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United States Department of Agriculture

Agricultural Research Service

Title: Real Time Monitoring of E. Coli O157:h7 Adherence to Beef Carcass Surface Tissue Using a Bioluminescence Reporter

Authors
item Siragusa, Gregory
item Nawotka, Kevin - STANFORD UNIV SCHOOL MED.
item Spilman, Stanley - STANFORD UNIV SCHOOL MED.
item Contag, Pamela - STANFORD UNIV SCHOOL MED.
item Contag, Christopher - STANFORD UNIV SCHOOL MED.

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 4, 1998
Publication Date: N/A

Interpretive Summary: E. coli O157:H7 is a foodborne disease-causing bacterium associated with consumption of undercooked beef, unpasteurized milk, and unpasteurized apple cider. We have constructed a strain of this bacteria that is bioluminescent; i.e., it has the ability to emit light or glow in the dark. This genetically engineered bioluminescent version of E. coli O157:H7 behaves exactly like the disease causing agent and was used to visualize the bacterium directly on the surface of beef carcass surface tissue using a highly sensitive ICCD video camera (intensified cooled charged couple device) system with a regular photographic lens. Observation of the organisms directly on carcass surface tissues enables the study of how these bacteria attach to or become associated with the surface of a beef carcass during the processing of the animal. Being able to study this process directly and in real time is important because it can be done without having to sample the carcass surface and subsequently culture or grow the bacteria to determine where they were located on the carcass surface. Sampling the complex surface of a carcass is often inconsistent and only samples a very small percentage of the total surface area of that carcass before it will be further processed into ground beef or other products. This research provides a potential means to assess decontamination protocols and study how the E. coli organism survives to cause disease in man.

Technical Abstract: A method of studying bacteria that are attached to a carcass surface would eliminate the need for repeated sampling and would facilitate understanding the interaction of potential human pathogens with tissue surfaces. We describe such a method using a bioluminescent reporter strain of E. coli O157:H7 that was constructed by transformation with the plasmid pCGLS1, an expression vector that encodes a complete bacterial luciferase (lux) operon. Beef carcass surface tissues were inoculated with the bioluminescent strain and adherent bacteria visualized in real time using a sensitive photon counting camera to image in situ. The reporter strain was found to luminesce from the tissue surfaces whether inoculated as a suspension in buffer or in a bovine fecal slurry. With this method, large areas of tissue inoculated with the reporter strain could be studied without the need for obtaining and culturing multiple samples from the tissue surface. Use of the complete lux operon as the bioluminescent reporter eliminated the need for exogenous addition of substrate. This allowed detection and quantitation of bacterial inocula, and rapid evaluation of adherence of this potential human pathogen to tissue surfaces. Following simple water rinses of inoculated carcass tissues, attachment duration varied with different carcass surface types. Bioluminescence and culture-derived viable bacterial counts were highly correlated. Real time assessment of microbial attachment to this complex menstruum will facilitate evaluation of washing and handling procedures that reduce bacterial contamination of beef.

Last Modified: 4/17/2014
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