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United States Department of Agriculture

Agricultural Research Service

Research Project: ENVIRONMENTALLY SOUND MANURE MANAGEMENT FOR REDUCTION OF GAS EMISSIONS, NUTRIENTS, AND PATHOGENS

Location: Agroecosystem Management Research

Title: Primary isolation of Shiga toxigenic Escherichia coli from environmental sources

Author
item Durso, Lisa

Submitted to: Journal of Environmental Quality
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 19, 2013
Publication Date: June 25, 2014
Repository URL: http://handle.nal.usda.gov/10113/59437
Citation: Durso, L.M. 2014. Primary isolation of Shiga toxigenic Escherichia coli from environmental sources. Journal of Environmental Quality. 42(5):1295-1307. DOI: 10.2134/JEQ2013.02.0035.

Interpretive Summary: There are many different kinds of bacteria that live naturally in the environment, in our intestines, and in close association with animals and insects. This article looks at how to find one specific kind of bacteria, and how to sort it out from the thousands of other bacteria in the same places. The target is a group of Escherichia coli that carry genes coding for Shiga-toxins. These Shiga toxigenic E. coli, otherwise known as “STEC”, are sometimes associated with disease in humans, especially foodborne illness associated with the most famous STEC, STEC O157. In addition to STEC O157, there are six other kinds of STEC that are associated with disease in humans, and that are regulated in food. In environmental and insect samples, the STEC are usually present in very low numbers, and are obscured by very high numbers of competing “background” bacteria. There are three main steps to isolating STEC from samples such as soil and insects. The first step is an enrichment to increase the number of target bacteria compared to background bacteria. The second step is to concentrate the target, and the final step is to grow the bacteria on differential agar, which ideally will result in the target being one color, and the background being a different color. The specific methods that are best will depend on which STEC is the target, and what kind of sample is being tested.

Technical Abstract: Since the time of the first microbe hunters, primary culture and isolation of bacteria has been a foundation of microbiology. Like other microbial methods, bacterial culture and isolation methodologies continue to develop. Although fundamental concepts like selection and enrichment are as relevant today as they were over 100 yr ago, advances in chemistry, molecular biology and bacterial ecology mean that today’s culture and isolation techniques serve additional supporting roles. The primary isolation of Shiga toxigenic Escherichia coli (STEC) from environmental sources relies on enriching the target while excluding extensive background flora. Due to the complexity of environmental substrates, no single method can be recommended; however, common themes are discussed. Brilliant Green Bile Broth, with or without antibiotics, is one of many broths used successfully for selective STEC enrichment. Stressed cells may require a pre-enrichment recovery step in a nonselective broth such as buffered peptone water. After enrichment, immunomagnetic separation with serotype specific beads drastically increases the chances for recovery of STEC from environmental or insect sources. Some evidence suggests that acid treating the recovered beads can further enhance isolation. Although it is common in human clinical, food safety, and water quality applications to plate the recovered beads on Sorbitol MacConkey Agar, other chromogenic media, such as modified CHROMagar, have proven helpful in field and outbreak applications, allowing the target to be distinguished from the numerous background flora. Optimum conditions for each sample and target must be determined empirically, highlighting the need for a better understanding of STEC ecology.

Last Modified: 12/27/2014
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