Enzyme-linked immunosorbent assay to detect Shiga toxin of Shigella dysenteriae and related toxins.

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detection of Shiga toxin. Four species of Shigella, Escherichia coli, and Vibro parahaemolyticus were tested for production of Shiga or Shiga-like toxin by ELISA and Vero cell bioassay. In the ELISA, most strains of S. dysenteriae and some strains of E. coli isolated from traveler's diarrhea were positive. These ELISA-positive strains were positive by Vero cell bioassay without exception. Some E. coli strains and most V. parahaemolyticus strains were toxic to Vero cells, although they were negative in the ELISA. Much of the cytotoxic activity was not neutralized by anti-Shiga toxin antiserum. The newly developed sandwich ELISA is specific and can be a substitute for the cumbersome Vero cell bioassay.     

Purification and some properties of Shiga-like toxin from Escherichia coli O157:H7 that is immunologically identical to Shiga toxin

A cytotoxin to Vero cells (Shiga-like toxin), which was neutralized by antibody against purified Shiga toxin produced by Shigella dysenteriae 1, was purified from Escherichia coli O157:H7, isolated from a patient with hemorrhagic colitis. The purification procedure consisted of ammonium sulfate fractionation, DEAE-cellulose column chromatography, chromatofocusing column chromatography and high performance liquid chromatography. About 200 micrograms of purified Shiga-like toxin was obtained from cell extracts of 14 liters of culture with a yield of about 15%. The purified Shiga-like toxin showed identical physicochemical, biological and immunological properties to those of Shiga toxin. Purified Shiga-like toxin and Shiga toxin also had the same mobilities on polyacrylamide disc gel electrophoresis and polyacrylamide gel isoelectrofocusing. On sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, purified Shiga-like toxin migrated as two bands corresponding to the A and B subunits, and these migrated to the same positions as A and B subunits of Shiga toxin. The amino acid composition of the purified Shiga-like toxin was also similar to that of Shiga toxin. The purified Shiga-like toxin showed various biological activities: lethal toxicity to mice when injected intraperitoneally, the LD50 being 30 ng per mouse; cytotoxicity to Vero cells, killing about 50% of the cells at 6 pg; and fluid accumulation in rabbit ileal loops at concentrations of more than 1.25 micrograms/loop. These values are comparable with those obtained with Shiga toxin. In an Ouchterlony double gel diffusion test, the lines formed by the purified Shiga-like toxin and Shiga toxin fused, indicating that the two toxins were immunologically identical.     

In vivo formation of hybrid toxins comprising Shiga toxin and the Shiga-like toxins and role of the B subunit in localization and cytotoxic activity.

Shiga toxin, Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) are cell-associated cytotoxins that kill both Vero cells and HeLa cells, whereas Shiga-like toxin II variant (SLT-IIv) is an extracellular cytotoxin that is more cytotoxic for Vero cells than for HeLa cells. The basis for these differences in cytotoxin localization and host cell specificity were examined in this study. The A and B subunit genes of Shiga toxin and the SLTs were recombined by two methods so that hybrid toxins would be formed in vivo. Complementation of heterologous subunits was accomplished by cloning the individual A and B subunit genes of SLT-I, SLT-II, and SLT-IIv on plasmid vectors of different incompatibility groups so that they could be maintained in double transformants of Escherichia coli. In addition, six operon fusions were constructed so that the A and B subunit genes of Shiga toxin, SLT-II, and SLT-IIv could be expressed as a single operon. The activities of the hybrid cytotoxins were assessed in three ways: (i) level of cytotoxicity, (ii) ratio of HeLa to Vero cell cytotoxicity, and (iii) ratio of extracellular to cell-associated cytotoxicity. Neither the A subunit of Shiga toxin nor SLT-I associated with a heterologous B subunit to form an active cytotoxin. However, in all other cases the hybrid molecules formed by subunit complementation or operon fusion were cytotoxic. Furthermore, the cytotoxic specificity and localization of the hybrid cytotoxins always corresponded to the activities of the native toxin possessing the same B subunit.     

Production of a unique cytotoxin by Campylobacter jejuni.

Campylobacter jejuni is an important diarrheal pathogen worldwide; the mechanisms by which it causes disease remain unclear. Because of its association with inflammatory diarrhea, we postulated that C. jejuni might produce a cytotoxin similar to that produced by Shigella sp., enterohemorrhagic Escherichia coli O157, or Clostridium difficile. Filtrates of 12 polymyxin-treated isolates of C. jejuni were placed on HeLa cells (sensitive to Shiga toxin cytotoxicity) and Chinese hamster ovary (CHO) cells. Of 12 isolates of C. jejuni tested, 5 killed 50% of the cells at greater than or equal to 1:4 dilutions of filtered suspensions of 10(9) bacteria per ml; killing was similar in HeLa and CHO cells (the CHO cells being insensitive to Shiga cytotoxin). One isolate produced a titer of 1:32 to 1:128. The relative potency in HeLa cells was comparable to that of E. coli strains that produce intermediate amounts of Shiga-like toxin. The other seven strains showed no cytotoxic effect, nor did the control diluents, polymyxin B, or supernatants of C. jejuni not treated with polymyxin B. Sonication also released active cytotoxin, but slightly less well than did polymyxin. The cytotoxic effect was dose dependent. Concentration of the C. jejuni in suspension by 10-fold before treatment with polymyxin B resulted in a 10-fold increase in the 50% cytotoxic dose. The cytotoxin effect was not neutralized by Shiga toxin immune serum against either Shiga-like toxin I or II or by anti-Clostridium difficile antiserum. The C jejuni cytotoxin was partially labile to trypsin (0.25%) and to heating to greater than or equal to 60 degrees C. Cytotoxicity was retained in Scientific Products dialysis tubing D1615-1 (Mr cutoff, 12,000 to 14,000). Some isolates of C. jejuni release a substance lethal to HeLa or CHO cells in vitro that is distinct from Shiga-like or Clostridium difficile toxin. This cytotoxin may contribute to the colonic mucosal invasive process that characterizes C. jejuni enteritis.     

Characterization of monoclonal antibodies against Shiga-like toxin from Escherichia coli

Three monoclonal antibodies, designated MAb 16E6, MAb 13C4, and MAb 19G8, were produced which recognize Shiga-like toxin (SLT) from Escherichia coli. All three monoclonal antibodies neutralized the cytotoxicity of E. coli SLT and were able to immunoprecipitate intact labeled toxin with Staphylococcus aureus protein A. The three antibodies were of the G1 heavy and kappa light chain classes. MAb 16E6 bound to the B subunit of SLT in Western blots and also neutralized the lethality of the toxin for mice and the enterotoxicity of the toxin in ligate rabbit ileal loops. The ability of MAb 16E6 to neutralize the cytotoxicity, lethality, and enterotoxicity of E. coli confirms the hypothesis that all three activities are associated with a single toxin. MAb 16E6 and MAb 13C4 also neutralized the cytotoxicity of purified Shiga toxin from Shigella dysenteriae type 1 and Shiga-like toxic activities in crude cell extracts from Shigella flexneri, Vibrio cholerae, Vibrio parahaemolyticus, and Salmonella typhimurium. Thus, Shiga toxin and the SLTs from E. coli, Shigella flexneri, V. cholerae, V. parahaemolyticus, and Salmonella typhimurium share a common B subunit epitope that is involved in neutralization. MAb 13C4 has been successfully used in a colony blot assay for the detection of bacterial colonies which produce high levels of SLT. Sixty-two different strains of bacteria were tested by both the cytotoxicity and colony blot assays for the presence of SLT. The colony blot assay detected all strains of bacteria which produce greater than or equal to 10(5) 50% cytotoxic doses of SLT per ml of sonic lysate. There were no false-positive results among the 62 samples tested.     

Rapid method to detect shiga toxin and shiga-like toxin I based on binding to globotriosyl ceramide (Gb3), their natural receptor.

Shiga toxin and the closely related Shiga-like toxins produced by Escherichia coli represent a group of very similar cytotoxins that may play an important role in diarrheal disease and hemolytic uremic syndrome. These toxins have the same biologic activities and according to recent studies also share the same binding receptor, globotriosyl ceramide (Gb3). They are currently detected, on the basis of their ability to damage several cell lines, by using expensive and tedious assays that require facilities for and experience with tissue cultures and are therefore most suitable for research laboratories. We have developed a rapid method to detect Shiga toxin and Shiga-like toxin I based on specific binding to their Gb3 natural receptor, which was coated onto microdilution plates. Bound toxin was then detected by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The sensitivity of the Gb3 ELISA was 0.2 ng (2 ng/ml) of purified toxin. The assay was positive with sonic extracts of Shigella dysenteriae serotype 1 strain 6OR (a Shiga toxin producer), E. coli serotype O26:H11 strain H30, and E. coli serotype O157:H7 (both Shiga-like toxin I producers). The assay was very specific in that no cross-reactivity was noted with purified cholera toxin, E. coli heat-labile and heat-stable enterotoxins, and Clostridium difficile cytotoxin, or sonic extracts of other cytotoxin-producing organisms, such as other shigellae, pathogenic and nonpathogenic E. coli, Salmonella spp., Campylobacter spp., and Aeromonas spp. These results were in complete agreement with a [3H]thymidine-labeled HeLa cell cytotoxicity assay and with detection of the structural genes by DNA hybridization studies with a Shiga-like toxin I probe. Quantitative analysis showed a high correlation between Gb3 ELISA and HeLa cell assay when fractions obtained at various stages of toxin purification were examined by both methods (r = 0.99, P < 0.01). This rapid Gb3 ELISA is sensitive and specific and may be diagnostically useful in cytotoxin-related infections.     

Purification and some properties of a Vero toxin from Escherichia coli O157:H7 that is immunologically unrelated to Shiga toxin

A cytotoxin to Vero cells (Vero toxin) was purified from Escherichia coli O157:H7 isolated from a patient with hemorrhagic colitis by ammonium sulfate fractionation, DEAE-cellulose column chromatography, repeated chromatofocusing column chromatography and repeated high performance liquid chromatography. About 440 micrograms of purified Vero toxin was obtained from 12 liters of culture with a yield of about 22%. The purified Vero toxin showed similar cytotoxic activity to that of Shiga toxin to Vero cells and killed about 50% of the Vero cells at 1 pg. The activity was lost on heating the toxin at 80 degrees C for 10 minutes, but not at 60 degrees C for 10 minutes. The toxin also showed lethal toxicity to mice when injected intraperitoneally, the LD50 being 1 ng per mouse. The purified Vero toxin consisted of A and B subunits with molecular weights of about 35,000 and 10,700, respectively, which were slightly larger than those of Shiga toxin. On polyacrylamide gel disc electrophoresis, the mobility of the purified Vero toxin differed from that of Shiga toxin. The isoelectric point of the toxin was 4.1, which was also different from that of Shiga toxin (pI = 7.0). Furthermore, Vero toxin and Shiga toxin were found to be immunologically unrelated; anti-Vero toxin did not react with Shiga toxin, and similarly anti-Shiga toxin did not react with the Vero toxin in either the Ouchterlony double gel diffusion test or enzyme-linked immunosorbent assay. The Vero toxin purified in this work was found to be immunologically identical to VT2 and Shiga-like toxin II reported previously.     

A method for detecting Shiga toxin and Shiga-like toxin-I in pure and mixed culture

Shiga toxin and Shiga-like toxins (SLTs, syn. Verotoxins) are currently detected by tissue culture assays that are expensive, time-consuming and require specialised facilities and experienced personnel. We have developed a rapid method to detect Shiga toxin and SLT-I (Verotoxin 1) based on their binding to globotriosyl ceramide (Gb3). Bound toxin was then detected by an enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The direct detection of cytotoxins from pure culture plates and from a mixed bacterial culture was studied. Using polymyxin extraction (0.1 g/L, 30 min, 37 degrees C) and Gb3-based ELISA we detected toxin from reference strains Shigella dysenteriae 1 strain 60R (Shiga toxin) and Escherichia coli O26:H11 strain H30 (SLT-I), and from clinical isolates of E. coli O157:H7 and O26:H11 (both SLT-I) from 11 patients with diarrhoea, haemorrhagic colitis or haemolytic uraemic syndrome. Toxin production by these strains was confirmed by a radiolabelled HeLa cell assay and the structural genes were detected by DNA hybridisation. The Gb3-based ELISA could detect SLT-I in extracts of a mixed culture even when the toxin-positive strains represented only 1% of the mixture. No cross-reactivity was found with bacteria that produce other cytotoxins, such as other E. coli and Shigella, Salmonella, Aeromonas and Campylobacter spp.     

Two toxin-converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities.

Escherichia coli O157:H7 strain 933 contains two distinct toxin-converting phages (933J and 933W). The biologic activities and antigenic relationship between the toxins produced by 933J and 933W lysogens of E. coli K-12, as well as the homology of the genes that encode the two toxins, were examined in this study. The 933J and 933W toxins, like Shiga toxin produced by Shigella dysenteriae type 1, were cytotoxic for the same cell lines, caused paralysis and death in mice, and caused fluid accumulation in rabbit ileal segments. The cytotoxic activity of 933J toxin for HeLa cells was neutralized by anti-Shiga toxin, whereas the activity of 933W toxin was not neutralized by this antiserum. In contrast, an antiserum prepared against E. coli K-12(933W) neutralized 933W toxin but not 933J toxin or Shiga toxin. For E. coli 933, most of the cell-associated cytotoxin was neutralized by anti-Shiga toxin, whereas most of the extracellular cytotoxin was neutralized by anti-933W toxin. However, a mixture of these antisera indicated the presence of both toxins in cell lysates and culture supernatants. Among 50 elevated cytotoxin-producing strains of E. coli, we identified 11 strains isolated from cases of diarrhea, hemorrhagic colitis, or hemolytic uremic syndrome that produced cell-associated cytotoxins which were neutralized by the 933W antitoxin. Southern hybridization studies showed that the cloned toxin structural genes from phage 933J hybridized with DNA from phage 933W under conditions estimated to allow no more than 26% base-pair mismatch. These findings indicate that E. coli produces two genetically related but antigenically distinct cytotoxins with similar biologic activities which we propose to name Shiga-like toxins I and II. Strains of E. coli that produce elevated levels of Shiga-like toxin I or Shiga-like toxin II, or both, have been associated with the clinical syndromes of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome.     

Effects of iron and temperature on Shiga-like toxin I production by Escherichia coli.

Iron is known to depress Shiga toxin production by Shigella dysenteriae 1, and temperature has been shown to regulate several genes required for Shigella invasiveness. In this study, the influence of iron and temperature on regulation of a highly related toxin, Shiga-like toxin I (SLT-I) of enterohemorrhagic Escherichia coli, was examined in strains lysogenic for the toxin-converting coliphage 933J and in strains carrying the cloned slt-I genes on a high-copy-number plasmid vector. For comparison, S. dysenteriae 1 was included in these studies. As expected, iron suppressed Shiga toxin synthesis, and reduced growth temperature was also found to decrease Shiga toxin production. Iron also suppressed SLT-I synthesis in E. coli lysogenized with phage 933J but did not demonstrably repress toxin synthesis in E. coli strains carrying the cloned slt-I genes. Temperature had no effect on SLT-I synthesis. Mini-Mu lac operon fusions were then isolated in the cloned slt-I genes and used to test for regulation of beta-galactosidase by iron. Iron did not decrease beta-galactosidase production in strains that harbored these operon fusion plasmids. Taken together, these results indicate that iron but not temperature represses SLT-I synthesis when the slt-I genes are phage associated but this suppression is not easily demonstrated when the slt-I genes are cloned on a high-copy-number plasmid.     

Production of Shiga-like toxin among Escherichia coli strains and other bacteria isolated from diarrhea in S?o Paulo, Brazil

An elevated level of Shiga-like toxin I (SLT-I) production was found in 1 of 466 Escherichia coli strains studied. Among the 34 sonic lysates obtained from classical enteropathogenic E. coli, 5 produced SLT-I. The Aeromonas, Citrobacter, Edwardsiella, Enterobacter, Klebsiella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Yersinia, and Vibrio strains also studied were not SLT producers, except for a Shigella dysenteriae type 1 strain. Although SLT-I-producing E. coli strains were isolated from diarrhea, they seem to be an uncommon cause of disease in children less than 1 year old in our community.     

Production of vero cytotoxin by Escherichia coli and Shiga toxin by Shigella dysenteriae 1 as related to the growth medium and availability of iron

Six strains of Escherichia coli producing Vero cytotoxin (VTEC) and six strains of Shigella dysenteriae 1 were examined for the production of extra- and intracellular Vero cytotoxin (VT) and Shiga toxin respectively, in relation to the growth medium and availability of iron. VTEC secreted less extracellular VT1 or VT2 when grown in trypticase soy broth (TSB) containing the iron chelator desferal, as compared to bacteria cultured in iron replete TSB. Growth in TSB containing desferal resulted in increased production of intracellular toxin by VT1 producing strains of E. coli; but had little effect on production of intracellular toxin by VT2 producing strains. Both extra- and intracellular levels of Shiga toxin were increased by growth in TSB containing desferal. Combining intra- and extracellular toxin titres, strains of E. coli producing VT1 gave highest titres following growth in TSB where iron was bound to the chelating agent desferal, while strains of S. dysenteriae 1 produced highest levels of toxin when grown in syncaseglucose broth made iron depleted using Chelex-100. Strains of S. dysenteriae 1 did not grow in syncase-glucose broth containing desferal.     

Shiga toxin-associated hemolytic-uremic syndrome: combined cytotoxic effects of Shiga toxin, interleukin-1 beta, and tumor necrosis factor alpha on human vascular endothelial cells in vitro.

This study explores the relationship between Shiga toxin-producing Shigella or Escherichia coli strains and the development of vascular complications in humans following bacillary dysentery. We propose that endotoxin-elicited interleukin-1 or tumor necrosis factor alpha (TNF) may combine with Shiga toxin to facilitate vascular damage characteristic of hemolytic-uremic syndrome. This study examines the cytotoxic effects of Shiga toxin, interleukin-1, and TNF on cultured human umbilical vein endothelial cells (HUVEC). Both Shiga toxin and TNF were cytotoxic to HUVEC, although HUVEC obtained from individual umbilical cords differed in their sensitivities to these agents. With Shiga toxin-sensitive HUVEC, combinations of TNF with Shiga toxin resulted in a synergistic cytotoxic effect. In contrast, interleukin-1 was not cytotoxic to HUVEC, nor did it enhance cell death in combination with Shiga toxin. The synergistic cytotoxic response of HUVEC to Shiga toxin and TNF was dose and time dependent for both agents and could be neutralized by monoclonal antibodies directed against either Shiga toxin or TNF. This synergistic response was delayed, being maximal on day 2. Preincubation (24 h) of HUVEC with TNF sensitized the cells to Shiga toxin. TNF alone had no effect on HUVEC protein synthesis but enhanced the inhibitory activity of Shiga toxin. These results are consistent with a role for Shiga toxin in the development of hemolytic-uremic syndrome at the level of the vascular endothelium in humans.     

Purification and characterization of an Escherichia coli Shiga-like toxin II variant.

A Shiga-like toxin II variant was purified to homogeneity from Escherichia coli TB1(pCG6), which contained the toxin genes cloned in multicopy plasmid pUC18. The purification scheme involved polymyxin B extraction of the toxin from bacterial cells, followed by differential (NH4)2SO4 precipitation, anion- and cation-exchange fast-protein liquid chromatography, and immunoaffinity exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified toxin revealed three protein bands that migrated with calculated molecular weights of 33,000, 27,500, and 7,500. These bands correspond to values for the A, A1, and B subunits, respectively, that would be expected on the basis of the nucleotide sequence and comparison with data for Shiga toxin and other Shiga-like toxins. Electrophoresis under nonreducing conditions resulted in disappearance of the 27,000-molecular-weight band. Western blot (immunoblot) analysis revealed three protein bands with molecular weights of 33,000, 27,500, and 7,500. The purified toxin induced typical signs of edema disease in pigs injected intravenously with doses as small as 3 ng/kg of body weight. The 50% cytotoxic doses for Vero, PK15, and Madin-Darby bovine and canine kidney cells were 0.5, 2.0, 8.0, and 8.0 pg, respectively. The 50% lethal dose of purified toxin for mice was 0.9 pg by the intraperitoneal route. Approximately 75 micrograms of purified toxin was required to induce a 1-ml/cm fluid response in rabbit ileal loops. Antiserum to the Shiga-like toxin II variant neutralized homologous toxin, Shiga-like toxin II, and Verotoxin 2 but not Shiga-like toxin I.     

Protein synthesis in HeLa or Henle 407 cells infected with Shigella dysenteriae 1, Shigella flexneri 2a, or Salmonella typhimurium W118.

The incorporation of [14C]leucine into protein was studied in two mammalian cell lines which had been infected with strains of Shigella dysenteriae 1, Shigella flexneri 2a, or Salmonella typhimurium W118. These cell lines differed in susceptibility to the effects of exogenously applied Shiga cytotoxin. All invasive shigella strains (which synthesize this toxin to a greater or lesser degree) were found to inhibit protein synthesis in both cell lines with equal efficiency. Leucine accumulation continued in these cells, but the labeled amino acid was preferentially incorporated into bacterial protein. S. typhimurium W118, which has not been shown to elaborate a Shiga-like toxin, had little effect on protein synthesis in infected host cells.     

Molecular detection of sorbitol-fermenting Escherichia coli O157 in patients with hemolytic-uremic syndrome.

Shiga-like toxin-producing Escherichia coli strains of serogroup O157 were identified in 26 of 104 patients with hemolytic-uremic syndrome and in 18 of 668 patients with diarrhea. All strains were identified by colony hybridization with DNA probes complementary to Shiga-like toxin I and Shiga-like toxin II gene sequences and characterized by biochemical tests and serotyping. Seventeen of these 44 patients had E. coli O157 strains which were unusual because they fermented sorbitol within 24 h of incubation and were positive for beta-glucuronidase activity. Culture filtrates of these sorbitol-fermenting strains were highly toxic to Vero cells in culture. Serological tests and DNA analysis performed by restriction endonuclease digestion of B-subunit toxin genes revealed that all 17 isolates produced Shiga-like toxin II. Although by using molecular probes we established a high frequency of sorbitol-fermenting E. coli O157 strains in the patients we examined, further studies on the prevalence of such isolates in other areas of endemic disease are clearly warranted.     

Cytotoxicity of Shiga toxin for primary cultures of human colonic and ileal epithelial cells.

Shiga toxin purified from Shigella dysenteriae 1 was cytotoxic to cultured epithelial cells from human colon and ileum. The cytotoxicity, which affected only about 50% of treated cells, was neutralized by rabbit antiserum monospecific for Shiga toxin and mediated by protein synthesis inhibition.     

Purification and some properties of a Vero toxin from a human strain of Escherichia coli that is immunologically related to Shiga-like toxin II (VT2)

A cytotoxin to Vero cells (Vero toxin), which was immunologically related to Shiga-like toxin II (SLT-II) (or VT2), was purified from a stain of Escherichia coli isolated from a patient with hemolytic uremic syndrome. The toxin was active on Vero cells but much less active on HeLa cells, a property similar to that of the recently identified SLT-II variant from E. coli strains that caused edema disease of swine. Thus the toxin purified in this report was tentatively named Shiga-like toxin II variant (Vero toxin 2 variant). The purification procedures consisted of ammonium sulfate fractionation, DEAE-Sepharose CL-6B column chromatography, chromatofocusing column chromatography, and repeated high performance liquid chromatography (HPLC) on TSK-gel G-2000SW column and on TSK-gel DEAE-5PW columns. About 90 micrograms of purified toxin was obtained from 451 of the culture supernatant with a yield of about 16%. The purified toxin consisted of A and B subunits of molecular sizes similar to those of SLT-II (VT2). The isoelectric point of the purified toxin was 6.1, which was different from that of SLT-II (VT2) (pI = 4.1). In an Ouchterlony double gel diffusion test, purified toxin and SLT-II (VT2) formed precipitin lines with spur formation against anti-purified toxin and anti-SLT-II (anti-VT2), respectively. The purified toxin was cytotoxic to Vero cells, about 6 pg of the toxin killing 50% of the Vero cells, and showed lethal toxicity to mice when injected intraperitoneally, the LD50 being about 2.7 ng per mouse.     

Enterobacter cloacae producing a Shiga-like toxin II-related cytotoxin associated with a case of hemolytic-uremic syndrome.

Two Shiga-like toxin-producing organisms were isolated from the feces of an infant with hemolytic-uremic syndrome by PCR followed by colony blot hybridization. One strain was identified as Escherichia coli OR:H9, while the other was identified as Enterobacter cloacae. Both isolates were highly cytotoxic for Vero cells, and Southern hybridization analysis of chromosomal DNA indicated that both contained a single slt-II-related gene and that these genes were located on similarly sized restriction fragments. Nucleotide sequence analysis indicated that the toxin encoded by the E. cloacae slt-II-related gene was very similar to Shiga-like toxin II variants from E. coli, differing from the most closely related toxin by 3 residues in the A subunit.     

Localization of stx, a determinant essential for high-level production of shiga toxin by Shigella dysenteriae serotype 1, near pyrF and generation of stx transposon mutants.

Hfr strains of Shigella dysenteriae serotype 1 were constructed by transient integration of an RP4 plasmid derivative carrying transposon Tn501 into the Shigella chromosome through Tn501-mediated cointegration. The Hfr strains were mated with Escherichia coli K-12 recipients carrying various auxotrophic markers, and E. coli recombinants which had received prototrophic Shigella genes were selected. Some of the E. coli transconjugants produced high levels of a cytotoxin which was neutralized by both polyclonal and monoclonal anti-Shiga toxin sera. The determinant for Shiga toxin production, designated stx, was first transferred to E. coli K-12 and then mapped by Hfr crosses to the trp-pyrF region located at 30 min on the E. coli chromosome. Bacteriophage P1-mediated transduction analysis of stx gave the following gene order: trp-pyrF-stx. The level of Shiga toxin production in E. coli Stx+ transconjugants and transductants was as high as that of the parental S. dysenteriae 1 strain. Stx- mutants of an Stx+ E. coli transductant were generated by random in vivo insertion mutagenesis with a Tn10 derivative transposon, Tn-mini-kan, followed by P1 cotransduction of the kanamycin resistance and PyrF+ markers into a pyrF Stx+ E. coli K-12 recipient. One stx::Tn-mini-kan transposon mutation was transferred by P1 transduction from this E. coli Stx- mutant to an E. coli K-12 Hfr strain and in turn transferred by conjugation to the original S. dysenteriae 1 strain plus two others. All kanamycin-resistant recombinants of S. dysenteriae 1 had lost their ability to produce high levels of Shiga toxin. A gene that specifies high-level Shiga toxin production is thus located near pyrF on the chromosome of S. dysenteriae 1. Stx- mutants of S. dysenteriae 1 exhibited full virulence in the Serény test.