RESUMEN
INTRODUCTION: We evaluated the presence of sIgA in saliva, versus Escherichia coli secreted proteins (Esp) related to the type III secretion system (T3SS), and its semi-quantitative concentration in children under 2 years-old (no longer breastfed) who were previously colonized or infected with enteropathogenic E. coli (EPEC). METHODS: We analyzed the presence of sIgA in 40 children, who previously had positive cultures for EPEC associated (n=17) or not associated (n=23) with diarrhea, using the Western Blot technique versus E. coli secreted proteins: EspABCD. A semi-quantitative measurement of the reaction for each protein was made by its density peaks (OD). RESULTS: We found sIgA versus all or some EspABCD proteins in both groups. However, the ill patients had higher concentrations of these antibodies than colonized patients. DISCUSSION: The presence of sIgA in saliva could reflect an intestinal immune response and their levels could be related to a greater exposure and/or bacterial load.
Asunto(s)
Escherichia coli Enteropatógena , Infecciones por Escherichia coli , Proteínas de Escherichia coli , Inmunoglobulina A Secretora/análisis , Factores de Virulencia/análisis , Escherichia coli Enteropatógena/inmunología , Infecciones por Escherichia coli/diagnóstico , Infecciones por Escherichia coli/inmunología , Proteínas de Escherichia coli/análisis , Humanos , Lactante , Saliva/inmunología , Sistemas de Secreción Tipo III/análisisRESUMEN
Abstract Objective: Intimins are protein adhesins of enteropathogenic Escherichia coli and enterohemorrhagic E. coli capable of inducing attachment and effacement lesions in enterocytes. Anti-intimin antibodies are important for the protection from enteropathogenic E. coli and enterohemorrhagic E. coli infections because these antibodies inhibit bacterial adhesion and impair the initial step of the pathogenesis. We studied the transfer of maternal anti-intimin antibodies from healthy Brazilian mothers to their newborns through the placenta and colostrum. Methods: Serum immunoglobulin G and secretory immunoglobulin A antibodies against conserved and variable regions of intimins α, β, and γ were analyzed using an enzyme linked-immunosorbent assay in the blood and colostrum from 45 healthy women as well as cord blood serum samples from their newborns. Results: The concentrations of antibodies reactive with α intimin were significantly lower than those of anti-γ and anti-conserved intimin antibodies in the colostrum samples. IgG serum antibodies reactive with all the subtypes of intimins were transferred to the newborns, but the concentrations of anti-conserved intimin serum antibodies were significantly higher in mothers and newborns than concentrations of antibodies against variable regions. The patterns of IgG transfer from mothers to newborns were similar for all anti-intimin antibodies. These values are similar to the percentage transference of total IgG. Conclusions: Anti-intimin antibodies are transferred from mothers to newborns through the placenta, and reinforce the protection provided by breastfeeding against diarrheagenic E. coli infections.
Resumo Objetivo: As intiminas são adesinas proteicas de Escherichia coli enteropatogênicas (EPEC) e enterro-hemorrágicas (EHEC) capazes de induzir as lesões attaching and effacing nos enterócitos. Anticorpos anti-intiminas são importantes para a proteção contra infecções por EPEC e EHEC porque esses anticorpos inibem a adesão bacteriana e impedem o passo inicial do mecanismo patogênico dessas bactérias. Nós estudamos a transferência de anticorpos maternos anti-intiminas de mães brasileiras saudáveis para os seus recém-nascidos através da placenta e do colostro. Métodos: Anticorpos séricos da classe IgG e secretórios da classe IgA (SIgA) reativos com as porções conservada (cons) e variáveis das intiminas α (vα), β (vβ) e γ (vγ) foram analisados pelo teste de ELISA no sangue e no colostro de 45 parturientes saudáveis e no sangue de cordão umbilical dos seus respectivos recém-nascidos. Resultados: As concentrações de anticorpos reativos com intimina vα foram significativamente mais baixas que as dos anticorpos anti-vγ e anti-cons nas amostras de colostro. Anticorpos IgG séricos reativos com todas as intiminas foram transferidos para os recém-nascidos, mas as concentrações de anti-cons foram significativamente mais altas tanto nas mães como nos recém-nascidos do que os anticorpos reativos com as regiões variáveis das intiminas. O padrão de transferência de IgG das mães para os recém-nascidos foi muito semelhante para todos os anticorpos anti-intiminas. Os valores de porcentagem de transferência foram semelhantes à transferência de IgG total. Conclusões: Anticorpos anti-intimina são transferidos das mães para os recém-nascidos pela placenta e corroboram a proteção contra infecções por Escherichia coli diarreiogênicas (DEC) conferida pelo aleitamento materno.
Asunto(s)
Humanos , Femenino , Recién Nacido , Autoanticuerpos/análisis , Inmunoglobulina A Secretora/análisis , Inmunoglobulina G/análisis , Calostro/inmunología , Escherichia coli Enteropatógena/inmunología , Sangre Fetal/inmunología , Ensayo de Inmunoadsorción Enzimática , Adhesinas Bacterianas/análisis , Adhesinas Bacterianas/inmunología , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/inmunologíaAsunto(s)
Humanos , Femenino , Recién Nacido , Infecciones por Escherichia coli/inmunología , Escherichia coli Enteropatógena/inmunología , Inmunidad Materno-Adquirida/inmunología , Adhesinas Bacterianas/metabolismo , Infecciones por Escherichia coli/metabolismo , Escherichia coli Enteropatógena/metabolismoAsunto(s)
Escherichia coli Enteropatógena/inmunología , Infecciones por Escherichia coli/inmunología , Inmunidad Materno-Adquirida/inmunología , Adhesinas Bacterianas/metabolismo , Escherichia coli Enteropatógena/metabolismo , Proteínas de Escherichia coli/metabolismo , Femenino , Humanos , Recién NacidoRESUMEN
OBJECTIVE: Intimins are protein adhesins of enteropathogenic Escherichia coli and enterohemorrhagic E. coli capable of inducing attachment and effacement lesions in enterocytes. Anti-intimin antibodies are important for the protection from enteropathogenic E. coli and enterohemorrhagic E. coli infections because these antibodies inhibit bacterial adhesion and impair the initial step of the pathogenesis. We studied the transfer of maternal anti-intimin antibodies from healthy Brazilian mothers to their newborns through the placenta and colostrum. METHODS: Serum immunoglobulin G and secretory immunoglobulin A antibodies against conserved and variable regions of intimins α, ß, and γ were analyzed using an enzyme linked-immunosorbent assay in the blood and colostrum from 45 healthy women as well as cord blood serum samples from their newborns. RESULTS: The concentrations of antibodies reactive with α intimin were significantly lower than those of anti-γ and anti-conserved intimin antibodies in the colostrum samples. IgG serum antibodies reactive with all the subtypes of intimins were transferred to the newborns, but the concentrations of anti-conserved intimin serum antibodies were significantly higher in mothers and newborns than concentrations of antibodies against variable regions. The patterns of IgG transfer from mothers to newborns were similar for all anti-intimin antibodies. These values are similar to the percentage transference of total IgG. CONCLUSIONS: Anti-intimin antibodies are transferred from mothers to newborns through the placenta, and reinforce the protection provided by breastfeeding against diarrheagenic E. coli infections.
Asunto(s)
Autoanticuerpos/análisis , Calostro/inmunología , Escherichia coli Enteropatógena/inmunología , Sangre Fetal/inmunología , Inmunoglobulina A Secretora/análisis , Inmunoglobulina G/análisis , Adhesinas Bacterianas/análisis , Adhesinas Bacterianas/inmunología , Autoanticuerpos/inmunología , Ensayo de Inmunoadsorción Enzimática , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/inmunología , Femenino , Humanos , Recién NacidoRESUMEN
The therapeutic action of phosphorylated mannanoligosaccharides (MOS) was investigated regarding its prebiotic activity on enteropathogenic Escherichia coli (EPEC). Diarrhea was induced in dogs by experimental infection with EPEC strains. Then MOS was supplied once a day, in water for 20 days. Immunological (IgA and IgG), hematological (lymphocytes, neutrophils and monocytes) and bacteriological variables (PCR detection of the eae gene of EPEC recovered from stool culture), as well as occurrence of diarrhea were evaluated. All strains caused diarrhea at 24, 48 and 72 h after infection. PCR results indicated that E. coli isolated from stool culture of all infected animals had the eae gene. There was no significant difference among groups as to number of blood cells in the hemogram and IgA and IgG production. MOS was effective in recovering of EPEC-infected dogs since prebiotic-treated animals recovered more rapidly from infection than untreated ones (p < 0.05). This is an important finding since diarrhea causes intense dehydration and nutrient loss. The use of prebiotics for humans and other animals with diarrhea can be an alternative for the treatment and prophylaxis of EPEC infections.
Asunto(s)
Sangre/inmunología , Diarrea/microbiología , Escherichia coli Enteropatógena/inmunología , Heces , Fármacos Gastrointestinales/metabolismo , Oligosacáridos/metabolismo , Prebióticos , Animales , Anticuerpos Antibacterianos/sangre , Fenómenos Químicos , Modelos Animales de Enfermedad , Perros , Escherichia coli , Fármacos Gastrointestinales/administración & dosificación , Fármacos Gastrointestinales/química , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Leucocitos/inmunología , Oligosacáridos/administración & dosificación , Oligosacáridos/químicaRESUMEN
Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to inject effectors into host cells and alter cellular physiology. The aim of the present study was to identify targets of human secretory immunoglobulin A (sIgA) antibodies from the proteins delivered by EPEC into HEp-2 cells after infection. Bacterial proteins delivered into EPEC-infected cells were obtained in sub-cellular fractions (cytoplasmic, membrane, and cytoskeleton) and probed with sIgA antibodies from human milk and analyzed by Western blotting. These sIgA antibodies reacted with Tir and EspB in the cytoplasmic and membrane fractions, and with intimin in the membrane fraction mainly. The sIgA also identified an EPEC surface-associated Heat-shock protein 70 (Hsp70) in HEp-2 cells infected with EPEC. Purified Hsp70 from EPEC was able to bind to HEp-2 cells, suggesting adhesive properties in this protein. EspC secreted to the medium reacted strongly with the sIgA antibodies. An EPEC 115 kDa protein, unrelated to the EspC protein, was detected in the cytoplasm of infected HEp-2 cells, suggesting that this is a new protein translocated by EPEC. The results suggest that there is a strong host antibody response to Tir and intimin, which are essential proteins for attaching and effacing (A/E) pathogen mediated disease.
Asunto(s)
Escherichia coli Enteropatógena/inmunología , Proteínas de Escherichia coli/inmunología , Inmunoglobulina A Secretora/inmunología , Leche Humana/inmunología , Células Hep G2 , Humanos , Factores de Virulencia/inmunologíaRESUMEN
The therapeutic action of phosphorylated mannanoligosaccharides (MOS) was investigated regarding its prebiotic activity on enteropathogenic Escherichia coli (EPEC). Diarrhea was induced in dogs by experimental infection with EPEC strains. Then MOS was supplied once a day, in water for 20 days. Immunological (IgA and IgG), hematological (lymphocytes, neutrophils and monocytes) and bacteriological variables (PCR detection of the eae gene of EPEC recovered from stool culture), as well as occurrence of diarrhea were evaluated. All strains caused diarrhea at 24, 48 and 72 h after infection. PCR results indicated that E. coli isolated from stool culture of all infected animals had the eae gene. There was no significant difference among groups as to number of blood cells in the hemogram and IgA and IgG production. MOS was effective in recovering of EPEC-infected dogs since prebiotic-treated animals recovered more rapidly from infection than untreated ones (p < 0.05). This is an important finding since diarrhea causes intense dehydration and nutrient loss. The use of prebiotics for humans and other animals with diarrhea can be an alternative for the treatment and prophylaxis of EPEC infections.
Asunto(s)
Animales , Perros , Sangre/inmunología , Diarrea/microbiología , Escherichia coli Enteropatógena/inmunología , Heces , Fármacos Gastrointestinales/metabolismo , Oligosacáridos/metabolismo , Prebióticos , Anticuerpos Antibacterianos/sangre , Fenómenos Químicos , Modelos Animales de Enfermedad , Escherichia coli , Fármacos Gastrointestinales/administración & dosificación , Fármacos Gastrointestinales/química , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Leucocitos/inmunología , Oligosacáridos/administración & dosificación , Oligosacáridos/químicaRESUMEN
A Escherichia coli enteropatogênica EPEC é um importante patógeno humano, que ao aderir nas células epiteliais do intestino, provoca redução na capacidade de absorção da mucosa intestinal causando diarréia. Os trabalhos desenvolvidos in vitro não mostram toda a complexidade do mecanismo de infecção por EPEC. A transferência passiva de anticorpos para o recém-nascido é importante para sua proteção durante o amadurecimento do seu sistema imunológico. As linhagens estudadas neste trabalho são camundongos selecionados geneticamente de acordo com a intensidade inflamatória aguda (AIRmin e AIRmax). Objetivo: O objetivo do presente projeto foi avaliar a resposta imune de mucosas destas linhagens após a inoculação de Escherichia coli enteropatogênica (EPEC) por via oral e observar a possível transferência passiva de anticorpos para a prole. Métodos: Camundongos de 8 semanas de idade receberam doses de EPEC de 1010 ufc por via oral. Foram feitas avaliações clínicas, pesagem e coleta de fluídos (sangue, fezes e leite) para pesquisa de anticorpos séricos (IgG) e secretores (IgA) por ensaio de ELISA. Para avaliar a transferência de anticorpos entre mãe e prole, também foram coletados sangue e leite dos recém-nascidos para pesquisa de anticorpos anti-EPEC através de ELISA. Resultados e Discussão: Não houve variação de peso nos animais tratados com a bactéria em comparação com os controles. A inoculação não provocou diarréia e nenhum outro sintoma de doença, mas foi capaz de estimular a produção de anticorpos anti-EPEC nos animais adultos. Os recém-nascidos de mães tratadas com a bactéria apresentaram níveis de anticorpos mais elevados em relação aos grupos controles. Esse modelo de infecção oral se mostrou adequado para o estudo de transferência passiva de anticorpos.
Asunto(s)
Masculino , Femenino , Animales , Adulto , Ratones , Ratones/inmunología , Escherichia coli Enteropatógena/inmunología , Formación de Anticuerpos , InmunidadRESUMEN
Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhea in children. EPEC adheres to the intestinal epithelium and causes attaching and effacing (A/E) lesions. Recombinant Mycobacterium smegmatis (Smeg) and Mycobacterium bovis BCG strains were constructed to express either BfpA or intimin. The entire bfpA gene and a portion of the intimin gene were amplified by PCR from EPEC genomic DNA and inserted into the pMIP12 vector at the BamHI/KpnI sites. The pMIP_bfpA and pMIP_intimin vectors were introduced separately into Smeg and BCG. Recombinant clones were selected based on kanamycin resistance and designated rSmeg_pMIP_(bfpA or intimin) and rBCG_pMIP_(bfpA or intimin). The expression of bfpA and intimin was detected by Immunoblotting using polyclonal anti-BfpA and anti-intimin antibodies. The immunogenicity of these proteins was assessed in C57BL/6 mice by assaying the feces and serum for the presence of anti-BfpA and anti-intimin IgA and IgG antibodies. TNF-α and INF-γ were produced in vitro by spleen cells from mice immunized with recombinant BfpA, whereas TNF-γ was produced in mice immunized with recombinant intimin. The adhesion of EPEC (E2348/69) to HEp-2 target cells was blocked by IgA or IgG antibodies from mice immunized with recombinant BfpA or intimin but not by antibodies from non-immunized mice. Immunogenic non-infectious vectors containing relevant EPEC virulence genes may be promising vaccine candidates.
Asunto(s)
Adhesinas Bacterianas/inmunología , Vacuna BCG/administración & dosificación , Proteínas de Escherichia coli/inmunología , Proteínas Fimbrias/inmunología , Mycobacterium smegmatis/inmunología , Adhesinas Bacterianas/genética , Animales , Anticuerpos Antibacterianos/sangre , Vacuna BCG/inmunología , Línea Celular , Escherichia coli Enteropatógena/inmunología , Escherichia coli Enteropatógena/patogenicidad , Células Epiteliales/citología , Células Epiteliales/microbiología , Proteínas de Escherichia coli/genética , Femenino , Proteínas Fimbrias/genética , Vectores Genéticos , Humanos , Interferón gamma/inmunología , Ratones , Ratones Endogámicos C57BL , Mycobacterium bovis/genética , Mycobacterium bovis/inmunología , Mycobacterium smegmatis/genética , Bazo/citología , Bazo/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
Intimin is an important virulence factor involved in the pathogenesis of enteropathogenic Escherichiacoli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). Both pathogens are still important causes of diarrhea inchildren and adults in many developing and industrialized countries. Considering the fact that antibodies areimportant tools in the detection of various pathogens, an anti-intimin IgG2b monoclonal antibody was previously raised in immunized mice with the conserved sequence of the intimin molecule (int388-667). In immunoblotting assays, this monoclonal antibody showed excellent specificity. Despite good performance, the monoclonal antibody failed to detect some EPEC and EHEC isolates harboring variant amino acids within the 338-667 regions of intimin molecules. Consequently, motivated by its use for diagnosis purposes, in this study we aimed to the cloning and expression of the single-chain variable fragment from this monoclonal antibody (scFv).Anti-intimin hybridoma mRNA was extracted and reversely transcripted to cDNA, and the light and heavy chains of the variable fragment of the antibody were amplified using commercial primers. The amplified chains were cloned into pGEM-T Easy vector. Specific primers were designed and used in an amplification and chain linkage strategy, obtaining the scFv, which in turn was cloned into pAE vector. E. coli BL21(DE3)pLys strainwas transformed with pAE scFv-intimin plasmid and subjected to induction of protein expression. Anti-intimin scFv,expressed as inclusion bodies (insoluble fraction), was denatured, purified and submitted to refolding. The proteinyield was 1 mg protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA andimmunofluorescence assays were performed, showing that 275 ng of scFv reacted with 2 mg of purified intimin,resulting in an absorbance of 0.75 at 492 nm.
Asunto(s)
Anticuerpos Monoclonales , Escherichia coli Enteropatógena/inmunología , Escherichia coli Enteropatógena/patogenicidad , Hibridomas/inmunología , Técnica del Anticuerpo Fluorescente/métodosRESUMEN
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.
Asunto(s)
Proteínas de Escherichia coli/análisis , Escherichia coli/inmunología , Proteínas Fimbrias/análisis , Anticuerpos Monoclonales , Antígenos Bacterianos/análisis , Antígenos Bacterianos/inmunología , Antígenos de Superficie/análisis , Antígenos de Superficie/inmunología , Adhesión Bacteriana , Niño , Diarrea/microbiología , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/inmunología , Escherichia coli Enteropatógena/metabolismo , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Humanos , Reacción en Cadena de la Polimerasa , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/inmunología , Escherichia coli Shiga-Toxigénica/metabolismoRESUMEN
INTRODUCTION: The identification of EPEC in clinical laboratories is based on the determination of the serotypes by agglutination with O and H antiserum. Currently the proper diagnosis of EPEC should be done by the identification of the intimin gen (eaeA) by PCR. OBJECTIVES: To compare the diagnosis of EPEC by serotyping and by PCR. MATERIALS AND METHODS: We collected EPEC strains, identify by their O antigen, from 4 clinical laboratories in Lima from diarrheal samples in children less than 5 years of age. In those strains we have searched for virulence genes by a real time multiplex PCR for the diarrheagenic E. coli. RESULTS: We collected 113 strains; 82% from children less than 2 years of age. Only 15 strains (13.3%) had the intimin gene and therefore a confirmatory diagnosis of EPEC. In addition we found 3 enterotoxigenic (ETEC), 3 shiga toxin-producing (STEC), 1 enteroagreggative (EAEC) and 1 enteroinvasive (EIEC) strains. CONCLUSIONS: PCR should be use for the proper identification of EPEC. However, molecular methods are still not easily available in clinical laboratories worldwide.
Asunto(s)
Adhesinas Bacterianas/genética , Pruebas de Aglutinación , Diarrea Infantil/diagnóstico , Escherichia coli Enteropatógena/aislamiento & purificación , Infecciones por Escherichia coli/diagnóstico , Proteínas de Escherichia coli/genética , Reacción en Cadena de la Polimerasa , Antígenos Bacterianos/sangre , Preescolar , ADN Bacteriano/aislamiento & purificación , Diarrea/diagnóstico , Diarrea/microbiología , Diarrea Infantil/microbiología , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/inmunología , Escherichia coli Enteropatógena/patogenicidad , Escherichia coli/clasificación , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , Humanos , Lactante , Antígenos O/sangre , Estudios Prospectivos , Estudios Retrospectivos , Escherichia coli Shiga-Toxigénica/genética , Especificidad de la Especie , Virulencia/genéticaRESUMEN
Shiga-like toxin 2 (Stx2)-producing enterohemorrhagic Escherichia coli (referred to as EHEC or STEC) strainsare the primary etiologic agents of hemolytic-uremic syndrome (HUS), which leads to renal failure and highmortality 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 therapeuticanti-HUS approaches. In the present report, we describe two Salmonella enterica serovar Typhimurium aroA vaccine strains expressing a nontoxic plasmid-encoded derivative of Stx2 (Stx2 AB) containing the complete nontoxic A2 subunit and the receptor binding B subunit. The two S. Typhimurium strains differ inthe expression of flagellin, the structural subunit of the flagellar shaft, which exerts strong adjuvant effects. Thevaccine strains expressed Stx2 AB, either cell bound or secreted into the extracellular environment, andshowed 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) andconferred 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 Stx2 AB 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.
Asunto(s)
Humanos , Ratones , Células Vero/microbiología , Escherichia coli Enteropatógena/inmunología , Vacunas , Escherichia coli/crecimiento & desarrollo , Escherichia coli/aislamiento & purificación , Escherichia coli/metabolismo , Salmonella entericaRESUMEN
Studies based on the analysis of housekeeping genes indicate that Escherichia coli and all Shigella species, except for Shigella boydii type 13, belong to a single species. This study analysed the phenotypic and genotypic characteristics of 23 E. coli strains isolated in different countries from faecal specimens taken from children with diarrhoea. Strains were identified using the VITEK system and typed with rabbit sera obtained against 186 somatic and 53 flagellar E. coli antigens and against 45 Shigella somatic antigens. Biochemical analysis of these strains showed a typical E. coli profile with a defined reaction against both E. coli O179 and S. boydii 16 somatic antisera. Agglutination assays for flagellar antigens showed a response against H2 in 7 (30 %) strains, H10 in 2 (9 %) strains, H32 in 12 (52 %) strains and H34 in 2 (9 %) strains, demonstrating 4 serotypes associated with this new somatic antigen 64474. A serum against one of these E. coli strains (64474) was prepared. Absorption assays of S. boydii 16 and E. coli 64474 antisera with E. coli O179 antigen removed the agglutination response against this O179 antigen completely, while the agglutination titres against both S. boydii 16 and E. coli 64474 remained the same. Four (17 %) E. coli strains showed antimicrobial resistance to piperacillin only, one (4 %) to piperacillin and trimethoprim/sulfamethoxazole, one (4 %) to ciprofloxacin, nitrofurantoin and piperacillin, and two (9 %) strains were resistant to ciprofloxacin, norfloxacin, ofloxacin, piperacillin and trimethoprim/sulfamethoxazole. With regards to PCR assays, one (4 %) of the strains was positive for Shigella gene ipaH, one (4 %) for ipaA, two (9 %) for ipaB, one (4 %) for ipaD, two (9 %) for sepA and three (13 %) for ospF. The rfb gene cluster in the E. coli strains was analysed by RFLP and compared with the gene cluster obtained from S. boydii 16. The rfb-RFLP patterns for all 23 E. coli strains were similar to those obtained for S. boydii 16. The results from PCR tests to detect rfb genes wzx (encoding O unit flippase) and wzy (encoding polymerase) belonging to a cluster related to the biosynthesis of the S. boydii 16-specific O antigen were positive in 21 (91 %) and 22 (96 %) of the strains, respectively. PCR assays to detect E. coli virulence genes were also performed. These assays detected enterotoxigenic E. coli genes ltA1 in 12 of the strains (52 %), st1a in 4 (17 %), cfa1 in 6 (26 %), cs1 in 1 (4 %), cs3 in 3 (13 %), cs13 in 9 (39 %) and cs14 in 5 (22 %) of the strains. Results from the PFGE analyses confirmed the wide geographical distribution of these strains suggesting that 64474 : H2, 64474 : H10, 64474 : H32 and 64474 : H34 are new serotypes of E. coli strains with a defined virulence capacity, and share a common O antigen with S. boydii 16.
Asunto(s)
Escherichia coli Enteropatógena/clasificación , Shigella boydii/clasificación , Niño , Electroforesis en Gel de Campo Pulsado , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/inmunología , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , Polimorfismo de Longitud del Fragmento de Restricción , Serotipificación , Shigella boydii/genética , Shigella boydii/inmunología , Virulencia/genéticaRESUMEN
The ability of some typical enteropathogenic Escherichia coli (EPEC) strains to adhere to, invade, and increase interleukin-8 (IL-8) production in intestinal epithelial cells in vitro has been demonstrated. However, few studies regarding these aspects have been performed with atypical EPEC (aEPEC) strains, which are emerging enteropathogens in Brazil. In this study, we evaluated a selected aEPEC strain (1711-4) of serotype O51:H40, the most prevalent aEPEC serotype in Brazil, in regard to its ability to adhere to and invade Caco-2 and T84 cells and to elicit IL-8 production in Caco-2 cells. The role of flagella in aEPEC 1711-4 adhesion, invasion, and IL-8 production was investigated by performing the same experiments with an isogenic aEPEC mutant unable to produce flagellin (FliC), the flagellum protein subunit. We demonstrated that this mutant (fliC mutant) had a marked decrease in the ability to adhere to T84 cells and invade both T84 and Caco-2 cells in gentamicin protection assays and by transmission electron microscopy. In addition, the aEPEC 1711-4 fliC mutant had a reduced ability to stimulate IL-8 production by Caco-2 cells in early (3-h) but not in late (24-h) infections. Our findings demonstrate that flagella of aEPEC 1711-4 are required for efficient adhesion, invasion, and early but not late IL-8 production in intestinal epithelial cells in vitro.
Asunto(s)
Adhesión Bacteriana , Enterocitos/inmunología , Enterocitos/microbiología , Escherichia coli Enteropatógena/inmunología , Escherichia coli Enteropatógena/patogenicidad , Flagelos/fisiología , Interleucina-8/metabolismo , Brasil , Línea Celular , Recuento de Colonia Microbiana , Citoplasma/microbiología , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli Enteropatógena/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Flagelina , Eliminación de Gen , Humanos , Microscopía Electrónica , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
A diarréia é um importante problema de saúde pública no mundo inteiro e a Escherichía coli é um dos mais freqüentes microorganismos causadores desta doença. A Escherichia coli enteropatogênica (EPEC), um dos principais agentes etiológicos das diarréias infantis no nosso país, é genética e fenotipicamente relacionada com a E. colí enterohemorrágica (EHEC) que além de provocar diarréia é responsável por complicações como síndrome hemolítica urêmica (HUS) e colite hemorrágica (HC). Embora a EHEC seja considerada emergente pela OMS, no Brasil poucos casos de complicações como HUS e HC foram reportados. O mecanismo de patogenicidade comum entre EPEC e EHEC é conhecido como a lesão "attaching and effacing" nos microvilos do enterócito. Esta lesão é mediada por um conjunto de fatores de virulência, dentre eles a intimina. A intimina é uma proteína de membrana externa, responsável pelo íntimo contato da bactéria com o enterócito, possui uma região N-terminal que é altamente conservada e uma região C-terminal que é variável. De acordo com a região variável, existem vários subtipos de intimina, dentre eles as intiminas , α, β e γ...
Asunto(s)
Humanos , Masculino , Femenino , Adulto , Anticuerpos/genética , Anticuerpos/inmunología , Diarrea/genética , Diarrea/inmunología , Escherichia coli Enteropatógena/fisiología , Escherichia coli Enteropatógena/inmunología , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Infecciones por Escherichia coli/inmunología , Calostro , Ensayo de Inmunoadsorción Enzimática , Métodos Analíticos de la Preparación de la Muestra , Suero , Interpretación Estadística de DatosRESUMEN
Enteropathogenic Escherichia coli (EPEC) are frequently isolated as a cause of infantile diarrhea in developing countries. Its pathogenicity is distinguished by histopathological alterations at the site of infection, known as attaching and effacing (A/E) lesions, in which bacterial virulence factors and host proteins participate. Intimin, a bacterial adhesin expressed by all EPEC described to date, is responsible for the intimate adherence of the bacteria to host cells and is essential for the formation of A/E lesions. Mucosal vaccination may represent an efficacious intervention to prevent EPEC infection and lower morbidity and mortality rates. Strategies for mucosal vaccinations that use lactic acid bacteria for the delivery of heterologous antigens rely on their safety profile and ability to stimulate the immune system. In the present work, we have constructed Lactobacillus casei strains expressing different fragments of intimin beta, a subtype that is frequently expressed by EPEC strains. Mucosal immunization of mice with L. casei expressing intimin fragments induced specific systemic and mucosal antibodies. These antibodies were able to recognize native intimin on the surface of EPEC and to inhibit in vitro EPEC binding to epithelial cells.
Asunto(s)
Adhesinas Bacterianas/genética , Adhesinas Bacterianas/inmunología , Anticuerpos Antibacterianos/inmunología , Adhesión Bacteriana , Escherichia coli Enteropatógena/fisiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Lacticaseibacillus casei/genética , Animales , Células Cultivadas , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/inmunología , Células Epiteliales , Inmunidad Mucosa , Inmunización , Lacticaseibacillus casei/inmunología , Ratones , Proteínas Recombinantes/inmunologíaRESUMEN
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.