Angela Melton-CElsa | Uniformed Services University (original) (raw)
Papers by Angela Melton-CElsa
ASM Press eBooks, May 26, 2015
Infection and Immunity, Mar 1, 2014
Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause food-borne outbreaks of hemorrh... more Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause food-borne outbreaks of hemorrhagic colitis and, less commonly, a serious kidney-damaging sequela called the hemolytic uremic syndrome (HUS). Stx, the primary virulence factor expressed by STEC, is an AB 5 toxin with two antigenically distinct forms, Stx1a and Stx2a. Although both toxins have similar biological activities, Stx2a is more frequently produced by STEC strains that cause HUS than is Stx1a. Here we asked whether Stx1a and Stx2a act differently when delivered orally by gavage. We found that Stx2a had a 50% lethal dose (LD 50) of 2.9 g, but no morbidity occurred after oral intoxication with up to 157 g of Stx1a. We also compared several biochemical and histological parameters in mice intoxicated orally versus intraperitoneally with Stx2a. We discovered that both intoxication routes caused similar increases in serum creatinine and blood urea nitrogen, indicative of kidney damage, as well as electrolyte imbalances and weight loss in the animals. Furthermore, kidney sections from Stx2a-intoxicated mice revealed multifocal, acute tubular necrosis (ATN). Of particular note, we detected Stx2a in kidney sections from orally intoxicated mice in the same region as the epithelial cell type in which ATN was detected. Lastly, we showed reduced renal damage, as determined by renal biomarkers and histopathology, and full protection of orally intoxicated mice with monoclonal antibody (MAb) 11E10 directed against the toxin A subunit; conversely, an irrelevant MAb had no therapeutic effect. Orally intoxicated mice could be rescued by MAb 11E10 6 h but not 24 h after Stx2a delivery.
The Journal of Infectious Diseases, Sep 1, 2003
Financial support: Centers for Disease Control and Prevention (cooperative agreement U50/CCU81440... more Financial support: Centers for Disease Control and Prevention (cooperative agreement U50/CCU814408, to fund laboratory surveillance for non-O157:H7 Shiga toxin-producing Escherichia coli); National Institutes of Health (grants AI-47499, to fund analysis of the isolates, and AI-20148, to fund work on stx 2d-activatable); Bundesministerium für Bildung und Forschung Verbundprojekt (Forschungsnetzwerk 01KI9903).
ASM Press eBooks, May 26, 2015
Shiga toxin (Stx) is one of the most potent bacterial toxins known. Stx is found in Shigella dyse... more Shiga toxin (Stx) is one of the most potent bacterial toxins known. Stx is found in Shigella dysenteriae 1 and in some serogroups of Escherichia coli (called Stx1 in E. coli). In addition to or instead of Stx1, some E. coli strains produce a second type of Stx, Stx2, that has the same mode of action as Stx/Stx1 but is antigenically distinct. Because subtypes of each toxin have been identified, the prototype toxin for each group is now designated Stx1a or Stx2a. The Stxs consist of two major subunits, an A subunit that joins noncovalently to a pentamer of five identical B subunits. The A subunit of the toxin injures the eukaryotic ribosome and halts protein synthesis in target cells. The function of the B pentamer is to bind to the cellular receptor, globotriaosylceramide, Gb3, found primarily on endothelial cells. The Stxs traffic in a retrograde manner within the cell, such that the A subunit of the toxin reaches the cytosol only after the toxin moves from the endosome to the Golgi and then to the endoplasmic reticulum. In humans infected with Stx-producing E. coli, the most serious manifestation of the disease, hemolytic-uremic syndrome, is more often associated with strains that produce Stx2a rather than Stx1a, and that relative toxicity is replicated in mice and baboons. Stx1a and Stx2a also exhibit differences in cytotoxicity to various cell types, bind dissimilarly to receptor analogs or mimics, induce differential chemokine responses, and have several distinctive structural characteristics.
... 34: 10581069. 44. Melton-Celsa, AR, SC Darnell, and AD O'Brien. 1996. Activation of Shi... more ... 34: 10581069. 44. Melton-Celsa, AR, SC Darnell, and AD O'Brien. 1996. Activation of Shiga-like toxins by mouse and human intestinal mucus correlates with virulence of enterohemorrhagic Escherichia coli O91: H21 isolates in orally infected, streptomycin-treated ...
Frontiers in Microbiology, Aug 13, 2019
An O104:H4 Shiga toxin (Stx)-producing enteroaggregative Escherichia coli (EAEC) strain caused a ... more An O104:H4 Shiga toxin (Stx)-producing enteroaggregative Escherichia coli (EAEC) strain caused a large outbreak of bloody diarrhea and the hemolytic uremic syndrome in 2011. We previously developed an ampicillin (Amp)-treated C57BL/6 mouse model to measure morbidity (weight loss) and mortality of mice orally infected with the prototype Stx-EAEC strain C227-11. Here, we hypothesized that mice fed C227-11 cured of the pAA plasmid or deleted for individual genes on that plasmid would display reduced virulence compared to animals given the wild-type (wt) strain. C227-11 cured of the pAA plasmid or deleted for the known pAA-encoded virulence genes aggR, aggA, sepA, or aar were fed to Amp-treated C57BL/6 mice at doses of 10 10-10 11 CFU. Infected animals were then either monitored for morbidity and lethality for 28 days or euthanized to determine intestinal pathology and colonization levels at selected times. The pAAcured, aggR, and aggA mutants of strain C227-11 all showed reduced colonization at various intestinal sites. However, the aggR mutant was the only mutant attenuated for virulence as it showed both reduced morbidity and mortality. The aar mutant showed increased expression of the aggregative adherence fimbriae (AAF) and caused greater systemic effects in infected mice when compared to the C227-11 wt strain. However, unexpectedly, both the aggA and aar mutants displayed increased weight loss compared to wt. The sepA mutant did not exhibit altered morbidity or mortality in the Amp-treated mouse model compared to wt. Our data suggest that the increased morbidity due to the aar mutant could possibly be via an effect on expression of an as yet unknown virulence-associated factor under AggR control.
Emerging Infectious Diseases, Oct 1, 2014
ASM Press eBooks, Apr 9, 2014
Shiga toxin-producing Escherichia coli (STEC) serotype O157:H7 is one of the most important human... more Shiga toxin-producing Escherichia coli (STEC) serotype O157:H7 is one of the most important human pathogenic microorganisms, which can cause life-threatening infections. Xanthium strumarium L. is a plant with anti-bacterial activity against gram-negative and gram-positive bacteria. This study aims to demonstrate in vitro efficacy of the essential oil (EO) extracted from Xanthium strumarium L. against E. coli O157:H7. Using the agar test diffusion, the effect of Xanthium strumarium L. EO (5, 10, 15, 30, 60, and 120 mg/mL) was verified at each of the four different growth phases of E. coli O157:H7. Cell counts of viable cells and colony forming unit (CFU) were determined at regular time points using Breed's method and colony counting method, respectively. No viable cell was detectable after the 1 hour-exposure to X. strumarium EO at 30, 60, and 120 mg/mL concentrations. No bacterial colony was formed after 1 h until the end of the incubation period at 24 h. At lower concentrations, the number of bacteria cells decreased and colonies could be observed only after incubation. At the exponential phase, the EO at 15 mg/mL was only bacteriostatic, while from 30 mg/mL started to be bactericidal. X. strumarium EO antibacterial activity against Shiga toxinproducing E. coli O157:H7 is dependent on EO concentration and physiological state of the microorganisms tested. The best inhibitory activity was achieved during the late exponential and the stationary phases.
Microbiology spectrum, Sep 19, 2014
Shiga toxin (Stx)-producing Escherichia coli (STEC) is an etiologic agent of bloody diarrhea. A s... more Shiga toxin (Stx)-producing Escherichia coli (STEC) is an etiologic agent of bloody diarrhea. A serious sequela of disease, the hemolytic uremic syndrome (HUS) may arise in up to 25% of patients. The development of HUS after STEC infection is linked to the presence of Stx. STEC strains may produce one or more Stxs, and the Stxs come in two major immunological groups, Stx1 and Stx2. A multitude of possible therapeutics designed to inhibit the actions of the Stxs have been developed over the past 30 years. Such therapeutics are important because antibiotic treatment of STEC infections is contraindicated due to an increased potential for development of HUS. The reason for the increased risk of HUS after antibiotic treatment is likely because certain antibiotics induce expression of the Stxs, which are generally associated with lysogenic bacteriophages. There are a few potential therapeutics that either try to kill STEC without inducing Stx expression or target gene expression within STEC. However, the vast majority of the treatments under development are designed to limit Stx receptor generation or to prevent toxin binding, trafficking, processing, or activity within the cell. The potential therapies described in this review include some that have only been tested in vitro and several that demonstrate efficacy in animals. The therapeutics that are currently the furthest along in development (completed phase I and II trials) are monoclonal antibodies directed against Stx1 and Stx2.
Microbiology spectrum, Jun 15, 2023
The release of Stx from STEC has been thought to be tied to phage-mediated lysis of the host bact... more The release of Stx from STEC has been thought to be tied to phage-mediated lysis of the host bacterial cell. In this study, we found that the stx 2a phage lytic genes are not required for the virulence of pathogenic O157:H7 clinical isolates in a murine model of STEC infection or for release of Stx2a into the supernatant of bacterial cultures. These results point to an alternate mechanism for Stx2a release from STEC strains.
The sharing of genome sequences in online data repositories, allows for large scale analyses of s... more 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...
Proceedings of the National Academy of Sciences of the United States of America, May 20, 2013
The likelihood that a single individual infected with the Shiga toxin (Stx)-producing, food-borne... more The likelihood that a single individual infected with the Shiga toxin (Stx)-producing, food-borne pathogen Escherichia coli O157:H7 will develop a life-threatening sequela called the hemolytic uremic syndrome is unpredictable. We reasoned that conditions that enhance Stx binding and uptake within the gut after E. coli O157:H7 infection should result in greater disease severity. Because the receptor for Stx, globotriaosylceramide, is up-regulated in the presence of butyrate in vitro, we asked whether a high fiber diet (HFD) that reportedly enhances butyrate production by normal gut flora can influence the outcome of an E. coli O157 infection in mice. To address that question, groups of BALB/c mice were fed high (10%) or low (2%) fiber diets and infected with E. coli O157:H7 strain 86-24 (Stx2+). Mice fed an HFD exhibited a 10-to 100-fold increase in colonization, lost 15% more body weight, exhibited signs of morbidity, and had 25% greater mortality relative to the low fiber diet (LFD)-fed group. Additionally, sections of intestinal tissue from HFD-fed mice bound more Stx1 and expressed more globotriaosylceramide than did such sections from LFD-fed mice. Furthermore, the gut microbiota of HFDfed mice compared with LFD-fed mice contained reduced levels of native Escherichia species, organisms that might protect the gut from colonization by incoming E. coli O157:H7. Taken together, these results suggest that susceptibility to infection and subsequent disease after ingestion of E. coli O157:H7 may depend, at least in part, on individual diet and/or the capacity of the commensal flora to produce butyrate. tubular necrosis | HCT-8 | microbiome S higa toxin (Stx)-producing Escherichia coli (STEC) infections continue to be a significant health burden in the United States. There are typically 15-20 outbreaks of STEC in the United States per year (1) that result in 265,000 illnesses, 3,600 hospitalizations, and 30 deaths annually (2). The United States Department of Agriculture estimates a total cost of about $500 million per year in health-related costs in the United States due to STEC infection (3). STEC, such as the most frequently isolated serotype in the United States, E. coli O157:H7 (1), are gut commensals of cattle. However, in humans, E. coli O157:H7 can cause diarrhea and hemorrhagic colitis after ingestion of as few as <50-300 organisms (4, 5). Such infections typically occur after individuals consume E. coli O157:H7-contaminated beef, fresh vegetables, water, or unpasteurized juice (6). On occasion, E. coli O157:H7 infections result from person-to-person spread of this low-infectious dose pathogen (6). The usual disease progression is as follows: 3 d after the ingestion of contaminated food or water, diarrhea, abdominal pain, and vomiting begin; frank, bloody diarrhea follows 2-3 d later. The bloody diarrhea generally resolves after 4 or 5 d. However, in 4-30% (7-10) of patients, the potentially life-threatening, hemolytic uremic syndrome (HUS) develops (6). HUS is characterized by acute kidney malfunction, microangiopathic hemolytic anemia, and thrombocytopenia (11). One aspect of STEC pathogenesis that is not clear is why a significant disparity in age and sometimes sex exists among those with STEC-related HUS. Children <10 y old are 10 times more likely to develop HUS following infection with STEC (12). In two recent large outbreaks, women developed HUS in disproportionate numbers (9, 13). Studies that address why children are more likely than adults to develop HUS suggest that a difference in complement activation (14, 15), platelet activation (16), or in nitric oxide production (17) may explain the disparity. The Stx produced by STEC is the critical virulence factor that not only may contribute to intestinal epithelia damage (18, 19) but is required for the development of HUS (20). STEC may produce Stx1, Stx2, or both toxins. Stx1 and Stx2 are highly related in both structure and function but cannot be neutralized by heterologous antisera. Although STEC that produce either Stx1 or Stx2 may cause severe disease in people, strains that make Stx2 are more likely to cause HUS (21). However, it is not clear how Stx gains access to the bloodstream from the intestinal lumen. Nevertheless, the preponderance of evidence indicates that Stx targets small vessel endothelial cells, and that toxin tropism, either directly or indirectly, leads to HUS (22). Cell-surface expression of the Stx receptor, globotriaosylceramide (Gb3), is required for cytotoxicity in vitro (23), and, in some models of STEC infection, Stx damages the intestinal epithelia (18, 19). Historically, it was presumed that Gb3 is not present on the gut epithelia (24, 25), and, thus, the mechanism by which the toxin might gain systemic access was unclear. However, contrary Significance We demonstrated that dietary fiber content affects susceptibility to Shiga toxin (Stx)-producing Escherichia coli (STEC) infection in mice. We showed that high fiber diet (HFD)-fed mice had elevated levels of butyrate, a beneficial gut metabolite that paradoxically enhances the cell-killing capacity of Stx. We also found that the amount of gut bacteria in HFD-fed mice increased whereas the percent of commensal Escherichia species (spp) decreased compared with animals fed a low fiber diet (LFD). These changes led to higher E. coli O157:H7 colonization levels, more weight loss, and greater rates of death in HFD-fed than in LFD-fed STEC-infected animals.
Microorganisms
Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne illness globall... more Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne illness globally, and infection with serotype O157:H7 is associated with increased risk of hospitalization and death in the U.S. The Stxs are encoded on a temperate bacteriophage (stx-phage), and phage induction leads to Stx expression; subtype Stx2a in particular is associated with more severe disease. Our earlier studies showed significant levels of RecA-independent Stx2 production by STEC O157:H7 strain JH2010 (stx2astx2c), even though activated RecA is the canonical trigger for stx-phage induction. This study aimed to further compare and contrast RecA-independent toxin production in Stx2-producing clinical isolates. Deletion of recA in JH2010 resulted in higher in vitro supernatant cytotoxicity compared to that from JH2016ΔrecA, and the addition of the chelator ethylenediaminetetraacetic acid (EDTA) and various metal cations to the growth medium exacerbated the difference in cytotoxicity exhibited ...
In a previous study, enterohemorrhagic Escherichia coli (EHEC) O157:H7 with a deletion and insert... more In a previous study, enterohemorrhagic Escherichia coli (EHEC) O157:H7 with a deletion and insertion in the eaeA gene encoding intimin was used to establish that intimin is required for the organism to attach to and efface microvilli in the piglet intestine (M. S. Donnenberg, S. Tzipori, M. L. McKee, A. D. O’Brien, J. Alroy, and J. B. Kaper, J. Clin. Invest. 92:1418–1424, 1993). However, in the same investigation, a role for intimin in EHEC adherence to HEp-2 cells could not be definitively demonstrated. To analyze the basis for this discrepancy, we constructed an in-frame deletion of eaeA and compared the adherence capacity of this mutant with that of the wild-type strain in vitro and in vivo. We observed a direct correlation between the requisite for intimin in EHEC O157:H7 colonization of the gnotobiotic piglet intestine and adherence of the bacterium to HEp-2 cells. The in vitro-in vivo correlation lends credence to the use of the HEp-2 cell adherence model for further study of ...
L'invention porte sur la preparation et l'utilisation d'anticorps monoclonaux humanis... more L'invention porte sur la preparation et l'utilisation d'anticorps monoclonaux humanises et a activite immunologique des toxines de Shiga, toxines associees au HC (colite hemorragique), et sur les sequelles de HUS (syndrome de Gasser) potentiellement thanatogenes transmises par des souches de bacteries pathogenes. L'invention decrit comment ces anticorps humanises peuvent servir a traiter ou a prevenir des maladies induites par les toxines de Shiga. L'un des aspects de l'invention porte sur un anticorps monoclonal humanise se fixant a la toxine de Shiga, dont les regions constantes sont des IgG1-kappa, et dont les regions variables sont d'origine murine. Un autre aspect porte sur des vecteurs d'expression et les cellules hotes transformees par lesdits vecteurs qui expriment les anticorps monoclonaux humanises de l'invention.
… coli 0157: H7 and other shiga …, 1998
... Immun. 61: 3832-3842. 16. Lindgren, SW, AR Melton-Celsa, and AD O&amp;amp;amp;amp;amp... more ... Immun. 61: 3832-3842. 16. Lindgren, SW, AR Melton-Celsa, and AD O&amp;amp;amp;amp;amp;#x27;Brien. Unpublished observation. 17. Lindgren, SW, JE Samuel, CK Schmitt, and AD O&amp;amp;amp;amp;amp;#x27;Brien. 1994. ... Infect. Immun. 62: 623-631. 18. Lindgren, SW, LD Teel, and AD O&amp;amp;amp;amp;amp;#x27;Brien. Unpublished observation. 19. ...
Springer eBooks, 2000
The Shiga toxins constitute a family of functionally and structurally related toxins produced by ... more The Shiga toxins constitute a family of functionally and structurally related toxins produced by Shigella dysenteriae type 1 (also called Shiga’s bacillus) and a subset of diarrheagenic Escherichia coli called STEC (for Shiga toxin-producing E. coli). Shiga toxins of STEC were formerly called Shiga-like toxins and are alternatively named Vero toxins (Calderwood et al. 1996; Karmali et al. 1996). We favor the Shiga-toxin nomenclature system based largely on historical precedence. Indeed, the prototype for the Shiga-toxin family, Shiga toxin produced by S. dysenteriae type 1, was first described in 1903 by Conradi (Conradi 1903) and, independently, by Neisser and Shiga (Neisser and Shiga 1903). Conradi made the seminal observation that lysates of Shiga’s bacillus caused hind-leg paralysis when injected into rabbits.
Several serotypes of Escherichia coli produce protein toxins closely related to Shiga toxin (Stx)... more Several serotypes of Escherichia coli produce protein toxins closely related to Shiga toxin (Stx) from Shigella dysenteriae serotype 1. These Stx-producing E. coli cause outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in humans, with the latter being more likely if the E. coli produce Stx2 than if they only produce Stx1. To investigate the differences among the Stxs, which are all AB5 toxins, the crystal structure of Stx2 from E. coli O157:H7 was determined at 1.8-Å resolution and compared with the known structure of Stx. Our major finding was that, in contrast to Stx, the active site of the A-subunit of Stx2 is accessible in the holotoxin, and a molecule of formic acid and a water molecule mimic the binding of the adenine base of the substrate. Further, the A-subunit adopts a different orientation with respect to the B-subunits in Stx2 than in Stx, due to interactions between the carboxyl termini of the B-subunits and neighboring regions of the A-subunit. Of the thre...
ASM Press eBooks, May 26, 2015
Infection and Immunity, Mar 1, 2014
Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause food-borne outbreaks of hemorrh... more Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause food-borne outbreaks of hemorrhagic colitis and, less commonly, a serious kidney-damaging sequela called the hemolytic uremic syndrome (HUS). Stx, the primary virulence factor expressed by STEC, is an AB 5 toxin with two antigenically distinct forms, Stx1a and Stx2a. Although both toxins have similar biological activities, Stx2a is more frequently produced by STEC strains that cause HUS than is Stx1a. Here we asked whether Stx1a and Stx2a act differently when delivered orally by gavage. We found that Stx2a had a 50% lethal dose (LD 50) of 2.9 g, but no morbidity occurred after oral intoxication with up to 157 g of Stx1a. We also compared several biochemical and histological parameters in mice intoxicated orally versus intraperitoneally with Stx2a. We discovered that both intoxication routes caused similar increases in serum creatinine and blood urea nitrogen, indicative of kidney damage, as well as electrolyte imbalances and weight loss in the animals. Furthermore, kidney sections from Stx2a-intoxicated mice revealed multifocal, acute tubular necrosis (ATN). Of particular note, we detected Stx2a in kidney sections from orally intoxicated mice in the same region as the epithelial cell type in which ATN was detected. Lastly, we showed reduced renal damage, as determined by renal biomarkers and histopathology, and full protection of orally intoxicated mice with monoclonal antibody (MAb) 11E10 directed against the toxin A subunit; conversely, an irrelevant MAb had no therapeutic effect. Orally intoxicated mice could be rescued by MAb 11E10 6 h but not 24 h after Stx2a delivery.
The Journal of Infectious Diseases, Sep 1, 2003
Financial support: Centers for Disease Control and Prevention (cooperative agreement U50/CCU81440... more Financial support: Centers for Disease Control and Prevention (cooperative agreement U50/CCU814408, to fund laboratory surveillance for non-O157:H7 Shiga toxin-producing Escherichia coli); National Institutes of Health (grants AI-47499, to fund analysis of the isolates, and AI-20148, to fund work on stx 2d-activatable); Bundesministerium für Bildung und Forschung Verbundprojekt (Forschungsnetzwerk 01KI9903).
ASM Press eBooks, May 26, 2015
Shiga toxin (Stx) is one of the most potent bacterial toxins known. Stx is found in Shigella dyse... more Shiga toxin (Stx) is one of the most potent bacterial toxins known. Stx is found in Shigella dysenteriae 1 and in some serogroups of Escherichia coli (called Stx1 in E. coli). In addition to or instead of Stx1, some E. coli strains produce a second type of Stx, Stx2, that has the same mode of action as Stx/Stx1 but is antigenically distinct. Because subtypes of each toxin have been identified, the prototype toxin for each group is now designated Stx1a or Stx2a. The Stxs consist of two major subunits, an A subunit that joins noncovalently to a pentamer of five identical B subunits. The A subunit of the toxin injures the eukaryotic ribosome and halts protein synthesis in target cells. The function of the B pentamer is to bind to the cellular receptor, globotriaosylceramide, Gb3, found primarily on endothelial cells. The Stxs traffic in a retrograde manner within the cell, such that the A subunit of the toxin reaches the cytosol only after the toxin moves from the endosome to the Golgi and then to the endoplasmic reticulum. In humans infected with Stx-producing E. coli, the most serious manifestation of the disease, hemolytic-uremic syndrome, is more often associated with strains that produce Stx2a rather than Stx1a, and that relative toxicity is replicated in mice and baboons. Stx1a and Stx2a also exhibit differences in cytotoxicity to various cell types, bind dissimilarly to receptor analogs or mimics, induce differential chemokine responses, and have several distinctive structural characteristics.
... 34: 10581069. 44. Melton-Celsa, AR, SC Darnell, and AD O'Brien. 1996. Activation of Shi... more ... 34: 10581069. 44. Melton-Celsa, AR, SC Darnell, and AD O'Brien. 1996. Activation of Shiga-like toxins by mouse and human intestinal mucus correlates with virulence of enterohemorrhagic Escherichia coli O91: H21 isolates in orally infected, streptomycin-treated ...
Frontiers in Microbiology, Aug 13, 2019
An O104:H4 Shiga toxin (Stx)-producing enteroaggregative Escherichia coli (EAEC) strain caused a ... more An O104:H4 Shiga toxin (Stx)-producing enteroaggregative Escherichia coli (EAEC) strain caused a large outbreak of bloody diarrhea and the hemolytic uremic syndrome in 2011. We previously developed an ampicillin (Amp)-treated C57BL/6 mouse model to measure morbidity (weight loss) and mortality of mice orally infected with the prototype Stx-EAEC strain C227-11. Here, we hypothesized that mice fed C227-11 cured of the pAA plasmid or deleted for individual genes on that plasmid would display reduced virulence compared to animals given the wild-type (wt) strain. C227-11 cured of the pAA plasmid or deleted for the known pAA-encoded virulence genes aggR, aggA, sepA, or aar were fed to Amp-treated C57BL/6 mice at doses of 10 10-10 11 CFU. Infected animals were then either monitored for morbidity and lethality for 28 days or euthanized to determine intestinal pathology and colonization levels at selected times. The pAAcured, aggR, and aggA mutants of strain C227-11 all showed reduced colonization at various intestinal sites. However, the aggR mutant was the only mutant attenuated for virulence as it showed both reduced morbidity and mortality. The aar mutant showed increased expression of the aggregative adherence fimbriae (AAF) and caused greater systemic effects in infected mice when compared to the C227-11 wt strain. However, unexpectedly, both the aggA and aar mutants displayed increased weight loss compared to wt. The sepA mutant did not exhibit altered morbidity or mortality in the Amp-treated mouse model compared to wt. Our data suggest that the increased morbidity due to the aar mutant could possibly be via an effect on expression of an as yet unknown virulence-associated factor under AggR control.
Emerging Infectious Diseases, Oct 1, 2014
ASM Press eBooks, Apr 9, 2014
Shiga toxin-producing Escherichia coli (STEC) serotype O157:H7 is one of the most important human... more Shiga toxin-producing Escherichia coli (STEC) serotype O157:H7 is one of the most important human pathogenic microorganisms, which can cause life-threatening infections. Xanthium strumarium L. is a plant with anti-bacterial activity against gram-negative and gram-positive bacteria. This study aims to demonstrate in vitro efficacy of the essential oil (EO) extracted from Xanthium strumarium L. against E. coli O157:H7. Using the agar test diffusion, the effect of Xanthium strumarium L. EO (5, 10, 15, 30, 60, and 120 mg/mL) was verified at each of the four different growth phases of E. coli O157:H7. Cell counts of viable cells and colony forming unit (CFU) were determined at regular time points using Breed's method and colony counting method, respectively. No viable cell was detectable after the 1 hour-exposure to X. strumarium EO at 30, 60, and 120 mg/mL concentrations. No bacterial colony was formed after 1 h until the end of the incubation period at 24 h. At lower concentrations, the number of bacteria cells decreased and colonies could be observed only after incubation. At the exponential phase, the EO at 15 mg/mL was only bacteriostatic, while from 30 mg/mL started to be bactericidal. X. strumarium EO antibacterial activity against Shiga toxinproducing E. coli O157:H7 is dependent on EO concentration and physiological state of the microorganisms tested. The best inhibitory activity was achieved during the late exponential and the stationary phases.
Microbiology spectrum, Sep 19, 2014
Shiga toxin (Stx)-producing Escherichia coli (STEC) is an etiologic agent of bloody diarrhea. A s... more Shiga toxin (Stx)-producing Escherichia coli (STEC) is an etiologic agent of bloody diarrhea. A serious sequela of disease, the hemolytic uremic syndrome (HUS) may arise in up to 25% of patients. The development of HUS after STEC infection is linked to the presence of Stx. STEC strains may produce one or more Stxs, and the Stxs come in two major immunological groups, Stx1 and Stx2. A multitude of possible therapeutics designed to inhibit the actions of the Stxs have been developed over the past 30 years. Such therapeutics are important because antibiotic treatment of STEC infections is contraindicated due to an increased potential for development of HUS. The reason for the increased risk of HUS after antibiotic treatment is likely because certain antibiotics induce expression of the Stxs, which are generally associated with lysogenic bacteriophages. There are a few potential therapeutics that either try to kill STEC without inducing Stx expression or target gene expression within STEC. However, the vast majority of the treatments under development are designed to limit Stx receptor generation or to prevent toxin binding, trafficking, processing, or activity within the cell. The potential therapies described in this review include some that have only been tested in vitro and several that demonstrate efficacy in animals. The therapeutics that are currently the furthest along in development (completed phase I and II trials) are monoclonal antibodies directed against Stx1 and Stx2.
Microbiology spectrum, Jun 15, 2023
The release of Stx from STEC has been thought to be tied to phage-mediated lysis of the host bact... more The release of Stx from STEC has been thought to be tied to phage-mediated lysis of the host bacterial cell. In this study, we found that the stx 2a phage lytic genes are not required for the virulence of pathogenic O157:H7 clinical isolates in a murine model of STEC infection or for release of Stx2a into the supernatant of bacterial cultures. These results point to an alternate mechanism for Stx2a release from STEC strains.
The sharing of genome sequences in online data repositories, allows for large scale analyses of s... more 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...
Proceedings of the National Academy of Sciences of the United States of America, May 20, 2013
The likelihood that a single individual infected with the Shiga toxin (Stx)-producing, food-borne... more The likelihood that a single individual infected with the Shiga toxin (Stx)-producing, food-borne pathogen Escherichia coli O157:H7 will develop a life-threatening sequela called the hemolytic uremic syndrome is unpredictable. We reasoned that conditions that enhance Stx binding and uptake within the gut after E. coli O157:H7 infection should result in greater disease severity. Because the receptor for Stx, globotriaosylceramide, is up-regulated in the presence of butyrate in vitro, we asked whether a high fiber diet (HFD) that reportedly enhances butyrate production by normal gut flora can influence the outcome of an E. coli O157 infection in mice. To address that question, groups of BALB/c mice were fed high (10%) or low (2%) fiber diets and infected with E. coli O157:H7 strain 86-24 (Stx2+). Mice fed an HFD exhibited a 10-to 100-fold increase in colonization, lost 15% more body weight, exhibited signs of morbidity, and had 25% greater mortality relative to the low fiber diet (LFD)-fed group. Additionally, sections of intestinal tissue from HFD-fed mice bound more Stx1 and expressed more globotriaosylceramide than did such sections from LFD-fed mice. Furthermore, the gut microbiota of HFDfed mice compared with LFD-fed mice contained reduced levels of native Escherichia species, organisms that might protect the gut from colonization by incoming E. coli O157:H7. Taken together, these results suggest that susceptibility to infection and subsequent disease after ingestion of E. coli O157:H7 may depend, at least in part, on individual diet and/or the capacity of the commensal flora to produce butyrate. tubular necrosis | HCT-8 | microbiome S higa toxin (Stx)-producing Escherichia coli (STEC) infections continue to be a significant health burden in the United States. There are typically 15-20 outbreaks of STEC in the United States per year (1) that result in 265,000 illnesses, 3,600 hospitalizations, and 30 deaths annually (2). The United States Department of Agriculture estimates a total cost of about $500 million per year in health-related costs in the United States due to STEC infection (3). STEC, such as the most frequently isolated serotype in the United States, E. coli O157:H7 (1), are gut commensals of cattle. However, in humans, E. coli O157:H7 can cause diarrhea and hemorrhagic colitis after ingestion of as few as <50-300 organisms (4, 5). Such infections typically occur after individuals consume E. coli O157:H7-contaminated beef, fresh vegetables, water, or unpasteurized juice (6). On occasion, E. coli O157:H7 infections result from person-to-person spread of this low-infectious dose pathogen (6). The usual disease progression is as follows: 3 d after the ingestion of contaminated food or water, diarrhea, abdominal pain, and vomiting begin; frank, bloody diarrhea follows 2-3 d later. The bloody diarrhea generally resolves after 4 or 5 d. However, in 4-30% (7-10) of patients, the potentially life-threatening, hemolytic uremic syndrome (HUS) develops (6). HUS is characterized by acute kidney malfunction, microangiopathic hemolytic anemia, and thrombocytopenia (11). One aspect of STEC pathogenesis that is not clear is why a significant disparity in age and sometimes sex exists among those with STEC-related HUS. Children <10 y old are 10 times more likely to develop HUS following infection with STEC (12). In two recent large outbreaks, women developed HUS in disproportionate numbers (9, 13). Studies that address why children are more likely than adults to develop HUS suggest that a difference in complement activation (14, 15), platelet activation (16), or in nitric oxide production (17) may explain the disparity. The Stx produced by STEC is the critical virulence factor that not only may contribute to intestinal epithelia damage (18, 19) but is required for the development of HUS (20). STEC may produce Stx1, Stx2, or both toxins. Stx1 and Stx2 are highly related in both structure and function but cannot be neutralized by heterologous antisera. Although STEC that produce either Stx1 or Stx2 may cause severe disease in people, strains that make Stx2 are more likely to cause HUS (21). However, it is not clear how Stx gains access to the bloodstream from the intestinal lumen. Nevertheless, the preponderance of evidence indicates that Stx targets small vessel endothelial cells, and that toxin tropism, either directly or indirectly, leads to HUS (22). Cell-surface expression of the Stx receptor, globotriaosylceramide (Gb3), is required for cytotoxicity in vitro (23), and, in some models of STEC infection, Stx damages the intestinal epithelia (18, 19). Historically, it was presumed that Gb3 is not present on the gut epithelia (24, 25), and, thus, the mechanism by which the toxin might gain systemic access was unclear. However, contrary Significance We demonstrated that dietary fiber content affects susceptibility to Shiga toxin (Stx)-producing Escherichia coli (STEC) infection in mice. We showed that high fiber diet (HFD)-fed mice had elevated levels of butyrate, a beneficial gut metabolite that paradoxically enhances the cell-killing capacity of Stx. We also found that the amount of gut bacteria in HFD-fed mice increased whereas the percent of commensal Escherichia species (spp) decreased compared with animals fed a low fiber diet (LFD). These changes led to higher E. coli O157:H7 colonization levels, more weight loss, and greater rates of death in HFD-fed than in LFD-fed STEC-infected animals.
Microorganisms
Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne illness globall... more Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne illness globally, and infection with serotype O157:H7 is associated with increased risk of hospitalization and death in the U.S. The Stxs are encoded on a temperate bacteriophage (stx-phage), and phage induction leads to Stx expression; subtype Stx2a in particular is associated with more severe disease. Our earlier studies showed significant levels of RecA-independent Stx2 production by STEC O157:H7 strain JH2010 (stx2astx2c), even though activated RecA is the canonical trigger for stx-phage induction. This study aimed to further compare and contrast RecA-independent toxin production in Stx2-producing clinical isolates. Deletion of recA in JH2010 resulted in higher in vitro supernatant cytotoxicity compared to that from JH2016ΔrecA, and the addition of the chelator ethylenediaminetetraacetic acid (EDTA) and various metal cations to the growth medium exacerbated the difference in cytotoxicity exhibited ...
In a previous study, enterohemorrhagic Escherichia coli (EHEC) O157:H7 with a deletion and insert... more In a previous study, enterohemorrhagic Escherichia coli (EHEC) O157:H7 with a deletion and insertion in the eaeA gene encoding intimin was used to establish that intimin is required for the organism to attach to and efface microvilli in the piglet intestine (M. S. Donnenberg, S. Tzipori, M. L. McKee, A. D. O’Brien, J. Alroy, and J. B. Kaper, J. Clin. Invest. 92:1418–1424, 1993). However, in the same investigation, a role for intimin in EHEC adherence to HEp-2 cells could not be definitively demonstrated. To analyze the basis for this discrepancy, we constructed an in-frame deletion of eaeA and compared the adherence capacity of this mutant with that of the wild-type strain in vitro and in vivo. We observed a direct correlation between the requisite for intimin in EHEC O157:H7 colonization of the gnotobiotic piglet intestine and adherence of the bacterium to HEp-2 cells. The in vitro-in vivo correlation lends credence to the use of the HEp-2 cell adherence model for further study of ...
L'invention porte sur la preparation et l'utilisation d'anticorps monoclonaux humanis... more L'invention porte sur la preparation et l'utilisation d'anticorps monoclonaux humanises et a activite immunologique des toxines de Shiga, toxines associees au HC (colite hemorragique), et sur les sequelles de HUS (syndrome de Gasser) potentiellement thanatogenes transmises par des souches de bacteries pathogenes. L'invention decrit comment ces anticorps humanises peuvent servir a traiter ou a prevenir des maladies induites par les toxines de Shiga. L'un des aspects de l'invention porte sur un anticorps monoclonal humanise se fixant a la toxine de Shiga, dont les regions constantes sont des IgG1-kappa, et dont les regions variables sont d'origine murine. Un autre aspect porte sur des vecteurs d'expression et les cellules hotes transformees par lesdits vecteurs qui expriment les anticorps monoclonaux humanises de l'invention.
… coli 0157: H7 and other shiga …, 1998
... Immun. 61: 3832-3842. 16. Lindgren, SW, AR Melton-Celsa, and AD O&amp;amp;amp;amp;amp... more ... Immun. 61: 3832-3842. 16. Lindgren, SW, AR Melton-Celsa, and AD O&amp;amp;amp;amp;amp;#x27;Brien. Unpublished observation. 17. Lindgren, SW, JE Samuel, CK Schmitt, and AD O&amp;amp;amp;amp;amp;#x27;Brien. 1994. ... Infect. Immun. 62: 623-631. 18. Lindgren, SW, LD Teel, and AD O&amp;amp;amp;amp;amp;#x27;Brien. Unpublished observation. 19. ...
Springer eBooks, 2000
The Shiga toxins constitute a family of functionally and structurally related toxins produced by ... more The Shiga toxins constitute a family of functionally and structurally related toxins produced by Shigella dysenteriae type 1 (also called Shiga’s bacillus) and a subset of diarrheagenic Escherichia coli called STEC (for Shiga toxin-producing E. coli). Shiga toxins of STEC were formerly called Shiga-like toxins and are alternatively named Vero toxins (Calderwood et al. 1996; Karmali et al. 1996). We favor the Shiga-toxin nomenclature system based largely on historical precedence. Indeed, the prototype for the Shiga-toxin family, Shiga toxin produced by S. dysenteriae type 1, was first described in 1903 by Conradi (Conradi 1903) and, independently, by Neisser and Shiga (Neisser and Shiga 1903). Conradi made the seminal observation that lysates of Shiga’s bacillus caused hind-leg paralysis when injected into rabbits.
Several serotypes of Escherichia coli produce protein toxins closely related to Shiga toxin (Stx)... more Several serotypes of Escherichia coli produce protein toxins closely related to Shiga toxin (Stx) from Shigella dysenteriae serotype 1. These Stx-producing E. coli cause outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in humans, with the latter being more likely if the E. coli produce Stx2 than if they only produce Stx1. To investigate the differences among the Stxs, which are all AB5 toxins, the crystal structure of Stx2 from E. coli O157:H7 was determined at 1.8-Å resolution and compared with the known structure of Stx. Our major finding was that, in contrast to Stx, the active site of the A-subunit of Stx2 is accessible in the holotoxin, and a molecule of formic acid and a water molecule mimic the binding of the adenine base of the substrate. Further, the A-subunit adopts a different orientation with respect to the B-subunits in Stx2 than in Stx, due to interactions between the carboxyl termini of the B-subunits and neighboring regions of the A-subunit. Of the thre...