Direct detection of Shiga toxigenic Escherichia coli strains belonging to serogroups O111, O157, and O113 by multiplex PCR

Abstract

Shiga toxigenic Escherichia coli (STEC) strains are a diverse group of organisms associated with severe gastrointestinal and systemic diseases in humans. Within the STEC family, eae-positive STEC strains, particularly those belonging to serogroups O157 and O111, appear to have greater virulence for humans. However, in spite of being eae negative, STEC strains belonging to serogroup O113 have frequently been associated with cases of severe STEC disease, including hemolytic-uremic syndrome (HUS). We have developed a modified multiplex PCR assay for detection of STEC strains belonging to these three serogroups in cultures of feces by using primers specific for portions of the genetic loci (rfb) encoding biosynthesis of the respective O antigen. These primers direct amplification of PCR products of 259, 406, and 593 bp for serogroups O157, O111, and O113, respectively. The assay was validated by testing 40 previously characterized STEC strains, with 100% agreement. It also detected STEC strains of the appropriate genotype in primary fecal cultures from 13 patients with HUS or bloody diarrhea. Thirty other primary fecal cultures from patients without evidence of STEC infection were negative.

FIG. 1

Analysis of reference STEC strains by multiplex PCR. 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 to 7, O113:H21 STEC strains MW10, 98NK2, 98BN1, 97MW1, 1183, and 3848, respectively; 8 to 10, O111:H⁻ STEC strains 96RO1, 95JB1, and PH, respectively; 11 to 13, O157:H⁻ STEC strains 96GR1, 96/0629, and 95SF2, respectively. The expected mobilities for the various serogroup-specific PCR products are also indicated.

FIG. 2

Sensitivity of O113-specific PCR. A culture of E. coli K-12 was spiked with serial 10-fold dilutions of a culture of O113:H21 STEC strain 98NK2, and extracts of these samples were then subjected to the rfb-specific PCR assay. Lanes: M, DNA size markers (pUC19 DNA digested with HpaII; fragment sizes visible are 501/489, 404, 331, and 242 bp); 1, negative control (unspiked E. coli K-12 extract); 2 to 9, extracts of E. coli K-12 culture spiked with 10¹-, 10²-, 10³-, 10⁴-, 10⁵-, 10⁶-, 10⁷-, and 10⁸-fold-diluted 98NK2 cultures, respectively. The expected mobility for the O113-specific PCR product is also indicated.

FIG. 3

Multiplex PCR analysis of primary fecal cultures. (A) Crude DNA extracts of stx-positive primary fecal cultures analyzed by using the rfb-specific PCR assay. 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 to 4, extracts from three patients with culture-proven O113:H21 STEC infection; 5, extract from an stx₂-positive, but culture-negative HUS patient with serological evidence of O113 infection; 6 and 7, extracts from patients with culture-proven O157:H⁻ STEC infection; 8 and 9, extracts from patients with culture-proven O111:H⁻ STEC infection. (B) Crude DNA extracts of stx-negative primary fecal cultures analyzed by using the rfb-specific PCR assay. Lanes: M, DNA size markers; 2 to 10, extracts of stx-negative primary fecal cultures; 11, positive control (pooled DNA extracts from reference O157, O111, and O113 STEC strains). The expected mobilities for the various serogroup-specific PCR products are also indicated.