The Effect of Amiloride on Pigment Expression in a Clinical Isolate of Pseudomonas Aeruginosa

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Pigment expression by Pseudomonas aeruginosa may enhance the virulence of pulmonary infections. The diuretic amiloride, currently under investigation as adjunct aerosol therapy in cystic fibrosis (CF), appears to qualitatively diminish pigment expression in some isolates in vitro. This study was performed to determine the effect of amiloride in vitro on quantitative pigment expression. The minimum inhibitory concentration of amiloride against a CF clinical isolate of P. aeruginosa that produces pigment was initially determined by broth dilution technique. A standard inoculum was incubated for 0 to 50 hours at 37 degreeC both in Mueller-Hinton broth alone and with subinhibitory concentrations of amiloride. Pigment was extracted with chloroform and 0.2 N hydrochloric acid sequentially after incubation. Pigment expression was measured by optical density at 520 nm. Growth was determined by optical density at 650 nm or as grams of bacterial dry weight. Pigment expression as a function of growth rate for control and amiloride-treated cells was identical over the first 7.25 hours. By 21.25 hours, both growth and pigment expression in amiloride-treated cells began to taper. The mean ± SEM pigment production under control conditions was 2.85 ± 0.26; OD 520 nm/gm of bacterial dry weight (n=22). At 100 and 300 µg/ml amiloride pigment production was 1.68 ± 0.39, P=0.032 and 0.96 ± 0.20, P=0.0004, respectively. The results indicate that subinhibitory amiloride can suppress pigment expression in one clinical isolate of P. aeruginosa in vitro.

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