Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157

Abstract

Shiga toxigenic Escherichia coli (STEC) comprises a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, certain strains appear to be of greater virulence for humans, for example, those belonging to serogroups O111 and O157 and those with particular combinations of other putative virulence traits. We have developed two multiplex PCR assays for the detection and genetic characterization of STEC in cultures of feces or foodstuffs. Assay 1 utilizes four PCR primer pairs and detects the presence of stx1, stx2 (including variants of stx2), eaeA, and enterohemorrhagic E. coli hlyA, generating amplification products of 180, 255, 384, and 534 bp, respectively. Assay 2 uses two primer pairs specific for portions of the rfb (O-antigen-encoding) regions of E. coli serotypes O157 and O111, generating PCR products of 259 and 406 bp, respectively. The two assays were validated by testing 52 previously characterized STEC strains and observing 100% agreement with previous results. Moreover, assay 2 did not give a false-positive O157 reaction with enteropathogenic E. coli strains belonging to clonally related serogroup O55. Assays 1 and 2 detected STEC of the appropriate genotype in primary fecal cultures from five patients with hemolytic-uremic syndrome and three with bloody diarrhea. Thirty-one other primary fecal cultures from patients without evidence of STEC infection were negative.

FIG. 1

Characterization of reference STEC strains by multiplex PCR assay 1. Lanes: M, DNA size markers (pUC19 DNA digested with HpaII; fragment sizes visible are 501/489, 404, 331, 242, 190, 147, and 111 bp); 1, negative control; 2, O157:H⁻ strain 96/2998 (stx₁⁺ stx₂⁺ eaeA⁺ EHEC hlyA⁺); 3, O157 strain 94-8628 (stx₂⁺ eaeA⁺ EHEC hlyA⁺); 4, O157 strain 96/0052 (stx₁⁺ stx₂⁺ eaeA⁺); 5, O48:H21 strain 94CR (stx₁⁺ stx₂⁺ EHEC hlyA⁺); 6, O128 strain 95AS1 (stx₁⁺ stx₂⁺); 7, O91 strain 95HE4 (stx₁⁺ EHEC hlyA⁺); 8, O113 strain MW10 (stx₂⁺ EHEC hlyA⁺); 9, OX3:H21 strain O31 (stx₂⁺). The expected mobilities for the various specific PCR products are also indicated.

FIG. 2

Characterization of reference STEC strains by multiplex PCR assay 2. Lanes: M, DNA size markers (pUC19 DNA digested with HpaII; fragment sizes visible are 501/489, 404, 331, 242, 190, 147, and 111 bp); 1, negative control; 2, O157:H⁻ strain 95SF2; 3, O157:H⁻ strain 96GR1; 4, O157 strain 96/0052; 5, O157:H7 strain 90/103; 6, O55 EPEC strain 93/282; 7, O111:H⁻ strain 95NR1; 8, O111:H⁻ strain 96RO1; 9, O111:H⁻ strain PH; 10, O111:H⁻ strain CB168. The expected mobilities for the O111- and O157-specific PCR products are also indicated.

FIG. 3

Multiplex PCR analysis of primary fecal cultures. Crude DNA extracts of primary fecal cultures were analyzed by multiplex PCR assay 1 or assay 2, as indicated. Lanes: 1, negative control; 2, patient 1 (HUS); 3, patient 2 (HUS); 4, patient 3 (HUS); 5, patient 4 (HUS); 6, patient 5 (bloody diarrhea); 7, patient 6 (control); 8, patient 7 (control). The expected mobilities for the various specific PCR products are also indicated.

FIG. 4

Sensitivity of multiplex PCR assay 2. An STEC-negative fecal culture was spiked with serial 10-fold dilutions of cultures of STEC strains 95NR1 (O111:H⁻) or 95SF2 (O157:H⁻). Extracts of these samples were then subjected to assay 2. Lanes: M, DNA size markers (pUC19 DNA digested with HpaII; fragment sizes visible are 501/489, 404, 331, 242, 190, 147, and 111 bp); 1, negative control; 2, STEC-negative fecal culture (FC); 3, FC plus a 10-fold dilution of a strain 95SF2 culture (10⁻¹ 95SF2); 4, FC plus 10⁻² 95SF2; 5, FC plus 10⁻³ 95SF2; 6, FC plus 10⁻⁴ 95SF2; 7, FC plus 10⁻⁵ 95SF2; 8, FC plus 10⁻¹ 95NR1; 9, FC plus 10⁻² 95NR1; 10, FC plus 10⁻³ 95NR1; 11, FC plus 10⁻⁴ 95NR1; 12, FC plus 10⁻⁵ 95NR1; 13, FC plus 10⁻¹ 95SF2 plus 10⁻¹ 95NR1. The expected mobilities for the O111- and O157-specific PCR products are also indicated.