%0 Journal Article
%T Fluorogenic Membrane Overlays to Enumerate Total and Fecal Escherichia coli and Total Vibrionaceae in Shellfish and Seawater
%A Gary P. Richards
%A Michael A. Watson
%J International Journal of Microbiology
%D 2010
%I Hindawi Publishing Corporation
%R 10.1155/2010/910486
%X Three assays were developed to enumerate total and fecal Escherichia coli and total Vibrionaceae in shellfish, seawater, and other foods and environmental samples. Assays involve membrane overlays of overnight colonies on nonselective agar plates to detect -glucuronidase and lysyl aminopeptidase activities for E. coli and Vibrionaceae, respectively. Cellulose membranes containing the substrates 4-methylumbeferyl- -D-glucuronide (MUG) produced a bright blue fluorescence when overlaid onto E. coli, while L-lysyl-7-amino-4-trifluoromethylcoumarin produced green fluorescent foci when overlaid onto Vibrionaceae family members. A multiplex assay was also developed for simultaneously enumerating total E. coli and total Vibrionaceae in oysters and seawater. Overall, 65% of overlaid E. coli (non-O157:H7) were MUG-positive, compared with 62% as determined by the most-probable-number-MUG assay. The overlays are rapid, simple, and cost effective for quantification purposes. This research provides practical alternatives for monitoring bacterial indicators and potential pathogens in complex samples, including molluscan shellfish. 1. Introduction A technique known as the colony overlay procedure for peptidases (COPP assay) was previously developed as the first test to enumerate total Vibrionaceae in shellfish, seawater, and well water [1, 2]. This assay has been recommended for use in monitoring molluscan shellfish and for potentially regulating their harvest based on the levels of total Vibrionaceae [1]. This total Vibrionaceae approach is similar in concept to traditional sanitary surveys of shellfish harvesting areas, which are based on the total number of fecal coliforms or E. coli present in either the shellfish or their surrounding waters [3, 4]. In the original COPP assay, a substrate, L-lysyl-7-amino-4-trifluoromethylcoumarin (L-Lys-AFC), is bound to a cellulose acetate membrane and the membrane is overlaid for 10 minutes onto an overnight culture grown on nonselective agar media. A lysyl aminopeptidase that is present in all Vibrionaceae family members tested to date [5] cleaves the substrate to produce fluorescent foci on the membrane when viewed under longwave UV [1]. In the present study, we extended this overlay concept to produce similar assays for total and fecal E. coli. These assays are based on the presence of ¦Â-glucuronidase (GUD) activity, which is found in most non-O157:H7 E. coli and results in cleavage of the substrate 4-methyl- -D-glucuronide (MUG) into the fluorescent 4-methylumbelliferone. MUG-based fluorescence assays for E. coli have been
%U http://www.hindawi.com/journals/ijmicro/2010/910486/