Actin accumulation associated with clustered and localized adherence in Escherichia coli isolated from patients with diarrhea.

Escherichia coli D2 (serotype 07:H-) that was isolated from a child with diarrhea hybridized with an F1845 DNA probe used to detect diffuse adherence. Strain D2 adhered to tissue culture cells (HeLa and HEp-2 cells) in a clustered pattern but did not autoagglutinate on the cell surface and induced the elongation of microvilli after 3 h of incubation. After 6 h of incubation, the infected cells were positive for fluorescent-actin staining at the site of clustered adherence. When analyzed with a confocal laser scanning microscope, each D2 cell was surrounded by accumulated actin in a capsule-like formation. Capsule-like, accumulated actin was also observed with enteropathogenic E. coli (EPEC), although in this case, actin accumulation was associated with EPEC microcolonies in a localized pattern. Four other strains of F1845 DNA probe-positive, diffusely adhering E. coli were negative for actin accumulation. Strain D2 did not hybridize with EPEC attaching and effacing DNA or EPEC adherence factor DNA probes. In addition, clustered D2 cells were found inside tissue culture cells. The data suggest a novel infectious mechanism as well as genetic heterogeneity of F1845 DNA probe-positive E. coli. Capsule-like, accumulated actin may protect the bacteria from host defense mechanisms.

Tissue culture-adherent Escherichia coli in infantile diarrhea.

To determine the association of tissue culture-adherent Escherichia coli with diarrhea, serotyped E. coli strains isolated in a yearlong case-control study of infantile diarrhea in Bangkok, Thailand, were examined for adherence to HeLa cells and for hybridization with the enteropathogenic E. coli adherence factor, the F1845, and the enteroaggregative E. coli (EAggEC) DNA probes. E. coli that adhered to HeLa cells in a localized adherence (LA) pattern (LA E. coli) was isolated from 26 of 509 infants with diarrhea (cases) and 9 of 509 age-matched controls (P = .006); E. coli with diffuse or aggregative adherence (DA or AA) to HeLa cells or that hybridized with the F1845 or EAggEC probes was not associated with infantile diarrhea. LA E. coli of classical enteropathogenic E. coli (EPEC) serotypes was isolated from 11 cases and 1 control (P = .003). EPEC O44:H18 that adhered to HeLa cells in a DA pattern and hybridized with the F1845 DNA probe was the predominant E. coli (five of five colonies tested) isolated from a 5-month-old girl with diarrhea in whom no other enteric infections were identified. Although LA E. coli was highly associated with infantile diarrhea, the role of DA and AA E. coli was uncertain in this setting.

Diarrhoeal disease in children less than one year of age at a children's hospital in Guangzhou, People's Republic of China.

We performed a case-control study of diarrhoea to determine its causes in children less than 1 year old in Guangzhou, People's Republic of China, in April to September 1989. Stools were cultured for Salmonella, Shigella, Campylobacter and vibrios by standard techniques; rotavirus (RV) was identified by polyacrylamide gel electrophoresis; and specific deoxyribonucleic acid probes were used to identify Escherichia coli containing genes coding for Shiga-like toxin I and II, enteropathogenic E. coli adherence factor, and enteroinvasive E. coli (EIEC). E. coli isolates were tested for heat-labile toxin (LT) and heat-stable toxin (ST) production and mannose-resistant adherence to HeLa cells. Rotavirus was identified in 13 of 174 children with diarrhoea (cases) and in 2% of 174 age-matched children without diarrhoea (controls), P less than 0.001. C. jejuni was identified in 10% of cases and 2% of controls, P = 0.003. Giardia lamblia was identified in 4 cases, LT and ST enterotoxigenic E. coli (ETEC) in 2, and S. flexneri in 1 case; they were not found in controls. ETEC that produced LT only was isolated from 5 cases and 3 controls, P = 0.721; E. coli that adhered to HeLa cells in a diffuse pattern was isolated from 30 cases and 40 controls, P = 0.229; and E. coli that adhered in an aggregative pattern was isolated from 20 cases and 18 controls, P = 0.863. EIEC was not isolated from cases or controls. Nine cases (5%) developed persistent diarrhoea (greater than 14 d duration). C. jejuni and aggregative E. coli were isolated from different children with persistent diarrhoea.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of enterotoxigenic Escherichia coli with synthetic alkaline phosphatase-conjugated oligonucleotide DNA probes.

Alkaline phosphatase-conjugated (AP) 26-base oligonucleotide DNA probes were compared with the same probes labeled with gamma-32P for the identification of heat-labile (LT) and heat-stable (ST) enterotoxigenic Escherichia coli (ETEC). The AP oligonucleotide probes were as sensitive as the radiolabeled (RL) probes in detecting LT and STA-2 target cell DNA, but the AP ST probe, which differed from STA-1 by two bases, was less sensitive than the RL probe in detecting STA-1 DNA (6.25 versus 0.78 ng). Of 94 ETEC that were identified with the RL probe, the AP probes detected 93% (28 of 30) of ST, 73% (25 of 34) of LT, and 67% (20 of 30) of LTST ETEC. When colony lysates of these ETEC were examined, the AP probes identified all 94 ETEC. In examinations of stool blots, the RL and AP probes were shown to have sensitivities of 71 and 59%, specificities of 91 and 86%, positive predictive values of 87 and 73%, and negative predictive values of 86 and 74%, respectively. AP oligonucleotide probes to detect ETEC were less sensitive in detecting ETEC by colony or stool blot hybridization than the RL probes but could be used by laboratories without access to radioisotopes to examine colony lysates.

HeLa cell-adherent enteropathogenic Escherichia coli in children under 1 year of age in Thailand.

Enteropathogenic Escherichia coli (EPEC) was isolated from 11% of 148 Hmong children under 1 year old with diarrhea at a refugee camp in northern Thailand. Of 16 children with EPEC-associated diarrhea, 11 were infected with EPEC that adhered to HeLa cells in a diffuse pattern, 3 were infected with EPEC that adhered to HeLa cells in a localized adherence (LA) pattern, and 2 were infected with EPEC that were nonadherent. In Bangkok, EPEC was isolated from 6% of 64 children under 1 year old with diarrhea and 7% of 56 children of the same age without diarrhea. Of four children with diarrhea, two were infected with EPEC with an LA pattern, and two were infected with nonadherent EPEC. Of four children without diarrhea, one was infected with EPEC with an LA pattern, one was infected with EPEC that adhered in a diffuse pattern, and two were infected with nonadherent EPEC. The 21 EPEC isolates with an LA pattern hybridized with the EPEC adherence factor DNA probe. EPEC was the only enteric pathogen identified in 16 (80%) of 20 children with EPEC-associated diarrhea. EPEC was as frequently isolated from children under 1 year old as were other bacterial enteric pathogens. The problem of identifying EPEC with pools of polyvalent antisera are described, and the need to identify additional enteropathogenic determinants of EPEC is discussed.

Plasmids coding for colonization factor antigens I and II, heat-labile enterotoxin, and heat-stable enterotoxin A2 in Escherichia coli.

Colonization factor antigens I and II (CFA/I and CFA/II) are important in the pathogenesis of diarrhea in humans caused by some enterotoxigenic Escherichia coli (ETEC). Plasmid DNA from 16 CFA/I+ and five CFA/II+ ETEC were examined by Southern blot analysis with enterotoxin gene probes and were compared with plasmid DNA from derivatives of the same ETEC that had lost the ability to produce these colonization factors. Among the 16 CFA/I+ ETEC strains, the loss of CFA/I was accompanied by the loss of a plasmid of between 34 and 68 megadaltons (MDa) coding for heat-stable enterotoxin A2 (ST-A2) in 12 strains, by the loss of a 60-MDa plasmid coding for heat-labile enterotoxin (LT) and ST-A2 in one strain, or by deletions of a segment of DNA encoding for ST-A2 in three strains. Among five CFA/II+ ETEC strains, the loss of CFA/II was associated with the loss of a plasmid of 75 MDa coding for LT and ST-A2 in three strains, with the loss of genes coding for LT and ST-A2 from a 68-MDa plasmid in one strain, or with no discernible loss of a plasmid or DNA sequences coding for enterotoxins in the remaining strain. The loss of CFA/I and CFA/II production was associated with the loss of DNA sequences encoding for ST-A2 in 20 of 21 ETEC examined.

Colonization factors associated with enterotoxigenic Escherichia coli isolated in Thailand.

Eighty-six percent (72 of 84) of heat-labile and heat-stable, none of 141 heat-labile, and 24% (27 of 111) heat-stable enterotoxigenic Escherichia coli isolates from Thailand aggregated in less than 1 M (NH4)2SO4, hemagglutinated human group A and bovine erythrocytes in 1% D-mannose, and possessed either colonization factor I or colonization factor II. No other colonization factors were identified by these two methods.

Rotavirus as a cause of severe gastroenteritis in adults.

Rotavirus was identified as the only etiological agent in 5% of adults (28 of 526) with diarrhea who were admitted to Bamrasnaradura Hospital in Nonthaburi, Thailand, during a 1-year period. Infection was determined by detection of rotavirus in diarrheal stools by enzyme-linked immunosorbent assay accompanied by a greater than fourfold rise in serum complement fixation and radioimmunoassay antibody titers to rotavirus. Adults with clinical rotavirus infections were as severely ill as patients with most bacterial enteric infections; only patients with cholera passed more watery stools and were more dehydrated than those with rotavirus infections. Only 2 of the 28 adults with rotavirus infections had known recent contact with young children with diarrhea. Rotavirus infections in these adults occurred most frequently in the cooler, drier months in Thailand than during the rest of the year. In some settings, rotavirus should be considered in the differential diagnosis of severe diarrhea in adults as well as in young children.