Isolation and detection of Shiga toxin-producing Escherichia coli in clinical stool samples using conventional and molecular methods

Abstract

The isolation of Shiga toxin-producing Escherichia coli (STEC) other than serogroup O157 from clinical stool samples is problematic due to the lack of differential phenotypic characteristics from non-pathogenic E. coli. The development of molecular reagents capable of identifying both toxin and serogroup-specific genetic determinants holds promise for a more comprehensive characterization of stool samples and isolation of STEC strains. In this study, 876 stool samples from paediatric patients with gastroenteritis were screened for STEC using a cytotoxicity assay, commercial immunoassay and a conventional PCR targeting Shiga-toxin determinants. In addition, routine culture methods for isolating O157 STEC were also performed. The screening assays identified 45 stools presumptively containing STEC, and using non-differential culture techniques a total of 20 O157 and 22 non-O157 strains were isolated. These included STEC serotypes O157 : H7, O26 : H11, O121 : H19, O26 : NM, O103 : H2, O111 : NM, O115 : H18, O121 : NM, O145 : NM, O177 : NM and O5 : NM. Notably, multiple STEC serotypes were isolated from two clinical stool samples (yielding O157 : H7 and O26 : H11, or O157 : H7 and O103 : H2 isolates). These data were compared to molecular serogroup profiles determined directly from the stool enrichment cultures using a LUX real-time PCR assay targeting the O157 fimbrial gene lpfA, a microsphere suspension array targeting allelic variants of espZ and a gnd-based molecular Oantigen serogrouping method. The genetic profile of individual stool cultures indicated that the espZ microsphere array and lpfA real-time PCR assay could accurately predict the presence and provide preliminary typing for the STEC strains present in clinical samples. The gnd-based molecular serogrouping method provided additional corroborative evidence of serogroup identities. This toolbox of molecular methods provided robust detection capabilities for STEC in clinical stool samples, including co-infection of multiple serogroups.

Molecular Characterization of Escherichia coli O157:H7 and non-O157 Shiga Toxin Producing E. coli from Retail Meat and Humans

“…However, higher isolation rate (7.5%) was reported in a different study in South Africa [43]. The isolation of non-O157 strains from human stools (2.7%) in the present study was comparable to 2.5% reported in Canada [44] and USA, where 4.2% of stool sample were positive [45]. …”

Improved Detection of Five Major Gastrointestinal Pathogens by Use of a Molecular Screening Approach

“…Other studies have reported higher success rates for STEC isolation by culture if serotypes other than O157 are also searched for (14,15,40). But in the routine laboratory, STECs other than O157 cannot be detected by means other than PCR aimed at the stx 1 and/or stx 2 genes.…”

“…Therefore, methods which quickly identify the negative specimens would facilitate routine screening. Antigen detection is a fast and effective alternative for several enteric pathogens (11,15,23,27,46). However, this method has a limited sensitivity, requires one test per pathogen, and is not available for all relevant pathogens.…”

Comparison of Shiga Toxin-Producing Escherichia coli Detection Methods Using Clinical Stool Samples

“…All these stool samples had been previously identified 27 to contain positive cultures of the following: O157:H7; O26:H11; O121:H19; O26:NM; O5:NM; O111: NM; O145:NM; O103:H2; O177:NM; O1:H7; O8:H19; O2: H4; and O25:H1.…”

“…A total of 36 stool samples previously identified 27 to contain the following 41 STEC strains (co-infection was found in five samples) were included in the study: O157:H7 (n ϭ 18); O26:H11 (n ϭ 10); O121:H19 (n ϭ 3); O26:NM (n ϭ 1); O5:NM (n ϭ 1); O111:NM (n ϭ 1); O145:NM (n ϭ 1); O103:H2 (n ϭ 1); O177:NM (n ϭ 1); O1:H7 (n ϭ 1); O8:H19 (n ϭ 1); O2:H4 (n ϭ 1); and O25:H1 (n ϭ 1).…”