Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx 1 and stx 2 for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli.Escherichia coli strains associated with diarrhea have been classified into six groups, based on clinical, epidemiological, and molecular criteria. These E. coli strains are commonly isolated from children with gastroenteritis in the developing world. Recent data suggest that these strains are common in the United States in children less than 5 years of age with acute diarrhea (10, 17). However, diarrheagenic E. coli strains are not routinely sought as stool pathogens in clinical laboratories. Some of these pathogens respond to antimicrobial agents, while for others (e.g., Shiga toxin-producing E. coli [STEC]), antibiotics should be avoided. Because the time frame in which treatment choices must be made is short, there is a need for a rapid, sensitive, and inexpensive detection technique. We have developed a monochromatic, fluorescence-based real-time PCR procedure to simultaneously identify eight virulence genes associated with the six classes of diarrheagenic E. coli. In this assay, the post-PCR products are identified based on melting-point curve analysis. This assay is simple, rapid, inexpensive, and reliable. It is suitable for use in clinical laboratories as well as research facilities. MATERIALS AND METHODSBacterial strains. One hundred twenty-six E. coli strains (Table 1) were analyzed, including strains representing all six of the currently recognized classes of diarrheagenic E. coli as well as commensal organisms. The prototypical strains, used in laboratories worldwide, included enterotoxigenic E. coli (ETEC) H10407; enteropathogenic E. coli (EPEC) 2348/69; STEC strains H30, HW1, and 86-24; and enteroaggregative E. coli (EAEC) strains O42 and 221. Addit...

We previously demonstrated that lactoferrin inhibits adherence of enteropathogenic Escherichia coli to HEp-2 cells and decreases invasiveness of Shigella flexneri in HeLa cells by disruption of the type III secretory system (TTSS) of both enteropathogens. To determine whether these effects were specific to the TTSS, we assessed the activity of bovine lactoferrin on enteroaggregative E. coli (EAEC), enteropathogens whose virulence is not TTSS dependent. Bovine lactoferrin at a concentration of 1.0 and 0.1 mg/mL inhibited EAEC growth. Saturation with iron reversed the bacteriostatic effect. Lactoferrin under nonbacteriostatic conditions decreased EAEC adherence to HEp-2 cells as evaluated by microscopy and CFUs; this effect was not iron dependent. Lactoferrin inhibited EAEC biofilm formation and increased autoagglutination. Lactoferrin blocks EAEC adherence by inducing release and degradation of aggregative adherence fimbria, a key element of EAEC pathogenesis. We hypothesized that lactoferrin binding to lipid A of lipopolysaccharide disrupts the virulence proteins anchored to the bacterial outermembrane. These data suggest that the effect of lactoferrin on surface proteins is not restricted to organisms having a TTSS.