Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes

Abstract

We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx(1) or variant genes were detected in 88 (40.2%) strains and stx(2) and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx(1), stx(1c), and stx(1d)) and the Stx2 (stx(2), stx(2d), stx(2-O118), stx(2e), and stx(2g)) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx(2) and/or mucus-activatable stx(2d) genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx(2) and stx(2d) STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx(2e)), lamb, and wildlife meat (stx(1c)). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.

FIG. 1.

PCR/RFLP typing of genes of the Stx1 family. PCR amplification of Stx1 family genes with primers Stx1com-F and Stx1com-R results in a 282-bp (stx₁, stx_1-O48, stx_1-CB168, and stx_1d) or a 283-bp (stx_1c) fragment. The PCR fragments were digested separately with BstEII (lanes 1 to 3), HaeII (lanes 4 to 6), or PflMI (lanes 7 to 9), yielding RFLP patterns characteristic of stx₁, stx_1c, and stx_1d, respectively (Table 2). Lanes M, molecular size standards; lanes 1, 4, and 7, C600(H19) stx₁ (GenBank accession no. M16625); lanes 2, 5, and 8, DG131/3 stx_1c (GenBank accession no. Z36901); lanes 3, 6, and 9, MHI813 stx_1d (GenBank accession no. AY17085).

FIG. 2.

PCR/RFLP typing of genes of the Stx2 family. PCR amplification of Stx2 family genes was performed with primers GK5 and GK6, resulting in a 269-bp fragment. The PCR fragment was digested separately with HaeIII, RsaI, FokI, and NciI, yielding RFLP patterns characteristic of stx₂, stx_2v-ha, stx_2v-hb, stx_2g, stx_2-NV206, and stx_2-EC1586 (Table 3). Lanes M, molecular size standards; lanes 1, C600(W34) stx₂ (GenBank accession no. X07865); lanes 2, CB2851 stx_2v-ha (GenBank accession no. AJ605767); lanes 3, CB7753 stx_2v-hb (GenBank accession no. AF479829); lanes 4, 12422 stx_2g (GenBank accession no. AY286000); lanes 5, NV206 stx_2-NV206 (GenBank accession no. AF329817); lanes 6, RL481/06 stx_2-EC1586 (GenBank accession no. AM498375).