Fluorescence Assay for Evaluating Microbicidal Activity of Hand Antiseptics

Abstract

We developed a fluorescent ␤-D-glucuronidase activity (BGA)-based assay for detecting and quantifying Escherichia coli in samples to assess the biocide efficacy of hand antiseptics. The fluorescence level is proportional to the number of viable E. coli organisms present. We compared our assay results to those of the E. coli plate count method specified by the European standard for testing hygienic hand rub disinfectant products (EN1500). The plate count method requires excessive handling and materials and is not valid if the number of organisms per plate is too low or high for counting in many of the samples. We optimized the fluorescent assay based on the cleavage of 4-methylumbelliferyl-␤-D-glucuronide by adding 4-nitrophenyl-␤-D-glucuronide, a nonfluorogenic BGA substrate, to induce glucuronidase activity and reduce assay time. Furthermore, our method can be automated and eliminates the need for multiple dilutions. Fluorescence was temporally monitored, and the time required to reach a specific value of fluorescence was correlated with the initial number of viable E. coli organisms on the samples. There was a positive correlation (P < 0.05) with a high correlation coefficient (R 2 ‫؍‬ 0.82) between the E. coli counts by plate count and fluorescence methods. Reported effects in fluorescent BGA were compared to the EN1500 plate count method with five hand disinfectants. We found our method more advantageous, because it was as sensitive as the EN1500 method, requires less time to complete, and is less expensive and less laborious than conventional plating techniques.

Progress in methods for the detection of viable Escherichia coli

“…These metabolites can be esters, enzymes, acids, gases, etc ., which are produced when the bacteria survive. According to the reported studies related to the detection of viable E. coli , the main cellular metabolites used are β-galactosidase (GALase), 68–70 β-glucuronidase (GLUase), 71–75 resorufin, 76–80 dehydrogenase, 81 menadione oxidoreductase, 82 ATP, 83,84 endotoxin, 85 catalase, 86 dimethyl disulfide, 87 etc ., which can realize the detection of 1–10 CFU viable E. coli . In addition, the metabolic activity of certain microorganisms alters the pH of the culture medium, enabling the detection and measurement of viable E .…”

“…Lopez-Gigosos et al developed a GLUase-based fluorescence assay to detect and quantify E. coli in samples to evaluate the insecticidal efficacy of hand sanitizers. 73 Togo et al used a GLUase-based biosensor that could detect 2 × 10 −2 CFU mL −1 of viable E. coli in contaminated water samples in less than 20 min, which was 100 times more sensitive than the spectrophotometric method. 74 Burnet et al established an online fluorescence-based method for measuring GLUase activity for rapid monitoring of E. coli in field water, which was confirmed by an automated microbial detection system.…”