Ethambutol inhibited the growth of acid-fast bacteria and enhanced the detection of Legionella in environmental water samples.

The growth of acid-fast bacteria often hinders the detection of Legionella in water samples on agar plates by the plate culture method. We studied whether anti-tubercular agents inhibit acid-fast bacteria growth on agar plates. First, the antimicrobial activities of isoniazid, ethionamide, and ethambutol were evaluated against Mycobacterium and Legionella. We found that ethambutol at ≥ 100 µg/mL completely inhibited Mycobacterium growth, but ethambutol at 1,000 µg/mL did not inhibit Legionella growth. Next, the effect of ethambutol dissolved in acid buffer was examined. Cell suspensions of L. pneumophila and Mycobacterium spp. were mixed, and ethambutol-acid buffer was added. After 5 min, mixtures were inoculated on GVPC agar plates and incubated at 36℃ for 6 d. We found that ethambutol inhibited Mycobacterium growth on agar plates, but the Legionella colonies recovered. The effect of ethambutol was also significant in the evaluation using bathwaters. Comparing 1,302 bathwaters, the addition of ethambutol reduced the detection rate of acid-fast bacteria from 30.6% to 0% and increased the detection rate of Legionella from 7.1% to 7.5%. Ethambutol, which selectively inhibited acid-fast bacteria growth, enhanced the detection of Legionella on agar plates and will contribute to improving the accuracy of Legionella testing by the plate culture method.

Single-cell chemical lysis method for analyses of intracellular molecules using an array of picoliter-scale microwells.

Analyzing the intracellular contents and enzymatic activities of single cells is important for studying the physiological and pathological activities at the cellular level. For this purpose, we developed a simple single-cell lysis method by using a dense array of microwells of 10-30-pL volume fabricated by poly(dimethylsiloxane) (PDMS) and a commercially available cell lysis reagent. To demonstrate the performance of this single-cell lysis method, we carried out two different assays at the single-cell level: detection of proteins by antibody conjugated microbeads and measurement of protease activity by fluorescent substrates. The results indicated that this method readily enabled us to monitor protein levels and enzymatic activities in a single cell. Because this method required only an array of PDMS microwells and a fluorescence microscope, the simplicity of this platform opens a way to explore the biochemical characteristics of single cells even by those who are not familiar with microfluidic technology.