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Applied and Environmental Microbiology, September 2008, p. 5645-5652, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.00503-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of Shiga Toxin Type 2 Variants in Escherichia coli Isolates from Animals, Food, and Humans{triangledown}

Jie Zheng,1 Shenghui Cui,1 Louise D. Teel,2 Shaohua Zhao,3 Ruby Singh,3 Alison D. O'Brien,2 and Jianghong Meng1*

Department of Nutrition & Food Science, University of Maryland, College Park, Maryland 20742,1 Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814,2 Center for Veterinary Medicine, Office of Research, Food and Drug Administration, Laurel, Maryland 207083

Received 2 March 2008/ Accepted 6 July 2008

There is considerable heterogeneity among the Shiga toxin type 2 (Stx2) toxins elaborated by Shiga toxin-producing Escherichia coli (STEC). One such Stx2 variant, the Stx2d mucus-activatable toxin (Stx2dact), is rendered more toxic by the action of elastase present in intestinal mucus, which cleaves the last two amino acids of the A2 portion of the toxin A subunit. We screened 153 STEC isolates from food, animals, and humans for the gene encoding Stx2dact by using a novel one-step PCR procedure. This method targeted the region of stx2dact that encodes the elastase recognition site. The presence of stx2dact was confirmed by DNA sequencing of the complete toxin genes. Seven STEC isolates from cows (four isolates), meat (two isolates), and a human (one isolate) that carried the putative stx2dact gene were identified; all were eae negative, and none was the O157:H7 serotype. Three of the isolates (CVM9322, CVM9557, and CVM9584) also carried stx1, two (P1332 and P1334) carried stx1 and stx2c, and one (CL-15) carried stx2c. One isolate, P1130, harbored only stx2dact. The Vero cell cytotoxicities of supernatants from P1130 and stx1 deletion mutants of CVM9322, CVM9557, and CVM9584 were increased 13- to 30-fold after treatment with porcine elastase. Thus, Stx2dact-producing strains, as detected by our one-step PCR method, can be isolated not only from humans, as previously documented, but also from food and animals. The latter finding has important public health implications based on a recent report from Europe of a link between disease severity and infection with STEC isolates that produce Stx2dact.

* Corresponding author. Mailing address: Department of Nutrition and Food Science, 0112 Skinner Building, University of Maryland, College Park, MD 20742. Phone: (301) 405-1399. Fax: (301) 314-3313. E-mail: jmeng{at}umd.edu

{triangledown} Published ahead of print on 25 July 2008.

Applied and Environmental Microbiology, September 2008, p. 5645-5652, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.00503-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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