Detection, enumeration and isolation of strains carrying the stx2 gene from urban sewage

2003 ◽  
Vol 47 (3) ◽  
pp. 109-116 ◽  
Author(s):  
A.R. Blanch ◽  
C. García-Aljaro ◽  
M. Muniesa ◽  
J. Jofre
Keyword(s):  
Shiga Toxin ◽  
Colony Growth ◽  
Coliform Bacteria ◽  
Shiga Toxins ◽  
E Coli ◽  
Urban Sewage ◽  
Shiga Toxin 2 ◽  
Shiga Toxin Genes ◽  
Lateral Transmission ◽  
Stx2 Gene

Verotoxigenic Escherichia coli strains have been related with waterborne outbreaks. Besides 0157:H7, several serotypes of E. coli and other enterobacteria have been implicated in outbreaks and reported to carry the shiga toxin genes. Shiga toxins, stx1 and stx2, are important virulence factors of these strains. These genes have been linked to bacteriophages and consequently are susceptible to lateral transmission. To better understand the ecology of these genes a study of the presence of the shiga toxin 2 gene (stx2) among coliform bacteria present in sewage samples was carried out. A procedure based on colony hybridisation was developed for the isolation of enterobacteria carrying this gene. Colony growth on Chromocult® agar was transferred to a membrane and hybridised with a gene specific probe. The procedure allowed detection of about one colony carrying the gene among around 1,000 faecal coliform colonies. The numbers of bacteria carrying the gene in sewage were also estimated by PCR indicating that the numbers of bacteria carrying the stx2 gene were about 1/1,000 faecal coliforms. The detected numbers by both methods were similar. Positive colony hybridisation was detected in four sewage origins. Fifty-two colonies showing positive signal were isolated from the Chromocult® agar plates, confirmed to be stx2 positive by PCR and phenotypically characterised. Results of the characterisation showed certain diversity among the isolates even in isolates from the same sample. Most of these isolates would not have been isolated with the methods regularly used for the isolation of E. coli 0157:H7 strains. The method will allow study of the numbers and characteristics of bacteria carrying the stx2 gene in different water environments and isolate them in order to determine their role in the spread of the gene.

scholarly journals Emergence of Escherichia coli encoding Shiga toxin 2f in human Shiga toxin-producing E. coli (STEC) infections in the Netherlands, January 2008 to December 2011

2014 ◽  
Vol 19 (17) ◽  
Author(s):  
I Friesema ◽  
K van der Zwaluw ◽  
T Schuurman ◽  
M Kooistra-Smid ◽  
E Franz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
The Netherlands ◽  
Shiga Toxin ◽  
Distinct Group ◽  
Shiga Toxins ◽  
E Coli ◽  
Mild Disease ◽  
Shiga Toxin 2 ◽  
Almost All ◽  
Human Infections

The Shiga toxins of Shiga toxin-producing Escherichia coli (STEC) can be divided into Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) with several sub-variants. Variant Stx2f is one of the latest described, but has been rarely associated with symptomatic human infections. In the enhanced STEC surveillance in the Netherlands, 198 STEC O157 cases and 351 STEC non-O157 cases, including 87 stx2f STEC isolates, were reported between 2008 and 2011. Most stx2f strains belonged to the serogroups O63:H6 (n=47, 54%), O113:H6 (n=12, 14%) and O125:H6 (n=12, 14%). Of the 87 stx2f isolates, 84 (97%) harboured the E. coli attaching and effacing (eae) gene, but not the enterohaemorrhagic E. coli haemolysin (hly) gene. Stx2f STEC infections show milder symptoms and a less severe clinical course than STEC O157 infections. Almost all infections with stx2f (n=83, 95%) occurred between June and December, compared to 170/198 (86%) of STEC O157 and 173/264 (66%) of other STEC non-O157. Stx2f STEC infections in the Netherlands are more common than anticipated, and form a distinct group within STEC with regard to virulence genes and the relatively mild disease.

scholarly journals Contribution and Interaction of Shiga Toxin Genes to Escherichia coli O157:H7 Virulence

Toxins ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 607 ◽  
Author(s):  
Gillian A.M. Tarr ◽  
Taryn Stokowski ◽  
Smriti Shringi ◽  
Phillip I. Tarr ◽  
Stephen B. Freedman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Population Level ◽  
Escherichia Coli O157 ◽  
Additive Interaction ◽  
Shiga Toxins ◽  
E Coli ◽  
Uremic Syndrome ◽  
Shiga Toxin Genes

Escherichia coli O157:H7 is the predominant cause of diarrhea-associated hemolytic uremic syndrome (HUS) worldwide. Its cardinal virulence traits are Shiga toxins, which are encoded by stx genes, the most common of which are stx1a, stx2a, and stx2c. The toxins these genes encode differ in their in vitro and experimental phenotypes, but the human population-level impact of these differences is poorly understood. Using Shiga toxin-encoding bacteriophage insertion typing and real-time polymerase chain reaction, we genotyped isolates from 936 E. coli O157:H7 cases and verified HUS status via chart review. We compared the HUS risk between isolates with stx2a and those with stx2a and another gene and estimated additive interaction of the stx genes. Adjusted for age and symptoms, the HUS incidence of E. coli O157:H7 containing stx2a alone was 4.4% greater (95% confidence interval (CI) −0.3%, 9.1%) than when it occurred with stx1a. When stx1a and stx2a occur together, the risk of HUS was 27.1% lower (95% CI −87.8%, −2.3%) than would be expected if interaction were not present. At the population level, temporal or geographic shifts toward these genotypes should be monitored, and stx genotype may be an important consideration in clinically predicting HUS among E. coli O157:H7 cases.

scholarly journals Identification of Human-Pathogenic Strains of Shiga Toxin-Producing Escherichia coli from Food by a Combination of Serotyping and Molecular Typing of Shiga Toxin Genes

2007 ◽  
Vol 73 (15) ◽  
pp. 4769-4775 ◽  
Author(s):  
Lothar Beutin ◽  
Angelika Miko ◽  
Gladys Krause ◽  
Karin Pries ◽  
Sabine Haby ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Severe Illness ◽  
Shiga Toxins ◽  
E Coli ◽  
Food Borne ◽  
Length Polymorphisms ◽  
Beef Products ◽  
Pcr Products ◽  
Shiga Toxin Genes

ABSTRACT We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx 1 or variant genes were detected in 88 (40.2%) strains and stx 2 and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx 1, stx 1c, and stx 1d) and the Stx2 (stx 2, stx 2d, stx 2-O118, stx 2e, and stx 2g) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx 2 and/or mucus-activatable stx 2d genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx 2 and stx 2d STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx 2e), lamb, and wildlife meat (stx 1c). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.

Escherichia coliShiga toxins induce apoptosis in epithelial cells that is regulated by the Bcl-2 family

2000 ◽  
Vol 278 (5) ◽  
pp. G811-G819 ◽  
Author(s):  
Nicola L. Jones ◽  
Avinash Islur ◽  
Rizwan Haq ◽  
Mariola Mascarenhas ◽  
Mohamed A. Karmali ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Shiga Toxin ◽  
T84 Cells ◽  
Shiga Toxins ◽  
E Coli ◽  
Proapoptotic Protein ◽  
Enhanced Expression ◽  
Shiga Toxin 2 ◽  
Induced Apoptosis

Human intestinal cells lack globotriaosylceramide (Gb3), the receptor for Shiga toxin-1 (Stx1) and Shiga toxin-2 (Stx2). Therefore, the role of these toxins in mediating intestinal disease during infection with Shiga toxin-producing Escherichia coli is unclear. The aims of this study were to determine whether Stx1 and Stx2 induce apoptosis in epithelial cells expressing (HEp-2, Caco-2) or lacking (T84) Gb3and to characterize the role of the Bcl-2 family. Stx1 (12.5 ng/ml) induced apoptosis in both HEp-2 (21.9 ± 7.9% vs. 0.8 ± 0.3%, P = 0.01) and Caco-2 (10.1 ± 1.2% vs. 3.1 ± 0.4%, P = 0.006) cells but not in Gb3-deficient T84 cells. Toxin-mediated apoptosis of HEp-2 cells was associated with enhanced expression of the proapoptotic protein Bax. Inhibition of caspase activation prevented toxin-stimulated apoptosis. In addition, overexpression of Bcl-2 by transient transfection blocked Stx1-stimulated cell death. These findings indicate that Shiga toxins produced by E. coli signal Gb3-expressing epithelial cells to undergo apoptosis in association with enhanced Bax expression, thereby resulting in activation of the caspase cascade.

Shiga toxin 2 and lipopolysaccharide cause monocytic THP-1 cells to release factors which activate platelet function

2005 ◽  
Vol 94 (11) ◽  
pp. 1019-1027 ◽  
Author(s):  
Fadila Guessous ◽  
Renata Polanowska-Grabowska ◽  
Tiffany Keepers ◽  
Tom Obrig ◽  
Adrian Gear ◽  
...  
Keyword(s):  
Platelet Function ◽  
Shiga Toxin ◽  
Escherichia Coli O157 ◽  
Monocytic Cells ◽  
Shiga Toxins ◽  
E Coli ◽  
Release Factors ◽  
Serotonin Secretion ◽  
Shiga Toxin 2 ◽  
Uremic Syndrome

SummaryPlatelet and monocyte activation may contribute to hemolytic anemia, thrombocytopenia and renal failure associated with the hemolytic uremic syndrome (HUS) caused by Escherichia coli O157:H7. Since Shiga toxins (Stxs) and lipopolysaccharide (LPS) from this bacterium are implicated in the pathogenesis of HUS, we examined whether stimulation of THP-1 human monocytic cells by Shiga toxin 2 (Stx2) and LPS can lead to the activation of platelet function. We now show that Stx2 caused THP-1 cells to release the chemokines IL-8, MDC, and RANTES and that the presence of LPS further stimulated this release. IL-8 was produced in greatest amount and was an effective co-agonist for inducing platelet aggregation. Primary human monocytes also released large amounts of IL-8 in response to LPS and Stx2. Factors released by THP-1 cells exposed to Stx2 and LPS activated platelet function as evidenced by increased aggregation, serotonin secretion, P-selectin exposure and by the formation of stable platelet-monocyte aggregates. Our data therefore show that monocytes exposed to E. coli-derived Stx2 and LPS release factors which activate platelet function.

scholarly journals Role for a Phage Promoter in Shiga Toxin 2 Expression from a Pathogenic Escherichia coliStrain

2001 ◽  
Vol 183 (6) ◽  
pp. 2081-2085 ◽  
Author(s):  
Patrick L. Wagner ◽  
Melody N. Neely ◽  
Xiaoping Zhang ◽  
David W. K. Acheson ◽  
Matthew K. Waldor ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Transfer ◽  
Shiga Toxin ◽  
Virulence Gene ◽  
Prophage Induction ◽  
Transcription Terminator ◽  
Shiga Toxins ◽  
E Coli ◽  
Shiga Toxin 2 ◽  
Transcription Complexes

ABSTRACT Shiga toxins (Stxs), encoded by the stxA andstxB genes, are important contributors to the virulence ofEscherichia coli O157:H7 and other Stx-producing E. coli (STEC) strains. The stxA and stxBgenes in STEC strains are located on the genomes of resident prophages of the λ family immediately downstream of the phage late promoters (p R′). The phage-encoded Q proteins modify RNA polymerase initiating transcription at the cognatep R′ promoter which creates transcription complexes that transcend a transcription terminator immediately downstream of p R′ as well as terminator kilobases distal to p R′. To test if this Q-directed processive transcription plays a role instx 2 AB expression, we constructed a mutant prophage in an O157:H7 clinical isolate from whichp R′ and part of Q were deleted but which has an intact pStx, the previously describedstx 2 AB-associated promoter. We report that production of significant levels of Stx2 in this O157:H7 isolate depends on the p R′ promoter. Since transcription initiating at p R′ ultimately requires activation of the phage lytic cascade, expression ofstx 2 AB in STEC depends primarily on prophage induction. By showing this central role for the prophage instx 2 AB expression, our findings contradict the prevailing assumption that phages serve merely as agents for virulence gene transfer.

scholarly journals Identification and Characterization of a Newly Isolated Shiga Toxin 2-Converting Phage from Shiga Toxin-ProducingEscherichia coli

1998 ◽  
Vol 66 (9) ◽  
pp. 4100-4107 ◽  
Author(s):  
Masahisa Watarai ◽  
Toshio Sato ◽  
Midori Kobayashi ◽  
Takeshi Shimizu ◽  
Shinji Yamasaki ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Plating Efficiency ◽  
Restriction Maps ◽  
Shiga Toxins ◽  
E Coli ◽  
Shiga Toxin 2 ◽  
Formal Science ◽  
K 12 ◽  
Identification And Characterization

ABSTRACT 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 Stx2Φ-I and Stx2Φ-II, from two clinical strains of STEC associated with the outbreaks in Japan in 1996 and found that Stx2Φ-I resembled 933W, the previously reported Stx2-converting phage, in its infective properties for E. coli K-12 strain C600 while Stx2Φ-II was distinct from them. The sizes of the plaques of Stx2Φ-I and Stx2Φ-II in C600 were different; the former was larger than the latter. The restriction maps of Stx2Φ-I and Stx2Φ-II were not identical; rather, Stx2Φ-II DNA was approximately 3 kb larger than Stx2Φ-I DNA. Furthermore, Stx2Φ-I and Stx2Φ-II showed different phage immunity, with Stx2Φ-I and 933W belonging to the same group. Infection of C600 by Stx2Φ-I or 933W was affected by environmental osmolarity differently from that by Stx2Φ-II. When C600 was grown under conditions of high osmolarity, the infectivity of Stx2Φ-I and 933W was greatly decreased compared with that of Stx2Φ-II. Examination of the plating efficiency of the three phages for the defined mutations in C600 revealed that the efficiency of Stx2Φ-I and 933W for the fadL mutant decreased to less than 10−7 compared with that for C600 whereas the efficiency of Stx2Φ-II decreased to 0.1% of that for C600. In contrast, while the plating efficiency of Stx2Φ-II for thelamB mutant decreased to a low level (0.05% of that for C600), the efficiencies of Stx2Φ-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 Stx2Φ-I and Stx2Φ-II whereas LamB acts as the receptor only for Stx2Φ-II.

scholarly journals Shiga Toxins, and the Genes Encoding Them, in Fecal Samples from Native Idaho Ungulates

2008 ◽  
Vol 75 (3) ◽  
pp. 862-865 ◽  
Author(s):  
Jeremy J. Gilbreath ◽  
Malcolm S. Shields ◽  
Rebekah L. Smith ◽  
Larry D. Farrell ◽  
Peter P. Sheridan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Wild Ungulates ◽  
Fecal Samples ◽  
Shiga Toxins ◽  
Toxin Genes ◽  
Genes Encoding ◽  
Shiga Toxin Genes

ABSTRACT Cattle are a known reservoir of Shiga toxin-producing Escherichia coli. The prevalence and stability of Shiga toxin and/or Shiga toxin genes among native wild ungulates in Idaho were investigated. The frequency of both Shiga genes and toxin was similar to that reported for Idaho cattle (∼19%).

scholarly journals Molecular analysis of Escherichia coli O157 strains isolated from cattle and pigs by the use of PCR and pulsed-field gel electrophoresis methods

2012 ◽  
Vol 47 (No. 6) ◽  
pp. 149-158 ◽  
Author(s):  
J. Osek ◽  
P. Gallien
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Genetic Relatedness ◽  
Rflp Analysis ◽  
Toxin Gene ◽  
Escherichia Coli O157 ◽  
Chromosomal Dna ◽  
E Coli ◽  
Shiga Toxin Genes ◽  
Porcine Origin

Fourteen Escherichia coli O157 strains isolated from cattle and pigs in Poland and in Germany were investigated, using PCR, for the genetic markers associated with Shiga toxin-producing E. coli (STEC). Only two strains, both of cattle origin, were positive for the fliC (H7) gene and could be classified as O157 : H7. Nine isolates had stx shiga toxin genes, either stx1 (1 strain), stx2 (4 isolates) or both (4 strains). The stx2-carrying samples were further subtyped by PCR for the stx2c, stx2d, and stx2e toxin variants. It was shown that all but one stx2-positive bacteria possessed the stx2c Shiga toxin gene type and one stx2 STEC isolate had the stx2d virulence factor sub-type. The eaeA (intimin) gene was found in 9 strains (8 isolates from cattle and one strain from pigs); all of them harboured the genetic marker characteristic of the gamma intimin variant. The translocated intimin receptor (tir) gene was detected in 7 isolates tested and among them only one tir-positive strain was recovered from pigs. The ehly E. coli enterohemolysin gene was amplified in all but one strains obtained from cattle and only in one isolate of porcine origin. The genetic relatedness of the analysed E. coli O157 strains was examined by restriction fragment length polymorphism (RFLP) of chromosomal DNA digested with XbaI. Two distinct but related RFLP pattern clusters were observed: one with 9 strains (8 isolates of bovine origin and one strain obtained from pigs) and the other one comprises the remaining 5 E. coli isolates (4 of porcine origin and one strain recovered from cattle). The results suggest that pigs, besides cattle, may be a reservoir of E. coli O157 strains potentially pathogenic to humans. Moreover, epidemiologically unrelated isolates of the O157 serogroup, recovered from different animal species, showed a clonal relationship as demonstrated by the RFLP analysis.

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