scholarly journals AnIn VitroCombined Antibiotic-Antibody Treatment Eliminates Toxicity from Shiga Toxin-Producing Escherichia coli

2015 ◽  
Vol 59 (9) ◽  
pp. 5435-5444 ◽  
Author(s):  
Craig Skinner ◽  
Guodong Zhang ◽  
Stephanie Patfield ◽  
Xiaohua He
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Shiga Toxin ◽  
Neutralizing Antibodies ◽  
Vero Cells ◽  
Antibody Treatment ◽  
Gastrointestinal Infections ◽  
Content Type ◽  
Life Threatening ◽  
Uremic Syndrome

ABSTRACTTreating Shiga toxin-producingEscherichia coli(STEC) gastrointestinal infections is difficult. The utility of antibiotics for STEC treatment is controversial, since antibiotic resistance among STEC isolates is widespread and certain antibiotics dramatically increase the expression of Shiga toxins (Stxs), which are some of the most important virulence factors in STEC. Stxs contribute to life-threatening hemolytic uremic syndrome (HUS), which develops in considerable proportions of patients with STEC infections. Understanding the antibiotic resistance profiles of STEC isolates and the Stx induction potential of promising antibiotics is essential for evaluating any antibiotic treatment of STEC. In this study, 42 O157:H7 or non-O157 STEC isolates (including the “big six” serotypes) were evaluated for their resistance against 22 antibiotics by using an antibiotic array. Tigecycline inhibited the growth of all of the tested STEC isolates and also inhibited the production of Stxs (Stx2 in particular). In combination with neutralizing antibodies to Stx1 and Stx2, the tigecycline-antibody treatment fully protected Vero cells from Stx toxicity, even when the STEC bacteria and the Vero cells were cultured together. The combination of an antibiotic such as tigecycline with neutralizing antibodies presents a promising strategy for future STEC treatments.

scholarly journals A Novel Tail-Associated O91-Specific Polysaccharide Depolymerase from a Podophage Reveals Lytic Efficacy of Shiga Toxin-Producing Escherichia coli

2020 ◽  
Vol 86 (9) ◽  
Author(s):  
Yibao Chen ◽  
Xiangmin Li ◽  
Shuang Wang ◽  
Lingyu Guan ◽  
Xinxin Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hemolytic Uremic Syndrome ◽  
Proinflammatory Cytokines ◽  
Shiga Toxin ◽  
Hemorrhagic Colitis ◽  
Content Type ◽  
Increased Risk ◽  
Specific Polysaccharide ◽  
Life Threatening ◽  
Uremic Syndrome

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) strains are important zoonotic foodborne pathogens, causing diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome (HUS) in humans. However, antibiotic treatment of STEC infection is associated with an increased risk of HUS. Therefore, there is an urgent need for early and effective therapeutic strategies. Here, we isolated lytic T7-like STEC phage PHB19 and identified a novel O91-specific polysaccharide depolymerase (Dep6) in the C terminus of the PHB19 tailspike protein. Dep6 exhibited strong hydrolase activity across wide ranges of pH (pH 4 to 8) and temperature (20 to 60°C) and degraded polysaccharides on the surface of STEC strain HB10. In addition, both Dep6 and PHB19 degraded biofilms formed by STEC strain HB10. In a mouse STEC infection model, delayed Dep6 treatment (3 h postinfection) resulted in only 33% survival, compared with 83% survival when mice were treated simultaneously with infection. In comparison, pretreatment with Dep6 led to 100% survival compared with that of the control group. Surprisingly, a single PHB19 treatment resulted in 100% survival in all three treatment protocols. Moreover, a significant reduction in the levels of proinflammatory cytokines was observed at 24 h postinfection in Dep6- or PHB19-treated mice. These results demonstrated that Dep6 or PHB19 might be used as a potential therapeutic agent to prevent STEC infection. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen worldwide. The Shiga-like toxin causes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome (HUS) in humans. Although antibiotic therapy is still used for STEC infections, this approach may increase the risk of HUS. Phages or phage-derived depolymerases have been used to treat bacterial infections in animals and humans, as in the case of the “San Diego patient” treated with a phage cocktail. Here, we showed that phage PHB19 and its O91-specific polysaccharide depolymerase Dep6 degraded STEC biofilms and stripped the lipopolysaccharide (LPS) from STEC strain HB10, which was subsequently killed by serum complement in vitro. In a mouse model, PHB19 and Dep6 protected against STEC infection and caused a significant reduction in the levels of proinflammatory cytokines. This study reports the use of an O91-specific polysaccharide depolymerase for the treatment of STEC infection in mice.

scholarly journals Cytotoxic and Apoptotic Effects of Recombinant Subtilase Cytotoxin Variants of Shiga Toxin-Producing Escherichia coli

2015 ◽  
Vol 83 (6) ◽  
pp. 2338-2349 ◽  
Author(s):  
J. Funk ◽  
N. Biber ◽  
M. Schneider ◽  
E. Hauser ◽  
S. Enzenmüller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Vero Cells ◽  
Toxic Activity ◽  
Content Type ◽  
B Subunits ◽  
Induced Apoptosis ◽  
The Individual ◽  
Apoptotic Effects ◽  
Subtilase Cytotoxin

In this study, the cytotoxicity of the recently described subtilase variant SubAB2-2of Shiga toxin-producingEscherichia coliwas determined and compared to the plasmid-encoded SubAB1and the chromosome-encoded SubAB2-1variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Their cytotoxicity on Vero cells was measured for the single A and B subunits, as well as for mixtures of both, to analyze whether hybrids with toxic activity can be identified. The results demonstrated that all three SubAB variants are toxic for Vero cells. However, the values for the 50% cytotoxic dose (CD50) differ for the individual variants. Highest cytotoxicity was shown for SubAB1. Moreover, hybrids of subunits from different subtilase toxins can be obtained which cause substantial cytotoxicity to Vero cells after mixing the A and B subunits prior to application to the cells, which is characteristic for binary toxins. Furthermore, higher concentrations of the enzymatic subunit SubA1exhibited cytotoxic effects in the absence of the respective B1subunit. A more detailed investigation in the human HeLa cell line revealed that SubA1alone induced apoptosis, while the B1subunit alone did not induce cell death.

scholarly journals Inhibition of Water Absorption and Selective Damage to Human Colonic Mucosa Are Induced by Subtilase Cytotoxin Produced by Escherichia coli O113:H21

2013 ◽  
Vol 81 (8) ◽  
pp. 2931-2937 ◽  
Author(s):  
Elizabeth Gerhardt ◽  
Mariana Masso ◽  
Adrienne W. Paton ◽  
James C. Paton ◽  
Elsa Zotta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Water Absorption ◽  
Shiga Toxin ◽  
Human Colon ◽  
Surface Epithelium ◽  
Inflammatory Infiltration ◽  
Content Type ◽  
Uremic Syndrome ◽  
Normal Water ◽  
Subtilase Cytotoxin

ABSTRACTShiga toxin-producingEscherichia coliO157:H7 (STEC) is by far the most prevalent serotype associated with hemolytic uremic syndrome (HUS) although many non-O157 STEC strains have been also isolated from patients with HUS. The main virulence factor of STEC is the Shiga toxin type 2 (Stx2) present in O157 and non-O157 strains. Recently, another toxin, named subtilase cytotoxin (SubAB), has been isolated from several non-O157 strains and may contribute to the pathogenesis of HUS. Here, we have demonstrated that an O113:H21 STEC strain expressing SubAB and Stx2 inhibits normal water absorption across human colon and causes damage to the surface epithelium, necrosis, mononuclear inflammatory infiltration, edema, and marked mucin depletion. This damage was less marked, but nevertheless significant, when purified SubAB orE. coliO113:H21 expressing only SubAB was assayed. This is the first study showing that SubAB may directly participate in the mechanisms of diarrhea in children infected with non-O157 STEC strains.

scholarly journals Shiga Toxin-Producing Serogroup O91 Escherichia coli Strains Isolated from Food and Environmental Samples

2017 ◽  
Vol 83 (18) ◽  
Author(s):  
Peter C. H. Feng ◽  
Sabine Delannoy ◽  
David W. Lacher ◽  
Joseph M. Bosilevac ◽  
Patrick Fach ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hemolytic Uremic Syndrome ◽  
Shiga Toxin ◽  
Severe Illness ◽  
Content Type ◽  
Virulence Potential ◽  
Short Palindromic Repeat ◽  
Uremic Syndrome ◽  
Severe Infections ◽  
Genetic Profiles

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) strains of the O91:H21 serotype have caused severe infections, including hemolytic-uremic syndrome. Strains of the O91 serogroup have been isolated from food, animals, and the environment worldwide but are not well characterized. We used a microarray and other molecular assays to examine 49 serogroup O91 strains (environmental, food, and clinical strains) for their virulence potential and phylogenetic relationships. Most of the isolates were identified to be strains of the O91:H21 and O91:H14 serotypes, with a few O91:H10 strains and one O91:H9 strain being identified. None of the strains had the eae gene, which codes for the intimin adherence protein, and many did not have some of the genetic markers that are common in other STEC strains. The genetic profiles of the strains within each serotype were similar but differed greatly between strains of different serotypes. The genetic profiles of the O91:H21 strains that we tested were identical or nearly identical to those of the clinical O91:H21 strains that have caused severe diseases. Multilocus sequence typing and clustered regularly interspaced short palindromic repeat analyses showed that the O91:H21 strains clustered within the STEC 1 clonal group but the other O91 serotype strains were phylogenetically diverse. IMPORTANCE This study showed that food and environmental O91:H21 strains have similar genotypic profiles and Shiga toxin subtypes and are phylogenetically related to the O91:H21 strains that have caused hemolytic-uremic syndrome, suggesting that these strains may also have the potential to cause severe illness.

scholarly journals Effects of Antibiotics on Shiga Toxin 2 Production and Bacteriophage Induction by Epidemic Escherichia coli O104:H4 Strain

2012 ◽  
Vol 56 (6) ◽  
pp. 3277-3282 ◽  
Author(s):  
Martina Bielaszewska ◽  
Evgeny A. Idelevich ◽  
Wenlan Zhang ◽  
Andreas Bauwens ◽  
Frieder Schaumburg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Therapy ◽  
Shiga Toxin ◽  
The Other ◽  
Emerging Pathogen ◽  
Outbreak Strain ◽  
Content Type ◽  
Shiga Toxin 2 ◽  
Uremic Syndrome

ABSTRACTThe role of antibiotics in treatment of enterohemorrhagicEscherichia coli(EHEC) infections is controversial because of concerns about triggering hemolytic-uremic syndrome (HUS) by increasing Shiga toxin (Stx) production. During the recent large EHEC O104:H4 outbreak, antibiotic therapy was indicated for some patients. We tested a diverse panel of antibiotics to which the outbreak strain is susceptible to interrogate the effects of subinhibitory antibiotic concentrations on induction ofstx2-harboring bacteriophages,stx2transcription, and Stx2 production in this emerging pathogen. Ciprofloxacin significantly increasedstx2-harboring phage induction and Stx2 production in outbreak isolates (Pvalues of <0.001 to <0.05), while fosfomycin, gentamicin, and kanamycin insignificantly influenced them (P> 0.1) and chloramphenicol, meropenem, azithromycin, rifaximin, and tigecycline significantly decreased them (P≤ 0.05). Ciprofloxacin and chloramphenicol significantly upregulated and downregulatedstx2transcription, respectively (P< 0.01); the other antibiotics had insignificant effects (P> 0.1). Meropenem, azithromycin, and rifaximin, which were used for necessary therapeutic or prophylactic interventions during the EHEC O104:H4 outbreak, as well as tigecycline, neither inducedstx2-harboring phages nor increasedstx2transcription or Stx2 production in the outbreak strain. These antibiotics might represent therapeutic options for patients with EHEC O104:H4 infection if antibiotic treatment is inevitable. We await further analysis of the epidemic to determine if usage of these agents was associated with an altered risk of developing HUS.

scholarly journals Enhanced Virulence of the Escherichia coli O157:H7 Spinach-Associated Outbreak Strain in Two Animal Models Is Associated with Higher Levels of Stx2 Production after Induction with Ciprofloxacin

2014 ◽  
Vol 82 (12) ◽  
pp. 4968-4977 ◽  
Author(s):  
T. Zangari ◽  
A. R. Melton-Celsa ◽  
A. Panda ◽  
M. A. Smith ◽  
I. Tatarov ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Animal Models ◽  
Shiga Toxin ◽  
Hemorrhagic Colitis ◽  
Escherichia Coli O157 ◽  
Outbreak Strain ◽  
Content Type ◽  
Intestinal Pathology ◽  
Phage Dna ◽  
Uremic Syndrome

ABSTRACTShiga toxin (Stx)-producingEscherichia coli(STEC) causes hemorrhagic colitis and the hemolytic-uremic syndrome (HUS). STEC strains may produce Stx1a and/or Stx2a or variants of either toxin. A 2006 spinach-associated outbreak of STEC O157:H7 resulted in higher hospitalization and HUS rates than previous STEC outbreaks. The spinach isolate, strain K3995, contains bothstx2aandstx2c. We hypothesized that the enhanced virulence of K3995 reflects the combination ofstx2alleles (carried on lysogenic phages) and/or the amount of Stx2 made by that strain. We compared the virulence of K3995 to those of other O157:H7 isolates and an isogenic Stx2 mutant in rabbits and mice. We also measured the relative levels of Stx2 produced from those strains with or without induction of thestx-carrying phage. Some rabbits infected with K3995 exhibited intestinal pathology and succumbed to infection, while none of those infected with O157:H7 strain 2812 (Stx1a+Stx2a+) died or showed pathological signs. Rabbits infected with the isogenic Stx2a mutant K3995stx2a::catwere not colonized as well as those infected with K3995 and exhibited no signs of disease. In the streptomycin-treated mouse model, more animals infected with K3995 died than did those infected with O157:H7 strain 86-24 (Stx2a+). Additionally, K3995 produced higher levels of total Stx2 and toxin phage DNA in cultures after phage induction than did 86-24. Our results demonstrate the greater virulence of K3995 compared to other O157:H7 strains in rabbits and mice. We conclude that this enhanced virulence is linked to higher levels of Stx2 expression as a consequence of increased phage induction.

Prevalence and Characterization of Shiga Toxin-Producing Escherichia coli Isolated from Cattle, Food, and Children during a One-Year Prospective Study in France

2000 ◽  
Vol 38 (3) ◽  
pp. 1023-1031 ◽  
Author(s):  
Nathalie Pradel ◽  
Valérie Livrelli ◽  
Christophe De Champs ◽  
Jean-Bernard Palcoux ◽  
Alain Reynaud ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Diarrheal Disease ◽  
Geographical Area ◽  
Vero Cells ◽  
Healthy Cattle ◽  
Uremic Syndrome ◽  
Stool Specimens ◽  
One Year

During a 1-year survey of Shiga toxin-producing Escherichia coli (STEC) prevalence in central France, 2,143 samples were investigated by PCR for Shiga toxin-encoding genes. A total of 330 (70%) of 471 fecal samples collected from healthy cattle at the Clermont-Ferrand slaughterhouse, 47 (11%) of 411 beef samples, 60 (10%) of 603 cheese samples, and 19 (3%) of 658 stool specimens from hospitalized children with and without diarrhea were positive for thestx gene(s). A STEC strain was isolated from 34% (162 of 471) of bovine feces, 4% (16 of 411) of beef samples, 1% (5 of 603) of cheese samples, and 1.5% (10 of 658) of stool specimens. Of the 220 STEC strains isolated, 34 (15%) harbored thestx 1 gene, 116 (53%) harbored thestx 2 gene, and 70 (32%) carried both thestx 1 and stx 2 genes. However, 32 (14.5%) were not cytotoxic for Vero cells. Theeae gene, found in 12 (5%) of the 220 strains, was significantly associated with the stx 1 gene and with isolates from children. Sequences homologous to ehxAwere found in 102 (46%) of the 220 strains. Thirteen serotypes, OX3:H2, O113:H21, O113:H4, OX3:H21, O6:H10, OX178:H19, O171:H2, O46:H38, O172:H21, O22:H16, O91:H10, O91:H21, and O22:H8, accounted for 102 (55%) of 186 typeable isolates, and only one strain (0.5% of the 186 STEC isolates from cattle), belonged to the O157:H7 serotype. We showed that the majority of the STEC isolates from cattle, beef, and cheese were not likely to be pathogenic for humans and that the STEC strains isolated from children in this study were probably not responsible for diarrheal disease. Finally, the strains associated with hemolytic-uremic syndrome in the same geographical area were shown to belong to particular subsets of the STEC population found in the bovine reservoir.

scholarly journals AB5Preassembly Is Not Required for Shiga Toxin Activity

2016 ◽  
Vol 198 (11) ◽  
pp. 1621-1630 ◽  
Author(s):  
Christine A. Pellino ◽  
Sayali S. Karve ◽  
Suman Pradhan ◽  
Alison A. Weiss
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Hemolytic Uremic Syndrome ◽  
Shiga Toxin ◽  
Target Cells ◽  
Content Type ◽  
B Subunit ◽  
Uremic Syndrome ◽  
A Subunit ◽  
B Subunits

ABSTRACTShiga toxin (Stx)-producingEscherichia coli(STEC) is a major cause of foodborne illness, including the life-threatening complication hemolytic-uremic syndrome. The German outbreak in 2011 resulted in nearly 4,000 cases of infection, with 54 deaths. Two forms of Stx, Stx1 and Stx2, differ in potency, and subtype Stx2a is most commonly associated with fatal human disease. Stx is considered to be an AB5toxin. The single A (enzymatically active) subunit inhibits protein synthesis by cleaving a catalytic adenine from the eukaryotic rRNA. The B (binding) subunit forms a homopentamer and mediates cellular association and toxin internalization by binding to the glycolipid globotriaosylceramide (Gb3). Both subunits are essential for toxicity. Here we report that unlike other AB5toxin family members, Stx is produced by STEC as unassembled A and B subunits. A preformed AB5complex is not required for cellular toxicity orin vivotoxicity to mice, and toxin assembly likely occurs at the cell membrane. We demonstrate that disruption of A- and B-subunit association by use of A-subunit peptides that lack enzymatic activity can protect mice from lethal doses of toxin. Currently, no treatments have been proven to be effective for hemolytic-uremic syndrome. Our studies demonstrate that agents that interfere with A- and B-subunit assembly may have therapeutic potential. Shiga toxin (Stx) produced by pathogenicEscherichia coliis considered to be an AB5heterohexamer; however, no known mechanisms ensure AB5assembly. Stx released byE. coliis not in the AB5conformation and assembles at the receptor interface. Thus, unassembled Stx can impart toxicity. This finding shows that preventing AB5assembly is a potential treatment for Stx-associated illnesses.IMPORTANCEComplications due to Shiga toxin are frequently fatal, and at present, supportive care is the only treatment option. Furthermore, antibiotic treatment is contraindicated due to the ability of antibiotics to amplify bacterial expression of Shiga toxin. We report, contrary to prevailing assumptions, that Shiga toxin produced by STEC circulates as unassembled A and B subunits at concentrations that are lethal to mice. Similar to the case for anthrax toxin, assembly occurs on receptors expressed on the surfaces of mammalian target cells. Disruption of Shiga toxin assembly by use of A-subunit peptides that lack enzymatic activity protects mice from lethal challenge with Shiga toxin, suggesting a new approach for development of therapeutics.

scholarly journals Retinoid Levels Influence Enterohemorrhagic Escherichia coli Infection and Shiga Toxin 2 Susceptibility in Mice

2014 ◽  
Vol 82 (9) ◽  
pp. 3948-3957 ◽  
Author(s):  
Gabriel Cabrera ◽  
Romina J. Fernández-Brando ◽  
María Jimena Abrey-Recalde ◽  
Ariela Baschkier ◽  
Alipio Pinto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Lethal Dose ◽  
Absolute Number ◽  
Enterohemorrhagic Escherichia Coli ◽  
Polymorphonuclear Cells ◽  
Intestinal Damage ◽  
Gastrointestinal Infections ◽  
Systemic Complications ◽  
Content Type

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) is a food-borne pathogen that produces Shiga toxin (Stx) and causes hemorrhagic colitis. Under some circumstances, Stx produced within the intestinal tract enters the bloodstream, leading to systemic complications that may cause the potentially fatal hemolytic-uremic syndrome. Although retinoids like vitamin A (VA) and retinoic acid (RA) are beneficial to gut integrity and the immune system, the effect of VA supplementation on gastrointestinal infections of different etiologies has been controversial. Thus, the aim of this work was to study the influence of different VA status on the outcome of an EHEC intestinal infection in mice. We report that VA deficiency worsened the intestinal damage during EHEC infection but simultaneously improved survival. Since death is associated mainly with Stx toxicity, Stx was intravenously inoculated to analyze whether retinoid levels affect Stx susceptibility. Interestingly, while VA-deficient (VA-D) mice were resistant to a lethal dose of Stx2, RA-supplemented mice were more susceptible to it. Given that peripheral blood polymorphonuclear cells (PMNs) are known to potentiate Stx2 toxicity, we studied the influence of retinoid levels on the absolute number and function of PMNs. We found that VA-D mice had decreased PMN numbers and a diminished capacity to produce reactive oxygen species, while RA supplementation had the opposite effect. These results are in line with the well-known function of retinoids in maintaining the homeostasis of the gut but support the idea that they have a proinflammatory effect by acting, in part, on the PMN population.

scholarly journals Genetic Diversity and Virulence Potential of Shiga Toxin-Producing Escherichia coli O113:H21 Strains Isolated from Clinical, Environmental, and Food Sources

2014 ◽  
Vol 80 (15) ◽  
pp. 4757-4763 ◽  
Author(s):  
Peter C. H. Feng ◽  
Sabine Delannoy ◽  
David W. Lacher ◽  
Luis Fernando dos Santos ◽  
Lothar Beutin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Diversity ◽  
Shiga Toxin ◽  
Food Sources ◽  
Content Type ◽  
Virulence Potential ◽  
Uremic Syndrome ◽  
Enterocyte Effacement ◽  
Human Infections ◽  
Pcr Microarray

ABSTRACTShiga toxin-producingEscherichia colistrains of serotype O113:H21 have caused severe human diseases, but they are unusual in that they do not produce adherence factors coded by the locus of enterocyte effacement. Here, a PCR microarray was used to characterize 65 O113:H21 strains isolated from the environment, food, and clinical infections from various countries. In comparison to the pathogenic strains that were implicated in hemolytic-uremic syndrome in Australia, there were no clear differences between the pathogens and the environmental strains with respect to the 41 genetic markers tested. Furthermore, all of the strains carried only Shiga toxin subtypes associated with human infections, suggesting that the environmental strains have the potential to cause disease. Most of the O113:H21 strains were closely related and belonged in the same clonal group (ST-223), but CRISPR analysis showed a great degree of genetic diversity among the O113:H21 strains.

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