presence of antibiotics. Protease secretion, including elastase, has been suppressed in vitro by sub-MICs of ciprofloxacin (7), gentamicin (30), tobramycin (11,30), azlocillin (11), ceftazidime (11), and tetracycline (27). Exotoxin A production in resistant isolates of P. aeruginosa has also been inhibited in vitro by ciprofloxacin (7).…”

Isolation of a broad spectrum antibiotic producer bacterium, Pseudoalteromonas piscicida PG-02, from the Persian Gulf

“…Serial samples were harvested from production medium at 2 hours intervals from 0 till 6 hours and 4 hours intervals from 6 hours till entering stationary and death phases of growth. Supernatants were harvested from these samples by centrifugation at 8,000 rpm at 4°C for 10 min and subsequently were sterilized by filtration 0.45 µm and then used for antibacterial assay (Shibl and Sowaygh, 1980). Antibiotic activity of samples was assessed against MRSA using disc diffusion method.…”