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United States Department of Agriculture

Agricultural Research Service

Research Project: Biology and Control of Human Pathogens on Fresh Produce

Location: Produce Safety and Microbiology Research

Title: Rapid detection of predation of Escherichia coli O157:H7 and sorting of bacterivorous tetrahymena by flow cytometry

Authors
item Ravva, Subbarao
item Hernlem, Bradley
item Sarreal, Chester

Submitted to: Frontiers in Cellular and Infection Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 15, 2014
Publication Date: May 5, 2014
Citation: Ravva, S.V., Hernlem, B.J., Sarreal, C.Z. 2014. Rapid Detection of predation of Escherichia coli O157:H7 and sorting of bacterivorous tetrahymena by flow cytometry. Frontiers in Cellular and Infection Microbiology. DOI:10.3389/fcimb.2014.000057.

Interpretive Summary: Pre-harvest contamination of produce is known to be associated with some foodborne illness outbreaks. In order to understand how this contamination occurs, it is important to know how pathogens survive in the environment, croplands, dairies and feedlots. It is known that pathogens can survive and even become more dangerous when associated with protozoa. In this work, we developed and applied a method to identify and separate protozoa that take in E. coli that were originally found in an apple juice foodborne illness outbreak. Because environmental protozoa are difficult to culture, which our method does not require, it may prove to be ideal in rapidly determining protozoa that harbor and transport enteric pathogens from livestock to produce crops grown nearby.

Technical Abstract: Protozoa are known to harbor bacterial pathogens, enhance their survival in the environment and make them hypervirulent. Rapid non-culture based detection methods are required to determine the environmental transport of enteric pathogens from point sources such as dairies and feedlots to food crops grown in proximity. Grazing studies were performed on a soilborne isolate of Tetrahymena fed green fluorescent protein (GFP) expressing Escherichia coli O157:H7 to determine the suitability of the use of such fluorescent prey bacteria to locate and sort bacterivorous protozoa by flow cytometry. In order to overcome autofluorescence of the target organism and to clearly discern Tetrahymena with ingested prey versus those without, a ratio of prey to host of at least 100:1 was determined to be preferable. Under these conditions, we successfully sorted the two populations and verified them by confocal microscopy. This technique can be easily adopted for environmental monitoring of rates of enteric pathogen destruction versus protection in protozoa.

Last Modified: 10/1/2014
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