Co-occurrence of Escherichia coli Shiga Toxin 2d-activatable Variant and AB5 Subtilase in STEC in Commercial Ground Beef

Abstract

Shiga toxin–producing E. coli (STEC) is a food- and water-borne pathogen that causes hemorrhagic colitis (bloody diarrhea), and in severe cases hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Shiga toxin (Stx) has two forms: Stx1 and Stx 2. A variant Stx2, called Stx2d-activatable is more virulent than othersubtypes and its detection in human isolates was shown to be a predictor for a more severe clinical outcome.

Purpose: The presence of Stx2d-activatable was determined in raw ground beef samples by PCR, PFGE analysis and sequencing of the Stx2A sub unit.

This study showed that certain strains of non-O157 EHECs such as those containing the variant Stx2d-activatable may be missed when testing for O157 only. These non-O157 EHECs may be as equally virulent as the O157 strains, hence equal importance in detecting these strains should be given to ensure consistent quality of beef supply.

Introduction

Shiga toxin-producing E. coli (STEC) is a food and water borne pathogen that causes hemorrhagic colitis (blody diarrhea), and in severe cases hemolytic uremic syndrome (HUS). Although the serotype O157 is responsible for most outbreaks, other serotypes O157 is responsible for most outbreaks, other serotypes such as O26,  O103, O111, O145 (4,5,14) have been involved in disease. The common factor is expression of shiga toxin (Stx). There are two forms, Stx1 and Stx2, which are not cross reactive. Stx2 is more often found in serious outbreaks of disease in E. coli infections. Stx2 has several subtypes. One variant, Stx2d-activatable originally found in a non-O157 E. coli serotype O91, interacts with elastase present in intestinal mucus, causing release of the active site (7-9), and making it more virulent than other subtypes. Detection of Stx2d-activatable in isolates from humans was shown to be a predictor for a more severe clinical outcome (2). It has subsequently been found in other non O157 serotypes.

Current testing protocols for E. coli contamination on food involve identifying O157 by plating on sorbitol MacConkey agar (SMAC) or by lateral flow assays and O157-specific PCR kits. Detecting non-O157 strains of E. coli containing the Stx2d-activatable toxin would be of value in improving the safety of our food supply. Here we present data showing the presence of stx2d-activatable in food samples containing non- O157 E. coli. These data suggest that more rigorous testing of food may be essential for preventing possible disease causing products from reaching the market.

Methods

Between September, 2008 and June, 2009 we tested 1800 ground beef samples for the presence of stx genes in E. coli infecting these foods. Finished raw ground beef products were purchased prepackaged from various retail stores in the Seattle, WA area. The products were transferred to the laboratory at ambient temperature and processed immediately. Briefly, 200 g of each sample was aseptically transferred to a sterile Whirl-Pak sample bag (Nasco, Modesto, CA) and 800 ml of IEH Media (Acumedia, Neogen Corp, Lansing, MI.) was added. Meat samples were homogenized in an IUL Masticator (IUL, S.A., Spain) for 60 s, and incubated at 35°C overnight for enrichment. Two microliters of enrichment culture were transferred to a tube containing commercially available multiplex PCR buffer for STEC detection (EC7 buffer, Molecular Epidemiology, Inc., Lake Forest Park, WA.). Amplification was performed using an Eppendorf Mastercycler (Eppendorf AG, Hamburg, Germany) according to the Technical Specifications for EC7, Molecular Epidemiology, Inc. Further PCR was run for specific detection of stx2d-activatable and cytolethal distending toxin 5 (cdt5) (6). Amplicons were resolved by 2% agarose gel electrophoresis (Bio-Rad Laboratories sub-cell model 192, Hercules, CA.), and ethidium bromide-stained gels were examined by UV illumination. Stx2d-activatable samples positive by PCR were then sequenced for confirmation. Serotypes defined by PCR were O26, O103, O111, O113, O121 and O145. Those strains that did not yield PCR signals were serotyped using conventional methods by the National Microbiology Laboratory, Winnipeg, MB, Canada. Isolates were analyzed by PFGE according to CDC Pulse Net protocol for molecular subtyping of E. coli  [http://www.cdc.gov/pulsenet/ protocols/ecoli_salmonella_shigella_protocols.pdf] with slight modifications (Specifications for PFGE, Molecular Epidemiology, Inc., Lake Forest Park, WA). PFGE gel images were analyzed with BioNumerics software (Applied Maths, Inc., Austin, Texas). Cluster analysis was performed using the Dice similarity coefficient and UPGMA (Unweighted Pair Group Method with Arithmetic mean) algorithm to construct a dendrogram.

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Results

We identified 38 ground beef samples positive for STEC contamination. The presence of stx2d-activatable was detected as described previously (16) using a stx2d activatable-specific primer homologous to the sequence that encodes the region of the A2 subunit recognized by elastase. Samples were also amplified using a stx2/2c primer (16) for comparison. Of the 38 STEC-positive samples, eight (21%) contained the stx2d-activatable variant. Three of these were positive for the stx2c variant as well (table 1). All of the strains containing stx2d-activatable were positive for subAB and negative for eae. Three stx2-positive E. coli cell lines obtained from ATCC also contained the subtype stx2d-activatable (data not shown). Cdt was identified in one of the stx2d-activatable samples and three of the stx2c–positive samples.

Discussion

The presence of stx2d-activatable in these food sources is alarming as this subtype is more virulent than other subtypes. It has a two amino acid residue mutation in its catalytic domain that allows cleavage by elastase in intestinal mucus (7-9). When cleaved, the active site is released. In an in vitro study, Stx2d-activatable had a CD50 of 101 compared with a CD50 of 103-104 for other Stx subtypes (16). This subtype has been also associated with sporadic cases of HC and HUS (9,12), and particularly with outbreaks in which the infecting E. coli was Intimin (eae) negative (2). One of the stx2d-activatable samples was also positive for cdt5, another toxin associated with diarrhea in patients (1,6,15). The serotypes varied for these positive samples, and were not of the serotype originally associated with Stx2d-activatable, O91. Recently, stx2d-activatable was identified in samples taken from cattle and sheep at slaughter, and these were also of various serotypes (13). These E. coli strains demonstrated increased Vero cell virulence when mouse intestinal mucus or purified elastase was added, indicating a possibility of more severe human disease if consumed within a contaminated food source.

Of concern here is that the stx2d-activatable subtype has occurred only in non-O157 strains of E. coli, and testing of both human disease samples and food samples commonly involves identification of the O157 serotype. Because only the O157 serotype does not ferment sorbitol, it can be easily distinguished from the others by growth on sorbitol MacConkey agar plates. This is fairly rapid and does not require skilled workers to run PCR and restriction digests. However, given our data, caution should be taken in declaring food safe when negative only for O157. Additional tests, such as ours, to determine the presence of Stx, and especially Stx2d-activatable in food should be performed before offering for public consumption.

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