RESUMO
UNLABELLED: Shiga toxins (Stx) are the main agent responsible for the development of hemolytic-uremic syndrome (HUS), the most severe and life-threatening systemic complication of infection with enterohemorrhagic Escherichia coli (EHEC) strains. We previously described Stx2 expression by eukaryotic cells after they were transfected in vitro with the stx2 gene cloned into a prokaryotic plasmid (pStx2). The aim of this study was to evaluate whether mammalian cells were also able to express Stx2 in vivo after pStx2 injection. Mice were inoculated by hydrodynamics-based transfection (HBT) with pStx2. We studied the survival, percentage of polymorphonuclear leukocytes in plasma, plasma urea levels, and histology of the kidneys and the brains of mice. Mice displayed a lethal dose-related response to pStx2. Stx2 mRNA was recovered from the liver, and Stx2 cytotoxic activity was observed in plasma of mice injected with pStx2. Stx2 was detected by immunofluorescence in the brains of mice inoculated with pStx2, and markers of central nervous system (CNS) damage were observed, including increased expression of glial fibrillary acidic protein (GFAP) and fragmentation of NeuN in neurons. Moreover, anti-Stx2B-immunized mice were protected against pStx2 inoculation. Our results show that Stx2 is expressed in vivo from the wild stx2 gene, reproducing pathogenic damage induced by purified Stx2 or secondary to EHEC infection. IMPORTANCE: Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC) infections are a serious public health problem, and Stx is the main pathogenic agent associated with typical hemolytic-uremic syndrome (HUS). In contrast to the detailed information describing the molecular basis for EHEC adherence to epithelial cells, very little is known about how Stx is released from bacteria in the gut, reaching its target tissues, mainly the kidney and central nervous system (CNS). In order to develop an efficient treatment for EHEC infections, it is necessary to understand the mechanisms involved in Stx expression. In this regard, the present study demonstrates that mammals can synthesize biologically active Stx using the natural promoter associated with the Stx-converting bacteriophage genome. These results could impact the comprehension of EHEC HUS, since local eukaryotic cells transduced and/or infected by bacteriophage encoding Stx2 could be an alternative source of Stx production.
Assuntos
Escherichia coli Êntero-Hemorrágica/metabolismo , Infecções por Escherichia coli/microbiologia , Regiões Promotoras Genéticas , Toxina Shiga II/biossíntese , Toxina Shiga II/genética , Animais , Encéfalo/metabolismo , Encéfalo/microbiologia , Encéfalo/patologia , Escherichia coli Êntero-Hemorrágica/genética , Infecções por Escherichia coli/patologia , Feminino , Humanos , Rim/metabolismo , Rim/microbiologia , Rim/patologia , Fígado/metabolismo , Fígado/microbiologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND AND AIMS: Shiga-like toxin (Stx)-producing Escherichia coli (STEC) infection is an ongoing health issue that can lead to serious complications, including hemolytic uremic syndrome (HUS) and death. This study assessed demographic and epidemiologic information of STEC infection among Argentinean children. METHODS: A prospective surveillance of 2435 screened children (age, 0.5-15 years) presenting with watery diarrhea and/or bloody diarrhea was undertaken to evaluate the clinical course of STEC infection. RESULTS: Prevalence of STEC infection was 4.1% among subjects presenting with watery diarrhea for ≤ 5 days' duration, bloody diarrhea for ≤ 36 hours' duration, or both. Incidence of STEC infection was significantly higher in the subjects with bloody diarrhea. Ninety-three STEC+ children underwent further evaluation, of whom 8 (8.6%) developed HUS. White blood cells, particularly neutrophils, were abnormally elevated at screening in 5 of 8 HUS subjects. Quantifiable serum Stx-2 values were noted within 24 to 48 hours after the onset of bloody diarrhea in 3 HUS subjects using a validated chemiluminescence assay, with levels quickly dissipating by HUS onset. CONCLUSIONS: Results suggest that young STEC-positive children with bloody diarrhea and exhibiting neutrophilic leukocytosis in the early course of their diarrhea are at risk for HUS progression. The observation of measurable concentrations of Stx-2 levels in the early post-bloody-diarrhea period and rapid dissipation at the time of HUS onset requires further evaluation.
Assuntos
Diarreia/epidemiologia , Infecções por Escherichia coli/epidemiologia , Síndrome Hemolítico-Urêmica/epidemiologia , Toxina Shiga II/biossíntese , Toxinas Shiga/biossíntese , Escherichia coli Shiga Toxigênica/isolamento & purificação , Adolescente , Argentina/epidemiologia , Criança , Diarreia/diagnóstico , Diarreia/microbiologia , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Feminino , Síndrome Hemolítico-Urêmica/diagnóstico , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Lactente , Masculino , Reação em Cadeia da Polimerase , Vigilância da População/métodos , Prevalência , Fatores de Risco , Toxina Shiga II/genética , Toxinas Shiga/genética , Escherichia coli Shiga Toxigênica/patogenicidadeRESUMO
Shiga-like toxin 2 (Stx2)-producing enterohemorrhagic Escherichia coli (referred to as EHEC or STEC) strains are the primary etiologic agents of hemolytic-uremic syndrome (HUS), which leads to renal failure and high mortality rates. Expression of Stx2 is the most relevant virulence-associated factor of EHEC strains, and toxin neutralization by antigen-specific serum antibodies represents the main target for both preventive and therapeutic anti-HUS approaches. In the present report, we describe two Salmonella enterica serovar Typhimurium aroA vaccine strains expressing a nontoxic plasmid-encoded derivative of Stx2 (Stx2DeltaAB) containing the complete nontoxic A2 subunit and the receptor binding B subunit. The two S. Typhimurium strains differ in the expression of flagellin, the structural subunit of the flagellar shaft, which exerts strong adjuvant effects. The vaccine strains expressed Stx2DeltaAB, either cell bound or secreted into the extracellular environment, and showed enhanced mouse gut colonization and high plasmid stability under both in vitro and in vivo conditions. Oral immunization of mice with three doses of the S. Typhimurium vaccine strains elicited serum anti-Stx2B (IgG) antibodies that neutralized the toxic effects of the native toxin under in vitro conditions (Vero cells) and conferred partial protection under in vivo conditions. No significant differences with respect to gut colonization or the induction of antigen-specific antibody responses were detected in mice vaccinated with flagellated versus nonflagellated bacterial strains. The present results indicate that expression of Stx2DeltaAB by attenuated S. Typhimurium strains is an alternative vaccine approach for HUS control, but additional improvements in the immunogenicity of Stx2 toxoids are still required.
Assuntos
Escherichia coli Êntero-Hemorrágica/imunologia , Infecções por Escherichia coli/prevenção & controle , Vacinas contra Escherichia coli/imunologia , Vetores Genéticos , Salmonella typhimurium/genética , Toxina Shiga II/imunologia , Administração Oral , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Antitoxinas/sangue , Chlorocebus aethiops , Creatinina/sangue , Infecções por Escherichia coli/imunologia , Vacinas contra Escherichia coli/administração & dosagem , Vacinas contra Escherichia coli/genética , Imunização Secundária , Camundongos , Camundongos Endogâmicos BALB C , Toxina Shiga II/biossíntese , Ureia/sangue , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Células VeroRESUMO
Shiga-like toxin-producing Escherichia coli (STEC) infection causes diarrhea, which is often bloody and which can result in potentially life-threatening hemolytic-uremic syndrome (HUS). Urtoxazumab, a humanized monoclonal antibody directed against the Shiga-like toxin 2 (Stx2) produced by STEC, has been developed as a promising agent for the prevention of HUS. Single randomized, intravenous, double-blind, placebo-controlled doses of urtoxazumab were administered to assess its safety and pharmacokinetics in healthy adults (0.1 to 3.0 mg/kg of body weight) and STEC-infected pediatric patients (1.0 and 3.0 mg/kg). No dose-related safety trends were noted, nor were antiurtoxazumab antibodies detected. The disposition of urtoxazumab showed a biexponential decline, regardless of the dose. In healthy adults, the mean terminal elimination half-life was consistent across the dose groups and ranged from 24.6 days (3.0-mg/kg dose group) to 28.9 days (0.3-mg/kg dose group). The mean maximum serum drug concentration (C(max)) ranged from 2.6 microg/ml at 0.1 mg/kg to 71.7 microg/ml at 3.0 mg/kg. The disposition of urtoxazumab following the administration of doses of 1.0 and 3.0 mg/kg in pediatric patients showed mean C(max)s of 19.6 and 56.1 microg/ml, respectively. Urtoxazumab was well tolerated, appears to be safe at doses of up to 3.0 mg/kg, and is a potential candidate for the prevention of HUS in pediatric patients.
Assuntos
Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli/metabolismo , Toxina Shiga II/antagonistas & inibidores , Toxina Shiga II/biossíntese , Adulto , Idoso , Antibacterianos/efeitos adversos , Antibacterianos/uso terapêutico , Anticorpos/análise , Anticorpos Monoclonais/efeitos adversos , Pré-Escolar , Relação Dose-Resposta a Droga , Método Duplo-Cego , Infecções por Escherichia coli/metabolismo , Feminino , Meia-Vida , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
The aim of this study was to characterize Shiga toxigenic Escherichia coli (STEC) by PCR using strains isolated from ham, beef, and cattle in Colombia. A total of 189 E. coli strains were tested for the presence of the uidA, stx1, and stx2 genes, and identification was confirmed by the automated PCR BAX system for E. coli O157:H7. Genes encoding Shiga-like toxins (stx) were found in eight (6.06%) of 132 strains previously isolated from minced beef; four (50%) of these strains yielded amplification products for both toxin genes (stx1 and stx2), and four (50%) yielded products only for the stx2 toxin. None of the strains analyzed were positive by PCR for the presence of the single base-pair mutation in the uidA gene from E. coli O157:H7; these results were confirmed by the BAX system analysis. A multiplex PCR assay was standardized for the three genes. Results from this study confirmed previous data about the low prevalence of E. coli O157:H7 and Shiga-like toxins in Colombia and is the first known report of the prevalence of non-O157 enterohemorrhagic E. coli in this country.
Assuntos
Contaminação de Alimentos/análise , Produtos da Carne/microbiologia , Reação em Cadeia da Polimerase/métodos , Toxinas Shiga/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Colômbia , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/metabolismo , Microbiologia de Alimentos , Mutação , Prevalência , Toxina Shiga I/biossíntese , Toxina Shiga I/genética , Toxina Shiga II/biossíntese , Toxina Shiga II/genética , Toxinas Shiga/biossíntese , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/metabolismoRESUMO
Current knowledge of the circulating enterohemorrhagic Escherichia coli (EHEC) strains as agents of severe infections has a significant impact on the improvement of diagnostic procedures and control strategies. This report describes a case of hemolytic uremic syndrome related to an uncommon EHEC O165:HNM serotype. As far as we know, this serotype has not been previously associated with human infections, nor has it been isolated from the animal reservoir in São Paulo, Brazil.
Assuntos
Escherichia coli Êntero-Hemorrágica/classificação , Síndrome Hemolítico-Urêmica/microbiologia , Toxina Shiga II/biossíntese , Animais , Brasil , Chlorocebus aethiops , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/isolamento & purificação , Escherichia coli Êntero-Hemorrágica/patogenicidade , Fezes/microbiologia , Feminino , Células HeLa , Síndrome Hemolítico-Urêmica/diagnóstico , Humanos , Lactente , Sorotipagem , Toxina Shiga II/genética , Toxina Shiga II/toxicidade , Células VeroRESUMO
A total of 107 Shiga toxin-producing Escherichia coli strains (STEC) isolated from different origins in São Paulo, Brazil, and belonging to different serotypes were characterized regarding stx subtypes and susceptibility to antimicrobial agents. Most of the human STEC strains harbored stx1 (85.7%), while stx2, associated or not to stx1, was identified preferentially in the animal and food strains. None of the STEC strains carried stx1c. Some genotypes occurred exclusively among strains of bovine origin as stx2c, stx1+2+2c (16.5% each), and stx2d (0.9%), whereas stx2+2c2vha) was only identified among the O157:H7 human strains. Moreover, the stx(2c2vhb) subtype was found more frequently among bovine than human strains (39% vs. 4.8%). The highest frequencies of susceptibility to antimicrobial agents were observed among bovine (87%) and food (100%) STEC strains, while 47.6% of the human isolates were resistant to at least one drug. Multiresistance occurred among O111 STEC strains from human and bovine origin. The antimicrobials to which resistance was most frequently observed were tetracycline (90%) and streptomycin (75%) among human strains, and also sulphazotrin (88%) in animal strains. A few serotypes were commonly identified among STEC strains isolated from diverse sources in Brazil, but in general the strains presented distinct stx subtypes and/or antimicrobial resistance profiles.
Assuntos
Escherichia coli/genética , Toxina Shiga I/genética , Toxina Shiga II/genética , Animais , Brasil , Bovinos , Farmacorresistência Bacteriana/genética , Escherichia coli/classificação , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Microbiologia de Alimentos , Genótipo , Humanos , Sorotipagem , Toxina Shiga I/biossíntese , Toxina Shiga II/biossínteseRESUMO
In order to determine the occurrence, serotypes and virulence markers of Shiga toxin-producing Escherichia coli (STEC) strains, 153 fecal samples of cattle randomly selected from six dairy farms in Sao Paulo State, Brazil, were examined for Shiga toxin (Stx) production by the Vero cell assay. Feces were directly streaked onto MacConkey Sorbitol Agar and incubated at 37 degrees C overnight. Sorbitol-negative colonies (maximum 20) and up to 10 sorbitol-positive colonies from each plate were subcultured onto presumptive diagnostic medium IAL. Sorbitol-negative isolates were screened with O157 antiserum for identification of O157:H7 E. coli. Isolates presenting cytotoxic activity were submitted to colony hybridization assays with specific DNA probes for stx1, stx2, eae, Ehly and astA genes. The isolation rate of STEC ranged from 3.8 to 84.6% depending on the farm analysed. STEC was identified in 25.5% of the animals, and most of them (64.1%) carried a single STEC serotype. A total of 202 STEC isolates were recovered from the animals, and except for the 2 O157:H7 isolates all the others expressed cytotoxic activity. The great majority of the STEC isolates carried both stx1 and stx2 genes (114/202, 56.4%) or stx2 (82/202, 40.6%); and whereas the Ehly sequence occurred in most of them (88%) eae was only observed in O157:H7 and O111:HNM isolates. Serotypes O113:H21, O178:H19 and O79:H14 were the most frequent STEC serotypes identified and widely distributed among animals from different farms, while others such as O77:H18, O88:H25 and O98:H17 occurred only in particular farms. This is the first report on the occurrence of STEC in dairy cattle in Sao Paulo State, and the results point to substantial differences in rate of isolation, serotypes and genetic profile of STEC that has been previously described among beef cattle in our community. Moreover, to our knowledge O79:H14 and O98:H17 represent new STEC serotypes, while O178:H19 has only been recently reported in Spain.
Assuntos
Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli O157/classificação , Toxina Shiga I/biossíntese , Toxina Shiga II/biossíntese , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Brasil , Bovinos , DNA Bacteriano/química , DNA Bacteriano/genética , Indústria de Laticínios , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/genética , Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fezes/microbiologia , Feminino , Hibridização de Ácido Nucleico , Antígenos O/metabolismo , Sorotipagem , Toxina Shiga I/genética , Toxina Shiga II/genéticaRESUMO
Shiga toxin producing-Escherichia coli (STEC), an important emerging foodborne pathogen, has been associated with bloody and non-bloody diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura. The cattle have been shown to be a major reservoir of STEC and raw foods such as ground beef and milk are the most common vehicles of infection. In the present study, the prevalence of STEC in 95 samples of frozen hamburgers and in 114 samples of soft cheese was established in 8.4% and 0.9%, respectively. The genotypic and phenotypic characteristics of the strains were determined. The virulence genes stx1, stx2, eaeA and EHEC-hlyA were identified by PCR and by colony blot hybridization assays. Serotyping, antimicrobial susceptibility and production of Stx using specific cytotoxicity assays on Vero cells were also determined. All STEC strains were characterized as eaeA-/EHEC-hlyA+. The stx2 genotype was prevalent (77.8%), and four different O:H serotypes were found, comprising: O8:H19 (5 strains), O113:H21 (1), O8:H16 (1), and O39:H49 (1). One STEC strain was nontypable. Although soft cheese complimented the microbiological quality controls for the coliform counts, the detection of STEC in one sample raises doubts concerning the effectiveness of the current quality controls. These data contribute to the implementation of strategies for the prevention and control of HUS.
Assuntos
Queijo/microbiologia , Escherichia coli/isolamento & purificação , Contaminação de Alimentos , Microbiologia de Alimentos , Carne/microbiologia , Toxina Shiga I/biossíntese , Toxina Shiga II/biossíntese , Adesinas Bacterianas/biossíntese , Adesinas Bacterianas/genética , Animais , Argentina , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Bovinos , Chlorocebus aethiops , Criopreservação , Resistência a Medicamentos/genética , Escherichia coli/classificação , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Proteínas de Escherichia coli/biossíntese , Inspeção de Alimentos , Conservação de Alimentos , Genótipo , Proteínas Hemolisinas/biossíntese , Fenótipo , Toxina Shiga I/genética , Toxina Shiga II/genética , Células Vero , Virulência/genéticaRESUMO
Different experimental approaches were evaluated for their ability to detect stx genes by PCR and identify Shiga toxin-producing Escherichia coli (STEC) in bovine fecal samples. One hundred and sixty fecal samples from steers in Argentina were processed by protocols that involved: (1) enrichment of fecal samples and DNA extraction using a commercially available kit (Protocol A); (2) plating on selective media after enrichment of the fecal sample followed by heat-lysis DNA extraction from the confluent growth zone (Protocol B); (3) analysis of individual colonies isolated from direct fecal culture on MacConkey agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (Protocol C), used as Gold Standard. PCR performed on bacteria from the confluent growth zone (Protocol B) proved to be the most sensitive methodology. In addition, enrichment for greater than 6h, enhanced sensitivity. Among eight STEC isolates, four were O8:H19 and four were stx2/eae-negative. An STEC isolate was characterized as O26:H11 with a stx1/eae/EHEC-hlyA genotype, often associated with human disease. Finally, no STEC O157 strains were isolated using these methods.