An Alternative Method to Concentrate E. coli O157:H7 from Meat Homogenates to Facilitate Detection by Cultural and Molecular Methods

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Title: An Alternative Method to Concentrate E. coli O157:H7 from Meat Homogenates to Facilitate Detection by Cultural and Molecular Methods
Author: Taylor, Thomas Matthew
Advisors: MaryAnne Drake, Committee Co-Chair
Donn Ward, Committee Member
LeeAnn Jaykus, Committee Co-Chair
Abstract: Escherichia coli O157:H7, first identified in 1982, is linked to approximately 73,500 cases of foodborne illness per year. Sensitive and efficient methods to detect this organism are crucial. Current bacterial concentration methods from foods, such as centrifugation and filtration, are problematic, often co-precipitating or becoming clogged with food particulates. Metal hydroxide concentration of bacterial cells has gained increased recognition as a new alternative method to non-specifically concentrate bacteria from foods (Lucore et al., 2000; Cullison and Jaykus, 2002). The objective of this research was to develop and optimize the application of a metal hydroxide concentration method for recovery of viable E. coli O157:H7 from ground beef with subsequent polymerase chain reaction (PCR) detection. Hypotheses that metal hydroxide concentration would lead to increased cell recoveries compared to controls and that subsequent detection by PCR would gain higher detection sensitivity were tested for E. coli O157:H7 in autoclaved and raw ground beef samples. In the study, 10 g autoclaved and raw ground beef samples were artificially contaminated with E. coli O157:H7 cells at 102, 104, or 106 CFU/ml; autoclaved and raw ground beef samples were also seeded with a low level inoculum (1 CFU/g beef) and allowed to incubate to previously mentioned inoculum levels. Bacterial concentration consisted of two primary steps: centrifugation (primary concentration) and metal hydroxide immobilization (secondary concentration). PCR primers targeted a 254-bp portion of the slt-II gene. Southern hybridization was conducted on nylon membranes to confirm presence of DNA amplicons and gain higher detection limits. Recoveries exceeded 70% for cooked beef samples without growth and 57% for cooked beef samples with growth. Further, recoveries for raw ground beef samples exceeded 66% for samples concentrated without growth and 55% for samples concentrated after growth in a selective medium. In all cases, loss to supernatant did not exceed 20%. Overall, there were no statistically significant differences in samples concentrated with the metal hydroxide and samples concentrated with a saline control. The PCR detection limit effectively was 102 CFU/ml for all samples except raw, no growth cell concentrates. Southern hybridization afforded confirmation and an increase in detection limits for all samples except from growth, raw beef cell concentrates. Data indicate that model food systems may not always be accurate indicators of a bacterium's behavior in a food matrix. Future research is recommended to investigate the mechanisms by which bacterial cells are less readily recovered following growth in a food.
Date: 2003-09-15
Degree: MS
Discipline: Food Science

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