Analysis of airborne actinomycete spores with fluorogenic substrates

Abstract

The reactions between seven fluorogenic substrates and different groups of enzymes, esterases, lipases, phosphatases, and dehydrogenases, were studied in a search for a new method for the detection of actinomycete spores. Fluorescence measurement was chosen as a fast and sensitive method for microbial analysis. The focus of the research was on the spores of important air contaminants: Streptomyces albus and Thermoactinomyces vulgaris. For the measurement of the enzymatic activity, the chosen fluorogenic substrate was added to a mixture of spores and nutrient media, and the resulting fluorescence was measured with a spectrofluorometer. Fluorogenic substrates were found to show enzymatic activities even for dormant spores. Comparison of the enzymatic activities of dormant spores with those of vegetative cells showed similarity of the enzymatic profiles but higher activity for vegetative cells. The increase of enzymatic activity from dormant spores to vegetative cells was not linear but fluctuating. The largest fluctuations were found after 4 to 5 h of incubation. The enzymatic activities of S. albus were 10 to 50 times lower than those of T. vulgaris, except for the dehydrogenase activity, which was seven times higher. These results indicate that analysis with fluorogenic substrates has the potential for becoming a fast and sensitive method for the enumeration and identification of airborne actinomycete spores.

FIG. 1

Enzymatic activities of S. albus and T. vulgaris spores with seven different fluorogenic substrates during spore development. Measurements at time zero were with dormant spores; other measurements were with activated spores after 2, 4, and 6 h of incubation. Error bars indicate the standard deviations from the means of four repeats. Statistical analysis with analysis of variance and Scheffe’s test indicated that a < b < c (P < 0.05).

FIG. 2

Lipase (FDPr) and dehydrogenase (RSZ) enzymatic activities of T. vulgaris measured every 30 min. The enzymatic activity of dormant spores is seen at t = 0; that of activated spores is seen at t = 0.5 h. The measurements between 1 and 5 h show the enzymatic activity during the spore development from activated spore status toward the vegetative cell status. Error bars indicate the standard deviations from the means of four repeats. Statistical analysis with analysis of variance and Scheffe’s test indicated that a < b (P < 0.05).

FIG. 3

Enzymatic activities of dormant spores and vegetative cells of S. albus. Error bars indicate the standard deviations from the means of four repeats.

FIG. 4

Enzymatic activities of dormant spores and vegetative cells of T. vulgaris. Error bars indicate the standard deviations from the means of four repeats.

FIG. 5

Background fluorescence and total fluorescence (background plus fluorescence resulting from enzymatic activity) of dormant spores of S. albus. Error bars indicate the standard deviations from the means of four repeats.

FIG. 6

Background fluorescence and total fluorescence (background plus fluorescence resulting from enzymatic activity) of dormant spores of T. vulgaris. Error bars indicate the standard deviations from the means of four repeats.