Cloning and sequencing of a Shiga-like toxin type II variant from Escherichia coli strain responsible for edema disease of swine.

A Shiga-like toxin type II variant (SLT-IIv) is produced by strains of Escherichia coli responsible for edema disease of swine and is antigenically related to Shiga-like toxin type II (SLT-II) of enterohemorrhagic E. coli. However, SLT-IIv is only active against Vero cells, whereas SLT-II is active against both Vero and HeLa cells. The structural genes for SLT-IIv were cloned from E. coli S1191, and the nucleotide sequence was determined and compared with those of other members of the Shiga toxin family. The A subunit genes for SLT-IIv and SLT-II were highly homologous (94%), whereas the B subunit genes were less homologous (79%). The SLT-IIv genes were more distantly related (55 to 60% overall homology) to the genes for Shiga toxin of Shigella dysenteriae type 1 and the nearly identical Shiga-like toxin type I (SLT-I) of enterohemorrhagic E. coli. (These toxins are referred to together as Shiga toxin/SLT-I.) The A subunit of SLT-IIv, like those of other members of this toxin family, had regions of homology with the plant lectin ricin. SLT-IIv did not bind to galactose-alpha 1-4-galactose conjugated to bovine serum albumin, which is an analog of the eucaryotic cell receptor for Shiga toxin/SLT-I and SLT-II. These findings support the hypothesis that SLT-IIv binds to a different cellular receptor than do other members of the Shiga toxin family but has a similar mode of intracellular action. The organization of the SLT-IIv operon was similar to that of other members of the Shiga toxin family. Iron did not suppress SLT-IIv or SLT-II production, in contrast with its effect on Shiga toxin/SLT-I. Therefore, the regulation of synthesis of SLT-IIv and SLT-II differs from that of Shiga toxin/SLT-I.     

Phenotypical characteristics of Shiga-like toxin Escherichia coli isolated from sheep dairy products

AIMS: To analyse phenotypical characteristics of Shiga toxin-producing Escherichia coli (STEC) strains from ovine origin. METHODS AND RESULTS: A total of 13 STEC strains (eight O157 and five non-O157) isolated from sheep dairy products were used in this study. Biochemical traits, motility, haemolytic activity, resistance to tellurite-cefixime, maximum growth temperature and antibiotic resistance were determined. The STEC strains were grouped into nine biochemical and physiological biotypes (five for the O157 and four for the non-O157 strains). All STEC strains showed resistance to bacitracin, cloxacilin, penicillin and tylosin. CONCLUSIONS: Different biotypes and antibiotic resistance patterns of STEC isolated from sheep dairy products were observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This work will be a contribution to the better characterization of STEC isolated from sheep dairy products, which have, to date, been scarcely studied, and to the better understanding of the risks associated with its consumption.     

Identification of Shiga-like toxin in Escherichia coli strains, etiological agents of diarrheal disease

Shiga-like toxin presence, in 20 E. coli strains, etiological agents of diarrheal diseases, is studied by preparing extracts at +4 degrees C, in the presence of chloroform and by i.v. inoculation in mice. In 4 out of 20 strains, Shiga-like toxin in high titres was identified. Most of the strains presented an inconstant and variable production of Shiga-like toxin in comparison with Shigella dysenteriae type 1 (Shiga) reference strain. The authors also confirm the existence of Shiga-like toxin under 2 forms (neutralizable with Shiga antitoxic serum and non-neutralizable). The importance of the obtained results is further discussed from the point of view of pathogeny and diagnosis of the infections produced by these germs.     

Yersinia HPI in septicemic Escherichia coli strains isolated from diverse hosts

High pathogenicity islands (HPIs), first identified in various Yersinia species, encode an iron uptake system. We have studied the occurrence of HPIs in septicemic strains of Escherichia coli isolated from a variety of hosts. The results presented in this communication indicate that most septicemic strains tested contained HPI sequences even though they already have the aerobactin encoding genes. We have also observed two types of HPI deletions, suggesting genetic instability of this element. Notable exceptions are several strains isolated from septicemia in sheep that lacked both iron acquisition systems.     

Genes coding for Shiga-like toxin and heat-stabile enterotoxin in porcine strains of Escherichia coli

Besides diarrheagenic enterotoxigenic Escherichia coli (ETEC) that produce classical heat stable and/or heat labile enterotoxins (STs, LTs) and the class of Shiga-like toxin-producing entero-hemorrhagic E. coli (EHEC), a new category of E. coli is defined sharing similarities with ETEC and EHEC. DNA hybridization studies indicate that some E. coli serovars from porcine origin harbor genes encoding cytotonic ST and cytotoxic Shiga-like toxin. The presence of two potent toxins might contribute to the virulence of such strains and should be taken into consideration when bio-assays are performed.     

Nucleotide sequence analysis and comparison of the structural genes for Shiga-like toxin I and Shiga-like toxin II encoded by bacteriophages from Escherichia coli 933

The nucleotide sequence of the Shiga-like toxin type II (SLT-II) structural genes cloned from bacteriophage 933W of the enterohemorrhagic Escherichia coli O157:H7 strain 933 was determined. This sequence was compared with the published sequence for the structural genes of the antigenically distinct Shiga-like toxin type I (SLT-I) encoded by bacteriophage 933J. The SLT-I and SLT-II structural genes shared 58% overall nucleotide and 56% amino acid sequence homologies. The A and B subunits of SLT-I and SLT-II were nearly identical in size and had similar secondary structures and hydropathy plots. The regulation proposed for the SLT-II operon is similar to that previously proposed for SLT-I.     

表达SLTEC保护性抗原的重组猪霍乱沙门氏菌C500株的构建及生物学特性

摘要:【目的】 利用平衡致死系统构建表达产类志贺氏毒素大肠杆菌(Shiga-like toxin Escherichia coli , SLTEC)保护性抗原的减毒猪霍乱沙门氏菌。【方法】 构建表达SLT-IIeB－FedF的重组质粒 ,再将其电转入终宿主菌减毒猪霍乱沙门氏菌ΔasdC500株中构建成口服活疫苗株 ,经聚丙烯酰胺凝胶电泳检测SLT-IIeB－FedF融合蛋白的表达情况,并观察重组菌体外培养的稳定性。【结果】 　利用宿主-载体平衡致死系统构建了表达SLTEC保护性抗原的重组减毒猪霍乱沙门氏菌     

Antimicrobial susceptibilities of Shiga toxin-producing Escherichia coli isolates from pigs with edema disease in Japan

Fifty-seven Shiga toxin-producing Escherichia coli (STEC) strains isolated from pigs with edema disease (ED) from 1997 to 2001 in Japan were examined for antimicrobial susceptibilities. The susceptibilities were compared with those of E. coli ATCC 23546 isolated from pig with ED in the 1950's. Consequently, the isolated STECs showed high susceptibility to peptides and bicozamycin in a way similar to the reference strain. On the other hand, the STECs showed low susceptibility to beta-lactams, tetracyclines, novobiocin, fosfomycin, trimethoprim, and old quinolones. It became clear that the susceptibilities of the isolated STECs had diminished in regard to antimicrobials.     

Aerobactin production linked to transferable antibiotic resistance in Escherichia coli strains isolated from sewage

Abstract The production of aerobactin has been suggested to be a virulence factor in Escherichia coli . We have studied the production of aerobactin in 155 E. coli strains, isolated from sewage, which carry conjugative antibiotic resistance plasmids, and 88 (57%) of these strains produced aerobactin, and 59 (38%) co-transferred production of the siderophore and antibiotic resistance. In 35 (22%) of the transconjugants, both characters seemed to be encoded into the same plasmid. Those aerobactin-producing-antibiotic resistance plasmids had different sizes and antibiotic resistance patterns. The ecological implications of these results are discussed.     

Genetic and immunological analysis of a novel variant of Shiga toxin 1 from bovine Escherichia coli strains and development of bead-ELISA to detect the variant toxin

A novel variant of Shiga toxin 1 (Stx1) was identified from bovine Escherichia coli strains. The stx1 variant genes designated as stx1v51 and stx1v52 were cloned and sequenced. The two variant genes differed each other by 2 bp, but the deduced amino acid sequences of the two Stx1 variant toxins were the same and had 94% and 92% homology to that of prototype A and B subunits of Stx1, respectively. The variant toxin designated as Stx1v52 was purified to homogeneity. Although inhibition of protein synthesis in vitro by purified Stx1v52 was almost equal to that of purified Stx1, Vero cell cytotoxicity and mouse lethality of Stx1v52 were several folds lower than those of prototype Stx1. In Ouchterlony double gel diffusion test, the precipitin line between Stx1v52 and Stx1 formed a spur against anti-Stx1 serum but was fused against anti-Stx1v52 serum. Stx1v52 and Stx1v52-specific-bead-ELISA was developed, and both Stx1 and Stx1v52 could be detected with high sensitivity using Stx1v52 conjugate. However, Stx1v52 but not Stx1 could be detected with Stx1v52-specific bead-ELISA.     

Characterization of shiga toxin type 2 variant B-subunit in Escherichia coli strains from asymptomatic human carriers by PCR-RFLP

Subtyping of shiga toxin type 2 variant B-subunit in 35 non-O157 and two O157 strains isolated from 37 asymptomatic human carriers yielded two strains with stx2, 10 strains with stx2c and 24 strains with stx2d genes. One isolate harboured stx2 and stx2c. The high Stx2d prevalence in asymptomatic carriers was conspicuous and may indicate a reduced pathogenicity of these toxin variants. Therefore, in order to appraise a positive STEC laboratory result, the strain must be isolated in every case. Shiga toxin types and further virulence-associated factors have to be investigated.     

Protection of Chinese hamster ovary cells from paraquat-mediated cytotoxicity by a low molecular weight mimic of superoxide dismutase (DF-Mn)

Paraquat exerts a cytotoxic effect of Chinese hamster ovary cells in culture via the superoxide radical (O₂⁻. We have described a superoxide dismutase (SOD) mimic based on manganese (DF-Mn) which consists of a one-to-one complex between desferrioxamine B (Desferal) and MnO₂. It is a small molecular weight molecule, easy to prepare and possesses considerable stability. It is now shown to protect mammalian cells from paraquat toxicity. Thus, 20 μM DF-Mn affords up to complete protection against the cytotoxicity of 200 μM paraquat in Chinese hamster ovary cells. Desferrioxamine B or MnO₂ alone gave no protection. MnCl₂ or catalase provided little or no protection against the paraquat, respectively. Equivalent amounts of human Cu-Zn SOD in terms of activity, also provided no protection. Copper diisopropylsalicylate (CuDIPS) provided limited, yet significant, protection, but this is explained in terms other than SOD activity. Finally, at higher concentrations, purified human SOD, exerts a limited toxicity as well as a protective ability against paraquat (similar to DF-Mn) both of which are eliminated upon heat denaturation of the enzyme. It appears that the SOD mimic, DF-Mn, can enter mammalian cells and can protect against the cytotoxic effects of O₂⁻.     

Application of polymerase chain reaction for determination of toxins in Escherichia coli strains isolated from pigs with diarrhea.

The PCR method was used for the determination of LTI, STI, STII enterotoxins and Stx2e toxin genes in E. coli strains isolated from pigs with diarrhea. It was shown that most of the strains (77.3%) were enterotoxigenic, producing mainly LTI and STII (30.8%) or STI and STII (21.3%) toxins. It was found that 10.6% of isolates possessed the stx2e genes responsible for the elaboration of shiga toxin 2e.     

Growth of Escherichia coli in the presence of trimethoprim-sulfamethoxazole facilitates detection of Shiga-like toxin producing strains by colony blot assay

Abstract Subinhibitory concentrations of trimethoprim-sulfamethoxazole increased the total yield of Shiga-like toxin (SLT), produced by Shigella dysenteria 1 and by enterophathogenic and enterohemorrhagic strains of Escherichia coli . Stimulation of SLT synthesis by trimethoprim-sulfamethoxazole was demonstrated by an increase in cytotoxic activity for HeLa cells and the diameter of the zone formed around bacterial colonies probed with monoclonal antibodies to SLT. Thus, supplementation of culture media with trimetroprimsulfamethoxazol will facilitate SLT purification and detection of SLT-producing bacteria.     

First isolation of Shiga toxin 1d producing Escherichia coli variant strains in shellfish from coastal areas in France

AIMS: This study was carried out to evaluate the presence of Shiga toxin-producing Escherichia coli (STEC) and E. coli O157:H7 in shellfish from French coastal environments. METHODS AND RESULTS: Shellfish were collected in six growing areas or natural beds (B category) and nonfarming areas (D category) from July 2002 to August 2004. PCR detection of stx genes was performed on homogenized whole shellfish and digestive gland tissues enrichments. STEC strains were detected by colony DNA hybridization using a stx-specific gene probe and E. coli O157 strains were additionally searched by immunomagnetic separation with O157-specific magnetic beads. Stx genes were detected in 40 of 144 (27.8%) sample enrichments from mussels, oysters or cockles, 32 of 130 enrichments (24.6%) were from B-category areas and eight of 14 (57.1%) from the D-category area. Five strains carrying stx(1) or stx(1d) genes and one stx negative, eae and ehxA positive E. coli O157:H7 were isolated from six of 40 stx-positive enrichments. No relation was found between the total E. coli counts in shellfish and the presence of STEC strains in the samples. CONCLUSIONS: The STEC strains of different serotypes and stx types are present in shellfish from French coastal environments. It is the first isolation of STEC stx1d strains in France. SIGNIFICANCE AND IMPACT OF THE STUDY: Shellfish collected in coastal environments can serve as a vehicle for STEC transmission.     

Escherichia coli strains of serogroup O139 isolated from oedema disease of swine bind fibronectin

Abstract 15 Escherichia coli strains of the serogroup O139, isolated from oedema disease of swine, were examined for their ability to interact with ¹²⁵I-labelled fibronectin. All strains were positive, and all except one showed higher fibronectin binding than Staphylococcus aureus strain Cowan 1 cells (to which fibronectin bound in the order of 15% of total protein added). 7 E. coli strains isolated from diarrhoea in young piglets were also tested, and 3 were positive. 2 of these strains showed higher binding than S. aureus Cowan 1 cells. E. coli strains expressing either K99 or K88 antigen were poor binders, comparable to cells of S. aureus strain Wood 46. There was no correlation between cell surface hydrophobicity, as determined by chromatography on Octyl-Sepharose, and the fibronectin-binding property.