A comparative study on the use of flow cytometry and colony forming units for assessment of the antibacterial effect of bacteriocins

Flow cytometry was investigated as a rapid method to determine the antibacterial effect of the bacteriocins nisin, pediocin PA-1, and sakacin A on the indicator organisms Lactobacillus reuteri DSM 12246, Lactobacillus sakei NCFB 2714 and Lactobacillus sakei DSM 20017, respectively. Fluorescence intensities of the cells were measured by flow cytometry upon exposure to bacteriocins after staining with carboxyfluorescein diacetate (cFDA) and were compared to the number of colony forming units (CFU). The fluorescence index (FI) of the bacterial populations decreased when exposed to the bacteriocins. For the different bacteriocins the pattern of decreases in FI and colony forming units differed at equal bacteriostatic concentrations. FI was the most sensitive measure of bacteriocin activity, i.e. the decrease in FI was observed at lower bacteriocin concentrations than decrease in CFU. It was demonstrated that the decrease in FI was caused by rapid leakage of carboxyfluorescein from cells exposed to pediocin. Cells showing severe leakage after pediocin treatment could be detected as CFU when transferred to a rich medium. Such a repair was less pronounced for cells exposed to sakacin and very limited for cells exposed to nisin. The influence of temperature and NaCl in combination with pediocin on FI and CFU of Lactobacillus sakei NCFB 2714 was examined at conditions relevant to foods. At all temperatures (5, 10, 20 and 37°C) and NaCl concentrations (0, 2 and 4% w/v) investigated the flow cytometric measurements were significantly more sensitive compared to CFU. Both methods showed that the inhibitory effect of pediocin increased with increasing temperatures and decreased with increasing NaCl concentrations.