Zastosowanie testu LAL do ilosciowego oznaczania endotoksyny Bacteroides fragilis

The aim of this study was the evaluation of LAL test with chromogenic substrate usefulness for the quantitative detection of B. fragilis endotoxin and the determination of the amount of endotoxin in culture filtrates of the strains of this species. Also, the trial was undertaken to determine the influence of clindamycin un endotoxin release from B. fragilis rods to the culture medium. Four B. fragilis strains were examined: one nonenterutoxigenic (NTBF) and three enterotoxigenic (ETBF). The growth of cultures was determined and endotoxin liberated to the culture medium during growth of strains was detected. BHI broth and BHI broth with addition of sub inhibitory doses (sub-MIC) of clindamycin were applied. Bacterial cultures were incubated for 48 hours at 37°C. Samples of bacterial cultures were collected after 4, 8, 16, 24 and 48 hours of cultivation, and the optical density was measured. Then the samples were centrifuged, supenatants were filtered through 0.45 µm filters and concentrated three times with 5000 D ultrafilters. Prepared samples were kept frozen at -70°C until used. The amount of endotoxin in samples was determined using quantitative LAL test with chromogenic substrate S- 2423. The results of the experiments indicate that LAL, test is the useful method for determination of B. fragilis endotoxin concentration. This endotoxin activates the enzymatic system present in Limulus polyphemus amcbocyte lysate. Endotoxin is shed spontaneously by B. fragilis rods to the culture medium during growth. Clindamycin at subinhibitory concentrations (sub-MIC) inhibits the growth of cultures of examined strains. The antibiotic caused increase in endotoxin amount in culture medium.