Evaluation of Multiplex PCRs for Diagnosis of Infection with Diarrheagenic Escherichia coli and Shigella spp

Abstract

We have developed two multiplex PCR assays that detect typical and atypical enteropathogenic Escherichia coli (EPEC) isolates, enteroaggregative E. coli (EAEC) isolates, enterotoxigenic E. coli (ETEC) isolates, enteroinvasive E. coli (EIEC) isolates, Shiga toxin-producing E. coli (STEC) isolates, and Shigella spp. The targets selected for each group were eae and bfpA for EPEC isolates, the target of probe CVD432 for EAEC isolates, the genes encoding heat-labile and heat-stable toxins for ETEC isolates, stx 1 and stx 2 for STEC isolates, and ipaH for EIEC isolates and Shigella spp. These PCRs were specific and sensitive for rapid detection of target isolates in stools. Among 150 stool specimens from the acute diarrhea tested, 9 samples (6%) had atypical EPEC, 9 (6%) had typical EPEC, 7 (4.7%) had EAEC, 3 (2%) had EIEC, 3 (2%) had Shigella spp., and 1 (0.7%) had an O26 STEC strain; we also detected mixed infections, 2 (1.3%) with EAEC and Shigella spp., 1 (0.7%) with atypical and typical EPEC strains, and another with atypical EPEC and EAEC strains. One of the multiplex PCRs directly applied to 36 stool specimens correctly identified 100% of EPEC and EAEC isolates.Five categories of Escherichia coli have been well associated with diarrhea in several epidemiological studies (9)and Shiga toxin-producing E. coli (STEC). The virulence mechanisms that characterize these categories of E. coli are genetically encoded by chromosomal, plasmid, and bacteriophage DNAs and are represented by the following genes: eae (attaching and effacing lesions), bfpA (localized adherence), the gene encoding enteroaggregative adherence, ipaH (enteroinvasive mechanism), the genes encoding heat-labile toxin (LT) and heat-stable toxin (ST), and stx 1 and stx 2 (Shiga toxins). To correctly identify diarrheagenic E. coli strains, these organisms must be differentiated from nonpathogenic members of the normal flora. Serotypic markers correlate, sometimes very closely, with specific categories of diarrheagenic E. coli; however, these markers are rarely sufficient in and of themselves to reliably identify a strain as diarrheagenic. Thus, the detection of diarrheagenic E. coli has focused increasingly on the identification of certain characteristics which themselves determine the virulence of these organisms. This identification process may include HEp-2 cell adherence, DNA hybridization, and PCR assays to detect the presence of specific virulence traits or the genes encoding these traits. The first two types of assays require special expertise, employ cell culture and radioactive material, and are time-consuming.We developed two multiplex PCR assays to detect the five categories of diarrheagenic E. coli organisms and Shigella spp.and assessed the direct application of those assays to human diarrheal stool samples.The targets selected for each category were eae and bfpA for EPEC isolates, the target of probe CVD432 for EAEC isolates, the LT and ST genes for ETEC isolates, stx 1 and stx 2 for STEC isolates, and ipaH for EIEC isolates and Shigella sp...

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

“…The genes encoding virulence factors are extensively studied and characterized, and several PCR methods have been developed to identify the virulence genes of DEC (Kimata et al 2005). Numerous multiplex PCR assays have been developed to identify DEC, and assays were recently described that are capable of distinguishing the five pathotypes of DEC (Aranda et al 2004, Nguyen et al 2005.…”

Characterization of the microbial community in a lotic environment to assess the effect of pollution on nitrifying and potentially pathogenic bacteria

“…Several virulence properties are well-characterised among E. coli populations, differentiating them from commensal and pathogenic strains. Considering the pathogenic E. coli, these virulence determinants are genetically encoded either by chromosomal, plasmid, or bacteriophage DNA and are represented by selected genes such as eae (attaching and effacing lesions), bfpA (localized adherence), ipaH (enteroinvasive mechanism), the genes encoding heat-labile toxin (elt), and heat-stable toxin (est), and the genes encoding the Shiga toxins, stx1 and stx2 (Aranda et al, 2004). Several studies have aimed at investigating the incidence of diarrheagenic E. coli in different water environments, and the majority of them have demonstrated that these lineages and the related virulence genes are present in urban and polluted environments.…”

“…The DNA used was the same as that used for the nitrifying bacteria analyses, and PCR amplifications were carried out as previously described by (Amann et al, 1990;Trebesius et al, 2000;Watanabe et al, 2001;Fukushima et al, 2003;Qin et al, 2003;Aranda et al, 2004;Rinttilä et al, 2004;Chotár et al, 2006). The negative control was the PCR reaction containing all the components needed to perform this analysis, except DNA.…”

Investigation of Pathogenic Escherichia coli Strains in Patients with Diarrhea

“…Bu nedenle E.coli'nin virülans faktörünün tayini, diyarejenik E.coli'nin ishal etkeni olarak tanımlanmasında giderek artan güvenilir bir laboratuvar yöntemi halini almıştır 6 . 7,8 .…”