Genetic variation in the flanking regions of Shiga toxin 2 gene in Shiga toxin-producing Escherichia coli O157:H7 isolated in Japan (original) (raw)
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Applied and Environmental Microbiology, 2004
Shiga toxins Stx1 and Stx2 play a prominent role in the pathogenesis of Shiga toxin-producing Escherichia coli (STEC) infections. Several variants of the stx 2 gene, encoding Stx2, have been described. In this study, we developed a PCR-restriction fragment length polymorphism system for typing stx 2 genes of STEC strains. The typing system discriminates eight described variants and allows the identification of new stx 2 variants and STEC isolates carrying multiple stx 2 genes. A phylogenetic tree, based on the nucleotide sequences of the toxin-encoding genes, demonstrates that stx 2 sequences with the same PvuII HaeIII HincII AccI type generally cluster together.
The sharing of genome sequences in online data repositories, allows for large scale analyses of specific genes or gene families. This can result in the detection of novel gene subtypes as well as development of improved detection methods. Here we used publicly available WGS data to detect a novel Stx subtype, Stx2n in two clinical E. coli strains isolated in the USA. During this process, additional Stx2 subtypes were detected; six Stx2j one Stx2m strain and one Stx2o, all were analyzed for variability from the originally described subtypes [1,2]. Complete genome sequences were assembled from short or long read sequencing and analyzed for serotype, and ST types. The stx2n and Stx2o WGS were further analyzed for virulence genes pro-phage analysis and phage insertion sites. Nucleotide and amino acid maximum parsimony trees showed expected clustering of the previously described subtypes and a clear separation of the novel Stx2n subtype. WGS data was used to design OMNI PCR primers for t...
Background/Purpose: Shiga-like toxin (Stx) is an important factor in the pathogenesis of Escherichia coli O157:H7 infection and is responsible for some severe complications. Stx2 is usually associated with hemolytic uremic syndrome in humans. Its expression is regulated by elements located upstream of the stx2 gene, including stx2-promoter sequence, ribosome binding site, and the antiterminator q gene. The present study aimed to find the correlation between regulatory elements and the expression level of Stx2 in two local isolates of E. coli O157:H7. Methods: Two local E. coli O157:H7 strains SM-25(1) and KL-48(2), originating from human and cattle feces, respectively, and an E. coli reference strain, ATCC 43894, were investigated. The complete stx2 gene covering the sequences of promoter, ribosome binding site, and open reading frame and q gene of each strain was analyzed. The magnitude of Stx2 production was detected with a reverse passive latex agglutination method and Stx mediated cellular damage was determined with the Vero cell assay. (I.W. Suardana).
Journal of medical microbiology, 2000
Three Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from patients with diarrhoea were identified, each of which contained three distinct stx genes (stx1, stx2 and stx2c). The strains belonged to the serotypes O52:H19, O75:H- and O157:H- and harboured eae and EHEC-hly sequences. Colony-blot immunoassay was used to demonstrate that both major types of Stx were expressed. The association of stx genes with either phage or phage DNA was demonstrated in all three strains. Isolated phage DNA from all strains contained stx1 sequences, but stx2 sequences were found only in phage DNA of two of these strains. The presence of three distinct stx genes may enhance the virulence of STEC strains and should be monitored. The observations demonstrate not only the potential of stx genes to spread within different serotypes, but also their capacity to accumulate within a single strain.
Journal of Microbiology, Immunology and Infection, 2016
Background/Purpose: Shiga-like toxin (Stx) is an important factor in the pathogenesis of Escherichia coli O157:H7 infection and is responsible for some severe complications. Stx2 is usually associated with hemolytic uremic syndrome in humans. Its expression is regulated by elements located upstream of the stx2 gene, including stx2-promoter sequence, ribosome binding site, and the antiterminator q gene. The present study aimed to find the correlation between regulatory elements and the expression level of Stx2 in two local isolates of E. coli O157:H7. Methods: Two local E. coli O157:H7 strains SM-25(1) and KL-48(2), originating from human and cattle feces, respectively, and an E. coli reference strain, ATCC 43894, were investigated. The complete stx2 gene covering the sequences of promoter, ribosome binding site, and open reading frame and q gene of each strain was analyzed. The magnitude of Stx2 production was detected with a reverse passive latex agglutination method and Stx mediated cellular damage was determined with the Vero cell assay.
Journal of Clinical Microbiology, 2003
Stx2d is a recently described Shiga toxin whose cytotoxicity is activated 10-to 1,000-fold by the elastase present in mouse or human intestinal mucus. We examined Shiga toxigenic Escherichia coli (STEC) strains isolated from food and livestock sources for the presence of activatable stx 2d . The stx 2 operons of STEC were first analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis and categorized as stx 2 , stx 2c vha , stx 2c vhb , or stx 2d EH250 . Subsequently, the stx 2c vha and stx 2c vhb operons were screened for the absence of a PstI site in the stx 2A subunit gene, a restriction site polymorphism which is a predictive indicator for the stx 2d (activatable) genotype. Twelve STEC isolates carrying putative stx 2d operons were identified, and nucleotide sequencing was used to confirm the identification of these operons as stx 2d . The complete nucleotide sequences of seven representative stx 2d operons were determined. Shiga toxin expression in stx 2d isolates was confirmed by immunoblotting. stx 2d isolates were induced for the production of bacteriophages carrying stx. Two isolates were able to produce bacteriophages 1662a and 1720a carrying the stx 2d operons. RFLP analysis of bacteriophage genomic DNA revealed that 1662a and 1720a were highly related to each other; however, the DNA sequences of these two stx 2d operons were distinct. The STEC strains carrying these operons were isolated from retail ground beef. Surveillance for STEC strains expressing activatable Stx2d Shiga toxin among clinical cases may indicate the significance of this toxin subtype to human health.
2010
Escherichia coli O157:H7 strains fall into three major genetic lineages that differ in their distribution among humans and cattle. Several recent studies have reported differences in the expression of virulence factors between E. coli O157:H7 strains from these two host species. In this study, we wished to determine if important virulence-associated "mobile genetic elements" such as Shiga toxin 2 (Stx2)-encoding prophage are lineage restricted or are host source related and acquired independently of the pathogen genotype. DNA sequencing of the stx 2 flanking region from a lineage II (LII) strain, EC970520, revealed that the transcriptional activator gene Q in LI strain EDL933 (upstream of stx 2) is replaced by a pphA (serine/threonine phosphatase) homologue and an altered Q gene in this and all other LII strains tested. In addition, nearly all LI strains carried stx 2 , whereas all LII strains carried variant stx 2c and 4 of 14 LI/II strains had copies of both stx 2 and variant stx 2c. Real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that LI and LI/II strains produce significantly more stx 2 mRNA and Stx2 than LII strains. However, among LI strains significantly more Stx2 is also produced by strains from humans than from cattle. Therefore, lineage-associated differences among E. coli O157:H7 strains such as prophage content, toxin type, and toxin expression may contribute to host isolation bias. However, the level of Stx2 production alone may also play an important role in the within-lineage association of E. coli O157:H7 strains with human clinical disease.
Mucus-Activatable Shiga Toxin Genotype stx2d in Escherichia coli O157:H7
Emerging infectious diseases, 2017
We identified the mucus-activatable Shiga toxin genotype stx2d in the most common hemolytic uremic syndrome-associated Escherichia coli serotype, O157:H7. stx2d was detected in a strain isolated from a 2-year-old boy with bloody diarrhea in Spain, and whole-genome sequencing was used to confirm and fully characterize the strain.
Applied and Environmental Microbiology, 2008
There is considerable heterogeneity among the Shiga toxin type 2 (Stx2) toxins elaborated by Shiga toxinproducing Escherichia coli (STEC). One such Stx2 variant, the Stx2d mucus-activatable toxin (Stx2dact), is rendered more toxic by the action of elastase present in intestinal mucus, which cleaves the last two amino acids of the A2 portion of the toxin A subunit. We screened 153 STEC isolates from food, animals, and humans for the gene encoding Stx2dact by using a novel one-step PCR procedure. This method targeted the region of stx 2dact that encodes the elastase recognition site. The presence of stx 2dact was confirmed by DNA sequencing of the complete toxin genes. Seven STEC isolates from cows (four isolates), meat (two isolates), and a human (one isolate) that carried the putative stx 2dact gene were identified; all were eae negative, and none was the O157:H7 serotype. Three of the isolates (CVM9322, CVM9557, and CVM9584) also carried stx 1 , two (P1332 and P1334) carried stx 1 and stx 2c , and one (CL-15) carried stx 2c . One isolate, P1130, harbored only stx 2dact . The Vero cell cytotoxicities of supernatants from P1130 and stx 1 deletion mutants of CVM9322, CVM9557, and CVM9584 were increased 13-to 30-fold after treatment with porcine elastase. Thus, Stx2dact-producing strains, as detected by our one-step PCR method, can be isolated not only from humans, as previously documented, but also from food and animals. The latter finding has important public health implications based on a recent report from Europe of a link between disease severity and infection with STEC isolates that produce Stx2dact.