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Forty pediatric patients with idiopathic hemolytic uremic syndrome (HUS) were investigated for evidence of infection by Verotoxin-producing Escherichia coli (VTEC). Fecal VTEC (belonging to at least six different O serogroups including O26, O111, O113, O121, O145, and O157) or specifically neutralizable free-fecal Verotoxin (VT) or both were detected in 24 (60%) patients but were not detected in 40 matched controls. Ten of 15 of the former developed fourfold or greater rises in VT-neutralizing antibody titers, as did six other patients who were negative for both fecal VTEC and VT. A total of 30 (75%) patients had evidence of VTEC infection by one or more criteria. We concluded that a significant association exists between idiopathic HUS and infection by VTEC. The detection of free-fecal VT was the most important procedure for the early diagnosis of this infection because, in our study, VTEC were never isolated in the absence of fecal VT, whereas fecal VT was often present even when VTEC were undetectable.

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High titers of Verotoxin (VT) were released from cell pellets of VT-producing Escherichia coli (VTEC; corresponding to E. coli strains producing "hikh" levels of Shiga-like toxin) after incubation in polymyxin B (0.1 mg/ml) for 30 min at 37°C. Maximal titers of polymyxin-releasable VT occurred in cells obtained from 5-h Penassay broth cultures and were up to eightfold higher than the peak culture supernatant VT titers which occurred in 8-h cultures. Polymyxin-releasable cell extracts of 5-h broth cultures inoculated with mixtures of VT-positive (VT') and VT-negative strains had easily detectable VT titers when the proportion of VT' cells in the mixture was about 1.0%, but culture supernatants were negative for VT even when this proportion was 20%. The results were the same whether the initial inoculum consisted of broth culture mixtures of VT' and VT-negative strains or colony sweeps (loopfuls of confluent bacterial growth) taken from solid plate media previously inoculated with the broth mixtures. In a clinical study, 80 stool cultures from patients with hemolytic uremic syndrome and family contacts with diarrhea were tested for free fecal VT, VT in polymyxin extracts of colony sweeps (VT/PECS), and VTEC (examination of 20 separate E. coli colonies from primary media for VT production). Of the 80 samples, 40 were positive for at least one of these three tests; all 40 were positive for free fecal VT, and 20 of these were positive for VT/PECS. VTEC (as few as 1 colony out of 20) were only isolated from 14 of the 20 cultures that were positive for VT/PECS. In six cases, the VT/PECS was positive even when none of 20 colonies tested were VT+, suggesting that the procedure was able to detect a proportion of VTEC that was less than one in 20 (5%). We conclude that the VT/PECS method is highly sensitive for detecting low concentrations of VTEC in stools and provides a rapid method for screening out stools that are negative for VTEC. The technique should also be of value in epidemiological studies for detecting low numbers of VTEC in animal feces, foods, and environmental samples.

ABSTRACT:A survey was conducted to determine the prevalence of spiral-shaped bacteria in animals as a possible source of pathogens causing chronic changes in the human and animal stomach as well as in other parts of the digestive tract. This study was carried out in three different groups of animals, pigs, cattle and dogs. Swabs from the oral cavity of dogs (n = 198) were stained using Gram's method to evaluate gastrospirillum-like organisms (GLOs) and revealed two different types of GLOs with an incidence of 23. 2% (46/198). The stomachs of the pigs (n = 104), cattle (n = 102), and dogs (n = 7) were collected for the urease test, brush cytology, light and electron microscopy, and PCR. A positive urease test was observed in 31.7% (33/104) of pigs, 90.2% (92/102) of cattle, and 85.7% (6/7) of dog samples. GLOs were detected in 37.5% (39/104) of pigs, 62.7% (64/102) of cattle, and 85.7% (6/7) of dog samples by brush cytology. Furthermore, positive PCR results were obtained in the stomach samples of dogs that had tested positive by both the urease test and brush cytology. The morphological study using brush cytology and scanning electron microscopy of a pig stomach revealed bacteria with the typical morphology of GLOs, which appeared to be similar to Helicobacter heilmanii. This study indicates that the urease test and brush cytology are useful tools for diagnosing GLOs in different animals. Moreover, the location of specimen collection can influence the diagnostic sensitivity of the examination.