Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods, and direct culture.

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RESUMO

We examined 30 children with classical hemolytic-uremic syndrome (HUS) for the presence of enterohemorrhagic Escherichia coli (EHEC) strains in stool samples and determined the specific immune response to O157 lipopolysaccharide in acute-phase serum samples from these patients. EHEC O157 strains were isolated from stool samples of 18 (60%) of the patients, and non-O157 EHEC strains were isolated from 5 (17%) of the patients. For O157 strain isolation from stools, we introduced a selective enrichment step using O157-specific antibodies attached to paramagnetic particles (immunomagnetic separation [IMS] method). This procedure allowed the detection of O157 strains at 10(2) CFU/g of stool in the presence of 10(7) coliform background flora organisms. By using IMS followed by plating on sorbitol MacConkey (SMAC) agar and cefixime-tellurite SMAC (CT-SMAC) agar, O157 strains were detected in 18 samples, whereas colony hybridization detected a subset of 12 positive samples and direct culture on CT-SMAC or SMAC agar detected only 7. Three of the 18 O157-positive stools were negative by cytotoxicity assay performed with stool filtrates and by direct PCR with DNA extracted from stools. The IMS technique allowed the isolation of O157 strains from 18 of 20 patients with serological evidence for O157 infection. Apart from the increase in sensitivity in O157 detection compared with that of direct culture, the IMS technique also has the advantage of being less labor-intensive and less time-consuming than the molecular methods. IMS can therefore be considered an efficient method for wide-spread use in the detection of O157 strains in clinical microbiology laboratories. However, because a significant number of HUS cases were attributable to non-O157 EHEC serogroups, the use of additional methods besides IMS in the bacteriological diagnosis of HUS is necessary.

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