Background Diarrheagenic E. coli are being recognized as important pediatric enteropathogens worldwide. However, it is unclear whether there are differences in age-related susceptibility to specific agents, especially among infants. Methods We conducted a passive surveillance diarrhea cohort study of 1034 children from 2 to 12 months of age in Lima, Perú. Control stool samples were collected from randomly selected children without diarrhea. All samples were analyzed for common enteric pathogens and for the diarrheagenic E. coli by a multiplex real-time PCR. Results The most commonly isolated pathogens from 1065 diarrheal episodes were the diarrheagenic E. coli (31%), including enteroaggregative (15.1%) and enteropathogenic E. coli (EPEC) (7.6%). Diarrheagenic E. coli, Campylobacter and rotavirus were more frequently isolated from infants ≥ 6m. Diffusely adherent E. coli and enterotoxigenic E. coli (ETEC) were more frequently isolated in diarrheal samples than in controls in older infants (p<0.05). Children ≥ 6m infected with ETEC had a 4.56-fold increased risk for diarrhea (95% CI, 1.20 to 17.28). Persistent diarrhea was more frequent in infants < 6m (13.5% vs. 3.6%, p<0.001). Among diarrheagenic E. coli positive samples, co-infections with other pathogens were more common in diarrhea than in controls (40.1% vs. 15.6%, p<0.001). Conclusions Diarrheagenic E. coli were more frequently isolated in older infants. In this setting with high frequency of pathogen exposure and high frequency of breastfeeding, we hypothesize that the major age-related differences result from decreased exposure to milk protective factors and with increased exposure to contaminated food and water.

In a prospective passive diarrhea surveillance cohort study of 1,034 infants of low socioeconomic communities in Lima, Peru, we determined the prevalence and antimicrobial drug susceptibility of the diarrheagenic Escherichia coli . The prevalence of diarrheagenic E. coli was 29% (161 of 557) in children with gastroenteritis and 30% (58 of 195) in the control group without diarrhea. The most common E. coli pathogens in diarrhea were enteroaggregative E. coli (EAEC) (14%), enteropathogenic E. coli (EPEC) (7%), diffusely adherent E. coli (DAEC) (4%), and enterotoxigenic E. coli (ETEC) (4%). Diarrheagenic E. coli as a group exhibited high levels of antimicrobial drug resistance in diarrheal cases to ampicillin (85%), cotrimoxazole (79%), tetracycline (65%), and nalidixic acid (28%). Among individual E. coli groups in patients with diarrhea, DAEC and EAEC exhibited significant higher frequencies of resistance to ampicillin, cotrimoxazole, tetracycline and nalidixic acid than EPEC and ETEC. Antimicrobial drug resistance to ampicillin and cotrimoxazole were more frequent in E. coli isolated from diarrheal samples than controls, which reflected greater antibiotic exposure in patients with gastroenteritis.

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...

Patients with diarrhea due to strains of enterohemorrhagic Escherichia coli (EHEC) (e. g. O157:H7) might be at a higher risk of developing hemolytic uremic syndrome when treated with antimicrobial agents. It has been suggested that this might be due to an increase of release or production of vero or shiga-like toxin from such organisms, possibly as a stress response to antimicrobial agents. The aim of this study was to detect such increases in extracellular toxin in vitro with a newly developed method that exposed EHEC to high sublethal concentrations followed by a recovery phase at progressively lower concentrations. Five strains of EHEC were exposed to continuously changing concentrations of ciprofloxacin, co-trimoxazole, cefixime and tetracycline. The amount of free shiga-like toxin I (SLT-I) released was compared to the amount released from inocula that were not exposed to antibiotics. There were significant differences between the five EHEC strains in the amount of toxin detected after exposure to antimicrobial agents (p less than 0.001). Equally important was the type of antibiotic (p less than 0.001), with ciprofloxacin inducing the largest increase ranging from 169 to 436%, followed by co-trimoxazole, cefixime and tetracycline. In addition, the increases in free toxin correlated with the concentration of the antibiotics (p less than 0.001). The association between antibiotic-induced increases in SLT-I produced by strains of EHEC and certain classes of antibiotics might influence the analysis of future epidemiological studies on risk factors for HUS.

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Much has been learned in recent years about the mechanisms by which breastfeeding improves child health and survival. However, there has been little progress in using these insights to improve pediatric care. Factors that are important for protecting the breast fed infant might be expected to decrease the adverse effects of weaning on diarrhea, growth, and development. Lactoferrin, an ironbinding protein with multiple physiological functions (anti-microbial, anti-inflammatory, and immunomodulatory), is one of the most important proteins present in mammalian milk. Protection against gastroenteritis is the most likely biologically relevant activity of lactoferrin. Multiple in vitro and animal studies have shown a protective effect of lactoferrin on infections with enteric microorganisms, including rotavirus, Giardia, Shigella, Salmonella and the diarrheagenic Escherichia coli. Lactoferrin has two major effects on enteric pathogens: it inhibits growth and it impairs function of surface expressed virulence factors thereby decreasing their ability to adhere or to invade mammalian cells. Thus, lactoferrin may protect infants from gastrointestinal infection by preventing the attachment by enteropathogens in the gut. Recently several clinical trials in children have started to address this issue. Whether lactoferrin can prevent a significant portion of diarrheal disease remains to be determined.

Shiga-like toxin-producing Escherichia coli have been associated with hemorrhagic colitis and the hemolytic uremic syndrome (HUS). Because Argentina has the highest reported frequency of HUS in the world, Argentine children were prospectively studied during the HUS seasons for evidence of Shiga-like toxin-related diseases. On the basis of serology, fecal cytotoxin neutralization, stool cultures, and DNA hybridization of colony lysates, most children with HUS had evidence of infection with Shiga-like toxin-producing organisms. Children with spring-summer diarrhea also commonly (32%, confidence interval 18%-46%) had clear-cut evidence of such infection. No controls (children without gastrointestinal, renal, or hemolytic disease) had free fecal cytotoxin, positive cultures for E. coli O157:H7, or DNA probe-positive organisms; 20% of them had low serum titers of antibodies to Shiga-like toxins. E. coli O157:H7 was not common in either HUS or diarrhea patients. The high frequency of Shiga-like toxin-induced diarrhea in young children in Argentina probably explains the high incidence of HUS in this country and suggests that HUS is a relatively uncommon complication of Shiga-like toxin-related disease.

SUMMARYAn experimental apparatus has been built to investigate the ignition of fuel beds as a result of impact with burning firebrands. The apparatus allowed the ignition and deposition of both single and multiple firebrands onto the target fuel bed. The moisture content of the fuel beds used was varied, and the fuels considered were pine needle beds, shredded paper beds and crevices constructed of cedar shingles. Firebrands were simulated by machining wood (Pinus ponderosa) into small disks of uniform geometry and the size of the disks was varied. Firebrand simulation was necessary because it is difficult to capture and characterize firebrands from an actual burning object. The firebrand ignition apparatus was installed into the fire emulator/detector evaluator to investigate the influence of an air flow on the ignition propensity of fuel beds. The results of this study are presented and compared with relevant studies in the literature.