20 results on '"Shiga Toxin 2"'
Search Results
2. Influence of RNase E deficiency on the production of stx2-bearing phages and Shiga toxin in an RNase E-inducible strain of enterohaemorrhagic Escherichia coli (EHEC) O157:H7
- Author
-
Thujitha Thuraisamy and Patricia B. Lodato
- Subjects
0301 basic medicine ,Microbiology (medical) ,RNase P ,viruses ,Prophages ,030106 microbiology ,Virulence ,Enzyme-Linked Immunosorbent Assay ,Viral Plaque Assay ,Biology ,medicine.disease_cause ,Microbiology ,Coliphages ,Shiga Toxin 2 ,03 medical and health sciences ,fluids and secretions ,Bacteriolysis ,STX2 ,hemic and lymphatic diseases ,Endoribonucleases ,medicine ,Humans ,Prophage ,Toxin ,Shiga toxin ,General Medicine ,biology.organism_classification ,Lytic cycle ,Enterohemorrhagic Escherichia coli ,biology.protein ,Bacteria ,Research Article - Abstract
Purpose. In enterohaemorrhagic Escherichia coli (EHEC), stx1 or stx2 genes encode Shiga toxin (Stx1 or Stx2, respectively) and are carried by prophages. The production and release of both stx phages and toxin occur upon initiation of the phage lytic cycle. Phages can further disseminate stx genes by infecting naive bacteria in the intestine. Here, the effect of RNase E deficiency on these two virulence traits was investigated. Methodology. Cultures of the EHEC strains TEA028-rne containing low versus normal RNase E levels or the parental strain (TEA028) were treated with mitomycin C (MMC) to induce the phage lytic cycle. Phages and Stx2 titres were quantified by the double-agar assay and the receptor ELISA technique, respectively. Results. RNase E deficiency in MMC-treated cells significantly reduced the yield of infectious stx2 phages. Delayed cell lysis and the appearance of encapsidated phage DNA copies suggest a slow onset of the lytic cycle. However, these observations do not entirely explain the decrease of phage yields. stx1 phages were not detected under normal or deficient RNase E levels. After an initial delay, high levels of toxin were finally produced in MMC-treated cultures. Conclusion. RNase E scarcity reduces stx2 phage production but not toxin. Normal concentrations of RNase E are likely required for correct phage morphogenesis. Our future work will address the mechanism of RNase E action on phage morphogenesis.
- Published
- 2018
3. Epidemiology and microbiology of Shiga toxin-producing Escherichia coli other than serogroup O157 in England, 2009–2013
- Author
-
Robin Smith, Gemma L. Vanstone, Neil T. Perry, Lisa Byrne, Kathie Grant, N. Launders, Claire Jenkins, Gauri Godbole, and Goutam K. Adak
- Subjects
Adult ,Male ,Microbiology (medical) ,Serotype ,Adolescent ,Genotype ,Biology ,Shiga Toxin 1 ,medicine.disease_cause ,Polymerase Chain Reaction ,Shiga Toxin 2 ,Microbiology ,law.invention ,Feces ,Young Adult ,fluids and secretions ,STX2 ,law ,medicine ,Humans ,Serotyping ,Adhesins, Bacterial ,Child ,Escherichia coli ,Escherichia coli Infections ,Polymerase chain reaction ,Aged ,Intimin ,Aged, 80 and over ,Shiga-Toxigenic Escherichia coli ,Escherichia coli Proteins ,Incidence ,Incidence (epidemiology) ,Infant ,General Medicine ,Middle Aged ,bacterial infections and mycoses ,Virology ,Bacterial adhesin ,England ,Case-Control Studies ,Child, Preschool ,Trans-Activators ,Female - Abstract
The implementation of direct testing of clinical faecal specimens for gastrointestinal (GI) pathogens by PCR offers a sensitive and comprehensive approach for the detection of Shiga toxin-producing Escherichia coli (STEC). The introduction of a commercial PCR assay, known as GI PCR, for the detection of GI pathogens at three frontline hospital laboratories in England between December 2012 and December 2013 led to a significant increase in detection of STEC other than serogroup O157 (non-O157 STEC). In 2013, 47 isolates were detected in England, compared with 57 in the preceding 4 years (2009–2012). The most common non-O157 STEC serogroup detected was O26 (23.2 %). A total of 47 (47.5 %) STEC isolates had stx2 only, 28 (28.3 %) carried stx1 and stx2, and the remaining 24 (24.2 %) had stx1 only. Stx2a (64.0 %) was the most frequently detected Stx2 subtype. The eae (intimin) gene was detected in 52 (52.5 %) non-O157 STEC isolates. Six strains of STEC O104 had aggR, but this gene was not detected in any other STEC serogroups in this study. Haemolytic ureamic syndrome was significantly associated with STEC strains possessing eae [odds ratio (OR) 5.845, P = 0.0235] and/or stx2a (OR 9.56, P = 0.0034) subtypes. A matched case–control analysis indicated an association between non-O157 STEC cases and contact with farm animals. Widespread implementation of the PCR approach in England will determine the true incidence of non-O157 STEC infection, highlight the burden in terms of morbidity and mortality, and facilitate the examination of risk factors to indicate whether there are niche risk exposures for particular strains.
- Published
- 2014
4. Differences in adherence and virulence gene expression between two outbreak strains of enterohaemorrhagic Escherichia coli O157 : H7
- Author
-
Thomas S. Whittam, Scott T. Henderson, Shannon D. Manning, Lindsey Ouellette, and Galeb Abu-Ali
- Subjects
Molecular Sequence Data ,Virulence ,Escherichia coli O157 ,medicine.disease_cause ,Shiga Toxin 2 ,Microbiology ,Bacterial Adhesion ,Cell Line ,Disease Outbreaks ,Species Specificity ,Gene expression ,medicine ,Animals ,Humans ,Gene ,Escherichia coli ,Escherichia coli Infections ,Oligonucleotide Array Sequence Analysis ,Genetics ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Microarray analysis techniques ,Escherichia coli Proteins ,Gene Expression Profiling ,Epithelial Cells ,Shiga toxin ,Gene Expression Regulation, Bacterial ,North America ,Cell and Molecular Biology of Microbes ,biology.protein ,Cattle ,DNA microarray ,Locus of enterocyte effacement - Abstract
The Escherichia coli O157 : H7 TW14359 strain was implicated in a multi-state outbreak in North America in 2006, which resulted in high rates of severe disease. Similarly, the O157 : H7 RIMD0509952 (Sakai) strain caused the largest O157 : H7 outbreak to date. Both strains were shown to represent divergent phylogenetic lineages. Here we compared global gene expression patterns before and after epithelial cell exposure, as well as the ability to adhere to and invade epithelial cells, between the two outbreak strains. Epithelial cell assays demonstrated a 2.5-fold greater adherence of the TW14359 strain relative to Sakai, while whole-genome microarrays detected significant differential expression of 914 genes, 206 of which had a fold change ≥1.5. Interestingly, most locus of enterocyte effacement (LEE) genes were upregulated in TW14359, whereas flagellar and chemotaxis genes were primarily upregulated in Sakai, suggesting discordant expression of these genes between the two strains. The Shiga toxin 2 genes were also upregulated in the TW14359 strain, as were several pO157-encoded genes that promote adherence, including type II secretion genes and their effectors stcE and adfO. Quantitative RT-PCR confirmed the expression differences detected in the microarray analysis, and expression levels were lower for a subset of LEE genes before versus after exposure to epithelial cells. In all, this study demonstrated the upregulation of major and ancillary virulence genes in TW14359 and of flagellar and chemotaxis genes in Sakai, under conditions that precede intimate bacterial attachment to epithelial cells. Differences in the level of adherence to epithelial cells were also observed, implying that these two phylogenetically divergent O157 : H7 outbreak strains vary in their ability to colonize, or initiate the disease process.
- Published
- 2010
5. Differential expression of stx2 variants in Shiga toxin-producing Escherichia coli belonging to seropathotypes A and C
- Author
-
Marjolaine Vareille, Christine Martin, J P Girardeau, Annie Garrivier, Alain P. Gobert, Yolande Bertin, and Thibaut de Sablet
- Subjects
DNA, Bacterial ,Transcription, Genetic ,DNA Footprinting ,Virulence ,Enzyme-Linked Immunosorbent Assay ,medicine.disease_cause ,Coliphages ,Shiga Toxin 2 ,Microbiology ,Bacterial genetics ,STX2 ,hemic and lymphatic diseases ,Gene expression ,medicine ,RNA, Messenger ,Serotyping ,Escherichia coli ,Gene ,Prophage ,Shiga-Toxigenic Escherichia coli ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Escherichia coli Proteins ,Genetic Variation ,Shiga toxin ,RNA, Bacterial ,biology.protein ,Polymorphism, Restriction Fragment Length - Abstract
Only a subset of Shiga toxin (Stx)-producing Escherichia coli (STEC) are human pathogens, but the characteristics that account for differences in pathogenicity are not well understood. In this study, we investigated the distribution of the stx variants coding for Stx2 and its variants in highly virulent STEC of seropathotype A and low-pathogenic STEC of seropathotype C. We analysed and compared transcription of the corresponding genes, production of Shiga toxins, and stx-phage release in basal as well as in induced conditions. We found that the stx(2) variant was mainly associated with strains of seropathotype A, whereas most of the strains of seropathotype C possessed the stx(2-vhb) variant, which was frequently associated with stx(2), stx(2-vha) or stx(2c). Levels of stx(2) and stx(2)-related mRNA were higher in strains belonging to seropathotype A and in those strains of seropathotype C that express the stx(2) variant than in the remaining strains of seropathotype C. The stx(2-vhb) genes were the least expressed, in basal as well as in induced conditions, and in many cases did not seem to be carried by an inducible prophage. A clear correlation was observed between stx mRNA levels and stx-phage DNA in the culture supernatants, suggesting that most stx(2)-related genes are expressed only when they are carried by a phage. In conclusion, some relationship between stx(2)-related gene expression in vitro and the seropathotype of the STEC strains was observed. A higher expression of the stx(2) gene and a higher release of its product, in basal as well as in induced conditions, was observed in pathogenic strains of seropathotype A. A subset of strains of seropathotype C shows the same characteristics and could be a high risk to human health.
- Published
- 2008
6. Differential binding of Shiga toxin 2 to human and murine neutrophils
- Author
-
Thomas P. Griener, George L. Mulvey, Paola Marcato, and Glen D. Armstrong
- Subjects
Microbiology (medical) ,Neutrophils ,Pentamer ,Protein subunit ,Globotriaosylceramide ,medicine.disease_cause ,Shiga Toxin 2 ,Microbiology ,Mice ,chemistry.chemical_compound ,Shiga-like toxin ,STX2 ,medicine ,Animals ,Humans ,Escherichia coli ,Cells, Cultured ,biology ,Trihexosylceramides ,Shiga toxin ,General Medicine ,biology.organism_classification ,Enterobacteriaceae ,Serum Amyloid P-Component ,chemistry ,biology.protein - Abstract
Shiga toxins (Stx1 and Stx2) are responsible for initiating haemolytic uraemic syndrome, a serious extraintestinal complication caused by enterohaemorrhagicEscherichia coliO157 : H7 infection in humans. Shiga toxins are classical AB5-type exotoxins, consisting of a globotriaosylceramide (Gb3)-binding B subunit pentamer and an enzymic A subunit. It is demonstrated in this study that Stx2 binds to human neutrophils by a non-classical mechanism that is independent of Gb3. In contrast, the investigation revealed that Stx2 binds to murine neutrophils by the classical Gb3-dependent mechanism. Moreover, whereas the human serum amyloid P (HuSAP) component inhibited Stx2 binding to murine neutrophils, HuSAP increased Stx2 binding to human neutrophils by 84.2 % (P≤0.002, Student'st-test). These observations may explain why HuSAP protects mice from the lethal effects of Stx2, whereas there is no indication that HuSAP plays a similar protective role in humans infected byE. coliO157 : H7.
- Published
- 2007
7. Mosaic structure of Shiga-toxin-2-encoding phages isolated from Escherichia coli O157:H7 indicates frequent gene exchange between lambdoid phage genomes
- Author
-
Per Einar Granum, Sigrid Brynestad, Birgit K. Johansen, and Yngvild Wasteson
- Subjects
Gene Transfer, Horizontal ,viruses ,Population ,Cattle Diseases ,Genome, Viral ,Escherichia coli O157 ,medicine.disease_cause ,Coliphages ,Shiga Toxin 2 ,Microbiology ,Genome ,chemistry.chemical_compound ,Chlorocebus aethiops ,medicine ,Animals ,Humans ,education ,Vero Cells ,Escherichia coli ,Gene ,Escherichia coli Infections ,Recombination, Genetic ,education.field_of_study ,biology ,Genetic Variation ,Shiga toxin ,biology.organism_classification ,Bacteriophage lambda ,Integrase ,chemistry ,Child, Preschool ,DNA, Viral ,biology.protein ,Cattle ,Female ,Virus Activation ,Polymorphism, Restriction Fragment Length ,Bacteria ,DNA - Abstract
Shiga-toxin-2 (stx(2))-encoding bacteriophages were isolated from Norwegian Escherichia coli O157:H7 isolates of cattle and human origin. The phages were characterized by restriction enzyme analysis, hybridization with probes for toxin genes and selected phage DNA such as the P gene, integrase gene and IS1203, and by PCR studies and partial sequencing of selected DNA regions in the integrase to stx(2) region of the phages. The stx(2)-phage-containing bacteria from which inducible phages were detected produced similar amounts of toxin, as shown by a Vero cell assay. The results indicate that the population of stx(2)-carrying phages is heterogeneous, although the phages from epidemiologically linked E. coli O157:H7 isolates were similar. There appears to have been frequent recombination of stx(2) phages with other lambdoid phages.
- Published
- 2001
8. Effects of interaction between Escherichia coli verotoxin and lipopolysaccharide on cytokine induction and lethality in mice
- Author
-
Shirou Tsujimoto, Keizo Yamaguchi, Kazuhiro Tateda, Fumio Gondaira, Kuri Suzuki, and Tetsuya Matsumoto
- Subjects
Lipopolysaccharides ,Male ,Microbiology (medical) ,Necrosis ,Lipopolysaccharide ,Colon ,Ratón ,medicine.medical_treatment ,Bacterial Toxins ,Enzyme-Linked Immunosorbent Assay ,Biology ,Escherichia coli O157 ,Kidney ,Shiga Toxin 2 ,Microbiology ,Proinflammatory cytokine ,Lethal Dose 50 ,Mice ,chemistry.chemical_compound ,medicine ,Animals ,Interleukin 6 ,Escherichia coli Infections ,Mice, Inbred BALB C ,Histocytochemistry ,Interleukin-6 ,Tumor Necrosis Factor-alpha ,General Medicine ,Specific Pathogen-Free Organisms ,Cytokine ,Gene Expression Regulation ,Liver ,chemistry ,Toxicity ,biology.protein ,Cytokines ,Tumor necrosis factor alpha ,medicine.symptom ,Interleukin-1 - Abstract
In Escherichia coli 0157 infections, verotoxins (VT) play a critical role in causing the disease, although other factors such as lipopolysaccharide (LPS) and inflammatory cytokines may affect the progression and course of the disease. The present study examined the roles of VT and LPS in induction of serum cytokines and lethality in mice. LD50 of VT2 (13 ng) was c. 10(4)-fold smaller than that of LPS (400 microg). Although the lethal toxicity of these toxins was examined in several experimental conditions, such as VT2 (5, 10, 20, 40 ng/mouse) alone or in combination with LPS (100 microg/mouse) at various times (-2 days to +2 days), no evidence of synergy was observed. VT2 did not augment LPS-induced tumour necrosis factor-alpha (TNF-alpha) or interleukin-6 production, and conversely suppressed TNF-alpha production when it was injected 2 days before LPS challenge. The data failed to indicate either synergic or additive effects of VT and LPS on cytokine production or lethality in mice. In contrast, antagonistic interactions were clearly observed in cytokine production in certain conditions. The results suggested that these toxins may be co-operatively involvedin the pathology of VT-related diseases, but not through synergic interactions.
- Published
- 2000
9. Comparative cytotoxicity of purified Shiga-like toxin-lle on porcine and bovine aortic endothelial and human colonic adenocarcinoma cells
- Author
-
C. L. Gyles, Clifford A. Lingwood, Beth Boyd, A. Valdivieso-Garcia, R. C. Clarke, A. Durette, and D. L. Macleod
- Subjects
Microbiology (medical) ,Neutral red ,Cell Survival ,Swine ,Bacterial Toxins ,Receptors, Cell Surface ,Adenocarcinoma ,Biology ,medicine.disease_cause ,Shiga Toxin 2 ,Microbiology ,Cell Line ,chemistry.chemical_compound ,Glycolipid ,Shiga-like toxin ,Chlorocebus aethiops ,Escherichia coli ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Cytotoxicity ,Vero Cells ,Aorta ,Cells, Cultured ,Chromatography, High Pressure Liquid ,Globosides ,Cytotoxins ,Toxin ,Trihexosylceramides ,General Medicine ,Flow Cytometry ,Molecular biology ,In vitro ,Endothelial stem cell ,chemistry ,Cell culture ,Colonic Neoplasms ,Immunology ,Cattle ,Endothelium, Vascular - Abstract
Porcine and bovine aortic endothelial cells and human colonic adenocarcinoma cells were compared for their susceptibility to the toxic effect of purified Shiga-like toxin IIe (SLT-IIe), measured by the neutral red cytotoxicity assay. Cytotoxicity correlated with toxin binding as indicated by fluorescence activated cell sorter analysis and with the globotriosylceramide (Gb3) and globotetraosylceramide (Gb4) content of cells determined by high pressure liquid chromatography. One line of porcine aortic endothelial cells was 1400-fold more susceptible than the line of bovine aortic endothelial cells that was tested, but a second line of porcine aortic endothelial cells was highly refractory to SLT-IIe. Human colonic adenocarcinoma cells lacked detectable levels of Gb4 and were least susceptible to SLT-IIe.
- Published
- 1996
10. Pheno-genotyping of verotoxin 2 (VT2)-producing Escherichia coli causing haemorrhagic colitis and haemolytic uraemic syndrome by direct analysis of patients' stools
- Author
-
Alessandra Gianviti, Ida Luzzi, Holger Rüssmann, Helge Karch, and Alfredo Caprioli
- Subjects
DNA, Bacterial ,Microbiology (medical) ,Genotype ,Bacterial Toxins ,Biology ,Polymerase Chain Reaction ,Shiga Toxin 2 ,Microbiology ,law.invention ,Feces ,chemistry.chemical_compound ,fluids and secretions ,Shiga-like toxin ,Neutralization Tests ,law ,Germany ,Chlorocebus aethiops ,Escherichia coli ,medicine ,Animals ,Humans ,Serotyping ,Colitis ,Child ,Vero Cells ,Genotyping ,Escherichia coli Infections ,Polymerase chain reaction ,General Medicine ,medicine.disease ,Subtyping ,Phenotype ,Italy ,chemistry ,VTEC ,Hemolytic-Uremic Syndrome ,Colitis, Ulcerative ,Restriction fragment length polymorphism ,Polymorphism, Restriction Fragment Length ,HeLa Cells - Abstract
The subtype of verotoxin 2 (VT2) found in 22 VT2-positive stool samples from severely diseased Italian and German children with haemorrhagic colitis or haemolytic uraemic syndrome, or both, and that produced by the corresponding VT-producing Escherichia coli (VTEC) strains isolated from the stools were studied by cytotoxicity sero-neutralisation assays and by polymerase chain reaction (PCR) amplification of the VT2 B-subunit gene, followed by restriction fragment length polymorphism (RFLP) analysis. The free faecal toxin was serotyped as the classical VT2 in 21 stool samples, and as the VT2 variant VT2c in one. For all but one of the VTEC isolates, the toxin phenotype was consistent with the type of VT produced in vivo and found in the corresponding stool samples. Genotyping was in agreement with phenotyping for those strains harbouring a single type of VT2 gene. Three O157: H7 isolates carrying both VT2 and VT2c genes had the VT2 phenotype, instead of the expected VT2c phenotype. Direct PCR analysis of stools detected VT genes in only 11 of 20 VT-positive stool samples suggesting that the Vero cell cytotoxicity assay is more sensitive in diagnosing VTEC infection. Immunological and genetic subtyping of VT2 performed directly on stool samples from patients with haemolytic uraemic syndrome could be a useful complementary approach to understanding the role of the different types of VT in this syndrome.
- Published
- 1995
11. Variants of Shiga-like toxin II constitute a major toxin component in Escherichia coli O157 strains from patients with haemolytic uraemic syndrome
- Author
-
Holger Rüssmann, Jürgen Heesemann, Alfredo Caprioli, Herbert Schmidt, and Helge Karch
- Subjects
Microbiology (medical) ,Genotype ,genetic structures ,Bacterial Toxins ,Molecular Sequence Data ,Enzyme-Linked Immunosorbent Assay ,Biology ,medicine.disease_cause ,Polymerase Chain Reaction ,Shiga Toxin 2 ,Microbiology ,law.invention ,HaeIII ,Enterotoxins ,chemistry.chemical_compound ,Shiga-like toxin ,law ,Escherichia coli ,medicine ,Animals ,Humans ,Child ,Vero Cells ,Escherichia coli Infections ,Polymerase chain reaction ,DNA Primers ,Base Sequence ,Gene Expression Regulation, Bacterial ,General Medicine ,biology.organism_classification ,Enterobacteriaceae ,Blotting, Southern ,Restriction enzyme ,Phenotype ,chemistry ,Hemolytic-Uremic Syndrome ,Restriction fragment length polymorphism ,Polymorphism, Restriction Fragment Length ,medicine.drug - Abstract
Summary The prevalence and genotype of Shiga-like toxins (SLTs) in Escherichia coli O157 strains from patients in Germany with haemolytic uraemic syndrome (HUS) were investigated. This was done by PCR amplification of the B-subunit genes with two primer pairs—one complementary to slt-IB, and the other homologous to both slt-IIB and slt-IIvB sequences. To distinguish between slt-II and slt-IIv, the amplified DNA was digested with restriction endonucleases HaeIII and FokI. Of the 38 strains examined, 17 harboured sequences for slt-IIv; four contained only slt-IIv, three carried both slt-IIv and slt-I, and 10 strains had slt-IIv and slt-II. A further three genotypes (slt-I, slt-II, slt-I/slt-II) were found in the remaining 21 strains resulting in a total of six slt genotypes. To determine whether the slt genes were expressed, and whether genotypes correlated with phenotypes, all strains were subjected to cytotoxicity assays and colony ELISA. All 38 strains displayed cytotoxic activity to Vero cells in similar quantities. The SLT-I-specific monoclonal antibody (MAb)13C4 reacted with all 10 strains in which slt-I sequences were identified. Colony blot ELISA with the SLT-II specific MAb11E10 detected 27 of 28 strains with slt-II sequences, but did not react with any of the seven strains that carried slt-IIv, or slt-I and slt-IIv. The high SLT variability shown here has diagnostic implications and may well have consequences for the host response in infections associated with these pathogens.
- Published
- 1994
12. Diagnosis of infections with Shiga-like toxin-producing Escherichia coli by use of enzyme-linked immunosorbent assays for Shiga-like toxins on cultured stool samples
- Author
-
H. Panigrahi, D. Law, A. A. Hamour, Leela A. Ganguli, David W. Denning, and D. W. K. Acheson
- Subjects
Diarrhea ,Microbiology (medical) ,Bacterial Toxins/biosynthesis ,genetic structures ,Bacterial Toxins ,Sorbitol-MacConkey agar ,Enzyme-Linked Immunosorbent Assay ,Shiga Toxin 1 ,medicine.disease_cause ,Sensitivity and Specificity ,Shiga Toxin 2 ,Microbiology ,Shiga Toxin ,Feces ,chemistry.chemical_compound ,Shiga-like toxin ,Colitis/diagnosis ,Escherichia coli ,medicine ,Humans ,Prospective Studies ,Escherichia coli Infections ,Faeces ,biology ,Toxin ,Shiga toxin ,General Medicine ,Colitis ,biology.organism_classification ,Virology ,Enterobacteriaceae ,chemistry ,Hemolytic-Uremic Syndrome ,biology.protein ,Colitis/microbiology ,Gastrointestinal Hemorrhage ,Bacteria ,Escherichia coli Infections/microbiology - Abstract
Shiga-like toxin-producing (SLT) Escherichia coli, particularly those belonging to serogroup O157, are responsible for haemorrhagic colitis, haemolytic uraemic syndrome and some cases of gastro-enteritis. The rapid and reliable diagnosis of all these infections is necessary for correct patient management and for epidemiological reasons, but is rarely possible with present methods. We compared the efficacy of two methods, (i) the culture of faeces in broth that contained mitomycin C followed by enzyme-linked immunosorbent assay (ELISA) for SLTs, and (ii) the culture of faeces on sorbitol MacConkey agar (SMA), in the detection of infections caused by SLT-producing E. coli. SLT-producing E. coli O157 strains were isolated on SMA from 42 of 475 faecal samples, but SLTs were detected by ELISA in culture supernates or lysates of 54 of 475 samples. SLT-producing E. coli strains were isolated subsequently from 11 of 12 ELISA-positive, SMA culture-negative samples by a colony blot technique. In four cases, SLT-producing E. coli of serogroups other than O157 were isolated and in seven cases E. coli O157 was isolated in small numbers. The ELISA is a rapid and sensitive technique for the diagnosis of SLT-producing E. coli infection, especially where low numbers of the organism are present in faeces and when the infection is caused by a serogroup other than O157.
- Published
- 1994
13. Effects of interaction between Escherichia coli verotoxin and lipopolysaccharide on cytokine induction and lethality in mice.
- Author
-
Suzuki K, Tateda K, Matsumoto T, Gondaira F, Tsujimoto S, and Yamaguchi K
- Subjects
- Animals, Colon pathology, Cytokines blood, Enzyme-Linked Immunosorbent Assay, Escherichia coli Infections blood, Histocytochemistry, Interleukin-1 biosynthesis, Interleukin-1 blood, Interleukin-6 biosynthesis, Interleukin-6 blood, Kidney pathology, Lethal Dose 50, Liver pathology, Male, Mice, Mice, Inbred BALB C, Shiga Toxin 2, Specific Pathogen-Free Organisms, Tumor Necrosis Factor-alpha analysis, Tumor Necrosis Factor-alpha biosynthesis, Bacterial Toxins immunology, Cytokines biosynthesis, Escherichia coli Infections immunology, Escherichia coli O157 pathogenicity, Gene Expression Regulation, Lipopolysaccharides immunology
- Abstract
In Escherichia coli 0157 infections, verotoxins (VT) play a critical role in causing the disease, although other factors such as lipopolysaccharide (LPS) and inflammatory cytokines may affect the progression and course of the disease. The present study examined the roles of VT and LPS in induction of serum cytokines and lethality in mice. LD50 of VT2 (13 ng) was c. 10(4)-fold smaller than that of LPS (400 microg). Although the lethal toxicity of these toxins was examined in several experimental conditions, such as VT2 (5, 10, 20, 40 ng/mouse) alone or in combination with LPS (100 microg/mouse) at various times (-2 days to +2 days), no evidence of synergy was observed. VT2 did not augment LPS-induced tumour necrosis factor-alpha (TNF-alpha) or interleukin-6 production, and conversely suppressed TNF-alpha production when it was injected 2 days before LPS challenge. The data failed to indicate either synergic or additive effects of VT and LPS on cytokine production or lethality in mice. In contrast, antagonistic interactions were clearly observed in cytokine production in certain conditions. The results suggested that these toxins may be co-operatively involvedin the pathology of VT-related diseases, but not through synergic interactions.
- Published
- 2000
- Full Text
- View/download PDF
14. Molecular characterisation of verocytotoxin-producing Escherichia coli of serogroup O111 from different countries.
- Author
-
Morabito S, Karch H, Schmidt H, Minelli F, Mariani-Kurkdjian P, Allerberger F, Bettelheim KA, and Caprioli A
- Subjects
- Animals, Bacterial Outer Membrane Proteins genetics, Cattle, DNA Fingerprinting, DNA, Bacterial genetics, Electrophoresis, Agar Gel, Electrophoresis, Gel, Pulsed-Field, Electrophoresis, Polyacrylamide Gel, Escherichia coli isolation & purification, Escherichia coli pathogenicity, Escherichia coli O157 isolation & purification, Escherichia coli O157 pathogenicity, Genotype, Humans, Microbial Sensitivity Tests, Plasmids genetics, Polymorphism, Restriction Fragment Length, RNA, Ribosomal genetics, Shiga Toxin 1, Shiga Toxin 2, Virulence genetics, Adhesins, Bacterial, Bacterial Toxins genetics, Carrier Proteins, Escherichia coli genetics, Escherichia coli O157 genetics, Escherichia coli Proteins
- Abstract
A collection of epidemiologically unrelated verocytotoxin (VT)-producing Escherichia coli (VTEC) strains of serogroup O111 isolated from human patients and cattle with diarrhoeal disease in five different countries were characterised by determination of their VT genotypes, the presence of other virulence factors such as the intimin-coding eae gene and the enterohaemorrhagic E. coli (EHEC) plasmid, and their antibiotic susceptibility patterns. The genetic relatedness among isolates was evaluated by genomic DNA fingerprinting techniques such as restriction fragment length polymorphism analysis of ribosomal RNA genes (ribotyping) and pulsed-field gel electrophoresis. The results indicated that the VTEC O111 examined belong to two distinct clonal lineages. The first group was constituted mainly of non-motile, eae-positive, EHEC plasmid-positive isolates from both man and cattle. The second lineage was represented by an O111:H2 epidemic strain, isolated during an outbreak of haemolytic uraemic syndrome in France and exhibiting an unusual combination of virulence factors: VT production and aggregative adhesion to HEp-2 cells associated with an enteroaggregative E. coli (EAEC) plasmid.
- Published
- 1999
- Full Text
- View/download PDF
15. Comparative cytotoxicity of purified Shiga-like toxin-IIe on porcine and bovine aortic endothelial and human colonic adenocarcinoma cells.
- Author
-
Valdivieso-Garcia A, MacLeod DL, Clarke RC, Gyles CL, Lingwood C, Boyd B, and Durette A
- Subjects
- Adenocarcinoma, Animals, Aorta, Cattle, Cell Line, Cell Survival drug effects, Cells, Cultured, Chlorocebus aethiops, Chromatography, High Pressure Liquid, Colonic Neoplasms, Escherichia coli, Flow Cytometry, Globosides analysis, Humans, Receptors, Cell Surface analysis, Shiga Toxin 2, Swine, Trihexosylceramides analysis, Tumor Cells, Cultured, Vero Cells, Bacterial Toxins toxicity, Cytotoxins toxicity, Endothelium, Vascular drug effects
- Abstract
Porcine and bovine aortic endothelial cells and human colonic adenocarcinoma cells were compared for their susceptibility to the toxic effect of purified Shiga-like toxin IIe (SLT-IIe), measured by the neutral red cytotoxicity assay. Cytotoxicity correlated with toxin binding as indicated by fluorescence activated cell sorter analysis and with the globotriosylceramide (Gb3) and globotetraosylceramide (Gb4) content of cells determined by high pressure liquid chromatography. One line of porcine aortic endothelial cells was 1400-fold more susceptible than the line of bovine aortic endothelial cells that was tested, but a second line of porcine aortic endothelial cells was highly refractory to SLT-IIe. Human colonic adenocarcinoma cells lacked detectable levels of Gb4 and were least susceptible to SLT-IIe.
- Published
- 1996
- Full Text
- View/download PDF
16. Pheno-genotyping of verotoxin 2 (VT2)-producing Escherichia coli causing haemorrhagic colitis and haemolytic uraemic syndrome by direct analysis of patients' stools.
- Author
-
Caprioli A, Luzzi I, Gianviti A, Russmann H, and Karch H
- Subjects
- Animals, Bacterial Toxins analysis, Bacterial Toxins genetics, Child, Chlorocebus aethiops, DNA, Bacterial analysis, Escherichia coli genetics, Escherichia coli metabolism, Feces chemistry, Feces microbiology, Genotype, Germany, HeLa Cells, Humans, Italy, Neutralization Tests, Phenotype, Polymerase Chain Reaction, Polymorphism, Restriction Fragment Length, Serotyping, Shiga Toxin 2, Vero Cells, Bacterial Toxins biosynthesis, Colitis, Ulcerative microbiology, Escherichia coli classification, Escherichia coli Infections microbiology, Hemolytic-Uremic Syndrome microbiology
- Abstract
The subtype of verotoxin 2 (VT2) found in 22 VT2-positive stool samples from severely diseased Italian and German children with haemorrhagic colitis or haemolytic uraemic syndrome, or both, and that produced by the corresponding VT-producing Escherichia coli (VTEC) strains isolated from the stools were studied by cytotoxicity seroneutralisation assays and by polymerase chain reaction (PCR) amplification of the VT2 B-subunit gene, followed by restriction fragment length polymorphism (RFLP) analysis. The free faecal toxin was serotyped as the classical VT2 in 21 stool samples, and as the VT2 variant VT2c in one. For all but one of the VTEC isolates, the toxin phenotype was consistent with the type of VT produced in vivo and found in the corresponding stool samples. Genotyping was in agreement with phenotyping for those strains harbouring a single type of VT2 gene. Three O157:H7 isolates carrying both VT2 and VT2c genes had the VT2 phenotype, instead of the expected VT2c phenotype. Direct PCR analysis of stools detected VT genes in only 11 of 20 VT-positive stool samples suggesting that the Vero cell cytotoxicity assay is more sensitive in diagnosing VTEC infection. Immunological and genetic subtyping of VT2 performed directly on stool samples from patients with haemolytic uraemic syndrome could be a useful complementary approach to understanding the role of the different types of VT in this syndrome.
- Published
- 1995
- Full Text
- View/download PDF
17. Genotyping of Shiga-like toxin genes in non-O157 Escherichia coli strains associated with haemolytic uraemic syndrome.
- Author
-
Rüssmann H, Kothe E, Schmidt H, Franke S, Harmsen D, Caprioli A, and Karch H
- Subjects
- Base Sequence, Blotting, Southern, Cytotoxins genetics, DNA Primers chemistry, Enterotoxins genetics, Escherichia coli classification, Genes, Bacterial, Genotype, Germany, Humans, Italy, Molecular Sequence Data, Neutralization Tests, Polymerase Chain Reaction, Polymorphism, Restriction Fragment Length, Sequence Analysis, Serotyping, Shiga Toxin 1, Shiga Toxin 2, Bacterial Toxins genetics, Escherichia coli genetics, Escherichia coli Infections microbiology, Hemolytic-Uremic Syndrome microbiology
- Abstract
The pheno- and genotypes of Shiga-like toxins (SLTs) in non-O157 Escherichia coli strains from patients with haemolytic uraemic syndrome were determined. The clinical isolates investigated were from Italy and Germany and belonged to serotypes O22:H8, O26:H-, O26:H11, O91:H-, O111:H- and O128:H-; one isolate was non-typable. SLT genotypes were analysed by complete nucleotide sequence analysis of the B-subunit genes. The results showed that 14 strains possessed slt-I alone, two contained slt-II alone and five isolates harboured both slt-I and slt-II genes. In only two strains were slt-II-related genes found, together with either slt-I or slt-II. These findings indicate that variants of SLT-II are rarely found in non-O157 E. coli isolates from patients with haemolytic uraemic syndrome. Polymerase chain reaction (PCR) with Taq cycle sequencing was found to be a suitable method for classification of slt genotypes.
- Published
- 1995
- Full Text
- View/download PDF
18. Variants of Shiga-like toxin II constitute a major toxin component in Escherichia coli O157 strains from patients with haemolytic uraemic syndrome.
- Author
-
Rüssmann H, Schmidt H, Heesemann J, Caprioli A, and Karch H
- Subjects
- Animals, Bacterial Toxins genetics, Base Sequence, Blotting, Southern, Child, DNA Primers chemistry, Enterotoxins genetics, Enzyme-Linked Immunosorbent Assay, Escherichia coli genetics, Escherichia coli Infections complications, Gene Expression Regulation, Bacterial, Genotype, Hemolytic-Uremic Syndrome etiology, Humans, Molecular Sequence Data, Phenotype, Polymerase Chain Reaction, Polymorphism, Restriction Fragment Length, Shiga Toxin 2, Vero Cells, Bacterial Toxins biosynthesis, Enterotoxins biosynthesis, Escherichia coli metabolism, Escherichia coli Infections microbiology, Hemolytic-Uremic Syndrome microbiology
- Abstract
The prevalence and genotype of Shiga-like toxins (SLTs) in Escherichia coli (O)157 strains from patients in Germany with haemolytic uraemic syndrome (HUS) were investigated. This was done by PCR amplification of the B-subunit genes with two primer pairs--one complementary to slt-IB, and the other homologous to both slt-IIB and slt-IIvB sequences. To distinguish between slt-II and slt-IIv, the amplified DNA was digested with restriction endonucleases HaeIII and FokI. Of the 38 strains examined, 17 harboured sequences for slt-IIv; four contained only slt-IIv, three carried both slt-IIv and slt-I, and 10 strains had slt-IIv and slt-II. A further three genotypes (slt-I, slt-II, slt-I/slt-II) were found in the remaining 21 strains resulting in a total of six slt genotypes. To determine whether the slt genes were expressed, and whether genotypes correlated with phenotypes, all strains were subjected to cytotoxicity assays and colony ELISA. All 38 strains displayed cytotoxic activity to Vero cells in similar quantities. The SLT-I-specific monoclonal antibody (MAb)13C4 reacted with all 10 strains in which slt-I sequences were identified. Colony blot ELISA with the SLT-II specific MAb11E10 detected 27 of 28 strains with slt-II sequences, but did not react with any of the seven strains that carried slt-IIv, or slt-I and slt-IIv. The high SLT variability shown here has diagnostic implications and may well have consequences for the host response in infections associated with these pathogens.
- Published
- 1994
- Full Text
- View/download PDF
19. Diagnosis of infections with Shiga-like toxin-producing Escherichia coli by use of enzyme-linked immunosorbent assays for Shiga-like toxins on cultured stool samples.
- Author
-
Law D, Hamour AA, Acheson DW, Panigrahi H, Ganguli LA, and Denning DW
- Subjects
- Bacterial Toxins biosynthesis, Colitis diagnosis, Colitis microbiology, Diarrhea diagnosis, Diarrhea microbiology, Enzyme-Linked Immunosorbent Assay, Escherichia coli metabolism, Escherichia coli Infections microbiology, Feces chemistry, Gastrointestinal Hemorrhage diagnosis, Gastrointestinal Hemorrhage microbiology, Hemolytic-Uremic Syndrome diagnosis, Hemolytic-Uremic Syndrome microbiology, Humans, Prospective Studies, Sensitivity and Specificity, Shiga Toxin 1, Shiga Toxin 2, Bacterial Toxins analysis, Escherichia coli isolation & purification, Escherichia coli Infections diagnosis, Feces microbiology
- Abstract
Shiga-like toxin-producing (SLT) Escherichia coli, particularly those belonging to serogroup O157, are responsible for haemorrhagic colitis, haemolytic uraemic syndrome and some cases of gastro-enteritis. The rapid and reliable diagnosis of all these infections is necessary for correct patient management and for epidemiological reasons, but is rarely possible with present methods. We compared the efficacy of two methods, (i) the culture of faeces in broth that contained mitomycin C followed by enzyme-linked immunosorbent assay (ELISA) for SLTs, and (ii) the culture of faeces on sorbitol MacConkey agar (SMA), in the detection of infections caused by SLT-producing E. coli. SLT-producing E. coli O157 strains were isolated on SMA from 42 of 475 faecal samples, but SLTs were detected by ELISA in culture supernates or lysates of 54 of 475 samples. SLT-producing E. coli strains were isolated subsequently from 11 of 12 ELISA-positive, SMA culture-negative samples by a colony blot technique. In four cases, SLT-producing E. coli of serogroups other than O157 were isolated and in seven cases E. coli O157 was isolated in small numbers. The ELISA is a rapid and sensitive technique for the diagnosis of SLT-producing E. coli infection, especially where low numbers of the organism are present in faeces and when the infection is caused by a serogroup other than O157.
- Published
- 1994
- Full Text
- View/download PDF
20. Detection by ELISA of low numbers of Shiga-like toxin-producing Escherichia coli in mixed cultures after growth in the presence of mitomycin C.
- Author
-
Law D, Ganguli LA, Donohue-Rolfe A, and Acheson DW
- Subjects
- Bacterial Toxins analysis, Enterococcus faecalis growth & development, Enzyme-Linked Immunosorbent Assay, Escherichia coli drug effects, Escherichia coli metabolism, Escherichia coli Infections diagnosis, Feces microbiology, Humans, Predictive Value of Tests, Proteus mirabilis growth & development, Shiga Toxin 1, Shiga Toxin 2, Bacterial Toxins biosynthesis, Enterotoxins biosynthesis, Escherichia coli growth & development, Mitomycin pharmacology
- Abstract
Techniques currently available to detect Shiga-like toxin (SLT)-producing Escherichia coli lack sensitivity or require specialised equipment and facilities, and in some cases detect only strains belonging to serotype O157. We have used an ELISA technique, capable of detecting both SLTI and SLTII with crude P1 glycoprotein from hydatid cysts, in combination with enhancement of toxin production by culture with mitomycin C. Supernates of Tryptone Soya Broth cultures containing mitomycin C 200 ng/ml were tested for SLTII. For SLTI, cell lysates pre-treated with polymyxin B were tested. In tests with E. coli O157:H7 in mixed culture with E. coli strain C600 alone, or with E. coli C600, Proteus mirabilis and Enterococcus faecalis, SLTI could be detected when the proportion of toxigenic organisms represented 1% of the mixture, and SLTII when the proportion was 0.025%. When faecal samples with added E. coli O157:H7 were examined in this system, SLTII-producing strains were detected when they comprised less than 0.1% of the coliform population. This technique is a sensitive and specific assay for detecting low numbers of SLT-producing organisms in mixed culture such as occurs in cases of haemolytic uraemic syndrome and haemorrhagic colitis.
- Published
- 1992
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.