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. 2003 Aug;41(8):3777-83.
doi: 10.1128/JCM.41.8.3777-3783.2003.

Presence of activatable Shiga toxin genotype (stx(2d)) in Shiga toxigenic Escherichia coli from livestock sources

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VSports app下载 - Presence of activatable Shiga toxin genotype (stx(2d)) in Shiga toxigenic Escherichia coli from livestock sources

Kari S Gobius et al. J Clin Microbiol. 2003 Aug.

Abstract

Stx2d is a recently described Shiga toxin whose cytotoxicity is activated 10- to 1000-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 stx(2d). The stx(2) operons of STEC were first analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis and categorized as stx(2), stx(2c vha), stx(2c vhb), or stx(2d EH250) VSports手机版. Subsequently, the stx(2c vha) and stx(2c vhb) operons were screened for the absence of a PstI site in the stx(2A) subunit gene, a restriction site polymorphism which is a predictive indicator for the stx(2d) (activatable) genotype. Twelve STEC isolates carrying putative stx(2d) operons were identified, and nucleotide sequencing was used to confirm the identification of these operons as stx(2d). The complete nucleotide sequences of seven representative stx(2d) operons were determined. Shiga toxin expression in stx(2d) isolates was confirmed by immunoblotting. stx(2d) isolates were induced for the production of bacteriophages carrying stx. Two isolates were able to produce bacteriophages phi1662a and phi1720a carrying the stx(2d) operons. RFLP analysis of bacteriophage genomic DNA revealed that phi1662a and phi1720a were highly related to each other; however, the DNA sequences of these two stx(2d) 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. .

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Figures

FIG. 1.
FIG. 1.
Predicted amino acid sequences of the stx2d A-subunit genes from isolates identified in this study aligned with the published sequences of Stx2d1 from STEC isolate B2F1 (11, 17) and Stx2 (GenBank accession no. AB035143). Dots indicate residues identical to those in Stx2d1. Residues Ser291 and Glu297, which distinguish the Stx2d1A-subunit carboxy terminus from that of Stx2A, are boldface and underlined.
FIG. 2.
FIG. 2.
Predicted amino acid sequences of the stx2d B-subunit genes from isolates identified in this study aligned with the published sequences of Stx2d1 from STEC isolate B2F1 (11, 17) and Stx2 (GenBank accession no. AB035143). Dots indicate residues identical to those of Stx2d1. Residues Asn16 and Asp24, which distinguish the Stx2d1B subunit from that of Stx2B, are boldface and underlined.
FIG. 3.
FIG. 3.
RFLP analysis of stx2d bacteriophages. Bacteriophage genomic DNA was isolated, digested with AvaI, and electrophoresed. Lanes: 1, undigested φ1720a; 2, φ1720a; 4, undigested φ1662a; and 5, φ1662a. Lane 3, DNA size markers of 23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kb.
FIG. 4.
FIG. 4.
Immunoblot analysis of Stx expression from stx2d-containing isolates. Equivalent inocula of stx2d-containing isolates were grown on nitrocellulose membranes placed on tryptic soy agar plates and grown overnight. The Stx expressed by bacterial colonies was captured on a capture membrane precoated with rabbit anti-stx antibodies. The capture membrane was probed with alkaline phosphatase-labeled rabbit anti-mouse immunoglobulin G. Triplicate inocula were made for each isolate, as indicated.

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