Use of DNA Probes and HEp-2 Cell Adherence Assay to Detect Diarrheagenic Escherichia coli

Levine, Myron M.; Prado, Valeria; Robins‐Browne, Roy M.; Lior, H.; Kaper, James B.; Moseley, Steve L.; Gicquelais, K G; Nataro, James P.; Víal, Pablo A.; Tall, Ben D.

doi:10.1093/infdis/158.1.224

Cited by 160 publications

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“…Enteroaggregative Escherichia coli (EAggEC) are the most recently recognized category of diarrheagenic E. coli (1)(2)(3)(4)(5)(6). EAggEC are defined by their distinctive, aggregative pattern of adherence to cultured human epithelial cells in vitro (7).…”

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confidence: 99%

“…EAggEC are defined by their distinctive, aggregative pattern of adherence to cultured human epithelial cells in vitro (7). Several epidemiological studies have found a close association between EAggEC and diarrhea in young children (2)(3)(4)(5)(6). Of particular interest, prospective studies in India, Mexico, and Brazil have noted a strong correlation with persistent childhood diarrhea (3)(4)(5)(6).…”

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confidence: 99%

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Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin.

Savarino

Fasano

Watson

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

245

195

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Enteroaggregative Escherichia coi (EAggEC) are associated with persistent diarrhea in young children. Some of these organisms produce a low-molecular-weight, heat-stable, plasmid-encoded enterotoxin that has been named EAggEC heat-table enterotoxin 1 (EAST1). We have cloned a 4.4-kb DNA fragment from the virulence plasmid of prototype EAggEC strain 17-2, which expresses enterotoxic activity as measured by electrogenic response in Ussing chambers mounted with rabbit ileal tissue. DNA-sequence analysis ofthis fragment identified an open reading frame (ORF) encoding a cysteine-rich polypeptide of 38 amino acids (Mr, 4100). Insertional and deletional mutations in this ORF resulted in loss of enterotoxic activity. The ORF was cloned into a T7 expression vector, and postinduction culture filtrates exhibited enterotoxic activity and increased ileal tissue cGMP levels. A synthetic peptide consisting of predicted amino acid residues 8-29 also showed enterotoxic activity. These data indicate that this ORF, named astA (EAggEC heat-table enterotoxin), represents the EASTI structural gene. EAST1 shows significant homology with the enterotoxic domain of heat-stable enterotoxin a (STa) of enterotoxigenic E. coil and with guanylin, a mammalian analog of STa. Unlike STa, which requires six cysteines and three disulfide linkages for ful biological activity, both EAST1 and guanylin contain four cysteine residues. Based on the cGMP data and the sequence homology to STa and guanylin, it is predicted that EAST1 stimulates the particulate form ofguanylate cyclase through the same receptorbinding region as STa and guanylin.

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confidence: 99%

mentioning

confidence: 99%

Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin.

Savarino

Fasano

Watson

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

245

195

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“…Localized adherence was observed in one of the 7. Levine et al reported that adherent E. coli strains were isolated from children without diarrhea, including strains with an aggregative adherence pattern (15). According to LD50 assay, however, these strains seemed to be significantly more virulent compared with laboratory strains such as E. coli HB101 or the strains confirmed to be normal enteric flora E. coli (unpublished data).…”

Section: Discussionmentioning

confidence: 96%

Adherence Patterns and DNA Probe Types of Escherichia coli Isolated from Diarrheal Patients in China

Xu¹,

Cheng²,

Wu³

et al. 1996

Microbiology and Immunology

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One hundred and seventy-two strains of Escherichia coli isolated from diarrheal patients in Beijing, P. R. China, were analyzed for plasmid DNA profile, HEp-2 cell adherence ability and reactivity to 10 previously described DNA probes. They had not been recognized as pathogenic E. coli in China. Of the 110 strains tested, 76 (69%) contained one or multiple large plasmids. Of the 71 strains with the large plasmids 64 could adhere to HEp-2 cells. Of the 172 strains, 102 (59.3%) were hybridized with at least one of the 10 probes. Of those, seven strains hybridized with enteroaggregative E. coli (EAggEC) probe. Their serotypes were O128 (two strains), 06 (one strain), and 0111 (one strain). Three strains were untypable. Six and three strains were hybridized with enteropathogenic E. coli (EPEC) attaching and effacing genes (eae) or EPEC adherence factor (EAF) probe, respectively. Two non-O157 : H7 strains hybridized with enterohemorrhagic E. coli (EHEC) probe. Seventy-two strains (41.9%) hybridized with shiga-like toxin 2 or 1 (SLT2 or SLT1) probes. Among the SLT1 or SLT2 probe-positive strains, 54 hybridized with invasive (INV) plasmid probe developed for identification of enteroinvasive E. coli (EIEC) and Shigella species. The INV and SLT probe-positive strains might represent a new variety of verotoxin-producing E. coli (VTEC).

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“…Several case-control studies including eaeA-positive EPEC indicated that EAF plasmid-positive isolates were significantly associated with diarrhoea [32,33]. However, atypical EPEC have also been reported as being the sole causative agent of acute diarrhoeal disease in several countries [32,[34][35][36].…”

Section: Discussionmentioning

confidence: 99%

Isolation from a sheep of an attaching and effacing Escherichia coli O115:H− with a novel combination of virulence factors

Hayes

Pearson

Roe

et al. 2002

Journal of Medical Microbiology

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Attaching and effacing (AE) lesions were observed in the caecum, proximal colon and rectum of one of four lambs experimentally inoculated at 6 weeks of age with Escherichia coli O157:H7. However, the attached bacteria did not immunostain with O157-specific antiserum. Subsequent bacteriological analysis of samples from this animal yielded two E. coli O115:H 2 strains, one from the colon (CO) and one from the rectum (RC), and those bacteria forming the AE lesions were shown to be of the O115 serogroup by immunostaining. The O115:H 2 isolates formed microcolonies and attaching and effacing lesions, as demonstrated by the fluorescence actin staining test, on HEp-2 tissue culture cells. Both isolates were confirmed by PCR to encode the epsilon (å) subtype of intimin. Supernates of both O115:H 2 isolates induced cytopathic effects on Vero cell monolayers, and PCR analysis verified that both isolates encoded EAST1, CNF1 and CNF2 toxins but not Shiga-like toxins. Both isolates harboured similar sized plasmids but PCR analysis indicated that only one of the O115:H 2 isolates (CO) possessed the plasmid-associated virulence determinants ehxA and etpD. Neither strain possessed the espP, katP or bfpA plasmid-associated virulence determinants. These E. coli O115:H 2 strains exhibited a novel combination of virulence determinants and are the first isolates found to possess both CNF1 and CNF2.

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Use of DNA Probes and HEp-2 Cell Adherence Assay to Detect Diarrheagenic Escherichia coli

Cited by 160 publications

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Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin.

Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin.

Adherence Patterns and DNA Probe Types of Escherichia coli Isolated from Diarrheal Patients in China

Isolation from a sheep of an attaching and effacing Escherichia coli O115:H− with a novel combination of virulence factors

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