生物発光法による抗らい菌活性測定法の開発 [in Japanese] Basic evaluation for new antimicrobial susceptibility testing of Mycobacterium leprae by bioluminescence assay (ATP method) [in Japanese]
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Antimicrobial susceptibility testing of <i>Mycobacterium leprae</i> by non-radioactive bioluminescence assay was developed. Optimization of the assay conditions such as temperature and time for ATP extraction, bacteria dose, preparation of bacteria suspension and pH of culture medium was carried out using <i>M. leprae</i> Thai 53 strain. Samples of bacterial suspension of <i>M. leprae</i> were first treated with filamentous cell treatment reagent at room temperature for 30 minutes and ATP was extracted from the leprosy bacilli by heating at 60 degrees for five minutes. Luciferin luciferase was added to the extract after cooling to room temperature followed by measurement of relative light units (RLU) of each sample using a luminometer. The concentrations of the drugs used for the evaluation of antimicrobial activities of rifampin (RFP), clofazimine (CLF), ofloxacin (OFLX) and clarithromycin (CAM) were 0.125, 0.50, 2.0 and 8.0μg/ml respectively. Middlebrook 7H9 broth medium was used (pH6.6) as the basal medium and the bacilli were cultivated at 32°C for 0-14 days. ATP was extracted from 0.1ml of culture suspension and inhibition of the luminescent activity was calculated. The results were compared to that obtained by radio-active CO<sub>2</sub> detection system, Buddemeyer method which is commonly used for measuring anti-<i>M. leprae</i> activity.<br>There was a good correlation between the results obtained by ATP method on the tenth day of culture and the results obtained by Buddemeyer method on the seventh day of culture. ATP method may be useful for the determination of drug susceptibility of <i>M. leprae</i>.
- Japanese journal of leprosy
Japanese journal of leprosy 75(3), 227-237, 2006-10-01
Japanese Leprosy Association