RESUMO

The latex agglutination test using single-chain antibody fragments (scFvStx1 and scFvStx2) coupled to latex particles, was compared with the gold standard Vero cell assay for Shiga toxin (Stx) detection, aiming to estimate the diagnosis potential of these scFv fragments in a rapid and straightforward test. The latex complexes identified the presence of the toxins up to a 1:8 dilution in the majority of the evaluated strains. Moreover, the Stx concentration was indirectly determined in Stx-producing Escherichia coli (STEC) strains, allowing detection limit inference. A Stx dilution curve was constructed, and the data was analyzed in a non-linear model by second-order polynomial regression for prediction (p-value of 0.001 and a R2 above 0.98 were considered for correlations). The detection limit was 30 ng/mL for Stx1 and 10 ng/mL for Stx2. The scFvStx1 and scFvStx2 coupled to latex nanoparticles provide a toxin assay with a competitive Stx detection limit, which has a low cost and short execution time. The diagnostic method proposed here, using, for the first time, recombinant antibody fragments, raises the possibility of developing a more affordable test to be used in the routine detection and surveillance of STEC infections.

Assuntos

Infecções por Escherichia coli/diagnóstico , Testes de Fixação do Látex , Toxina Shiga I/isolamento & purificação , Toxina Shiga II/isolamento & purificação , Escherichia coli Shiga Toxigênica , Anticorpos de Cadeia Única/imunologia , Animais , Chlorocebus aethiops , Proteínas Recombinantes/imunologia , Escherichia coli Shiga Toxigênica/isolamento & purificação , Células Vero

RESUMO

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.

Assuntos

Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Doenças dos Ovinos/microbiologia , Ovinos/microbiologia , Escherichia coli Shiga Toxigênica/genética , Fatores de Virulência/genética , Animais , Reservatórios de Doenças , Infecções por Escherichia coli/microbiologia , Variação Genética , Doenças dos Ovinos/epidemiologia , Toxina Shiga II/metabolismo , Escherichia coli Shiga Toxigênica/imunologia , Escherichia coli Shiga Toxigênica/isolamento & purificação

RESUMO

countries. Intimate adhesion of the bacteria to intestinal cells occurs via binding of the adhesin intimin to theTIR receptor exposed on cell surfaces. Here, Lactobacillus casei expressing a fragment of -intimin (L.casei-Intcv) was tested as mucosal vaccines in mice against intestinal colonization with the murine pathogenCitrobacter rodentium. Oral or sublingual immunization of C57BL/6 mice with L. casei-Intcv induced anti-IntcvIgA in feces but no IgG in sera. Conversely, anti-Intcv IgG was induced in the sera of mice after sublingualimmunization with purified Intcv. All vaccines were able to decrease C. rodentium recovery from feces. However,this reduction was more evident and sustained over time in mice immunized with L. casei-Intcv by thesublingual route. These mice also displayed an increase in interleukin 6 (IL-6) and gamma interferon (IFN- )secretion by spleen cells 10 days after infection. Additionally, oral or sublingual immunization of C3H/HePasmice, which are highly susceptible to C. rodentium infection, with L. casei-Intcv induced anti-Intcv antibodiesand significantly increased survival after challenge. Immunohistological analysis of colon sections revealedthat C. rodentium was located in deep fractions of the tissue from C3H/HePas mice immunized with L. casei whereas superficial staining was observed in colon sections from mice immunized with L. casei-Intcv. The results indicate that vaccines composed of L. casei expressing intimin may represent a promising approach and that the C3H/HePas infection model with C. rodentium can be used to evaluate potential vaccines against EPEC.

Assuntos

Ratos , Administração Oral , Administração Sublingual , Interferon gama , Baço/anatomia & histologia , Baço/imunologia , Citrobacter rodentium/patogenicidade , Lacticaseibacillus casei/genética

RESUMO

Enteropathogenic Escherichia coli (EPEC) is a common cause of diarrhea in children from developing countries. Intimate adhesion of the bacteria to intestinal cells occurs via binding of the adhesin intimin to the TIR receptor exposed on cell surfaces. Here, Lactobacillus casei expressing a fragment of ß-intimin (L. casei-Int(cv)) was tested as mucosal vaccines in mice against intestinal colonization with the murine pathogen Citrobacter rodentium. Oral or sublingual immunization of C57BL/6 mice with L. casei-Int(cv) induced anti-Int(cv) IgA in feces but no IgG in sera. Conversely, anti-Int(cv) IgG was induced in the sera of mice after sublingual immunization with purified Int(cv). All vaccines were able to decrease C. rodentium recovery from feces. However, this reduction was more evident and sustained over time in mice immunized with L. casei-Int(cv) by the sublingual route. These mice also displayed an increase in interleukin 6 (IL-6) and gamma interferon (IFN-γ) secretion by spleen cells 10 days after infection. Additionally, oral or sublingual immunization of C3H/HePas mice, which are highly susceptible to C. rodentium infection, with L. casei-Int(cv) induced anti-Int(cv) antibodies and significantly increased survival after challenge. Immunohistological analysis of colon sections revealed that C. rodentium was located in deep fractions of the tissue from C3H/HePas mice immunized with L. casei whereas superficial staining was observed in colon sections from mice immunized with L. casei-Int(cv.) The results indicate that vaccines composed of L. casei expressing intimin may represent a promising approach and that the C3H/HePas infection model with C. rodentium can be used to evaluate potential vaccines against EPEC.

Assuntos

Adesinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Citrobacter rodentium/imunologia , Portadores de Fármacos , Infecções por Enterobacteriaceae/prevenção & controle , Proteínas de Escherichia coli/imunologia , Vetores Genéticos , Lacticaseibacillus casei/genética , Adesinas Bacterianas/genética , Administração Oral , Administração Sublingual , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Citrobacter rodentium/genética , Colo/patologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/mortalidade , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Feminino , Humanos , Imunização/métodos , Interferon gama/metabolismo , Interleucina-6/metabolismo , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Baço/imunologia , Análise de Sobrevida , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia

RESUMO

AIMS: To evaluate the sensitivity and specificity of polyclonal and monoclonal antibodies (Mabs) against intimin in the detection of enteropathogenic and enterohaemorrhagic Escherichia coli isolates using immunoblotting. METHODS AND RESULTS: Polyclonal and Mabs against the intimin-conserved region were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti-intimin IgG-enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of 1.3x10(-8) mol l(-1), failed in the detection of some of these isolates. CONCLUSION: All employed antibodies showed 100% specificity, not reacting with any of the eae-negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti-intimin IgG-enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. SIGNIFICANCE AND IMPACT OF THE STUDY: The rabbit anti-intimin IgG-enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.

Assuntos

Adesinas Bacterianas/imunologia , Anticorpos Monoclonais/imunologia , Escherichia coli Êntero-Hemorrágica/classificação , Escherichia coli Enteropatogênica/classificação , Proteínas de Escherichia coli/imunologia , Animais , Especificidade de Anticorpos , Feminino , Soros Imunes/imunologia , Immunoblotting/métodos , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Ratos , Sensibilidade e Especificidade

RESUMO

AIMS: The aim of study was to develop a colony immunoblot assay to differentiate typical from atypical enteropathogenic Escherichia coli (EPEC) by detection of bundle-forming pilus (BFP) expression. METHODS AND RESULTS: Anti-BFP antiserum was raised in rabbits and its reactivity was confirmed by immunoelectron microscopy and by immunoblotting recognizing bundlin, the major pilus repeating subunit. The bacterial isolates tested in the colony immunoblot assay were grown in different media. Proteins from bacterial isolates were transferred to nitrocellulose membrane after treatment with phosphate buffer containing Triton X-100, EDTA and sodium chloride salts. When 24 typical EPEC and 96 isolates including, 72 atypical EPEC, 13 Gram-negative type IV-expressing strains and 11 enterobacteriaceae were cultivated in Dulbecco's Modified Eagle's Medium agar containing fetal bovine serum or in blood agar in the presence of CaCl(2) , they showed a positivity of 92 and 83%, and specificity of 96 and 97%, respectively. CONCLUSION: The assay enables reliable identification of BFP-expressing isolates and contributes to the differentiation of typical and atypical EPEC. SIGNIFICANCE AND IMPACT OF THE STUDY: The colony immunoblot for BFP detection developed in this study combines the simplicity of an immunoserological assay with the high efficiency of testing a large number of EPEC colonies.

Assuntos

Técnicas de Tipagem Bacteriana/métodos , Escherichia coli Enteropatogênica/classificação , Fímbrias Bacterianas/química , Immunoblotting/métodos , Animais , Escherichia coli Enteropatogênica/isolamento & purificação , Coelhos

RESUMO

The aim of study was to develop a colony immunoblot assay to differentiatetypical from atypical enteropathogenic Escherichia coli (EPEC) by detectionof bundle-forming pilus (BFP) expression. Anti-BFP antiserum was raised in rabbits and itsreactivity was confirmed by immunoelectron microscopy and by immunoblotting recognizing bundlin, the major pilus repeating subunit. The bacterial isolates tested in the colony immunoblot assay were grown in different media. Proteins from bacterial isolates were transferred to nitrocellulose membrane after treatment with phosphate buffer containing Triton X-100, EDTA and sodium chloride salts. When 24 typical EPEC and 96 isolates including, 72 atypical EPEC, 13 Gram-negative type IV-expressing strains and 11 enterobacteriaceae were cultivated in Dulbecco’s Modified Eagle’s Medium agar containing fetal bovine serum or in blood agar in the presence of CaCl2, they showed a positivity of 92 and 83%, and specificity of 96 and 97%, respectively. The assay enables reliable identification of BFP-expressing isolatesand contributes to the differentiation of typical and atypical EPEC.The colony immunoblot for BFP detectiondeveloped in this study combines the simplicity of an immunoserologicalassay with the high efficiency of testing a large number of EPECcolonies.

Assuntos

Humanos , Escherichia coli Enteropatogênica , Escherichia coli Enteropatogênica/genética , Immunoblotting/métodos , Polietilenoglicóis/análise

RESUMO

To evaluate the sensitivity and specificity of polyclonal and monoclonalantibodies (Mabs) against intimin in the detection of enteropathogenic andenterohaemorrhagic Escherichia coli isolates using immunoblotting.Polyclonal and Mabs against the intimin-conservedregion were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti-intimin IgG-enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of1Æ3 · 10)8 mol l)1, failed in the detection of some of these isolates. All employed antibodies showed 100% specificity, not reacting with any of the eae-negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti-intimin IgG-enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. Significance and Impact of the Study: The rabbit anti-intimin IgG-enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.

Assuntos

Coelhos , Ratos , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/classificação , Anticorpos Monoclonais/imunologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Bactérias/classificação , Bactérias/crescimento & desenvolvimento , Immunoblotting/métodos

RESUMO

Members of the genera Bacteroides and Parabacteroides are important constituents of both human and animal intestinal microbiota, and are significant facultative pathogens. In this study, the ability of Bacteroides spp. and Parabacteroides distasonis isolated from both diarrhoeal and normal stools (n = 114) to adhere to and invade HEp-2 cells was evaluated. The presence of putative virulence factors such as capsule and fimbriae was also investigated. Adherence to HEp-2 cells was observed in 75.4% of the strains, which displayed non-localized clusters. Invasion was observed in 37.5% and 26% of the strains isolated from diarrhoeal and non-diarrhoeal stools, respectively. All strains displayed a capsule, whereas none of them showed fimbriae-like structures. This is the first report of the ability of Bacteroides spp. and P. distasonis to adhere to and invade cultured HEp-2 epithelial cells.

Assuntos

Aderência Bacteriana , Bacteroidetes/fisiologia , Bacteroidetes/patogenicidade , Diarreia/microbiologia , Trato Gastrointestinal/microbiologia , Animais , Cápsulas Bacterianas/análise , Bacteroidetes/citologia , Bacteroidetes/isolamento & purificação , Linhagem Celular , Criança , Pré-Escolar , Contagem de Colônia Microbiana , Citosol/microbiologia , Células Epiteliais/microbiologia , Fezes/microbiologia , Fímbrias Bacterianas , Humanos , Lactente , Microscopia Eletrônica , Microscopia Imunoeletrônica , Fatores de Virulência/análise

RESUMO

AIMS: To determine the suitability of eight different commercial broth media for Shiga toxin (Stx) production. METHODS AND RESULTS: Shiga toxin-producing Escherichia coli (STEC) strains producing Stx1 or Stx2 were grown at 37 degrees C (250 rev min(-1)) for 24 h in brain heart infusion broth, E. coli broth, Evans medium, Luria-Bertani broth, Penassay broth, buffered-peptone water, syncase broth and trypticase soy broth. Toxin production was measured by enzyme-linked immunosorbent assay (ELISA) in polymyxin-treated cell pellets and/or supernatants of cultures, ELISA optical densities reached 1 when isolates were grown for 2-4 h in E. coli broth in the presence of antibiotic. Besides, a collection of STEC-expressing Stx strains was evaluated and the Stx production was assayed in the supernatants and in polymyxin-treated pellets of bacterial growth after 4 h of cultivation in E. coli broth in the presence of antibiotic. CONCLUSIONS: The most suitable medium for Stx production was E. coli broth when the bacterial isolates were grown for 4 h in the presence of ciprofloxacin and the Stx production is detected in the supernatant. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents the first comprehensive comparison of different broth media with regard to Stx production to establish optimal culture conditions for STEC detection in routine diagnostic laboratories.

Assuntos

Meios de Cultura , Escherichia coli/metabolismo , Toxinas Shiga/biossíntese , Animais , Bovinos , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática , Escherichia coli/patogenicidade , Células Vero

RESUMO

1. Trypanosoma cruzi epimastigote forms are very rapidly removed from the circulation of normal and C5-deficient mice. Depletion of C3 by cobra venom factor results in a significant delay in parasite clearance. 2. During parasite clearance there is a significant decrease in the number of circulating platelets and parasite clearance is considerably delayed in thrombocytopenic animals. 3. In vitro incubation of epimastigote forms with normal mouse serum leads to the formation of parasite clumps provided that platelets are present. Innactivation of factor B or depletion of C3 prevents this phenomenon. 4. When epimastigotes are incubated with normal mouse serum they absorb one or more factors required for their aggregation with platelets. 5. It is suggested that in mice T. cruzi epimastigote forms are removed from circulation by the alternative pathway of complement activation and that both C3 and platelets are required for parasite clearance

Assuntos

Animais , Camundongos , Plaquetas/parasitologia , Complemento C3/metabolismo , Complemento C5/deficiência , Trypanosoma cruzi/fisiologia , Ativação do Complemento , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Agregação Plaquetária