SL440 Shiga Toxin-Producing Escherichia coli: Detection, Differentiation, and Implications for Food Safety1 William J. Zaragoza, Max Teplitski, and Clifton K. Fagerquist2 Introduction for the effective detection of the Shiga-toxin-producing pathogens in a variety of food matrices. Shiga toxin is a protein found within the genome of a type of virus called a bacteriophage. These bacteriophages can There are two types of Shiga toxins—Shiga toxin 1 (Stx1) integrate into the genomes of the bacterium E. coli, giving and Shiga toxin 2 (Stx2). Stx1 is composed of several rise to Shiga toxin-producing E. coli (STEC). Even though subtypes knows as Stx1a, Stx1c, and Stx1d. Stx2 has seven most E. coli are benign or even beneficial (“commensal”) subtypes: a–g. Strains carrying all of the Stx1 subtypes affect members of our gut microbial communities, strains of E. humans, though they are less potent than that of Stx2. Stx2 coli carrying Shiga-toxin encoding genes (as well as other subtypes a,c, and d are frequently associated with human virulence determinants) are highly pathogenic in humans illness, while the other subtypes affect different animals. and other animals. When mammals ingest these bacteria, Subtypes Stx2b and Stx2e affect neonatal piglets, while STECs can undergo phage-driven lysis and deliver these target hosts for Stx2f and Stx2g subtypes are not currently toxins to mammalian guts. The Shiga toxin consists of an known (Fuller et al. 2011). Stx2f was originally isolated A subunit and 5 identical B subunits. The B subunits are from feral pigeons (Schmidt et al. 2000), and Stx2g was involved in binding to gut epithelial cells. The A subunit isolated from cow feces (Leung and Peiris 2003). Because is composed of the catalytically active A1 subunit and the of the diversity of Shiga toxins in nature and their broad A2 fragment, which stabilizes the AB5 holotoxin structure. range of hosts, detection and differentiation of these toxins In humans, these toxins can cause hemolytic uremic is critical. Researchers have developed numerous methods syndrome (HUS). Such infections involve the production of for detecting and distinguishing between the various types substances that destroy blood vessels and may cause severe and subtypes of these toxins. One such method involves kidney damage. The first documented case of clinical HUS polymerase chain reaction (PCR) using a multiplex system, associated with Shiga toxin was in 1983. Since then, STECs which is intended to distinguish between many types and have been recognized as a common cause of acute renal subtypes of Stx at the same time (Scheutz et al. 2012). failure among children in the United States. In the past, Another nucleic acid-based method is quantitative real- raw or undercooked beef was a common source of STEC time PCR (qPCR), which, like PCR, relies on the ability to contamination. More recently, STEC infections associated measure the presence of a given gene in a sample (Bustin et with fresh produce have significantly increased, and STEC al. 2009). Reverse Transcription qPCR (RT-qPCR) (Bustin infections from milk, juice, soft cheeses, and contaminated et al. 2009), which measures gene expression, can be used water have also been reported, thus underscoring the need in clinical, food testing, and research labs; although, due 1. This document is SL440, one of a series of the Department of Soil and Water Sciences, UF/IFAS Extension. Original publication date July 2016. Visit the EDIS website at http://edis.ifas.ufl.edu. 2. William J. Zaragoza, Ph.D. student, Department of Soil and Water Sciences, USDA Agricultural Research Service, Albany, CA; Max Teplitski, professor, Department of Soil and Water Science, UF/IFAS Extension; and Clifton K. Fagerquist, research chemist, USDA-ARS, Albany, CA. The Institute of Food and Agricultural Sciences (IFAS) is an Equal Opportunity Institution authorized to provide research, educational information and other services only to individuals and institutions that function with non-discrimination with respect to race, creed, color, religion, age, disability, sex, sexual orientation, marital status, national origin, political opinions or affiliations. For more information on obtaining other UF/IFAS Extension publications, contact your county’s UF/IFAS Extension office. U.S. Department of Agriculture, UF/IFAS Extension Service, University of Florida, IFAS, Florida A & M University Cooperative Extension Program, and Boards of County Commissioners Cooperating. Nick T. Place, dean for UF/IFAS Extension. to the instability of RNA, once extracted, RT-qPCR has CT-SMAC agar because the bacteria are susceptible to limited applications in diagnostics and food testing. tellurite (Gould et al. 2014). A third approach has been developed using monoclonal To isolate non-O157 STEC, the CDC recommends that the antibodies that can bind to the toxins and differentiate them Shiga-toxin-positive liquid cultures are streaked to a less based on their immunological properties (Skinner et al. selective agar (e.g., MacConkey agar, SMAC, Statens Serum 2015). A fourth approach involves the use of mass spec- Institut [SSI] enteric medium, or blood agar) (Gould et al. trometry to detect these toxins (Fagerquist and Zaragoza 2014). Traditional enteric media, such as Hektoen agar, 2016; Fagerquist et al. 2014). Each approach has benefits xylose-lysine-desoxycholate (XLD) agar, and Salmonella- and caveats, so it is important to use them in a compli- Shigella agar, may inhibit many E. coli and are, therefore, mentary fashion for robust detection and differentiation. less desirable (Blom et al. 1999). Most non-O157 STEC In fact, the Centers for Disease Control and Prevention ferment both sorbitol and lactose, although exceptions have (CDC) highlights the need for complementary approaches been reported (Gould et al. 2014). Representative results of in the identification of STEC in clinical samples. The CDC culture-based tests are shown in Figure 1. suggests a combination of culture-based techniques and a simultaneous assay for the Shiga toxins or the genes encod- ing these toxins (Gould et al. 2009). Even though this CDC guideline was created for clinical samples, it is prudent to follow similar approaches for environmental and food testing. This EDIS document was developed for laboratory scientists and technicians working on the detection and identification of STEC in food matrices and environmental samples. This document will also be useful for clients of such laboratories to assist with the determination of which tool set is most appropriate for the detection of STEC. Our goal is to compare and contrast the advantages and disad- vantages of common laboratory techniques used for this Figure 1. STEC isolation from various selective media. (A) Cells from purpose. Assessment of sample data sets is also included in enrichment broth are plated on CT-SMAC. (B) Suspect colonies the document to assist with data interpretation. appear as pale on CT-SMAC and steel blue on NT-Rainbow Media, and non-O157 STECs appear as pink colonies on NT-Rainbow (C) Suspect STECS expressing b-galactosidase and hemolysin are indicated by Detection of STEC blue colonies with a zone of clearing on Sheeps blood agar (D) Typical non-O157 STECs are shown growing on CHROMagar and appear as Culture-Based Detection blue colonies. Credits: Mike Cooley O157 STEC can usually be distinguished from most commensal E. coli by their inability to ferment sorbitol Nucleic-Acid-Based Detection within 24 hours when plated on a sorbitol-containing agar. To isolate O157 STEC, samples are plated onto a selective Recently, a PCR-based method has been developed to and differential media, such as sorbitol-MacConkey agar test samples from various sources for the presence of stx (SMAC), cefixime tellurite-sorbitol MacConkey agar genes (Scheutz et al. 2012). PCR is easy to perform and (CT-SMAC), or CHROMagar O157. After incubation for relatively inexpensive. This protocol involves a multiplex 16 to 24 hours at 37°C (99°F), the plate should be examined PCR approach that employs multiple primer sets in a single for possible O157 colonies, which are colorless on SMAC or PCR reaction in order to detect different types and subtypes CT-SMAC and are mauve or pink on CHROMagar O157. of stx genes in a single sample. This approach was tested Both CT-SMAC and CHROMagar O157 are considered by multiple laboratories on various thermocyclers and was to be more selective than SMAC (Church et al. 2007). found to be robust. However, results indicate that caution Non-motile flagella-less (H-) sorbitol-fermenting STEC must be exercised when testing samples for the presence O157, which are fairly uncommon in the United States of stx2 c and d subtypes, because different laboratories and primarily reported in Europe, might not grow on reported instances of cross-reactivity between these two subtypes in PCR assays. In situations where cross-reactivity is suspected, researchers may have to repeat the analysis Shiga Toxin-Producing Escherichia coli: Detection, Differentiation, and Implications for Food Safety 2 without multiplexing. If cross-reactivity still occurs (e.g., For PCR-based detection, colonies to be tested must be a PCR product is seen for both stx2c and stx2d subtypes, grown on nonselective agars, such as tryptic soy agar even when the primers are used in separate reactions, (TSA), heart infusion agar (HIA), or blood agar (Gould et like in Figure 2), researchers may have to verify the result al. 2009); tests done on colonies grown on selective media with an orthologous method. In addition to these caveats, often interfere with the PCR reaction leading to poorly RT-qPCR may fail to detect stx mRNA because the genes reproducible results. According to the CDC, DNA-based are not being expressed, which creates the potential for a Shiga toxin gene detection is not currently approved for false negative. Analysts must also be aware of the presence the diagnosis of STEC infection in human clinical samples of cryptic bacteriophages.