Enteroaggregative Escherichia Coli Heat-stable Enterotoxin 1 Research Articles

Detection and Isolation of Escherichia coli with a Coding Gene for Enteroaggregative Escherichia coli Heat-Stable Enterotoxin 1 from Food and Comparison with Fecal Isolates

Enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) was originally regarded as a putative enterotoxin of enteroaggregative Escherichia coli. Although its etiological role has not yet been elucidated, it has been epidemiologically suggested that some strains of E. coli possessing EAST1-coding gene (astA) but no other identifiable pathogenic properties comprise a new group of diarrhea-associated E. coli (EAST1EC). However, the source of the organisms and their prevalence in foods are still obscure. In this study, methods for detection of the organisms in foods heavily contaminated with coliforms were evaluated and properties of the isolated strains were compared with those of fecal strains. Four enrichment methods (brilliant green lactose bile broth, E. coli, lauryl tryptose broth, and a combination of brain heart infusion broth and tryptone phosphate broth) were evaluated through inspection of 115 samples. PCR showed positive results in 26 samples after enrichment with a combination of brain heart infusion broth and tryptone phosphate broth, and EAST1EC was successfully isolated from 18 samples. Fifteen samples showed a positive reaction in the PCR test after enrichment by the other methods, and the organisms were isolated from only 10 specimens. The highest prevalence of EAST1EC was found in animal products (16 of 54, 29.6%); the organism was rarely found in foods of plant origin (2 of 45, 4.4%) or fishery products (1 of 16, 6.3%). Although EAST1EC is unexpectedly common in animal products, its potential as a human pathogen remains uncertain because the possession of some virulence properties differs significantly between strains from fecal specimens and those from foods. Some food isolates, however, possess the same characteristics as diarrheal isolates do. It is necessary to clarify the pathogenicity of EAST1EC and the significance of food as a source of infection.

Expression, purification, and biochemical characterization of enteroaggregative Escherichia coli heat-stable enterotoxin 1

Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) is a 4.1 kDa protein originally discovered in EAEC but known to be scattered in other diarrheagenic E. coli as well, possibly causing diarrhea in humans and animals. We report for the first time a method to express and purify EAST1 using the Glutathione S-transferase (GST) fusion system. The gst and astA genes were fused together on a pGEX-2T plasmid vector to produce a GST-EAST1 fusion protein. Using Glutathione Sepharose affinity chromatography and C 8 reverse phase high pressure liquid chromatography, EAST1 was purified to homogeneity with a yield of 0.29 mg/L of culture. The protein purified by this method was confirmed to be EAST1 by NH 2-terminal sequencing and mass spectrometry. The molecular weight of EAST1 is 4104.0 Da, confirming a 38 amino acid peptide as predicted by the astA gene sequence. Mass spectrometry analysis of EAST1 and of two generated peptides established the presence and suggested the position of two disulfide bridges of EAST1 in the conformations C1–C2 and C3–C4. Polyclonal antibodies were raised against EAST1 in New Zealand white rabbits to a titer of 1:8000 using affinity-purified GST-EAST1 fusion protein and to a titer of 1:100 using HPLC-purified EAST1. The biological activity of various EAST1 preparations was tested using the suckling mouse assay with CD-1 and CFW mice strains, but failed to produce fluid accumulation in the intestine.

Detection of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) gene and its relationship with fimbrial and enterotoxin markers in E. coli isolates from pigs with diarrhoea

The presence of the astA gene responsible for production of enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) was examined in E. coli strains isolated from pigs with postweaning diarrhoea. Two hundred and seven isolates were tested using PCR for the astA marker and for heat-labile I (LTI), heat-stable I (STI), and heat-stable II (STII) enterotoxin genes. Moreover, the isolates were also analysed for their serotypes (O and K antigens) as well as for fimbrial adhesins using agglutination methods. It was shown that 96 (46.4%) of the isolates possessed the astA genetic determinant. The most common EAST1-positive E. coli serotype was O149:K91 and these strains were mostly LTI/STII-positive. A close correlation between the presence of F4 fimbriae and the EAST1 gene was also observed: 88 of 96 (91.7%) astA + isolates tested possessed the F4 antigen. Thus, EAST1 enterotoxin may represent an additional virulence determinant playing a role in the pathogenesis of porcine colibacillosis.

Enteroaggregative Escherichia coli Heat-Stable Enterotoxin 1 (EAST1): A New Toxin with an Old Twist

Enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) is a small protein that was first detected more than a decade ago in an enteroaggregative E. coli (EAEC) strain isolated from the stools of a diarrheic child. The EAST1 gene, astA, is not solely present in EAEC, but also in other categories of diarrheagenic E. coli. Strains expressing EAST1 have been shown to induce diarrhea principally in humans, although they have also been associated with piglets and calves. EAST1 toxin has been proposed as a virulence factor implicated in the mechanism of pathogenesis of EAEC and could play a role in the pathogenicity of other enteropathogens as well. This toxin is often compared to E. coli STa enterotoxin because they share some physical and mechanistic similarities. This review summarizes the various observations on EAST1 since its discovery.

Prevalence of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) gene in isolates in weaned pigs with diarrhea and/or edema disease

A total of 476 Escherichia coli isolated from weaned pigs with diarrhea and/or edema disease were screened for the presence of the enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) gene by polymerase chain reaction (PCR). E. coli strains that carried EAST1 genes were also tested by PCR for the presence of genes for five fimbriae (F4, F5, F6, F18 and F41), two heat-stable (STa and STb) and one heat-labile (LT) enterotoxin, and Shiga toxin 2e (Stx2e). One hundred and forty nine (31.3%) of the 476 E. coli isolates carried the gene for EAST1. Of these 149 isolates, 66 (44.3%) carried the east1 gene only and 83 (55.7%) carried genes for the fimbrial adhesins or enterotoxins. E. coli which carried east1 gene also possessed genes for STa or F4 frequently. EAST1 may represent an additional determinant in the pathogenesis of E. coli diarrhea in weaned pigs.

The sequences of enterohemorrhagic Escherichia coli and Yersinia pestis that are homologous to the enteroaggregative E. coli heat-stable enterotoxin gene: cross-species transfer in evolution

The enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) gene is widely distributed among diarrheagenic E. coli. In this study, we examined the sequences of enterohemorrhagic E. coli (EHEC) strains by PCR and sequencing. All the EHEC strains possessed the EAST1 gene homologues but with two types of mutations. One of the mutation types was strongly associated with the large outbreak episodes in 1996 in Japan. Sequence comparison showed that the EHEC sequences are a branch of the EAST1 gene sequence family that showed the cross-species transfer in evolution among E. coli and Yersinia pestis.

Detection of enteroaggregative Escherichia coli from sporadic diarrhea patients

A study of enteroaggregative Escherichia coli (EAggEC) which was recently reported as a causative agent of diarrhea was attempted, by isolating these organisms from the fecal samples collected from sporadic diarrhea patients in Miyazaki Prefecture during the period from January 1993 to April 1998, and by investigating several characteristics of the isolates. By using the PCR method targetting aggR gene which was a transcriptional activator of aggregative adherence fimbria I expression, thirty four strains of aggR(+)-Escherichia coli (E. coli) were detected from 2,652 fecal samples. Twenty nine of these 34 isolates were confirmed as EAggEC by demonstration of aggregative adherence to HEp-2 cells, and the other 5 isolates were not EAggEC because they showed negative adherence to HEp-2 cells. The above mentioned, aggR-PCR method revealed that there were a few non-EAggEC strains with aggR gene. It has been reported that aggregative adherence fimbriae are encoded by the plasmid of about 60 Md. All of the 29 EAggEC isolates possessed plasmids of about 50 Md or more, and these plasmids were suggested to relate to aggregative adherence fimbriae. Sixteen (55%) of the 29 isolates were classified serologically into two serotypes, O111:H21 and O126:H27, and the other 13 isolates were classified into ten groups or more which included a few strains in a group. EAggEC heat-stable enterotoxin 1 (EAST1) gene was demonstrated in nineteen of 29 isolates. In drug susceptibility test, 72%, 59% and 21% of the 29 isolates showed resistance to Ampicillin, Cefazolin, and Streptomycin, respectively.

Comparison of the nucleotide sequence of enteroaggregative Escherichia coli heat-stable enterotoxin 1 genes among diarrhea-associated Escherichia coli

The presence of the enteroaggregative Escherichia coli (EAggEC) heat-stable enterotoxin 1 (EAST1) gene was investigated in 15 strains each of EAggEC, enteropathogenic E. coli (EPEC), EPEC-related strains of non-EPEC serotypes, diffusely adhering E. coli (type 1 DAEC) that carries F1845 adhesive pili (or a related adhesin), and enteroinvasive E. coli (EIEC) by PCR and colony hybridization. The EAST1 gene or its homologue was present in 53.3% of EAggEC, 20% of EPEC, 13.3% of the EPEC-related strains, and 6.7% of type 1 DAEC. EIEC and E. coli unrelated with diarrhea had no gene with sequence similarity to the EAST1 gene. Comparison of the EAST1 gene sequences analyzed in this study as well as those reported previously showed that EAggEC (including strain O42, which was shown to be pathogenic in volunteer experiments), EPEC, type 1 DAEC, type 2 DAEC (which carries the 57-kDa outer membrane protein as an adhesin), and enterotoxigenic E. coli shared a common sequence. A variant type of the EAST1 gene sequence was present in the EAggEC strain 17-2 (initially characterized for the EAST1 gene) and in an EPEC-related strain of a non-EPEC serotype. These data suggest that the EAST1 gene or its variant is a virulence gene widely distributed among diarrhea-associated E. coli.