Action of phosphorylated mannanoligosaccharides on immune and hematological responses and fecal consistency of dogs experimentally infected with enteropathogenic Escherichia coli strains

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.

Comparación entre el diagnóstico serológico y el diagnóstico por Reacción en Cadena de la Polimerasa (PCR) para Escherichia coli Enteropatogénica (EPEC) / Comparison of Enteropathogenic Escherichia Coli (EPEC) diagnosis by serology and by Polymerase Chain Reaction (PCR)

Introdución. En los laboratorios clínicos la identificación de EPEC se basa en la determinación de serotipos específicos por técnicas de aglutinación utilizando antisueros O y H. Actualmente la identificación del gen de intimina (eaeA) por PCR es el método diagnóstico de elección para EPEC. Objetivos. Comparar el diagnóstico por serología con el diagnóstico por PCR de cepasde EPEC. Materiales y Métodos. Se recolectaron cepas identificadas como EPEC en base al antígeno O, de 4 laboratorios clínicos de Lima, procedentes de muestras de diarrea de niños menores de 5 años. En estas cepas se buscaron genes relacionados a virulencia mediante un PCR múltiple a tiempo real para las E. coli diarreogénicas. Resultados. Se recolectaron 113 cepas; 82% de niños menores de 2 años. Únicamente15 cepas (13.3%) presentaron el gen de intimina con un diagnóstico confirmatorio de EPEC. Adicionalmente se encontraron 3 cepas enterotoxigénicas (ETEC), 3 productoras de shiga-toxina (STEC), 1 entero agregativa (EAEC) y 1 enteroinvasiva (EIEC). Conclusiones. Para la identificación correcta de EPEC se debe usar el PCR. Sin embargo, los métodos moleculares aún no están fácilmente disponibles en los laboratorios clínicos a nivel mundial.

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 serotypin 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.

Patogénesis molecular, epidemiología y diagnóstico de Escherichia coli enteropatógena / Molecular pathogenesis, epidemiology and diagnosis of enteropathogenic Escherichia coli

Escherichia coli enteropatógena (EPEC) es una de las principales causas de diarrea en niños menores de dos años en países en vías de desarrollo. La principal característica histopatológica de la infección es una lesión que induce la EPEC en el intestino conocida como la lesión A/E (adherencia y eliminación). Las bacterias se adhieren a los enterocitos y permiten la acumulación de la actina del citoesqueleto en la región apical de la célula, hasta formar una estructura de tipo "pedestal" y causar la eliminación de las microvellosidades intestinales. A pesar de que se conoce de modo detallado el proceso de formación de los pedestales de actina, aún no se ha esclarecido el mecanismo global de la diarrea que induce EPEC. La diarrea se ha vinculado con: a) la destrucción de las microvellosidades del enterocito, b) la salida masiva de iones hacia la luz intestinal y c) la secreción de alguna enterotoxina. En estudios realizados en países en vías de desarrollo se ha demostrado que EPEC es uno de los principales agentes participantes en la diarrea infantil, con elevadas tasas de morbilidad y mortalidad. El diagnóstico microbiológico de la infección se realiza con metodologías adicionales a las utilizadas con regularidad en el laboratorio de microbiología clínica, entre ellas las siguientes: a) serotipificación, b) ensayo de adherencia, c) prueba de FAS (tinción fluorescente para actina) y d) detección específica de genes que codifican a proteínas incluidas en la patogénesis, como el bfpA y eae. Un objetivo de esta revisión es actualizar los avances observados en la patogénesis molecular de la infección por EPEC, las metodologías para el diagnóstico microbiológico y la epidemiología en México y otros países en vías de desarrollo.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhea in infants less than two years of age in developing countries. To induce diarrhea EPEC uses several virulence factors acting on a still unknown and mysterious mechanism. The hallmark of EPEC infection is a histological intestinal alteration known as the attaching and effacing (A/E) lesion. The bacterium attaches intimately to the enterocyte and induces assembly of cytoskeleton intracellular actin on the cellular surface. Rearrangements of the actin cytoskeleton form a pedestal-like structure where bacterium tightly cups the cells, leading to degeneration of brush border microvilli. Although the mechanism of EPEC-induced pedestal formation has been dissected in detail, the overall mechanism of diarrhea is still obscure. It is believed that EPEC-mediated secretory diarrhea is related to a) intestinal microvilli effacement, b) massive loss of intracellular ions into the intestinal milieu and c) secretion of an EPEC enterotoxin. Epidemiological studies conducted in developing countries have shown that EPEC is one of the main bacteria frequently isolated from children with diarrhea, causing high morbidity and mortality rates. The microbiological diagnosis of EPEC-induced disease is performed with analytic methodologies different from those used by the standard microbiology laboratory, the most relevant being: a) serotypification, b) the adherence assay, c) FAS test, and d) the specific detection of virulence-involved genes (bfpA and eae genes) using molecular biology techniques. The purpose of this review is to update the most recent findings regarding the molecular pathogenesis of EPEC, its epidemiology in Mexico as well as other developing countries, and also the developed methodology for the diagnosis of EPEC infection.

Epidemiological Study and Virulence Associated Genes of Enteropathogenic Escherichia coli Isolated from Diarrheal Neonates

A total of 136 strains of Escherichia coli, isolated from rectal swabs of neonates at the neonatal intensive care unit of Pusan National University Hospital during the period from February to April of 2001 and March to April of 2002 were serotyped. The presence of eaeA, aggA, bfpA, astA, LT, and ST genes was test by PCR. Four enteroaggregative E. coli (EAggEC) strains were isolated in 2001 and eight in 2002. In 2001, three strains of enterotoxigenic E. coli (ETEC) were isolated and these strains were tested for antimicrobial susceptibility. ETEC isolates were detected by a reverse passive latex agglutination (RPLA) test for the production of heat-labile enterotoxin. The strains were analyzed by using plasmid profiling, random amplified polymorphic DNA (RAPD), and pulsed field gel electrophoresis (PFGE) methods. The O serotypes could be assigned to 29 isolates (21.3%): O166, 6; O167, 5; O86a, O6 and O127a, 4 each; O8, 2; and O28ac, O44, O158, O20, 1 each. There were 107 untypable isolates. EAggEC isolates were typed as O86a in 4, O127a in 4, and untypable in 4 strains. Three isolates of ETEC were typed as O6. The PCR detected aggA gene in 12 strains (8.8%), but bfpA, eaeA, EAST-1, and ST genes were not detected. LT gene was detected in 3 strains (2.2%) by PCR and DNA probe hybridization, LT production was confirmed in those strains by a latex bead aggregation method. Out of the 15 EAggEC and ETEC strains, eleven (80.0%) were multi-drug resistant to more than 3 antibiotics. Thirteen groups were classed by antibiogram and most strains were susceptible to gentamicin, kanamycin, and cefoperazone, but resistant to ampicillin (100%), cephalothin (66.7%), cefuroxime (46.6%), and tetracycline (46.7%). All isolates possessed a 60 kbp plasmid and 15 strains possessed smaller plasmids. Twelve EAggEC and 3 ETEC strains were differentiated into 5 and 3 groups by the plasmid profiling. RAPD and PFGE grouped the EAggEC isolates into 7 and 6 groups, respectively. The RAPD and PFGE patterns matched in 11 isolates (91.7%) among the 12 EAggEC strains, but the plasmid profile analysis and antibiogram showed no correlation. Three ETEC strains showed different plasmid profiles, and RAPD and PFGE patterns.