Excretion product of shigella dysenteriae (sdyep) induced cell death in early larval stage of zebrafish (danio rerio): acridine orange and ethidium bromide (AO/EB) in vivo staining / Producto de excreción de shigella dysenteriae (pesdy) induce muerte celular en larvas de pez cebra (danio rerio): marcaje in vivo con naranja de acridina y bromuro de etidio (NA/BE)

In the field of studies of acute toxicity induced by bacterial agents, Shiga toxins have been relevant due to the severity of the extra-intestinal diseases they cause. Numerous studies have shown that Shiga toxin induced apoptosis in different cell types; however, this important process has been little studied in vivo experimental models. In this study, the effects of excretion products of Shigella dysenteriae, in which Shiga toxin is present, were investigated on early larval stages of Zebrafish, an animal model with many advantages over other in vivo experimental models traditionally used. Both the collection of eggs and larvae of Zebrafish, and the product from excretion from Shigella dysenteriae (SdyEP) were performed according to laboratory standards. Also, toxicity bioassay, larvae treatment with pure and diluted solution, 10-1, 10-2, 10-3 , 10-4 and 10-5, v/v SdyEP and cell death in vivo using Acridine Orange (AO) and Ethidium Bromide (EB) were applied. The excretion product of Shigella dysenteriae (SdyEP) effect was expressed in terms of larval mortality and dependent dilution rather than incubation time. The larval population surviving treatment with Shigella excretion product presents severe morphological effects. The larval population generally presents notable severe morphological damage, the necrosis state is represented by the opacity of the larvae after being treated for 24 h (b) compared to control. Other changes associated with larval anatomy were also observed; particularly the caudal end curvature was significant into 10%. The use of AO/EB revealed a distribution pattern from fluorescence into green and orange in surviving larvae SdyEP poisoning, there was a large population of dead cells around the anal and caudal region as evidenced by the presence of orange nuclei in greater numbers as controls in the larvae. The results support the application of coloring AO/EB in Zebrafish experimental models for the evaluation of the toxic action of new molecules and new products with therapeutic potential.

En el campo de los estudios de toxicidad aguda inducida por agentes bacterianos, las toxinas Shiga resultan relevantes debido a la severidad de las enfermedades extra-intestinales que causan. Numerosos estudios han demostrado que la toxina Shiga induce la apoptosis en diferentes tipos de células, sin embargo, este importante proceso ha sido poco estudiado en modelos experimentales in vivo. En este estudio, fueron evaluados los efectos del productos de excreción de Shigella dysenteriae (PESdy), sobre estadios larvarios de pez cebra (Danio rerio), un modelo animal con muchas ventajas sobre otros modelos experimentales in vivo utilizados tradicionalmente. Tanto la recolección de los huevos y larvas de pez cebra, así como la obtención del producto de la excreción, se realizaron de acuerdo a los estándares de laboratorio. Poblaciones larvarias, fueron tratadas con distintas soluciones; pura y diluidas, 101, 10-2, 10-3, 10-4 y 10-5, v/v de PESdy. La muerte celular in vivo, usando naranja de acridina (NA) y bromuro de etidio (BE) fue evaluada. El efecto del SdyEP, se expresó como dependiente de la concentracion y del tiempo de exposición. La población de larvas sobrevivientes, presentaron curvatura troncal en un 10%, en relación a los controles. La necrosis se puso en evidencia a través de la opacidad de las larvas después de 24 h. El uso de NA/BE reveló un patrón de distribución de la fluorescencia en verde y naranja en larvas sobrevivientes al tratamiento. Una granpoblación de células muertas, alrededor de la región anal y caudal, se ponen en evidencia por la presencia de núcleos de naranja en mayor número que en los controles. Los resultados apoyan la aplicación de la coloración NA/BE en modelo experimental de pez cebra, para la evaluación de la acción tóxica de nuevas moléculas y nuevos productos de excreción bacterial con potencial terapéutico.

Survivability of Shigella dysenteriae type 1 & S. flexneri 2a in laboratory conditions simulating aquatic environment.

BACKGROUND & OBJECTIVES: Information about the duration of survival of Shigellae in water is speculative. The present study was undertaken to assess the duration of survival of Shigella dysenteriae type 1 and S. flexneri type 2a in the laboratory conditions simulating the aquatic environment, their invasive property and the association of different physico-chemical parameters in the survival process. METHODS: Five natural water sources were selected in a diarrhoea prone rural area. Collection of water, determination of physico-chemical parameters and bacteriology were carried out following standard procedures. Filter-sterilised water samples were inoculated with S. dysenteriae type 1 and S. flexneri 2a and survival was monitored by estimating total viable count at regular intervals. Bivariate correlations between the duration of survival and physico-chemical parameters were estimated. Multiple linear regression models were fitted for the duration of survival of the bacteria. RESULTS: All water sources were contaminated with faecal coliforms including Escherichia coli, S. dysenteriae type 1 survived for a mean duration of 3.33 days and S. flexneri 2a for mean 11.167 days in field water samples in laboratory condition. Duration of survival had positive correlation with the initial bacterial counts. In the multiple regression model the strongest predictor of the duration of survival of both S. dysenteriae type 1 and S. flexneri 2a was the concentration of bacteria. Other possible predictors for S. flexneri 2a were Mg and water temperature. INTERPRETATION & CONCLUSION: S. dysenteriae type 1 with epidemic potential survives for shorter duration than S. flexneri 2a. Although some of the physico-chemical parameters had positive relation with duration of survival, the variation of these in natural water samples studied has not caused much variation in the survival in case of S. dysenteriae type 1. In case of S. flexneri 2a, the observed variation in Mg concentration can cause up to 25 days difference in the duration of survival and thus could be a factor determining the endemicity of S. flexneri 2a infection.