Rapid Identification and Differentiation of Clinical Isolates of Enteropathogenic Escherichia coli (EPEC), Atypical EPEC, and Shiga Toxin-Producing Escherichia coli by a One-Step Multiplex PCR Method

Abstract

Enteropathogenic Escherichia coli (EPEC), atypical enteropathogenic E. coli, and Shiga toxin-producing E. coli differ in their virulence factor profiles, clinical manifestations, and prognosis, and they require different therapeutic measures. We developed and evaluated a robust multiplex PCR to identify these pathogroups based on sequences complementary to escV, bfpB, stx 1 , and stx 2 .Pathogenic Escherichia coli strains are responsible for a broad spectrum of intestinal and extraintestinal diseases, including diarrhea, urinary tract infections, septicemia, and neonatal meningitis (11). Enteropathogenic E. coli (EPEC) and the majority of clinical isolates of Shiga toxin (Stx)-producing E. coli (STEC) harbor the "locus of enterocyte effacement" (LEE), a pathogenicity island that is responsible for the phenotype of attaching-and-effacing (A/E) lesions (6, 11).EPEC are a major cause of human infantile diarrhea predominantly in less-developed countries but are also identified with increasing frequency in industrialized areas (1,11,17). These pathogens colonize the small intestine, induce the degeneration of epithelial microvilli, and intimately adhere to the host cell. Comparable to a "molecular syringe," the chromosomally encoded type III secretion system injects "effector" proteins into the host cell, inducing a characteristic rearrangement of the actin cytoskeleton resulting in the formation of "pedestals." These characteristic histopathological alterations are summarized as "A/E lesions."The genes responsible for the A/E lesions are located on an ϳ35-kb pathogenicity island, known as the locus of enterocyte effacement (LEE). Typical EPEC harbor an additional 60-MDa plasmid, the EPEC adherence factor (EAF) plasmid (16), that is not present in atypical EPEC (here abbreviated as ATEC) strains (3, 28). The EAF plasmid harbors the bundleforming pilus (bfp) operon, encoding the type IV pili responsible for localized adherence and the formation of microcolonies on host cells. ATEC strains harbor homologues of the LEE pathogenicity island but, due to the lack of the EAF plasmid (3, 8), they mostly adhere in a diffuse pattern to epithelial cells. Recent epidemiological evidence indicates an increasing prevalence of ATEC particularly in developed countries (see, for example, references 1, 17, 21, and 28) but also in developing countries (see, for example, references 9 and 27). This also indicates that in the field the EAF plasmid is not essential to cause disease.Like ATEC strains, the closely related STEC responsible for sporadic infections as well as serious outbreaks worldwide, mostly harbor the LEE pathogenicity island and lack the BFP-encoding EAF plasmid. STEC strains differ genoand phenotypically from ATEC by their production of Stx. These pathogens cause an acute inflammation of the colon, resulting in hemorrhagic colitis with rare but serious sequelae including neurological disorders and the hemolyticuremic syndrome (HUS), the leading cause of acute renal failure in children (11,12).Identification of EPEC, ATEC,...

Comparison of the EntericBio Multiplex PCR System with Routine Culture for Detection of Bacterial Enteric Pathogens

“…When the 12 samples with positive results for E. coli O157 obtained with the EntericBio system were tested by the National E. coli Reference Laboratory, 10 samples with positive results for E. coli O157 were generated; 9 of those samples with positive results were found to be positive for VT2. The EntericBio system results for these remaining two samples were not confirmed either by the method of Müller et al (10) or by the National E. coli Reference Laboratory.…”

“…For E. coli O157, the method of Müller et al (10) was used to investigate the samples for the presence of the verotoxin 1 (VT1) and/or VT2 gene among the specimens found to be positive with the EntericBio system. Furthermore, each of the fecal samples for which positive results were derived was sent for confirmatory testing to the National E. coli Reference Laboratory.…”

Shiga Toxin, Cytolethal Distending Toxin, and Hemolysin Repertoires in Clinical Escherichia coli O91 Isolates

“…The O91 strains from the other countries were kindly provided by D. Orth (Austrian Reference Laboratory for Enterohemorrhagic E. coli, Innsbruck, Austria), A. Siitonen (the National Public Health Institute, Helsinki, Finland), and M. A. Karmali (Laboratory for Food-Borne Zoonoses, Public Health Agency of Canada, Guelph, Ontario, Canada). A subset of the strains (n ϭ 35) was described previously (4,7,13,17,26,27,29,30). The ages of patients from whom the O91 STEC strains originated ranged from 4 months to 89 years (mean, 26.8 years; median, 18 years).…”

Identification of Unconventional Intestinal Pathogenic Escherichia coli Isolates Expressing Intermediate Virulence Factor Profiles by Using a Novel Single-Step Multiplex PCR

“…Additional strains are from our collection. A subset of these strains was analyzed for stx genotypes, bfpB, and escV in a preceding study (29) and described previously (5,40,41). The nine reference strains used as controls in the MPCR included EPEC strain E2348/69 (LEE positive, bfp positive), ATEC strain 9812 (LEE positive), STEC strain EDL933 (LEE positive, stx 1 positive, stx 2 positive), STEC strain 04-3175 (stx 1 positive, stx 2 positive), ETEC strain 164/82 (elt positive, estIa positive), ETEC strain 117/86 (estIb positive), EIEC strain 99-10282 (invE positive), and EAEC strain 02-1850 (astA positive, aggR positive, pic positive).…”