Epidemiological relatedness and clonal types of natural populations of Escherichia coli strains producing Shiga toxins in separate populations of cattle and sheep.

Two separate animal populations consisting of a herd of cattle (19 animals) and a flock of sheep (25 animals) were investigated for strains of Escherichia coli producing Shiga toxins (STEC) over a time period of 6 months. Thirty-three STEC were isolated from 63.2% of cattle and grouped into 11 serotypes and eight electrophoretic types (ETs) by multilocus enzyme analysis. In sheep, 88% of the animals excreted STEC (n = 67 isolates) belonging to 17 different serotypes and 12 different ETs. STEC from cattle and sheep differed with respect to serotype, and only 4 of the 16 ETs occurred in both animal populations. In cattle, ET14 (O116:H21) strains predominated, whereas other STEC serotypes occurred only sporadically. The predominating STEC types in sheep were ET4 (O125 strains), ET11 (O128:H2 and others), and ET14 (O146:H21). In contrast to their diversity, STEC originating from the same animal population were similar with respect to Shiga toxin (stxy genes. Almost all STEC isolated from cattle were positive for stx2 and stx2c; only one was positive for stx1. In sheep, almost all STEC isolated were positive for stx1 and stx2, whereas stx2c was not found. XbaI-digested DNAs of genetically closely related O146:H21 strains have different restriction profiles which were associated with size alterations in XbaI fragments hybridizing with stx1- and stx2-specific DNA probes. Our results indicate that stx-encoding bacteriophages might be the origin of the genetic heterogeneity in STEC from animals.     

Functional Capacity of Shiga-Toxin Promoter Sequences in Eukaryotic Cells

Shiga toxins (Stx) are the main virulence factors in enterohemorrhagic Escherichia coli (EHEC) infections, causing diarrhea and hemolytic uremic syndrome (HUS). The genes encoding for Shiga toxin-2 (Stx2) are located in a bacteriophage. The toxin is formed by a single A subunit and five B subunits, each of which has its own promoter sequence. We have previously reported the expression of the B subunit within the eukaryotic environment, probably driven by their own promoter. The aim of this work was to evaluate the ability of the eukaryotic machinery to recognize stx2 sequences as eukaryotic-like promoters. Vero cells were transfected with a plasmid encoding Stx2 under its own promoter. The cytotoxic effect on these cells was similar to that observed upon incubation with purified Stx2. In addition, we showed that Stx2 expression in Stx2-insensitive BHK eukaryotic cells induced drastic morphological and cytoskeletal changes. In order to directly evaluate the capacity of the wild promoter sequences of the A and B subunits to drive protein expression in mammalian cells, GFP was cloned under eukaryotic-like putative promoter sequences. GFP expression was observed in 293T cells transfected with these constructions. These results show a novel and alternative way to synthesize Stx2 that could contribute to the global understanding of EHEC infections with immediate impact on the development of treatments or vaccines against HUS.     

The effect of probiotics and organic acids on Shiga-toxin 2 gene expression in enterohemorrhagic Escherichia coli O157:H7

Probiotics are known to have an inhibitory effect against the growth of various foodborne pathogens, however, the specific role of probiotics in Shiga-toxin-producing Escherichia coli (STEC) virulence gene expression has not been well defined. Shiga toxins are members of a family of highly potent bacterial toxins and are the main virulence marker for STEC. Shiga toxins inhibit protein synthesis in eukaryotic cells and play a role in hemorrhagic colitis and hemolytic uremic syndrome. STEC possesses Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), both of which have A and B subunits. Although STEC containing both Stx1 and Stx2 has been isolated from patients with hemorrhagic colitis, Stx2 is more frequently associated with human disease complications. Thus, the effect of Lactobacillus, Pediococcus, and Bifidobacterium strains on stx2A expression levels in STEC was investigated. Lactic acid bacteria and bifidobacteria were isolated from farm animals, dairy, and human sources and included L. rhamnosus GG, L. curvatus, L. plantarum, L. jensenii, L. acidophilus, L. casei, L. reuteri, P. acidilactici, P. cerevisiae, P. pentosaceus, B. thermophilum, B. boum, B. suis and B. animalis. E. coli O157:H7 (EDL 933) was coincubated with sub-lethal concentrations of each probiotic strain. Following RNA extraction and cDNA synthesis, relative stx2A mRNA levels were determined according to a comparative critical threshold (Ct) real-time PCR. Data were normalized to the endogenous control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the level of stx2A expression between treated and untreated STEC was compared. Observed for all probiotic strains tested, stx2A was down-regulated, when compared to the control culture. Probiotic production of organic acids, as demonstrated by a decrease in pH, influenced stx2A gene expression.     

Complete nucleotide sequence of the prophage VT1-Sakai carrying the Shiga toxin 1 genes of the enterohemorrhagic Escherichia coli O157:H7 strain derived from the Sakai outbreak

Shiga toxins 1 and 2 (Stx1 and Stx2) are encoded by prophages lysogenized in enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains. Lytic growth of the phage particles carrying the stx1 genes (stx1A and stx1B) of the EHEC O157:H7 strain RIMD 0509952, which was derived from the Sakai outbreak in 1996 in Japan, was induced after treatment with mitomycin C, but the plaque formation of the phage was not detected. We have determined the complete nucleotide sequence of the prophage VT1-Sakai. The integration site of the prophage was identified within the yehV gene at 47.7 min on the chromosome. The stx1 genes were downstream of the Q gene in the prophage genome, suggesting that their expression was regulated by the Q protein, the regulator of the late gene expression of the phage, which is similar to that of the stx1 or stx2 genes carried by the lambdoid phages reported previously. The sequences of the N gene and its recognition sites, nutL and nutR, were not homologous to those of the phages carrying the stx genes thus far reported, but they were very similar to those of bacteriophage phi21. The sequences of the repressor proteins, CI and Cro, that regulate expression of the early genes had low similarities with those of the known repressors of other phages, and their operator sequences were different from any sequence reported. These data suggest that multiple genetic recombination among bacteriophages with different immunities took place to generate the prophage VT1-Sakai. Comparison between the sequences of VT1-Sakai and lambda suggests that the ancestor of VT1-Sakai was produced by illegitimate excision, like lambda gal and bio phages.     

Expression of the amino terminal part of synthetic human growth hormone gene and somatomedin-like activity of expressed protein

We have constructed three different plasmids containing parts of the human growth hormone gene using chemically synthesized oligomers and cloned them for the purpose of expressing them in Escherichia coli. AB, B and BC gene segments corresponding to ABhGH (residue 1-138), BhGH (residue 44-138) and BChGH (residue 44-192) were placed under the control of a tryptophan promoter in the expression vector. Upon induction with 3-indolylacrylic acid, ABhGH accumulated in cells but the BhGH and BChGH segments were not detected appreciably. Northern blotting analysis showed that the amount of mRNA transcribed from the AB gene segment was about ten-fold higher than that from the B or BC gene segment. ABhGH was found to have insulin-like growth factor I (IGF-I) activity, which could be explained by the hydrophilicity curves of these proteins.     

Spontaneous tandem amplification and deletion of the shiga toxin operon in Shigella dysenteriae 1

Only one species of Shigella, Shigella dysenteriae 1, has been demonstrated to produce Shiga toxin (Stx). Stx is closely related to the toxins produced by Shiga toxin-producing Escherichia coli (STEC). In STEC, these toxins are often encoded on lambdoid bacteriophages and are major virulence factors for these organisms. Although the bacteriophage-encoded stx genes of STEC are highly mobile, the stx genes in S. dysenteriae 1 have been believed to be chromosomally encoded and not transmissible. We have located the toxin genes of S. dysenteriae 1 to a region homologous to minute 30 of the E. coli chromosome, within a 22.4 kbp putative composite transposon bracketed by IS600 insertion sequences. This region is present in all the S. dysenteriae 1 strains examined. Tandem amplification occurs via the flanking insertion sequences, leading to increased toxin production. The global regulatory gene, fnr, is located within the stx region, allowing deletions of the toxin genes to be created by anaerobic growth on chlorate-containing medium. Deletions occur by recombination between the flanking IS600 elements. Lambdoid bacteriophage genes are found both upstream and within the region, and we demonstrate the lysogeny of Shigella species with STEC bacteriophages. These observations suggest that S. dysenteriae 1 originally carried a Stx-encoding lambdoid prophage, which became defective due to loss of bacteriophage sequences after IS element insertions and rearrangements. These insertion sequences have subsequently allowed the amplification and deletion of the stx region.     

A plasmid encoding a combination of mosquito-larvicidal genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus confers toxicity against a broad range of mosquito larvae when expressed in Gram-negative bacteria

A recombinant plasmid harboring cry4A, cry4B and cry11A from Bacillus thuringiensis subsp. israelensis and binary toxin genes from Bacillus sphaericus has been constructed. The three cry genes were placed under the control of the cry4B promoter whereas the binary toxin gene was controlled by its native promoter. The expression of toxins in Escherichia coli harboring the resulting plasmid, p4BDA-5142, was investigated. Cry4B expression was highest compared to other toxins. Although the level of toxin expression was low compared with E. coli expressing single toxins, the recombinant E. coli strain harboring p4BDA-5142 exhibited broad range mosquito-larvicidal activity against all Aedes, Culex and Anopheles larvae. This work has shown that the development of the recombinant plasmid can be used to broaden the host range spectrum of the appropriate bacterial host for mosquito control.     

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.     

江苏某地健康绵羊群产志贺毒素大肠杆菌体内分离株的分子流行病学及致病力

【目的】探讨江苏某羊场健康绵羊体内产志贺毒素大肠杆菌的带菌和流行情况,同时就分离株的致病力和对Vero细胞的毒性作用作了研究。【方法】基于本实验室已经建立的EHEC O157:H7 EDL933W株的stx1、stx2、eaeA、hlyA四个基因的多重PCR检测并配合选择性增菌、平板筛选等方法对STEC进行分离鉴定。【结果】在为期6个月的连续跟踪调查中,共分离到STEC菌株107株,分离率为19.4%(107/550)。分离株属于41种O血清型、62种O:H血清型,未定型(ONT)有22株,粗糙型(OR)1株。其中属于绵羊STEC的优势血清型有O5(2株)、O91(1株)、O103(1株)。本文检测到的优势血清型为O93,stx2阳性菌株的分离率较stx1阳性菌株的分离率高,LD50测定结果表明分离株对小鼠致病力不高,受试的3个分离株均不能致小鼠死亡。对107株stx阳性分离株噬菌斑试验表明,71株阳性菌株携带噬菌体(66.3%,71/109)。受试分离株进行Vero细胞毒性试验,其中有一个菌株stx基因阳性但不能使Vero细胞产生病变。【结论】绵羊是STEC的天然宿主,可健康带菌。虽然STEC分离株对小鼠的致病力较弱,但不能排除其对人类安全的威胁。STEC携带志贺毒素基因并不意味着一定表达志贺毒素,需对志贺毒素的表达及调控机理做进一步的研究。     

Characterisation of Shiga toxin-producing Escherichia coli (STEC) isolated from seafood and beef

Shiga toxin-producing Escherichia coli (STEC) strains isolated in Mangalore, India, were characterised by bead-enzyme-linked immunosorbent assay (bead-ELISA), Vero cell cytotoxicity assay, PCR and colony hybridisation for the detection of stx1 and stx2 genes. Four strains from seafood, six from beef and one from a clinical case of bloody diarrhoea were positive for Shiga toxins Stx1 and Stx2 and also for stx1and stx2 genes. The seafood isolates produced either Stx2 alone or both Stx1 and Stx2, while the beef isolates produced Stx1 alone. The stx1 gene of all the beef STEC was found to be of recently reported stx1c type. All STEC strains and one non-STEC strain isolated from clam harboured EHEC-hlyA. Interestingly, though all STEC strains were negative for eae gene, two STEC strains isolated from seafood and one from a patient with bloody diarrhoea possessed STEC autoagglutinating adhesion (saa) gene, recently identified as a gene encoding a novel autoagglutinating adhesion.     

Insertion site occupancy by stx2 bacteriophages depends on the locus availability of the host strain chromosome

Shiga toxin-producing Escherichia coli (STEC) is an emergent pathogen characterized by the expression of Shiga toxins, which are encoded in the genomes of lambdoid phages. These phages are infectious for other members of the Enterobacteriaceae and establish lysogeny when they integrate into the host chromosome. Five insertion sites, used mainly by these prophages, have been described to date. In the present study, the insertion of stx(2) prophages in these sites was analyzed in 168 STEC strains isolated from cattle. Additionally, insertion sites were determined for stx(2) phages which (i) converted diverse laboratory host strains, (ii) coexisted with another stx(2) prophage, and (iii) infected a recombinant host strain lacking the most commonly used insertion site. Results show that depending on the host strain, phages preferentially use one insertion site. For the most part, yehV is occupied in STEC strains while wrbA is preferentially selected by the same stx phages in E. coli laboratory strains. If this primary insertion site is unavailable, then a secondary insertion site is selected. It can be concluded that insertion site occupancy by stx phages depends on the host strain and on the availability of the preferred locus in the host strain.     

Prevalence of the stx2 gene in coliform populations from aquatic environments

Shiga toxin-producing Escherichia coli strains are human pathogens linked to hemorrhagic colitis and hemolytic uremic syndrome. The major virulence factors of these strains are Shiga toxins Stx1 and Stx2. The majority of the genes coding for these toxins are borne by bacteriophages. Free Stx2-encoding bacteriophages have been found in aquatic environments, but there is limited information about the lysogenic strains and bacteria present in the environment that are susceptible to phage infection. The aim of this work was to study the prevalence and the distribution of the stx(2) gene in coliform bacteria in sewage samples of different origins. The presence of the stx(2) gene was monitored every 2 weeks over a 1-year period in a municipal sewage treatment plant. A mean value of 10(2) genes/ml was observed without significant variation during the study period. This concentration was of the same order of magnitude in raw municipal sewage of various origins and in animal wastewater from several slaughterhouses. A total of 138 strains carrying the stx(2) gene were isolated by colony hybridization. This procedure detected approximately 1 gene-carrying colony per 1,000 fecal coliform colonies in municipal sewage and around 1 gene-carrying colony per 100 fecal coliform colonies in animal wastewaters. Most of the isolates belonged to E. coli serotypes other than E. coli O157, suggesting a low prevalence of strains of this serotype carrying the stx(2) gene in the wastewater studied.     

Eudragit E100(®) potentiates the bactericidal action of ofloxacin against fluoroquinolone-resistant Pseudomonas aeruginosa

Sorbitol-fermenting Escherichia coli O157:NM (SF O157) is an emerging pathogen suggested to be more virulent than nonsorbitol-fermenting Escherichia coli O157:H7 (NSF O157). Important virulence factors are the Shiga toxins (stx), encoded by stx1 and/or stx2 located within prophages integrated in the bacterial genome. The stx genes are expressed from p(R) (') as a late protein, and anti-terminator activity from the Q protein is necessary for read through of the late terminator t(R) (') and activation of p(R) (') . We investigated the regulation of stx2(EDL933) expression at the genomic level in 17 Norwegian SF O157. Sequencing of three selected SF O157 strains revealed that the anti-terminator q gene and genes upstream of stx2(EDL933) were identical or similar to the ones observed in the E.?coli O111:H- strain AP010960, but different from the ones observed in the NSF O157 strain EDL933 (AE005174). This suggested divergent stx2(EDL933) -encoding bacteriophages between NSF O157 and the SF O157 strains (FR874039-41). Furthermore, different DNA structures were detected in the SF O157 strains, suggesting diversity among bacteriophages also within the SF O157 group. Further investigations are needed to elucidate whether the q(O111:H) (-) gene observed in all our SF O157 contributes to the increased virulence seen in SF O157 compared to NSF O157. An assay for detecting q(O111:H) (-) was developed.