Evaluación de la viabilidad y caracterización bacteriana anaeróbica estricta de la flora intestinal humana para ensayos in-vitro

Abstract

This thesis was conducted to assess the feasibility and to characterize the strict anaerobic bacteria of the human intestinal flora (Bacteroides fragilis, Clostridium perfringens and Clostridium difficile). The staff of the Laboratory of Food and Nutrition Project VLIR-IUC “Food, Nutrition and Health”, who were responsible for collection procedures, provided the samples. The total amount of sample came from three individuals who collected the feces over a period of 24 hours for three consecutive days under standardized conditions and controlled diet and collection according to the research needs of the project. The microbial characterization was performed by comparing bacterial populations of fecal samples from each individual by culturing and counting using selective media. Bacteroides fragilis was grown on Bacteroides Bile Agar Esculin, Clostridium perfringens was grown on Sulphite Polymyxin Sulfadiazine Agar, and Clostridium difficile was grown on Agar Cycloserine Cefoxitin Fructose 7% sheep blood. The bacterial suspension was elaborated from fecal samples and with a standardized solution for anaerobic phosphate buffer; it was preserved by freezing at - 80 ⁰C with 25% glycerol as cryoprotectant. The bacterial viability was assessed over time by culturing and counting on selective medias for five weeks. Of the three bacteria studied, Bacteroides fragilis was the predominant microorganism (49±0.8%), followed by Clostridium perfringens (26±0.8%) and Clostridium difficile finally (25±0.8%) for three individuals. Microbial counts of Clostridium perfringens and Clostridium difficile present significant difference between individuals (p < 0,001 y p = 0.0001 respectively); while, microbial count of Bacteroides fragilis were not significantly different (p = 0.068). The analysis of bacterial viability shows that the bacterial suspension prepared and preserved under conditions established keeps viable to Clostridium perfringens and Clostridium difficile during 5 weeks of study, but this does not maintain viable Bacteroides fragilis.