RESUMO
The presence of E. coli producer of shiga toxin and rotavirus was investigated in 90 stool samples from children less than 3 years old with diarrhea. Three aliquots were separated from each sample: the first one underwent previous enrichment for E. coli O157, the second one was plated on agar MacConkey-Sorbitol and Red Eosine with MUG, and the last one was frozen at -70 degrees C for the later analysis of rotavirus. The search of the antigen O157 of E. coli was carried out by immunochromatography in vitro of Coris Bioconcept (Belgium). The presence of the antigen O157 (rbf O157) and the genes that code for the shiga toxin (stx1 and stx2) were determined by PCR. Rotavirus were detected by electrophoresis in polyacrylamide gels. The 90 samples analyzed by immunochromatography were negative for the antigen O157. The isolates were STEC strains non O157 and contained the gene sx1, showing a 10% of positivity. The electrophoresis for the viral RNA detected rotavirus in 21 (23.33%) samples. This result confirms the rotavirus prevalence and suggests, that the circulation of STEC strains non O157 is an indication of the involvement of these strains in the ethiology of acute diarrheas.
Assuntos
Diarreia Infantil/microbiologia , Escherichia coli/isolamento & purificação , Fezes/microbiologia , Rotavirus/isolamento & purificação , Pré-Escolar , Diarreia Infantil/virologia , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/metabolismo , Fezes/virologia , Humanos , Lactente , Toxina Shiga I/análise , Toxina Shiga I/biossínteseRESUMO
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
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.
Assuntos
Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/metabolismo , Reação em Cadeia da Polimerase/veterinária , Toxina Shiga I/biossíntese , Toxina Shiga II/biossíntese , Animais , Argentina , Bovinos , DNA Bacteriano/química , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Masculino , Antígenos O/sangue , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Toxina Shiga I/genética , Toxina Shiga II/genética , VirulênciaRESUMO
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.(AU)