RESUMEN

Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.

Asunto(s)

Animales Domésticos/microbiología , Animales Salvajes/microbiología , Técnicas de Tipificación Bacteriana , Dermatoglifia del ADN , ADN Bacteriano/genética , Escherichia coli Enteropatógena/clasificación , Escherichia coli Enteropatógena/genética , Animales , Brasil , Análisis por Conglomerados , ADN Bacteriano/química , Electroforesis en Gel de Campo Pulsado , Escherichia coli Enteropatógena/aislamiento & purificación , Proteínas de Escherichia coli/genética , Genotipo , Humanos , Epidemiología Molecular , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Serotipificación , Factores de Virulencia/genética

RESUMEN

This study characterized 76 atypical enteropathogenic Escherichia coli (aEPEC) strains, previously classified by the eae(+) EAF-negative stx(-) genotype, isolated from children with diarrhea in Brazil. Presence of bfpA and bfpA/perA was detected in 2 and 6 strains, respectively. The expression of bundle-forming pilus (BFP), however, was observed by immunofluorescence in 1 bfpA and 3 bfpA/perA strains, classifying them as typical EPEC (tEPEC). The remaining 72 aEPEC strains were characterized by serotyping, intimin typing, adherence patterns to HEp-2 cells, capacity to induce actin aggregation (fluorescent actin staining test), and antimicrobial resistance. Our results show that aEPEC comprise a very heterogeneous group that does not present any prevalence or association regarding the studied characteristics. It also suggest that tEPEC and aEPEC must not be classified only by the reactivity with the EAF probe, and that the search of other markers present in pEAF, as well as the BFP expression, must be considered for this matter.

Asunto(s)

Adhesinas Bacterianas/genética , Diarrea/microbiología , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/patogenicidad , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Toxina Shiga/genética , Adhesión Bacteriana , Técnicas de Tipificación Bacteriana , Brasil , Línea Celular , Niño , Escherichia coli Enteropatógena/clasificación , Escherichia coli Enteropatógena/aislamiento & purificación , Proteínas Fimbrias/genética , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Reacción en Cadena de la Polimerasa/métodos , Proteínas Represoras/genética , Serotipificación , Virulencia

RESUMEN

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.

Asunto(s)

Adhesión Bacteriana , Células Epiteliales/microbiología , Escherichia coli/aislamiento & purificación , Escherichia coli/fisiología , Línea Celular , Escherichia coli/clasificación , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Factores de Virulencia/genética

RESUMEN

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.

Asunto(s)

Anticuerpos Antibacterianos/biosíntesis , Ensayo de Inmunoadsorción Enzimática/métodos , Infecciones por Escherichia coli/microbiología , Toxina Shiga I/análisis , Toxina Shiga II/análisis , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Animales , Anticuerpos Antibacterianos/análisis , Supervivencia Celular , Chlorocebus aethiops , ADN Bacteriano/química , ADN Bacteriano/genética , Humanos , Immunoblotting , Reacción en Cadena de la Polimerasa , Conejos , Toxina Shiga I/inmunología , Toxina Shiga II/inmunología , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/patogenicidad , Células Vero , Virulencia

RESUMEN

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/inmunología

RESUMEN

The genetic relationship among the Escherichia coli pathotypes was investigated. We used random amplified polymorphic DNA (RAPD) data for constructing a dendrogram of 73 strains of diarrheagenic E. coli. A phylogenetic tree encompassing 15 serotypes from different pathotypes was constructed using multilocus sequence typing data. Phylogram clusters were used for validating RAPD data on the clonality of enteropathogenic E. coli (EPEC) O serogroup strains. Both analyses showed very similar topologies, characterized by the presence of two major groups: group A includes EPEC H6 and H34 strains and group B contains the other EPEC strains plus all serotypes belonging to atypical EPEC, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). These results confirm the existence of two evolutionary divergent groups in EPEC: one is genetically and serologically very homogeneous whereas the other harbors EPEC and non-EPEC serotypes. The same situation was found for EAEC and EHEC.

Asunto(s)

Técnicas de Tipificación Bacteriana , Escherichia coli/genética , Variación Genética/genética , ADN Bacteriano/genética , Escherichia coli/clasificación , Escherichia coli/patogenicidad , Humanos , Fenotipo , Filogenia , Técnica del ADN Polimorfo Amplificado Aleatorio , Análisis de Secuencia de ADN , Virulencia/genética

RESUMEN

The genetic relationship among the Escherichia coli pathotypes was investigated. We used random amplified polymorphic DNA (RAPD) data for constructing a dendrogram of 73 strains of diarrheagenic E. coli. A phylogenetic tree encompassing 15 serotypes from different pathotypes was constructed using multilocus sequence typing data. Phylogram clusters were used for validating RAPD data on the clonality of enteropathogenic E. coli (EPEC) O serogroup strains. Both analyses showed very similar topologies, characterized by the presence of two major groups: group A includes EPEC H6 and H34 strains and group B contains the other EPEC strains plus all serotypes belonging to atypical EPEC, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). These results confirm the existence of two evolutionary divergent groups in EPEC: one is genetically and serologically very homogeneous whereas the other harbors EPEC and non-EPEC serotypes. The same situation was found for EAEC and EHEC.

Asunto(s)

Humanos , Técnicas de Tipificación Bacteriana , Escherichia coli/genética , Variación Genética , ADN Bacteriano/genética , Escherichia coli/clasificación , Escherichia coli/patogenicidad , Fenotipo , Filogenia , Técnica del ADN Polimorfo Amplificado Aleatorio , Análisis de Secuencia de ADN , Virulencia/genética

RESUMEN

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

Asunto(s)

Humanos , Animales , Niño , Ratones , Conejos , Anticuerpos Monoclonales/biosíntesis , Toxinas Bacterianas/inmunología , Enterotoxinas/biosíntesis , Escherichia coli/inmunología , Inmunoglobulina G/biosíntesis , Anticuerpos Monoclonales , Enterotoxinas/genética , Enterotoxinas/inmunología , Escherichia coli/genética , Técnicas para Inmunoenzimas , Inmunoglobulina G , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Serotipificación

RESUMEN

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.

Asunto(s)

Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Escherichia coli/genética , Escherichia coli/fisiología , Virulencia/genética , Animales , Bovinos , Enfermedades de los Bovinos/microbiología , Dermatoglifia del ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/clasificación , Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/genética , Enfermedades de los Caballos/microbiología , Caballos/microbiología , Humanos , Proteína 2 Reguladora de Hierro/genética , Epidemiología Molecular , Hibridación de Ácido Nucleico , Filogenia , Ribotipificación , Serotipificación , Porcinos/microbiología , Enfermedades de los Porcinos/microbiología , Transactivadores/genética , Factores de Virulencia/genética

RESUMEN

Community-based monitoring was conducted in order to investigate the occurrence of diarrhea in 'sentinel areas' of Salvador, Brazil, and to establish a preliminary profile of the most common pathogens present in children's diarrhea by screening stool samples. This report describes the results obtained from twice weekly home visits to identify and follow diarrhea episodes and testing of carer-requested stool sample collection over a 6-month period. Participants were selected from a large longitudinal study in 21 areas representing the city's poorer socioeconomic and sanitary conditions. Fecal samples were examined for the presence of pathogenic bacteria, viruses and protozoa. The mean incidence of diarrhea was 4.97 episodes per child-year, and longitudinal prevalence was 13.6 days per child-year (3.7%). Pathogens were found in 44% of the fecal samples examined. Bacteria were the most frequently encountered pathogens (isolated in 22% of samples), followed by protozoa (19.5%) and viruses (16%). Viral and bacterial pathogens were associated with episodes of severe diarrhea, while viral and protozoan pathogens were associated with longer episodes. The study demonstrated the importance of a public health monitoring system based on 'sentinel areas'.

Asunto(s)

Diarrea/epidemiología , Brasil/epidemiología , Preescolar , Diarrea/microbiología , Diarrea/parasitología , Diarrea Infantil/epidemiología , Diarrea Infantil/microbiología , Diarrea Infantil/parasitología , Heces/microbiología , Heces/parasitología , Femenino , Humanos , Incidencia , Lactante , Estudios Longitudinales , Masculino , Salud Urbana

RESUMEN

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Toxinas Bacterianas/inmunología , Enterotoxinas/biosíntesis , Escherichia coli/inmunología , Inmunoglobulina G/biosíntesis , Animales , Niño , Enterotoxinas/genética , Enterotoxinas/inmunología , Escherichia coli/genética , Proteínas de Escherichia coli , Humanos , Técnicas para Inmunoenzimas , Ratones , Reacción en Cadena de la Polimerasa , Conejos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Serotipificación

RESUMEN

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathogens that colonize the gut through the formation of attaching and effacing lesions, which depend on the translocation of effector proteins via a locus of enterocyte effacement-encoded type III secretion system. Recently, two effector proteins, EspJ and TccP, which are encoded by adjacent genes on prophage CP-933U in EHEC O157:H7, have been identified. TccP consists of a unique N-terminus region and several proline-rich domains. In this project we determined the distribution of tccP in O157:H7, in non-O157 EHEC, and in typical and atypical EPEC isolates. All the EHEC O157:H7 strains tested were tccP(+). Unexpectedly, tccP was also found in non-O157 EHEC, and in typical and atypical EPEC isolates, particularly in strains belonging to serogroups O26 (EHEC), O119 (typical EPEC), and O55 (atypical EPEC). We recorded some variation in the length of tccP, which reflects diversity in the number of the proline-rich repeats. These results show the existence of a class of "attaching and effacing" pathogens which express a combination of EPEC and EHEC virulence determinants.

Asunto(s)

Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Animales , Australia , Brasil , China , Escherichia coli/química , Infecciones por Escherichia coli/veterinaria , Escherichia coli O157/química , Escherichia coli O157/genética , Microbiología de Alimentos , Variación Genética , Humanos , Datos de Secuencia Molecular , Reino Unido

RESUMEN

Bacterial diarrheal diseases remain a major cause of morbidity and mortality in developing countries. Diffusely adhering Escherichia coli (DAEC) is a newly proposed category of diarrheagenic E. coli based on epidemiological studies. Sat, a new virulence factor of some uropathogeic Escherichia coli, was described with a vacuolating cytotoxic action in bladder and kidney tissues. In the present study, we analyzed the Sat effects, produced by a DAEC strain in rabbit ileal intestinal tissue and cultured epithelial cells. We observed enterotoxic activity in rabbit ileum tissues by Ussing chamber assays, a pronounced fluid accumulation in rabbit ileum loops with villous necrosis observed in the histopathologic examination, and morphological changes in monolayer cultures of Y1 adrenal cells. Our results suggest that DAEC strains may be involved in diarrhea.

Asunto(s)

Toxinas Bacterianas/toxicidad , Diarrea/microbiología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/toxicidad , Escherichia coli/patogenicidad , Íleon/patología , Animales , Adhesión Bacteriana , Línea Celular , Diarrea/patología , Modelos Animales de Enfermedad , Escherichia coli/metabolismo , Infecciones por Escherichia coli/patología , Ratones , Conejos , Factores de Virulencia/toxicidad

RESUMEN

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathogens that colonize the gut through the formation of attaching and effacing lesions, which depend on the translocation of effector proteins via a locus of enterocyte effacement-encoded type III secretion system. Recently, two effector proteins, EspJ and TccP, which are encoded by adjacent genes on prophage CP-933U in EHEC O157:H7, have been identified. TccP consists of a unique N-terminus region and several proline-rich domains. In this project we determined the distribution of tccP in O157:H7, in non-O157 EHEC, and in typical and atypical EPEC isolates. All the EHEC O157:H7 strains tested were tccP+. Unexpectedly, tccP was also found in non-O157 EHEC, and in typical and atypical EPEC isolates, particularly in strains belonging to serogroups O26 (EHEC), O119 (typical EPEC), and O55 (atypical EPEC). We recorded some variation in the length of tccP, which reflects diversity in the number of the proline-rich repeats. These results show the existence of a class of “attaching and effacing” pathogens which express a combination of EPEC and EHEC virulence determinants.

Asunto(s)

Escherichia coli Enteropatógena

RESUMEN

The so called enteropathogenic Escherichia coli (EPEC) O serogroups include typical and atypical EPEC, enterohaemorrragic E. coli, enterotoxigenic E. coli, and enteroaggregative E. coli. The aim of this article is to review the composition of each O serogroup and the major serotypes, clones, and additional virulence characteristics of each of these diarrheagenic categories. Their adherence patterns and genetic relationships are also presented. The review is based on the study of 805 strains of serogroups O26, O55, O86, O111, O114, O119, O125, O126, O1127, O128, and O142 most of which isolated in Sao Paulo from children with diarrhea between 1970 and 1990. Since some O serogroups include more than one diarrheagenic category O serogrouping only should be abandoned as a diagnostic method. However serotyping is a reliable method for those serotypes that correspond to clones.

Asunto(s)

Adhesinas de Escherichia coli , Escherichia coli/clasificación , Serotipificación , Escherichia coli/genética , Escherichia coli/patogenicidad , Humanos , Virulencia

RESUMEN

The socalled enteropathogenic Escherichia coli (EPEC) O serogroups include typical and atypical EPEC, enterohaemorrragic E. coli, enterotoxigenic E. coli, and enteroaggregative E. coli. The aim of this article is to review the composition of each O serogroup and the major serotypes, clones, and additional virulence characteristics of each of these diarrheageniccategories. Their adherence patterns and genetic relationships are also presented. The review is based on the study of 805 strains of serogroups O26, O55, O86, O111, O114, O119, O125, O126, O1127, O128, and O142 most of which isolated in São Paulo from children with diarrhea between 1970 and 1990. Since some O serogroups include more than one diarrheageniccategory O serogrouping only should be abandoned as a diagnostic method. However serotyping is a reliable method for those serotypes that correspond to clones.

Asunto(s)

Humanos , Serotipificación , Adhesinas de Escherichia coli , Escherichia coli , Virulencia , Escherichia coli

RESUMEN

The LEE 4 genes sepL, espA, espD, espB, and espF were detected in 50 strains of typical and atypical enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli by PCR. sepL was amplified in 90%, espA in 94%, espB in 50%, espD in 40%, and espF in 78% of all strains, employing prototype EPEC-based primers. With O26:H(-)-based primers, espB was detected in all O26 strains, and O157:H7-specific primers amplified espD and espB among all O55:H7 and O157:H7 strains. Our results indicated that espA and sepL should be more conserved between different EPEC and EHEC serotypes, while espB, espD, and espF should be more diverse. Apparently this variation is related to serogroup or serotype, but sequencing assays are necessary to confirm such conservation/diversity and their association with serogroup or serotype. Secreted protein analyses of espA, espD, and espB PCR-negative strains demonstrated that their encoded proteins present distinct immunological types, reflecting the genetic variability of those genes.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Genes Bacterianos , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Escherichia coli/patogenicidad , Escherichia coli O157/genética , Proteínas de Escherichia coli/metabolismo , Antígenos O/inmunología , Fosfoproteínas/genética , Reacción en Cadena de la Polimerasa , Serotipificación

RESUMEN

Enteroaggregative Escherichia coli (EAEC) is distinguished by its characteristic aggregative adherence (AA) pattern to cultured epithelial cells. In this study we investigated the role of type I fimbriae (TIF) in the AA pattern to HEp-2 cells and in biofilm formation. Accentuation of this pattern was observed when the adherence assay was performed in the absence of mannose. This effect was observed in the prototype EAEC strain 042 (O44:H18), O128:H35 strains and for other EAEC serotypes. Antiserum against TIF decreased AA by 70% and 90% for strains 042 and 18 (O128:H35 prototype strain), respectively. A non-polar knockout of fimD, the TIF usher, in strains 042 and 18 resulted in inhibition of the accentuated AA pattern of approximately 80% and 70% respectively, and biofilm formation diminution of 49% for 042::fimD and 76% for 18::fimD. Our data evidence a role for TIF in the AA pattern and in EAEC biofilm formation, demonstrating that these phenotypes are multifactorial.

Asunto(s)

Adhesión Bacteriana/fisiología , Proteínas Bacterianas , Biopelículas/crecimiento & desarrollo , Proteínas de Escherichia coli , Escherichia coli/fisiología , Fimbrias Bacterianas/fisiología , Adhesinas de Escherichia coli/genética , Adhesinas de Escherichia coli/fisiología , Anticuerpos Antibacterianos , Antígenos Bacterianos , Adhesión Bacteriana/genética , Línea Celular Tumoral , Recuento de Colonia Microbiana , Escherichia coli/patogenicidad , Proteínas Fimbrias/genética , Proteínas Fimbrias/fisiología , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/inmunología , Humanos , Manosa/metabolismo , Microscopía , Microscopía Inmunoelectrónica , Mutagénesis Insercional

RESUMEN

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin gene (fliC) was performed in 233 strains of enteropathogenic Escherichia coli (EPEC) O serogroups for determining their flagellar antigen (H) status. The serological detection of flagellin is the basis for the H-codes typing system in E. coli. Thus, it is impossible to serotype nonmotile bacteria (i.e. to assign H-codes). Twenty-eight fliC restriction patterns were obtained for motile (H2, H4, H6, H7, H8, H9, H10, H11, H12, H18, H21, H27, H32, H34, H35, H40 and H51) and nonmotile serotypes (H(-)). Each motile serotype was characterized by one or two fliC specific restriction patterns. The only exception was serogroup O128ab, where a common restriction pattern was found for serotypes O128ab:H2 and O128ab:H35, even after digestion with RsaI, AluI and Sau3AI endonucleases. These two serotypes were, however, discriminated by single strand conformation polymorphism (SSCP) analysis of RsaI restriction fragments. Nonmotile strains showed fliC restriction patterns identical to some known H serotypes. The PCR-RFLP analysis of fliC gene proved to be a useful method for identifying the H variants in motile and nonmotile EPEC O serogroups.

Asunto(s)

Técnicas de Tipificación Bacteriana , Escherichia coli/clasificación , Flagelina/genética , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Secuencia de Aminoácidos , Antígenos Bacterianos/genética , ADN Bacteriano/análisis , Desoxirribonucleasas de Localización Especificada Tipo II , Diarrea/microbiología , Escherichia coli/genética , Escherichia coli/patogenicidad , Escherichia coli/fisiología , Infecciones por Escherichia coli/microbiología , Humanos , Datos de Secuencia Molecular , Polimorfismo Conformacional Retorcido-Simple , Análisis de Secuencia de ADN , Serotipificación

RESUMEN

Enteropathogenic Escherichia coli (EPEC) has been associated with infantile diarrhea and mortality in humans in developing countries. While diarrhea is also a major problem among primates kept in captivity, the role of E. coli is unclear. This study was designed to characterize diarrheagenic E. coli recovered from the feces of 56 New World nonhuman primates, primarily marmosets (Callithrix spp.). Seventeen of the 56 primates had signs of diarrhea and/or enteritis. E. coli recovered from feces from these animals was tested by PCR for genes encoding virulence factors of diarrheagenic E. coli and for patterns of adherence to HeLa cells. In addition, isolates were characterized by the fluorescence actin staining test and by their ability to induce attaching and effacing lesions. PCR for the eae gene was positive in 10 of the 39 (27%) apparently healthy animals and in 8 of the 17 (47%) animals with diarrhea and/or enteritis. Colonies of eae(+) E. coli were serotyped and examined by PCR for genes encoding EPEC virulence markers. The eae(+) E. coli isolates recovered from both healthy and sick nonhuman primates demonstrated virulence-associated attributes similar to those of EPEC strains implicated in human disease and are designated monkey EPEC. The results presented here indicate that EPEC may be a significant pathogen for nonhuman primates, deserving further investigation. The similarities between the affected animals investigated in this study and human EPEC infections suggest that marmosets may represent an important model for EPEC in humans.

Asunto(s)

Diarrea/microbiología , Escherichia coli/clasificación , Actinas/metabolismo , Animales , Adhesión Bacteriana , Diarrea/patología , Modelos Animales de Enfermedad , Escherichia coli/aislamiento & purificación , Femenino , Haplorrinos , Humanos , Masculino , Serotipificación , Especificidad de la Especie , Factores de Virulencia/análisis