RESUMO
More than 400 serotypes of Shiga toxin-producing Escherichia coli (STEC) have been implicated in outbreaks and sporadic human diseases. In recent years STEC strains belonging to serogroup O178 have been commonly isolated from cattle and food of bovine origin in South America and Europe. In order to explore the significance of these STEC strains as potential human pathogens, 74 German and Argentinean E. coli O178 strains from animals, food and humans were characterized phenotypically and investigated for their serotypes, stx-genotypes and 43 virulence-associated markers by a real-time PCR-microarray. The majority (n = 66) of the O178 strains belonged to serotype O178:H19. The remaining strains divided into O178:H7 (n = 6), O178:H10 (n = 1), and O178:H16 (n = 1). STEC O178:H19 strains were mainly isolated from cattle and food of bovine origin, but one strain was from a patient with hemolytic uremic syndrome (HUS). Genotyping of the STEC O178:H19 strains by pulsed-field gel electrophoresis revealed two major clusters of genetically highly related strains which differ in their stx-genotypes and non-Stx putative virulence traits, including adhesins, toxins, and serine-proteases. Cluster A-strains including the HUS-strain (n = 35) carried genes associated with severe disease in humans (stx2a, stx2d, ehxA, saa, subAB1, lpfAO113 , terE combined with stx1a, espP, iha). Cluster B-strains (n = 26) showed a limited repertoire of virulence genes (stx2c, pagC, lpfAO113 , espP, iha). Among O178:H7 strains isolated from deer meat and patients with uncomplicated disease a new STEC variant was detected that is associated with the genotype stx1c/stx2b/ehxA/subAB2/espI/[terE]/espP/iha. None of the STEC O178 strains was positive for locus of enterocyte effacement (LEE)- and nle-genes. Results indicate that STEC O178:H19 strains belong to the growing group of LEE-negative STEC that should be considered with respect to their potential to cause diseases in humans.
Assuntos
Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/veterinária , Microbiologia de Alimentos , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/isolamento & purificação , Animais , Argentina/epidemiologia , Bovinos , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , Cervos , Eletroforese em Gel de Campo Pulsado , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Genótipo , Alemanha/epidemiologia , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Sorotipagem , Toxina Shiga/genética , Escherichia coli Shiga Toxigênica/genética , Fatores de Virulência/genéticaRESUMO
Escherichia coli strains of serogroup O26 comprise two distinct groups of pathogens, characterized as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among the several genes related to type III secretion system-secreted effector proteins, espK was found to be highly specific for EHEC O26:H11 and its stx-negative derivative strains isolated in European countries. E. coli O26 strains isolated in Brazil from infant diarrhea, foods, and the environment have consistently been shown to lack stx genes and are thus considered atypical EPEC. However, no further information related to their genetic background is known. Therefore, in this study, we aimed to discriminate and characterize these Brazilian O26 stx-negative strains by phenotypic, genetic, and biochemical approaches. Among 44 isolates confirmed to be O26 isolates, most displayed flagellar antigen H11 or H32. Out of the 13 nonmotile isolates, 2 tested positive for fliCH11, and 11 were fliCH8 positive. The identification of genetic markers showed that several O26:H11 and all O26:H8 strains tested positive for espK and could therefore be discriminated as EHEC derivatives. The presence of H8 among EHEC O26 and its stx-negative derivative isolates is described for the first time. The interaction of three isolates with polarized Caco-2 cells and with intestinal biopsy specimen fragments ex vivo confirmed the ability of the O26 strains analyzed to cause attaching-and-effacing (A/E) lesions. The O26:H32 strains, isolated mostly from meat, were considered nonvirulent. Knowledge of the virulence content of stx-negative O26 isolates within the same serotype helped to avoid misclassification of isolates, which certainly has important implications for public health surveillance.
Assuntos
Escherichia coli Êntero-Hemorrágica/isolamento & purificação , Escherichia coli Enteropatogênica/isolamento & purificação , Fenótipo , Adesinas Bacterianas/genética , Aderência Bacteriana/genética , Brasil , Células CACO-2 , Escherichia coli Êntero-Hemorrágica/classificação , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Enteropatogênica/classificação , Escherichia coli Enteropatogênica/genética , Proteínas de Escherichia coli/genética , Marcadores Genéticos , Células HeLa , Proteínas Hemolisinas/genética , Humanos , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Fatores de Virulência/genéticaRESUMO
O125 is an enteropathogenic Escherichia coli (EPEC) serogroup, which includes the O125ac:H6 serotype, defined as atypical EPEC. Strains of this serotype displayed the aggregative adherence (AA) pattern with HEp-2, Caco-2, T84, and HT-29 cells, possessed all the LEE region genes, and expressed intimin, Tir, and EspABD, although the attaching-effacing lesion was not detected in vitro. These results confirm that E. coli O125ac:H6 is atypical EPEC that displays the AA pattern and indicate the necessity of testing for EPEC genes combined with the determination of the adherence pattern for atypical EPEC identification.
Assuntos
Aderência Bacteriana , Células Epiteliais/microbiologia , Escherichia coli/isolamento & purificação , Escherichia coli/fisiologia , Linhagem Celular , Escherichia coli/classificação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Fatores de Virulência/genéticaRESUMO
STEC has emerged as an important group of enteric pathogens worldwide. In this study, rabbit polyclonal Stx1 and Stx2 antisera were raised and employed in the standardization of immunoassays for STEC detection. Using their respective antisera, the limit of detection of the toxin was 35.0 pg for Stx1 and 5.4 pg for Stx2. By immunoblotting, these antisera recognized both toxin subunits. Cross-reactivity was observed in the A subunit, but only Stx2 antiserum was able to neutralize the cytotoxicity of both toxins in the Vero cell assay. Six stx-harboring E. coli isolates were analyzed for their virulence traits. They belonged to different serotypes, including the O48:H7, described for the first time in Brazil. Only three strains harbored eae, and the e-hly gene and hemolytic activity was detected in five strains. Three isolates showed new stx2 variants (stx(2v-ha) and stx(2vb-hb)). The ELISA assay detected all six isolates, including one VCA-negative isolate, while the immunodot assay failed to detect one isolate, which was VCA-positive. In contrast, the colony-immunoblot assay detected only one VCA-positive isolate. Our results demonstrate that among the immunoassays developed in this study, the immunodot, and particularly the ELISA, appear as perspective for STEC detection in developing countries.
Assuntos
Anticorpos Antibacterianos/biossíntese , Ensaio de Imunoadsorção Enzimática/métodos , Infecções por Escherichia coli/microbiologia , Toxina Shiga I/análise , Toxina Shiga II/análise , Escherichia coli Shiga Toxigênica/isolamento & purificação , Animais , Anticorpos Antibacterianos/análise , Sobrevivência Celular , Chlorocebus aethiops , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Immunoblotting , Reação em Cadeia da Polimerase , Coelhos , Toxina Shiga I/imunologia , Toxina Shiga II/imunologia , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/patogenicidade , Células Vero , VirulênciaRESUMO
Enteroaggregative Escherichia coli (EAEC) is characterized by the expression of the aggregative adherence pattern to cultured epithelial cells. In this study, we determined the phenotypic and genotypic relationships among 86 EAEC strains of human and animal (calves, piglets and horses) feces. Serotypes and the presence of EAEC virulence markers were determined, and these results were associated with ribotyping. Strains harboring aggR (typical EAEC) of human origin were found carrying several of the searched markers, while atypical EAEC harbored none or a few markers. The strains of animal origin were classified as atypical EAEC (strains lacking aggR) and harbored only irp2 or shf. Strains from humans and animals belonged to several different serotypes, although none of them prevailed. Sixteen ribotypes were determined, and there was no association with virulence genes profiles or serotypes. Relationship was not found among the strains of this study, and the assessed animals may not represent a reservoir of human pathogenic typical EAEC.
Assuntos
Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/genética , Escherichia coli/fisiologia , Virulência/genética , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Impressões Digitais de DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/genética , Doenças dos Cavalos/microbiologia , Cavalos/microbiologia , Humanos , Proteína 2 Reguladora do Ferro/genética , Epidemiologia Molecular , Hibridização de Ácido Nucleico , Filogenia , Ribotipagem , Sorotipagem , Suínos/microbiologia , Doenças dos Suínos/microbiologia , Transativadores/genética , Fatores de Virulência/genéticaRESUMO
We report the frequency of the different diarrheagenic Escherichia coli (DEC) categories isolated from children with acute endemic diarrhea in Salvador, Bahia. The E. coli isolates were investigated by colony blot hybridization with the following genes probes: eae, EAF, bfpA, Stx1, Stx2, ST-Ih, ST-Ip, LT-I, LT-II, INV, and EAEC, as virulence markers to distinguish typical and atypical EPEC, EHEC/STEC, ETEC, EIEC, and EAEC. Seven of the eight categories of DEC were detected. The most frequently isolated was atypical EPEC (10.1%) followed by ETEC (7.5%), and EAEC (4.2%). EHEC, STEC, EIEC, and typical EPEC were each detected once. The strains of ETEC, EAEC, and atypical EPEC belonged to a wide variety of serotypes. The serotypes of the others categories were O26:H11 (EHEC), O21:H21 (STEC), O142:H34 (typical EPEC), and O:H55 (EIEC). We also present the clinical manifestations and other pathogenic species observed in children with DEC. This is the first report of EHEC and STEC in Salvador, and one of the first in Brazil.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Diarreia/microbiologia , Doenças Endêmicas , Infecções por Escherichia coli/microbiologia , Escherichia coli/classificação , Brasil/epidemiologia , Pré-Escolar , Diarreia/epidemiologia , Escherichia coli/genética , Infecções por Escherichia coli/epidemiologia , Fezes/microbiologia , Genótipo , Humanos , Fenótipo , PrevalênciaRESUMO
We report the frequency of the different diarrheagenic Escherichia coli (DEC) categories isolated from children with acute endemic diarrhea in Salvador, Bahia. The E. coli isolates were investigated by colony blot hibridization whit the following genes probes: eae, EAF, bfpA, Stx1, Stx2, ST-Ih, ST-Ip, LT-I, LT-II, INV, and EAEC, as virulence markers to distinguish typical and atypical EPEC, EHEC/STEC, ETEC, EIEC, and EAEC. Seven of the eight categories of DEC were detected. The most frequently isolated was atypical EPEC (10.1 percent) followed by ETEC (7.5 percent), and EAEC (4.2 percent). EHEC, STEC, EIEC, and typical EPEC were each detected once. The strains of ETEC, EAEC, and atypical EPEC belonged to a wide variety of serotypes. The serotypes of the others categories were O26:H11 (EHEC), O21:H21 (STEC), O142:H34 (typical EPEC), and O?H55 (EIEC). We also present the clinical manifestations and other pathogenic species observed in children with DEC. This is the first report of EHEC and STEC in Salvador, and one of the first in Brazil.