ABSTRACT
The aim of the present study was to gain further insight on the reliability of the colorimetric determination of the activity of bacterial nitrate reductase to evaluate bacterial concentrations and interaction between microorganisms and enterocyte-like cells. Nitrite produced after incubation of the samples with a nitrate-formate solution was determined with a diazotization reaction with sulphanilic acid and N-naphthyl-ethylene-diamonium dichloride. Cell association assays were performed with differentiated Caco-2 cells. A biphasic relationship was found between nitrite concentration and bacterial densities. This behavior seems to be due to the sigmoideal character of the kinetics of nitrate reduction. Association to Caco-2 cells was strongly strain dependent being Staphylococcus aureus ATCC 25923 the strain showing the highest values of association. For some strains, percentages of association calculated on the basis of the colorimetric assay were significantly higher than those calculated in terms of viable counts. Bacterial association with enterocyte-like cells can be evaluated by measures of the activity of bacterial nitrate reductase provided that the biphasic relationship between bacterial and nitrite concentrations is taken into account for the calculations. Results presented in this paper show the applicability of the colorimetric method to assess the amount of microorganisms associated to human enterocytes in culture.