Use of DNA probes and HEp-2 cell adherence assay to detect diarrheagenic Escherichia coli.
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Biomedical subjects
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Escherichia coli that exhibit the aggregative pattern of adherence to HEp-2 cells (enteroadherent-aggregative E. coli [EA-AggEC]) have been epidemiologically incriminated as a cause of diarrhea. We undertook a preliminary microbiological and pathogenetic characterization of 42 isolates of this putative pathogen. The strains were negative by tests with DNA probes for enteropathogenic, enterotoxigenic, enteroinvasive, and enterohemorrhagic E. coli and, by serotype, did not fit these categories. Thirty-nine of 42 strains had a 55-65-megadalton plasmid; many shared DNA homology. With one representative strain, plasmid transfer was accompanied by transfer of smooth lipopolysaccharide, fimbriae expression, and the aggregative property. EA-AggEC caused characteristic lesions in rabbit and rat ileal loops. The intestinal lesions and (Shiga-like) limb paralysis and death in rabbits inoculated with live organisms suggest toxin involvement; assays for Shiga-like toxins were negative. These preliminary results support the contention that EA-AggEC may represent a distinct category of diarrheagenic E. coli.
The newly described stable enterotoxin producing, enterotoxigenic Escherichia coli, serotype O153:H45, capable of expressing colonizing factor antigen I, is frequently isolated as a cause of diarrhea among Chilean children. Hybridization studies of five new strains confirmed previous results which indicated that the stable enterotoxin genes are contained in nonconjugative plasmids ranging in size from 81 to 87 kilobases. The strains expressed similar antibiotic resistance and metabolic properties but differed in their plasmid content.
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Enterohemorrhagic Escherichia coli (EHEC) cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), make potent cytotoxins (Verotoxins [VT] or Shiga-like toxins), and possess a plasmid (approximately 60 megadaltons) that encodes a new fimbrial antigen and promotes attachment to epithelial cells. We evaluated the use of a DNA probe, prepared from a 3.4-kilobase segment of the EHEC plasmid, to detect EHEC. The probe hybridized with 106 (99%) of 107 O157:H7 and 34 (77%) of 44 O26:H11, VT-positive strains from patients with colitis, HUS, and diarrheal disease and hybridized with 21 (81%) of 26 VT-positive E. coli of serotypes other than O157:H7 or O26:H11 from patients with hemorrhagic colitis and HUS. We examined 601 other strains, including 18 serotype O26 isolates of H types other than H11, 306 enteropathogenic E. coli, 60 enteroinvasive E. coli, 119 enterotoxigenic E. coli, and 20 isolates from the urinary tract and 77 isolates from the normal intestinal flora; only one (O127:H-) was positive (specificity, 99.8%). Serotype O26:H11, previously considered a classic enteropathogenic E. coli serotype, is now shown to be EHEC.
A total of 516 Escherichia coli strains randomly isolated from coprocultures of 154 Chilean children with diarrhea and 66 controls were examined with DNA probes and tested for adherence to HEp-2 cells. Three adherence patterns were distinguished, localized, true diffuse and "aggregative." Enteropathogenic E. coli (EPEC) were detected by EPEC adherence factor probe among 86 of the 372 isolates (23%) from patients with diarrhea vs. 14 of 144 (10%) strains from controls (P less than 0.0002). Of 95 strains that manifested localized adherence, 97% were EPEC adherence factor probe-positive; thus the HEp-2 assay may serve as an alternative to the probe in identifying EPEC adherence factor-positive EPEC. True diffuse adherence was not associated with diarrhea. In contrast the aggregative pattern appears to signify a new, distinct class of diarrheagenic E. coli (enteroadherent-aggregative E. coli). The aggregative pattern was found in only 3 of 27 enterotoxigenic, 0 of 4 enteroinvasive, 0 of 2 enterohemorrhagic and 2 of 86 EPEC strains but in 84 of 253 probe-negative strains (P less than 0.00001) from patients with diarrhea; in comparison only 20 of 134 probe-negative strains from controls were aggregative E. coli (P less than 0.00001 vs. probe-negative strains from diarrhea patients).
A total of 24 pediatric patients with a diagnosis of meningitis, confirmed by positive CFS culture, were enrolled in the study. All patients received ceftriaxone in different doses. No difference in clinical outcome was found between the patients who received ceftriaxone twice a day and the 11 who received the drug as a single daily dose. The latter dosage proves to be preferable. The therapeutic outcome in this series of patients was 22 cured and 2 failures. Bacteriological and pharmacokinetic findings are discussed.
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The incidence of enterotoxigenic Escherichia coli (ETEC) was investigated in 95 E. coli strains isolated from 48 infants with diarrhea in Santiago, Chile. By using standard biological assays and DNA-DNA hybridization procedures, ETEC was found in 31.2% of the cases: 14 strains produced heat-stable enterotoxin (ST) only, three strains produced heat-labile enterotoxin (LT) and ST, and two strains produced LT only. DNA probes detected all enterotoxin producers except one ST-producing strain. The ST strains hybridized with one or both of the human ST probes (ST Ib and ST A2). Two of the LT-ST strains hybridized with the ST Ia and ST Ib probes, and the third strain did not hybridize with any of the ST probes. Only the ST group expressed multiple resistance (85.7%) and colonization factor antigen I (CFA I) (92.8%); CFA II was found in two of three LT-ST strains. The O153:H45 serotype was found in 10 of 14 ST strains, and O6:K15:H16 was found in one LT strain and in two LT-ST strains. These findings suggest that ETEC, especially strains that produce ST, may be an important cause of diarrhea among Chilean infants.
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