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 β-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
References
[1]
G. P. Richards, M. A. Watson, and S. Parveen, “Development of a simple and rapid fluorogenic procedure for identification of Vibrionaceae family members,” Applied and Environmental Microbiology, vol. 71, no. 7, pp. 3524–3527, 2005.
[2]
G. P. Richards and M. A. Watson, “A simple fluorogenic method to detect Vibrio cholerae and Aeromonas hydrophila in well water for areas impacted by catastrophic disasters,” American Journal of Tropical Medicine and Hygiene, vol. 75, no. 3, pp. 516–521, 2006.
[3]
Council Directive 91/492/EEC, “Laying down the health conditions for the production and placing on the market of live bivalve mollusks,” Official Journal of the European Community, vol. L268, pp. 1–14, 1991.
[4]
“National Shellfish Sanitation Program Model Ordinance IV, Shellstock growing areas,” Department of Health and Human Services, U.S. Food and Drug Administration, Washington, DC, USA, 1999.
[5]
G. P. Richards and S. Parveen, “A survey for phosphoglucose isomerase with lysyl aminopeptidase activity in Vibrionaceae and non-Vibrio pathogens,” Biochimica et Biophysica Acta, vol. 1748, no. 1, pp. 128–133, 2005.
[6]
S. M. Holt, P. A. Hartman, and C. W. Kaspar, “Enzyme-capture assay for rapid detection of Escherichia coli in oysters,” Applied and Environmental Microbiology, vol. 55, no. 1, pp. 229–232, 1989.
[7]
J. A. Koburger and M. L. Miller, “Evaluation of a fluorogenic MPN procedure for determining Escherichia coli in oysters,” Journal of Food Protection, vol. 48, no. 3, pp. 244–245, 1985.
[8]
S. R. Rippey, L. A. Chandler, and W. D. Watkins, “Fluorometric method for enumeration of Escherichia coli in molluscan shellfish,” Journal of Food Protection, vol. 50, no. 8, pp. 685–690, 1987.
[9]
W. H. Andrews, C. R. Wilson, and P. L. Poelma, “Glucuronidase assay in a rapid MPN determination for recovery of Escherichia coli from selected foods,” Journal of the Association of Official Analytical Chemists, vol. 70, no. 1, pp. 31–34, 1987.
[10]
L. Fiksdal, M. Pommepuy, M.-P. Caprais, and I. Midttun, “Monitoring of fecal pollution in coastal waters by use of rapid enzymatic techniques,” Applied and Environmental Microbiology, vol. 60, no. 5, pp. 1581–1584, 1994.
[11]
C. H？ller, G. Havemeister, and K. O. Gundermann, “Comparison of BGB-MUG and LSTB-MUG in microbiological surveillance of recreational waters,” Zentralblatt fur Hygiene und Umweltmedizin, vol. 198, no. 2, pp. 138–151, 1995.
[12]
P. Villari, M. Iannuzzo, and I. Torre, “An evaluation of the use of 4-methylumbelliferyl- -D-glucuronide (MUG) in different solid media for the detection and enumeration of Escherichia coli in foods,” Letters in Applied Microbiology, vol. 24, no. 4, pp. 286–290, 1997.
[13]
U.S. Food and Drug Administration, “Enumeration of Escherichia coli and the coliform bacteria,” chapter 4. Bacteriological Analytical Manual, 2002, http://www.cfsan.fda.gov/~ebam/bam-4.html.
[14]
J. S. Thompson, D. S. Hodge, and A. A. Borczyk, “Rapid biochemical test to identify verocytotoxin-positive strains of Escherichia coli serotype O157,” Journal of Clinical Microbiology, vol. 28, no. 10, pp. 2165–2168, 1990.
[15]
P. S. Hayes, K. Blom, P. Feng, J. Lewis, N. A. Strockbine, and B. Swaminathan, “Isolation and characterization of a -D-glucuronidase-producing strain of Escherichia coli serotype O157:H7 in the United States,” Journal of Clinical Microbiology, vol. 33, no. 12, pp. 3347–3348, 1995.
[16]
R. Kirby and K. L. Ruoff, “Cost-effective, clinically relevant method for rapid identification of beta-hemolytic streptococci and enterococci,” Journal of Clinical Microbiology, vol. 33, no. 5, pp. 1154–1157, 1995.
[17]
B. H. Himelbloom and R. C. Pfutzenreuter, “False-positive fluorescence by pink salmon tissue and Staphylococci in a rapid test for Escherichia coli,” Journal of Food Protection, vol. 61, no. 9, pp. 1119–1123, 1998.
[18]
G. W. Chang, J. Brill, and R. Lum, “Proportion of -D-glucuronidase-negative Escherichia coli in human fecal samples,” Applied and Environmental Microbiology, vol. 55, no. 2, pp. 335–339, 1989.
[19]
E. W. Rice, M. J. Allen, and S. C. Edberg, “Efficacy of -glucuronidase assay for identification of Escherichia coli by the defined-substrate technology,” Applied and Environmental Microbiology, vol. 56, no. 5, pp. 1203–1205, 1990.
[20]
M. Killian and P. Bülow, “Rapid diagnosis of enterobacteriaceae. I. Detection of bacterial glycosidases,” Acta Pathologica et Microbiologica Scandinavica B, vol. 84, no. 5, pp. 245–251, 1976.
[21]
P. Feng and K. A. Lampel, “Genetic analysis of uidA expression in enterohaemorrhagic Escherichia coli serotype O157:H7,” Microbiology, vol. 140, no. 8, pp. 2101–2107, 1994.
[22]
S. R. Monday, T. S. Whittam, and P. C. H. Feng, “Genetic and evolutionary analysis of mutations in the gusA gene that cause the absence of -glucuronidase activity in Escherichia coli O157:H7,” Journal of Infectious Diseases, vol. 184, no. 7, pp. 918–921, 2001.
