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Journal of Clinical Microbiology, August 2003, p. 3777-3783, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3777-3783.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Presence of Activatable Shiga Toxin Genotype (stx2d) in Shiga Toxigenic Escherichia coli from Livestock Sources

Kari S. Gobius,* Glen M. Higgs, and Patricia M. Desmarchelier

Food Science Australia, Tingalpa DC, Queensland 4173, Australia

Received 4 February 2003/ Returned for modification 21 March 2003/ Accepted 14 April 2003

Stx2d is a recently described Shiga toxin whose cytotoxicity is activated 10- to 1,000-fold by the elastase present in mouse or human intestinal mucus. We examined Shiga toxigenic Escherichia coli (STEC) strains isolated from food and livestock sources for the presence of activatable stx2d. The stx2 operons of STEC were first analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis and categorized as stx2, stx2c vha, stx2c vhb, or stx2d EH250. Subsequently, the stx2c vha and stx2c vhb operons were screened for the absence of a PstI site in the stx2A subunit gene, a restriction site polymorphism which is a predictive indicator for the stx2d (activatable) genotype. Twelve STEC isolates carrying putative stx2d operons were identified, and nucleotide sequencing was used to confirm the identification of these operons as stx2d. The complete nucleotide sequences of seven representative stx2d operons were determined. Shiga toxin expression in stx2d isolates was confirmed by immunoblotting. stx2d isolates were induced for the production of bacteriophages carrying stx. Two isolates were able to produce bacteriophages {phi}1662a and {phi}1720a carrying the stx2d operons. RFLP analysis of bacteriophage genomic DNA revealed that {phi}1662a and {phi}1720a were highly related to each other; however, the DNA sequences of these two stx2d operons were distinct. The STEC strains carrying these operons were isolated from retail ground beef. Surveillance for STEC strains expressing activatable Stx2d Shiga toxin among clinical cases may indicate the significance of this toxin subtype to human health.

* Corresponding author. Mailing address: Food Science Australia, Cnr Wynnum and Creek Rd., Cannon Hill 4170, Australia. Phone: 61-7-3214-2036. Fax: 61-7-3214-2062. E-mail: Kari.Gobius{at}csiro.au.

Journal of Clinical Microbiology, August 2003, p. 3777-3783, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3777-3783.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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