Shiga toxin-producing Escherichia coli (STEC) has been recognized as an important foodborne pathogen. Of this group, O91 is one of the common serogroups frequently isolated from patients and food in some countries, with O91:H21 being previously implicated in hemolytic uremic syndrome (HUS). Cattle are principle reservoirs for STEC, and studies examining STEC shedding in cattle often include experimental inoculation of strains of interest using antibiotic resistance markers for identifiable recovery. However, indigenous fecal microbes exhibiting similar resistance patterns can confound such studies. Such was the case in a study by our group when attempting to characterize shedding patterns of O91:H21 in calves, leading us to seek other, more effective, markers. Among our strategies was the development of a chromosomally integrated bioluminescence marker via transposon mutagenesis using a luxCDABE cassette from Photorhabdus luminescens and a plasmid borne GFP marker via transformation of the pGFP vector. The luxCDABE marker was inserted on host chromosome at a site that was 27 nucleotides before the stop codon of gene yihL and confirmed to have little impact on important virulence genes and growth rate with a very high stability. In contrast, plasmid borne GFP marker showed poor stability without the application of appropriate antibiotic selection pressure. For calves receiving luxCDABE-marked O91:H21, the fecal counts of the organismranged from 1.2 x 10 3 to 1.3 x 10 4CFU/g at two days post inoculation and decreased to 5.8 to 8.7 x 10 2 CFU/g or undetectable level after two weeks.Intestinal contents sampled from various positions at day 14 post inoculation indicated that cecum and descending colon may be the primary localization sites of this O91:H21 strain. Compared to antibiotic resistance markers, the use of bioluminescence markers does not require the restricted pre-inoculation screening of animals. The enumeration of luxCDABE-marked O91:H21 from feces and intestinal contents was easily accomplished and confirmed reliable by M-PCR analysis under the presence of indigenous bacteria which cannot be eliminated by antibiotic-supplemented selective plates. Therefore, the chromosomal integrated luxCDABE marker may be a better model for the study of STEC colonization and shedding in cattle.

Shiga toxin-producing E. coli (STEC) cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). All STEC produce one or both of two Shiga toxins, Stx1 and Stx2. STEC strains that produce Stx2 are more strongly associated with HUS than strains that produce Stx1 or both Stx1 and Stx2. Epidemiologic evidence indicates a recent increase in the rate of HUS among STEC outbreaks. The increasing rate of HUS could be explained by a shift in the toxin profiles of STEC strains. The purpose of this study was to examine trends in toxin profiles of human STEC O157 isolates from 1996 to 2008 and to assess whether an increase in the number of Stx2-only-producing strains could be correlated with a recent increase in HUS cases. Data from three independent datasets, collected from PulseNet, eFORS and NARMS, were used. Additionally, trends such as seasonal variations, geographical variations, gender differences, and age differences were examined for each toxin profile. Results from this study show a shift in the toxin profile of human STEC O157 strains in the United States, in that the proportion of Stx2-only producing strains has increased dramatically since 1996.

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Hemolytic uremic syndrome (HUS) induced by Shiga toxin-producing E. coli (STEC) is the most common causes of acute kidney injury in children. The therapy of the disease is symptomatic and the main factors leading to the development of severe course of a STEC-HUS are still unknown. In our study, we dealt with factors leading to development of a severe course of STEC-HUS in pediatric patients on both the host and pathogen side. Using retrospective analysis of the courses in children in the Czech Republic, we found that the most common cause of STEC-HUS was serotype O26 and HUS most often affected children under 3 years of age. 63,8 % required dialysis and mortality was 8.62 %. On the host side we focused on the relationship between the activation of the alternative complement pathway and the severity of the course of HUS. We found a significant difference in the level of the C3 part of complement in patients who required dialysis and patients for whom dialysis was not necessary. We also a cut-off value for the C3 part of complement and its reduction below 0.825 g / l was associated with the need for dialysis treatment and a higher incidence of extrarenal complications. Based not only on our results, it can be assumed that the therapeutic effect of complement could affect the severity of the disease....