Stx)-producing Escherichia Coli Research Articles

Responses of human intestinal microvascular endothelial cells to Shiga toxins 1 and 2 and pathogenesis of hemorrhagic colitis.

Endothelial damage is characteristic of infection with Shiga toxin (Stx)-producing Escherichia coli (STEC). Because Stx-mediated endothelial cell damage at the site of infection may lead to the characteristic hemorrhagic colitis of STEC infection, we compared the effects of Stx1 and Stx2 on primary and transformed human intestinal microvascular endothelial cells (HIMEC) to those on macrovascular endothelial cells from human saphenous vein (HSVEC). Adhesion molecule, interleukin-8 (IL-8), and Stx receptor expression, the effects of cytokine activation and Stx toxins on these responses, and Stx1 and Stx2 binding kinetics and bioactivity were measured. Adhesion molecule and IL-8 expression increased in activated HIMEC, but these responses were blunted in the presence of toxin, especially in the presence of Stx1. In contrast to HSVEC, unstimulated HIMEC constitutively expressed Stx receptor at high levels, bound large amounts of toxin, were highly sensitive to toxin, and were not further sensitized by cytokines. Although the binding capacities of HIMEC for Stx1 and Stx2 were comparable, the binding affinity of Stx1 to HIMEC was 50-fold greater than that of Stx2. Nonetheless, Stx2 was more toxic to HIMEC than an equivalent amount of Stx1. The decreased binding affinity and increased toxicity for HIMEC of Stx2 compared to those of Stx1 may be relevant to the preponderance of Stx2-producing STEC involved in the pathogenesis of hemorrhagic colitis and its systemic complications. The differences between primary and transformed HIMEC in these responses were negligible. We conclude that transformed HIMEC lines could represent a simple physiologically relevant model to study the role of Stx in the pathogenesis of hemorrhagic colitis.

Detection and Genetical Characterization of Shiga Toxin‐Producing Escherichia coli from Wild Deer

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains isolated from wild deer in Japan were examined. A total of 43 fecal samples were collected 4 times from 4 different sites around Obihiro City, Hokkaido, Japan, in June and July 1997. Seven STEC strains were isolated by PCR screening, all of them were confirmed by ELISA and Vero cell cytotoxicity assay to be producing only active Stx type 2 (Stx2). Moreover, they seemed to carry the hemolysin and eaeA genes of STEC O157:H7, and some isolates harbored large plasmids which were similar to the 90-kilobase virulence plasmid of STEC O157:H7. Based on their plasmid profiles, antibiotic resistance patterns, PCR-based DNA fingerprinting data obtained by using random amplified polymorphic DNA (RAPD) and the stx2 gene sequences, all isolates were divergent from each other except for 3 isolates from the first and second samplings. A DNA sequence analysis of representative isolates revealed that deer originating STEC strains were closely related to each other, but not to the Stx2-producing STEC strains isolated from a mass outbreak in Obihiro at the same time. A phylogenic analysis of the deduced Stx2 amino acid sequences demonstrated that three distinct clusters existed in the deer originating STEC strains and that the Stx of deer originating STEC was closely associated with that originating from humans, but not those of STEC originating from other animals. These results suggest that STEC contamination of deer carcasses should be considered as a potential source of human infection and adequate sanitary inspection of meat for human consumption is also essential for wild animals.

Detection and long-term existence of Shiga toxin (Stx)-producing Escherichia coli in sheep.

The isolation and characterization of Shiga-like toxin (Stx)-producing Escherichia coli (STEC) from sheep are described. The distribution of stx genes in E. coli isolates was detected by PCR. When brain heart infusion (BHI) broth and novobiocin supplemented m-EC broth (N-mEC) were used as enrichment culture for the isolation of STEC, N-mEC, compared to BHI, showed clearly lower efficiency. Finally, 5 STEC isolates from 4 sheep were isolated and characterized by biochemical and genetical analysis. All of them were confirmed by ELISA and Vero cell cytotoxicity assay for the production of Stx. Moreover, some strains carried hemolysin and eaeA genes and harbored large plasmids. Based on their plasmid profiles, antibiotic patterns and PCR-based DNA fingerprinting analysis using random amplified polymorphic DNA (RAPD), all isolates were different from each other. Three of the isolates were identified to belong to serogroups O2, O153 and O165, respectively, and the STEC strains belonging to these serogroups had been isolated from STEC outbreaks in humans. Four months after the first isolation in July 1997, STEC from sheep #1 was isolated again. A new isolate, HI-11, was identified as STEC O2:Hnt. Simultaneously, 2 STEC, which were genetically and phenotypically different from each other, were isolated from the same sheep at intervals of 4 months. These results demonstrate that sheep may be an important animal for studying human STEC infections, and that further epidemiological surveys on STEC are necessary.

Identification and characterization of a newly isolated shiga toxin 2-converting phage from shiga toxin-producing Escherichia coli.

Shiga toxins 1 (Stx1) and 2 (Stx2) are encoded by toxin-converting bacteriophages of Stx-producing Escherichia coli (STEC), and so far two Stx1- and one Stx2-converting phages have been isolated from two STEC strains (A. D. O'Brien, J. W. Newlands, S. F. Miller, R. K. Holmes, H. W. Smith, and S. B. Formal, Science 226:694-696, 1984). In this study, we isolated two Stx2-converting phages, designated Stx2Phi-I and Stx2Phi-II, from two clinical strains of STEC associated with the outbreaks in Japan in 1996 and found that Stx2Phi-I resembled 933W, the previously reported Stx2-converting phage, in its infective properties for E. coli K-12 strain C600 while Stx2Phi-II was distinct from them. The sizes of the plaques of Stx2Phi-I and Stx2Phi-II in C600 were different; the former was larger than the latter. The restriction maps of Stx2Phi-I and Stx2Phi-II were not identical; rather, Stx2Phi-II DNA was approximately 3 kb larger than Stx2Phi-I DNA. Furthermore, Stx2Phi-I and Stx2Phi-II showed different phage immunity, with Stx2Phi-I and 933W belonging to the same group. Infection of C600 by Stx2Phi-I or 933W was affected by environmental osmolarity differently from that by Stx2Phi-II. When C600 was grown under conditions of high osmolarity, the infectivity of Stx2Phi-I and 933W was greatly decreased compared with that of Stx2Phi-II. Examination of the plating efficiency of the three phages for the defined mutations in C600 revealed that the efficiency of Stx2Phi-I and 933W for the fadL mutant decreased to less than 10(-7) compared with that for C600 whereas the efficiency of Stx2Phi-II decreased to 0.1% of that for C600. In contrast, while the plating efficiency of Stx2Phi-II for the lamB mutant decreased to a low level (0.05% of that for C600), the efficiencies of Stx2Phi-I and 933W were not changed. This was confirmed by the phage neutralization experiments with isolated outer membrane fractions from C600, fadL mutant, or lamB mutant or the purified His6-tagged FadL and LamB proteins. Based on the data, we concluded that FadL acts as the receptor for Stx2Phi-I and Stx2Phi-II whereas LamB acts as the receptor only for Stx2Phi-II.

Perspective: the problem of non-O157:H7 shiga toxin (Verocytotoxin)-producing Escherichia coli.

Escherichia coli 0157:H7 causes diarrhea, hemorrhagic colitis, and the hemolytic uremic syndrome (HUS) [1, 2]. The cardinal virulence trait of E. coli 0157:H7 is the production of Shiga toxins (Stx, also termed Verocytotoxins) [3,4]. Unlike most coliforms in human feces, E. coli 0157:H7 does not ferment sorbitol after overnight incubation [5]. Therefore, it is easily and economically detected by laboratories using sorbitolMacConkey (s-Mac) agar screening to identify sorbitol-nonfermenting candidate E. coli 0157:H7 [6, 7]. Such colonies are then tested for the presence of the 0157 antigen, which, if detected, presumptively identifies Stx-producing E. coli (STEC) 0157:H7. In contrast, it is not known which of the many STEC belonging to serotypes other than 0157:H7 (non0157:H7 STEC) are pathogens; with rare exceptions [8, 9], such organisms ferment sorbitol and would be overlooked on an s-Mac agar plate. The same difficulty applies to the detection of sorbitol-fermenting Stx-producing E. coli 0157:NM, a pathogen in Germany [10]. In this issue, Ludwig et al. [11], report that 6 of 8 HUS patients from whom non-0157:H7 STEC were isolated produced antibodies to the O-specific lipopolysaccharide of the putatively causative strains. Ludwig et al. also determined that patients infected with E. coli expressing O antigens 26, 55, and 111 did not make antibodies to the 0157 lipopolysaccharide. Humans infected with E. coli 0157:H7 respond briskly with antibodies to the 0157 antigen [12-16], so absence of anti0157 antibodies probably exonerates E. coli 0157:H7 as the pathogen that precipitated the HUS. This is an important point, because children from whom such non-0157:H7 STEC have been isolated sometimes produce convalescent antibodies to the 0157 lipopolysaccharide [12-14], suggesting that E. coli 0157:H7 was missed in the stool culture. Furthermore, simultaneous enteric infections with E. coli 0157:H7 and non-