Figure 1

The conditioned media of Enterobacter sp. and Escherichia coli K1 exhibited amoebistatic and amoebicidal activities. (A) A. castellanii (10⁵) were incubated with 50% bacterial conditioned media (BCM) and 50% PYG at 30°C for 48 h. For controls, A. castellanii were incubated with PYG alone, 100% PBS, and 50% PBS plus 50% PYG. After incubation, amoebae were counted using a haemocytometer. The initial inoculum of 10⁵ amoebae was used as baseline. Note that the incubation in the non-nutritive 100% PBS showed no growth, while incubation in the growth medium resulted in over 8-fold increase in the number of amoebae compared with the original inoculum. P-values were calculated by comparing results of 100% PYG with BCM using student’s t-test. (*) indicates p<0.001. (B) Enterobacter sp. and E. coli K1 BCM were diluted using PYG, where 0% dilution represents neat BCM, while 50% dilution represents BCM and PYG in 1:1 ratio. A. castellanii (10⁵) were incubated in various dilutions of BCM of Enterobacter sp. and E. coli K1 and incubated at 30°C for 48 h. After incubation, amoebae were counted using a haemocytometer. The initial inoculum of 10⁵ amoebae increased to over 8-fold in PYG alone (0% dilution). P-values were calculated by comparing results of 0% BCM (i.e., PYG alone) with different concentrations of BCM. (*), (**) and (***) indicate p- values of <0.05, <0.01 and <0.001, respectively. (C) A. castellanii (10⁶) were incubated with neat (0%) or diluted (50%) BCM at 30°C for 24 h. After incubation, 0.1% Trypan blue dye was added and amoebae along with the blue coloured amoebae (dead cells) were counted using a haemocytometer. Note that cells treated with neat BCM produced A. castellanii death. The data are presented as the mean ± standard error of three independent experiments performed in duplicate.