Effects of furazolidone pretreatment of Salmonella enteritidis PT4 at sub- and suprainhibitory concentrations on phagocytosis and intracellular survival in chicken macrophages

The antimicrobial effect of the nitrofuran derivative furazolidone at sub- and suprainhibitory concentrations on Salmonella enteritidis PT4 and the influence with regard to interaction with avian macrophages was investigated in this study. Phagocytosis of furazolidone-sensitive (FzS) S. enteritidis with chicken macrophages in the presence of furazolidone at concentrations of 1/8, 1/4, 1/2 and 8× MIC resulted in an increase in the rate of phagocytic killing of approximately 3-, 6-, 6.5- and 9-fold, respectively, with 1/2 and 8× MIC concentrations producing statistically significant (P<0.05) increases in phagocytosis. Treatment of the FzSSalmonella with furazolidone at concentrations of 4× and 10× MIC, for 15 min prior to phagocytosis, also significantly (P<0.005) increased phagocytic uptake when compared with untreated bacteria. The rate of phagocytosis monitored over 90 min was highest between 30 and 60 min with the furazolidone pretreated salmonella, compared with the delayed rate of the control between 60 and 90 min. Exposure of FzS and FzR strains with suprainhibitory concentrations of furazolidone at 4×, 8× and 10× MIC for 30 min prior to phagocytosis demonstrated an increase in bacterial killing. Exposure of strains to sub-inhibitory concentrations of furazolidone led to an increase in chemiluminescence during phagocytosis with macrophages, suggesting an increase in oxidative metabolism in the macrophages as a result of an increase in activation and phagocytosis. Pretreatment of the strains with suprainhibitory concentrations of furazolidone for 30 min prior to phagocytosis demonstrated a similar increase in oxidative metabolism in the macrophages. Measurement of the amount of -furazolidone associated with chicken macrophages was determined over 20 h incubation. The level of radioactivity of -furazolidone alone was used to estimate the amount of cell-associated nitrofuran when incubated with the macrophages by means of regression analysis. Incubation with concentrations of 16, 32 and 64 μg/ml for 20 h resulted in the cell association of ≥1 μg/ml of furazolidone, which is the concentration required for the agent to exhibit bactericidal activity on furazolidone-sensitive Salmonella strains. Furazolidone was able to reduce intracellular salmonella viability at all concentrations, but total killing was achieved only with concentrations of ≥8 μg/ml, which supports the results for furazolidone association with the macrophages. This substantiates that the bioactivity of the nitrofuran was not inhibited or diminished in the intracellular environment of the macrophage and that exposure of salmonella to nitrofurans enhances phagocytosis.