The Deleterious Effects of Shiga Toxin Type 2 Are Neutralized In Vitro by FabF8:Stx2 Recombinant Monoclonal Antibody.

Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic Escherichia coli (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC50) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes.

OMV-based vaccine formulations against Shiga toxin producing Escherichia coli strains are both protective in mice and immunogenic in calves.

Strains of Shiga toxin-producing Escherichia coli (STEC) can cause the severe Hemolytic Uremic Syndrome (HUS). Shiga toxins are protein toxins that bind and kill microvascular cells, damaging vital organs. No specific therapeutics or vaccines have been licensed for use in humans yet. The most common route of infection is by consumption of dairy or farm products contaminated with STEC. Domestic cattle colonized by STEC strains represent the main reservoir, and thus a source of contamination. Outer Membrane Vesicles (OMV) obtained after detergent treatment of gram-negative bacteria have been used over the past decades for producing many licensed vaccines. These nanoparticles are not only multi-antigenic in nature but also potent immunopotentiators and immunomodulators. Formulations based on chemical-inactivated OMV (OMVi) obtained from a virulent STEC strain (O157:H7 serotype) were found to protect against pathogenicity in a murine model and to be immunogenic in calves. These initial studies suggest that STEC-derived OMV has a potential for the formulation of both human and veterinary vaccines.

Pathogenic role of inflammatory response during Shiga toxin-associated hemolytic uremic syndrome (HUS).

Hemolytic uremic syndrome (HUS) is defined as a triad of noninmune microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. The most frequent presentation is secondary to Shiga toxin (Stx)-producing Escherichia coli (STEC) infections, which is termed postdiarrheal, epidemiologic or Stx-HUS, considering that Stx is the necessary etiological factor. After ingestion, STEC colonize the intestine and produce Stx, which translocates across the intestinal epithelium. Once Stx enters the bloodstream, it interacts with renal endothelial and epithelial cells, and leukocytes. This review summarizes the current evidence about the involvement of inflammatory components as central pathogenic factors that could determine outcome of STEC infections. Intestinal inflammation may favor epithelial leakage and subsequent passage of Stx to the systemic circulation. Vascular damage triggered by Stx promotes not only release of thrombin and increased fibrin concentration but also production of cytokines and chemokines by endothelial cells. Recent evidence from animal models and patients strongly indicate that several immune cells types may participate in HUS physiopathology: neutrophils, through release of proteases and reactive oxygen species (ROS); monocytes/macrophages through secretion of cytokines and chemokines. In addition, high levels of Bb factor and soluble C5b-9 (sC5b-9) in plasma as well as complement factors adhered to platelet-leukocyte complexes, microparticles and microvesicles, suggest activation of the alternative pathway of complement. Thus, acute immune response secondary to STEC infection, the Stx stimulatory effect on different immune cells, and inflammatory stimulus secondary to endothelial damage all together converge to define a strong inflammatory status that worsens Stx toxicity and disease.

A recombinant O-polysaccharide-protein conjugate approach to develop highly specific monoclonal antibodies to Shiga toxin-producing Escherichia coli O157 and O145 serogroups.

Shiga toxin-producing Escherichia coli (STEC) is the major etiologic agent of hemolytic-uremic syndrome (HUS). The high rate of HUS emphasizes the urgency for the implementation of primary prevention strategies to reduce its public health impact. Argentina shows the highest rate of HUS worldwide, being E. coli O157 the predominant STEC-associated HUS serogroup (>70%), followed by E. coli O145 (>9%). To specifically detect these serogroups we aimed at developing highly specific monoclonal antibodies (mAbs) against the O-polysaccharide (O-PS) section of the lipopolysaccharide (LPS) of the dominant STEC-associated HUS serogroups in Argentina. The development of hybridomas secreting mAbs against O157 or O145 was carried out through a combined immunization strategy, involving adjuvated-bacterial immunizations followed by immunizations with recombinant O-PS-protein conjugates. We selected hybridoma clones that specifically recognized the engineered O-PS-protein conjugates of O157 or O145 serogroups. Indirect ELISA of heat-killed bacteria showed specific binding to O157 or O145 serogroups, respectively, while no cross-reactivity with other epidemiological important STEC strains, Brucella abortus, Salmonella group N or Yersinia enterocolitica O9 was observed. Western blot analysis showed specific recognition of the sought O-PS section of the LPS by all mAbs. Finally, the ability of the developed mAbs to bind the surface of whole bacteria cells was confirmed by flow cytometry, confocal microscopy and agglutination assays, indicating that these mAbs present an exceptional degree of specificity and relative affinity in the detection and identification of E. coli O157 and O145 serogroups. These mAbs may be of significant value for clinical diagnosis and food quality control applications. Thus, engineered O-PS specific moieties contained in the recombinant glycoconjugates used for combined immunization and hybridoma selection are an invaluable resource for the development of highly specific mAbs.

ppGpp and cytotoxicity diversity in Shiga toxin-producing Escherichia coli (STEC) isolates.

Shiga toxin-producing Escherichia coli (STEC) is a known food pathogen, which main reservoir is the intestine of ruminants. The abundance of different STEC lineages in nature reflect a heterogeneity that is characterised by the differential expression of certain genotypic characteristics, which in turn are influenced by the environmental conditions to which the microorganism is exposed. Bacterial homeostasis and stress response are under the control of the alarmone guanosine tetraphosphate (ppGpp), which intrinsic levels varies across the E. coli species. In the present study, 50 STEC isolates from healthy sheep were evaluated regarding their ppGpp content, cytotoxicity and other relevant genetic and phenotypic characteristics. We found that the level of ppGpp and cytotoxicity varied considerably among the examined strains. Isolates that harboured the stx2 gene were the least cytotoxic and presented the highest levels of ppGpp. All stx2 isolates belonged to phylogroup A, while strains that carried stx1 or both stx1 and stx2 genes pertained to phylogroup B1. All but two stx2 isolates belonged to the stx2b subtype. Strains that belonged to phylogroup B1 displayed on average low levels of ppGpp and high cytotoxicity. Overall, there was a negative correlation between cytotoxicity and ppGpp.

Antibodies to Shiga toxins in Brazilian cattle.

Cattle are considered a reservoir of Shiga toxin-producing Escherichia coli (STEC). There is no information about the presence of antibodies against Shiga toxins in Brazilian bovine serum. Using ELISA, all sera tested showed antibodies against the two main STEC virulence factors; Stx1 and Stx2. Neutralizing antibodies against Stx1 and/or Stx2 were detected in all but one serum. In conclusion, our results indicated that these animals had been exposed to STEC producing both toxins.

Development and characterization of recombinant antibody fragments that recognize and neutralize in vitro Stx2 toxin from Shiga toxin-producing Escherichia coli.

BACKGROUND: Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes. METHODS AND FINDINGS: In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA. CONCLUSION: In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro.

Diversity of Shiga toxin-producing Escherichia coli in sheep flocks of Paraná State, southern Brazil.

Sheep constitute an important source of zoonotic pathogens as Shiga toxin-producing Escherichia coli (STEC). In this study, the prevalence, serotypes and virulence profiles of STEC were investigated among 130 healthy sheep from small and medium farms in southern Brazil. STEC was isolated from 65 (50%) of the tested animals and detected in all flocks. A total of 70 STEC isolates were characterized, and belonged to 23 different O:H serotypes, many of which associated with human disease, including hemolytic-uremic syndrome (HUS). Among the serotypes identified, O76:H19 and O65:H- were the most common, and O75:H14 and O169:H7 have not been previously reported in STEC strains. Most of the STEC isolates harbored only stx1, whereas the Stx2b subtype was the most common among those carrying stx2. Enterohemolysin (ehxA) and intimin (eae) genes were detected in 61 (87.1%) and four (5.7%) isolates, respectively. Genes encoding putative adhesins (saa, iha, lpfO113) and toxins (subAB and cdtV) were also observed. The majority of the isolates displayed virulence features related to pathogenesis of STEC, such as adherence to epithelial cells, high cytotoxicity and enterohemolytic activity. Ovine STEC isolates belonged mostly to phylogenetic group B1. PFGE revealed particular clones distributed in some farms, as well as variations in the degree of genetic similarity within serotypes examined. In conclusion, STEC are widely distributed in southern Brazilian sheep, and belonged mainly to serotypes that are not commonly reported in other regions, such as O76:H19 and O65:H-. A geographical variation in the distribution of STEC serotypes seems to occur in sheep.

Serogroup-specific bacterial engineered glycoproteins as novel antigenic targets for diagnosis of shiga toxin-producing-escherichia coli-associated hemolytic-uremic syndrome.

Human infection with Shiga toxin-producing Escherichia coli (STEC) is a major cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening condition characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. E. coli O157:H7 is the dominant STEC serotype associated with HUS worldwide, although non-O157 STEC serogroups can cause a similar disease. The detection of anti-O157 E. coli lipopolysaccharide (LPS) antibodies in combination with stool culture and detection of free fecal Shiga toxin considerably improves the diagnosis of STEC infections. In the present study, we exploited a bacterial glycoengineering technology to develop recombinant glycoproteins consisting of the O157, O145, or O121 polysaccharide attached to a carrier protein as serogroup-specific antigens for the serological diagnosis of STEC-associated HUS. Our results demonstrate that using these antigens in indirect ELISAs (glyco-iELISAs), it is possible to clearly discriminate between STEC O157-, O145-, and O121-infected patients and healthy children, as well as to confirm the diagnosis in HUS patients for whom the classical diagnostic procedures failed. Interestingly, a specific IgM response was detected in almost all the analyzed samples, indicating that it is possible to detect the infection in the early stages of the disease. Additionally, in all the culture-positive HUS patients, the serotype identified by glyco-iELISAs was in accordance with the serotype of the isolated strain, indicating that these antigens are valuable not only for diagnosing HUS caused by the O157, O145, and O121 serogroups but also for serotyping and guiding the subsequent steps to confirm diagnosis.

Oral administration of Shiga toxin-producing Escherichia coli induces intestinal and systemic specific immune response in mice.

Hemolytic uremic syndrome (HUS) is the major complication of gastrointestinal infections with enterohemorrhagic Escherichia coli (EHEC) and is mediated by the production of Shiga toxins (Stx). Although it has been previously reported that not only HUS patients but healthy children have anti-Stx antibodies, very little is known about how these infections impact on mucosal immune system to generate a specific immune response. This work aimed to evaluate the immune responses elicited after a single oral dose of EHEC in a mouse model of HUS at weaning. We found sequential activation of T and B lymphocytes together with an increased percentage of IgA-bearing B cells in Peyer's patches and mesenteric lymph nodes. We also found fecal anti-EHEC IgA and serum anti-Stx2 IgG in EHEC-inoculated mice. Besides, these mice were partially protected against an intravenous challenge with Stx2. These data demonstrate that one episode of EHEC infection is enough to induce activation in the gut-associated lymphoid tissue, especially the B cell compartment, and lead to the production of specific IgA in mucosal tissue and the generation of systemic protection against Stx2 in a percentage of intragastrically inoculated mice. These data also support the epidemiologic observation that a second episode of HUS is very rare.

Molecular mechanisms that mediate colonization of Shiga toxin-producing Escherichia coli strains.

Shiga toxin-producing Escherichia coli (STEC) is a group of pathogens which cause gastrointestinal disease in humans and have been associated with numerous food-borne outbreaks worldwide. The intimin adhesin has been considered for many years to be the only colonization factor in these strains. However, the rapid progress in whole-genome sequencing of different STEC serotypes has accelerated the discovery of other adhesins (fimbrial and afimbrial), which have emerged as important contributors to the intestinal colonization occurring during STEC infection. This review summarizes recent progress to identify and characterize, at the molecular level, novel adhesion and colonization factors in STEC strains, with an emphasis on their contribution to virulence traits, their host-pathogen interactions, the regulatory mechanisms controlling their expression, and their role as targets eliciting immune responses in the host.

Nitric oxide-mediated apoptosis in rat macrophages subjected to Shiga toxin 2 from Escherichia coli.

Shiga toxin-producing Escherichia coli are important food-borne pathogens. The main factor conferring virulence on this bacterium is its capacity to secrete Shiga toxins (Stxs), which have been reported to induce apoptosis in several cell types. However, the mechanisms of this apoptosis have not yet been fully elucidated. In addition, Stxs have been shown to stimulate macrophages to produce nitric oxide (NO), a well-known apoptosis inductor.The aim of this study was to investigate the participation of NO in apoptosis of rat peritoneal macrophages induced by culture supernatants or Stx2 from E. coli. Peritoneal macrophages incubated in the presence of E. coli supernatants showed an increase in the amounts of apoptosis and NO production. Furthermore, inhibition of NO synthesis induced by addition of aminoguanidine (AG) was correlated with a reduction in the percentage of apoptotic cells, indicating participation of this metabolite in the apoptotic process. Similarly, treatment of cells with Stx2 induced an increase in NO production and amount of apoptosis, these changes being reversed by addition of AG. In summary, these data show that treatment with E. coli supernatants or Stx2 induces NO-mediated apoptosis of macrophages.

Detection of the CS20 colonization factor antigen in diffuse-adhering Escherichia coli strains.

We analyzed a randomly selected group of 30 diffusely adherent (DAEC), 30 enteropathogenic, 30 enteroaggregative, and five Shiga toxin-producing Escherichia coli strains isolated from children with diarrhea. Enterotoxigenic E. coli (ETEC) colonization factors (CFs) were evaluated by a dot-blot assay using 21 CF-specific monoclonal antibodies. Out of 95 non-ETEC strains, three DAEC were found to express coli surface antigen 20 (CS20). No other E. coli expressed CFs. We confirmed the three CS20-positive strains as ETEC-negative by repeat PCR and as toxin-negative by ganglioside-GM1-enzyme-linked immunosorbent assay. To our knowledge, this is the first study that has identified currently recognized CFs in non-ETEC diarrheagenic E. coli strains identified using molecular methods. CFs may be an unrecognized relevant adherence factor in other E. coli, which may then play a role in pathogenesis and the immune response of the host.

Antimicrobial resistance in Brazilian isolates of Shiga toxin-encoding Escherichia coli from cows with mastitis / Resistência a agentes antimicrobianos em cepas brasileiras de Escherichia coli codificadora de Shiga toxina isoladas de vacas com mastite

Antimicrobial resistance is a matter of growing concern in public and animal health. Public health can be threatened by the transfer of pathogens from animals to people via indirect contact through food or directly by contact with animals. The potential transfer of resistance determinants from animals to humans through Escherichia coli strains is an additional cause of concern. From February to November 2004, 670 samples of bovine mastitic milk were collected in two Brazilian states. The 231 E. coli strains isolated from the samples were screened for the presence of genes encoding Shiga toxin (stx 1 and stx 2) and intimin (eae). Twenty (8.6%) strains were shown by PCR to harbor the Shiga toxin genes (8 harbored the stx 1 gene, 12 the stx 2 gene and none both of them). Two (0.8%) strains were eae positive; however, they did not carry the stx 1 or stx 2. These strains were also screened for resistance to 12 antimicrobial agents. Predominant resistance was to amikacin (60.0%), gentamicin (50.0%), streptomycin (50.0%) and ampicillin (45.0%). Multidrug resistance was found among 7 isolates (35.0%). These results indicate that dairy cattle from the region surveyed may be a source of STEC potentially pathogenic for humans.

A existência de resistência aos agentes antimicrobianos entre as bactérias tem-se revelado um sério problema para a saúde publica e para a saúde animal. A saúde publica pode ser comprometida pela transferência de bactéria patogênica proveniente dos animais para as pessoas através de um contato indireto pelos alimentos ou através de um contato direto com os animais. A possível transferência de genes de resistência a antimicrobianos entre a microbiota animal e a microbiota humana através da bactéria Escherichia coli representa um motivo adicional de apreensão. De Fevereiro a Novembro de 2004 foram coletadas 670 amostras de leite obtido de bovinos que apresentavam mastite provenientes de dois estados brasileiros. As 231 cepas de E. coli isoladas destas amostras de leite foram analisadas para a verificação da presença dos genes codificadores de Shiga toxina (stx 1 e stx 2) e da intimina (eae). Através da técnica de PCR foram detectadas 20 cepas (8.6%) de E. coli carregando os genes de Shiga toxina (8 apresentavam o gene stx 1 e 12 apresentavam o gene stx 2, e nenhuma das cepas apresentava os dois genes). Duas cepas carregavam o gene eae (0,8%), no entanto elas não apresentavam o gene stx 1 nem stx 2. Estas linhagens também foram analisadas em relação a sua resistência frente a 12 agentes antimicrobianos. Resistência foi predominantemente determinada para amicacina (60,0%), gentamicina (50,0%), estreptomicina (50.0%) e ampicilina (45,0%). Resistência a múltiplas drogas foi determinada em 7 destas cepas. Estes resultados indicaram que o gado leiteiro das regiões avaliadas pode representar uma fonte de disseminação de cepas STEC potencialmente patogênicas para os seres humanos.

In vitro reactivity and growth inhibition of EPEC serotype O111 and STEC serotypes O111 and O157 by homologous and heterologous chicken egg yolk antibody.

IgY is a chicken egg yolk antibody which has been used for treatment and prophylaxis of gastrointestinal infections. Our aim was to verify if IgY obtained from chickens immunized with EPEC O111, STEC O111 and STEC O157 is able to show in vitro reactivity and biological activity towards the three bacteria. IgY was obtained from eggs laid before and after immunization with each bacterium. The preparations of IgY anti-EPEC O111 and anti-STEC O111 shared high reactivity detected by ELISA and growth inhibition ability towards both bacteria EPEC O111 and STEC O111. Nevertheless, the preparation of IgY anti-STEC O157 showed high reactivity and growth inhibitory effect only towards the homologous strain. Our results showing in vitro biological activity of IgY reinforce its use as an alternative for the treatment or prophylaxis of E. coli infections and encourage the development of in vivo studies for a possible future human therapeutic use.