Efectos del medio de cultivo y de la metodología aplicada sobre la formación de biopelículas de 2 cepas de Escherichia coli diarreagénicas / Effects of the culture medium and the methodology applied on the biofilm formation of 2 diarrheagenic Escherichia coli strains

La capacidad de formar biopelículas de los microorganismos patógenos en gran variedad de ambientes, superficies y condiciones trae consigo un importante riesgo, tanto para la industria alimentaria como para la salud pública. Este trabajo tuvo como objetivo evaluar y comparar los efectos de la metodología empleada y de los medios de cultivo utilizados, sobre la capacidad de una cepa de Escherichia coli verotoxigénica no O157 y una enteropatogénica de formar biopelículas sobre una superficie de poliestireno. Se ensayaron 2 variantes metodológicas en cultivo estático y se utilizaron medios de cultivo con diferente composición. Los resultados mostraron que ambas cepas formaron una mayor cantidad de biopelícula en cultivo en LB suplementado con glucosa, con recambio del medio a las 24 h y la cuantificación de la biopelícula realizada a las 48 h de incubación. Dichas condiciones podrían ser utilizadas en futuros estudios sobre formación de biopelícula.

The ability to form biofilms of pathogenic microorganisms in a wide variety of environments, surfaces and conditions constitute an important risk, both for the food industry and for public health. The aim of this work was to evaluate and to compare the effects of the methodology applied and the culture medium used on the ability of a non-O157 verotoxigenic Escherichia coli strain and an enteropathogenic strain to form biofilm on polystyrene surface. Two methodological variants were tested in static culture and culture mediums with different composition were used. The results showed that both strains were able to form a greater biofilm under culture in LB supplemented with glucose, with medium replacement at 24 h and the quantification of the biofilm carried out at 48 h of incubation. These conditions could be used in future studies on biofilm formation.

Intestinal cell migration damage induced by enteropathogenic Escherichia coli strains

Epithelial cell migration is an essential response to enteric pathogens such as enteropathogenic Escherichia coli (EPEC). This study aimed to investigate the effects of EPEC infection on intestinal epithelial cell migration in vitro, as well as the involvement of type III secretion system (T3SS) and Rho GTPases. Crypt intestinal epithelial cells (IEC-6) were infected with EPEC strains (E2348/69, ΔescF, and the LDI001 strain isolated from a malnourished Brazilian child) and commensal E. coli HS. Wound migration and cell death assays were performed at different time-points. Transcription and expression of Rho GTPases were evaluated using real-time PCR and western blotting. Overall, EPEC E2348/69 reduced migration and increased apoptosis and necrosis levels compared to EPEC LDI001 and E. coli HS strains. Moreover, EPEC LDI001 impaired cell migration at a higher level than E. coli HS and increased necrosis after 24 hours compared to the control group. The different profiles of virulence genes between the two wild-type EPEC strains, characterized by the absence of espL and nleE genes in the LDI001, might explain the phenotypic results, playing significant roles on cell migration impairment and cell death-related events. Moreover, the type III secretion system is determinant for the inhibition of intestinal epithelial cell migration by EPEC 2348/69, as its deletion prevented the effect. Active Rac1 concentrations were increased in E2348/69 and LDI001-infected cells, while the T3SS-deficient strain did not demonstrate this activation. This study contributes with valuable insight to characterize the mechanisms involved in the impairment of intestinal cell migration induced by EPEC.

Mecanismos de virulencia de Escherichia coli enteropatógena / Virulence mechanisms of enteropathogenic Escherichia coli

Acute diarrheal disease (ADD) is a global public health problem, especially in developing countries and is one of the causes of mortality in children under five. ADD etiologic agents include viruses, bacteria and parasites in that order. Escherichia coli bacteria it is classified as a major diarrheagenic agent and transmitted by consuming contaminated water or undercooked foods. This review compiled updates on information virulence factors and pathogenic mechanisms involved in adhesion and colonization of seven pathotypes of E. coli called enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), shigatoxigenic E. coli (STEC), enteroaggregative E. coli (EAEC) and diffusely-adherent E. coli (DAEC). A final pathotype, adherent-invasive E. coli (AIEC) associated with Crohn's disease was also reviewed. The diarrheagenic pathotypes of E. coli affect different population groups and knowledge of the molecular mechanisms involved in the interaction with the human is important to guide research towards the development of vaccines and new tools for diagnosis and control.

La enfermedad diarreica aguda (EDA) es un problema de salud pública mundial, especialmente en los países en vía de desarrollo y es una de las causas de mortalidad en niños bajo cinco años de edad. Los agentes etiológicos de EDA incluyen virus, bacterias y parásitos, en ese orden. Dentro de las bacterias, Escherichia coli está clasificada como uno de los principales agentes diarreagénicos y se trasmite por el consumo de agua y alimentos contaminados o mal cocidos. Esta revisión recopiló información actualizada sobre los factores de virulencia y los mecanismos de patogenicidad implicados en la adhesión y colonización de siete patotipos de E. coli denominados, E. coli enteropatógena (ECEP), E. coli enterotoxigénica (ECET), E. coli enteroinvasora (ECEI), E. coli shigatoxigénica (ECST), E. coli enteroagregativa (ECEA) y E. coli de adherencia difusa (ECAD). Un último patotipo, E. coli adherente invasor (ECAI) asociado a la enfermedad de Crohn también fue revisado. Los patotipos diarreagénicos de E. coli afectan a diferentes grupos poblacionales y el conocimiento de los mecanismos moleculares implicados en la interacción con el humano es importante para orientar las investigaciones hacia el desarrollo de vacunas o nuevas herramientas para su diagnóstico y control.

Occurrence and molecular characterization of enteropathogenic Escherichia coli serotypes isolated from children with diarrhoea in Najaf, Iraq.

Purpose: Enteropathogenic Escherichia coli (EPEC) are among the most important pathogens infecting children worldwide and are one of the main causes of diarrhoea. The study was carried out to investigate the occurrence of EPEC as a cause of infectious diarrhoea in children younger than 2 years of age and characterize their virulence genes. Materials and Methods: During the study period, a total of 656 faecal specimens from children with diarrhoea and 54 from healthy children were analyzed. E. coli isolates were serotypically identified with EPEC polyvalent and monovalent antisera. The isolated EPEC were examined for the presence of the attaching and effacing (eaeA), bundle-forming pilus (bfpA), Shiga like toxins (stx1 and stx2 ), enterohaemorrhagic E. coli enterohaemolysin (EHEC hlyA) and EPEC adherence factor (EAF) genes by the PCR assay. Results: The study has shown that 22 (3.4%) had diarrhoea due to EPEC, while no EPEC isolates were detected in asymptomatic children. The highest number of the EPEC isolated belonging to polyvalent 2. The primers encoding virulence genes were subjected to all the EPEC isolates. Only 9.1%, 27.3%, and 9.1% isolates gave positive re sults with intimin (eaeA), bfbA and (EAF) genes, respectively. None of the isolates were positive for stx 1, stx 2, and hlyA genes. Typical EPEC (eaeA +, bfpA +) was diagnosed in two isolates, while, atypical EPEC was manifested in four isolates. Conclusions: According to the results, the frequency of EPEC isolates in Najaf was lower than what has been suspected and the investigation including the use of molecular technique and serotyping, are necessary to allow precise identification and epidemiological study of these pathogens.

Detection & characterization of Shiga toxin producing Escherichia coli (STEC) & enteropathogenic Escherichia coli (EPEC) in poultry birds with diarrhoea.

Background & objectives Limited information is available on shiga toxin producing Escherichia coli (STEC) in animals and birds from India. An outbreak of acute diarrhoea in poultry birds at Aizawl, Mizoram was investigated for detection and characterization of STEC and enteropathogenic E. coli (EPEC). Methods E. coli was isolated and identified from rectal swabs, intestinal contents, heart blood and spleen of 19 poultry birds that died due to acute diarrhoea during the outbreak. Phenotypic characterization was done by standard bacteriological and biochemical techniques. All the isolates were serotyped based on their somatic antigens. Virulence genes (stx1, stx2, eaeA and hlyA) were detected by multiplex PCR assay. Results A total of 42 E. coli isolates were obtained, of which 24 belonged to 3 serogroups (O64, O89 and O91) and the remaining 18 were untypable (UT). Altogether, 14 (33.33%) isolates carried at least 1 virulence gene, of which 10 (23.81%) and 4 (9.52%) were recorded as STEC and EPEC, respectively. Of the 10 STEC isolates, one carried only stx2, one carried stx2 and hlyA, four carried stx1, stx2 and hlyA, two carried stx1, eaeA and hlyA genes and two carried stx1 and eaeA. Of the four EPEC isolates, two carried eaeA and hlyA, one carried only eaeA gene and 1 carried only hlyA gene. Interpretation & conclusions This is the first report on the involvement of STEC in poultry in India.

[ frequency of cryptosporidiosis among gastroenteritic patients in western cities of Mazandaran Province [2007-2009][

Gastroenteritis is caused by parasitic and non-parasitic microorganisms. Cryptosporidiosis is one of the parasitic diseases leading to acute or chronic gastroenteritis caused by Cryptosporidium spp. Self-limiting gastroenteritis is observed in immunocompetent individuals, but in immunocompromised patients it causes a sever disease. High humidity, ecological conditions, water supplies, domestic and industrial animal husbandry and the rate of raining have made the Mazandaran regions as a province for transmission of parasites. The objective of this study was to determine the frequency of cryptosporidiosis among gastroenteritic patients in western cities of Mazandaran Province, during 2007-2009. This analytical study was conducted in cities of Chalus, Tonekabon, and Ramsar located in west Mazandaran province, northern Iran. Stool samples from patients with gastroenteritis and healthy individuals were collected, fixed and examined by direct method [DM] for the diagnosis of enteropathogenic and non-pathogenic parasites; acid-fast staining [AFS] and auramine phenol fluorescence [APF] for detection of Cryptosporidium oocysts and analysed using ANOVA and t-ests. The mean prevalence rate of parasitic infections in three cities was 2.38% with the highest rate of infection associated with Giardia lamblia [1.43%], Blastocystis hominis [0.71%], and Entamoeba coli [0.24%], respectively. No Cryptospordium sp. was observed among the test and control groups. Based on our data, a low rate of parasitic infection and also an absence of cryptospordiosis, compared to the previous studies, in western part of Mazandaran province were established. This may be associated with improvements in public health education, water treatment environmental sanitation, and low animal contacts during recent years

[Endotoxemia and death caused by klebsiella pneumoniae [k1] and enteropathogenic escherichia coli in a red deer calf [cervus elaphus maral]]

A three month-old-male red deer calf [Cervus elaphus maral] was examined post mortem for the cause of death in Arasbaran preserved area in East Azerbaijan in September 2006. The main history of the case was the lack of colostrum intake after birth. The necropsy 6 hours after death, revealed severe general congestion especially in lungs and visceral organs [liver and jejunum]. The cut surface of lungs was moist and bronchial lumina contained a large amount of frothy pinkish edema fluid. Diffuse congestion of lung and porteinous exudates was prominent in examination. Histopathological examination revealed shock lung and hepatocytes dissociation with single cell necrosis in liver. Microscopic examination was in line with shock lung and alveolar edema. No parasites were observed within red blood cells. Bacteriological cultures gave rise to gram negative cocoobacilli and further biochemical tests performed on isolated colonies revealed the presence of pure Escherichia coli in liver and Klebsiella pnemoniae in lung. Using serological tests, E. coli serotypes O20 and O114 and Klebsiella pnemoniae serotype K1 were identified in purified bacterial cultures. This report presents endotoxemia and death in a red deer calf with lack of colostrum intake and transportation stress history

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.