Environmental Free-Living Amoebae Isolated from Soil in Khon Kaen, Thailand, Antagonize Burkholderia pseudomallei

Abstract

Presence of Burkholderia pseudomallei in soil and water is correlated with endemicity of melioidosis in Southeast Asia and northern Australia. Several biological and physico-chemical factors have been shown to influence persistence of B. pseudomallei in the environment of endemic areas. This study was the first to evaluate the interaction of B. pseudomallei with soil amoebae isolated from B. pseudomallei-positive soil site in Khon Kaen, Thailand. Four species of amoebae, Paravahlkampfia ustiana, Acanthamoeba sp., Naegleria pagei, and isolate A-ST39-E1, were isolated, cultured and identified based on morphology, movement and 18S rRNA gene sequence. Co-cultivation combined with a kanamycin-protection assay of B. pseudomallei with these amoebae at MOI 20 at 30°C were evaluated during 0-6 h using the plate count technique on Ashdown's agar. The fate of intracellular B. pseudomallei in these amoebae was also monitored by confocal laser scanning microscopy (CLSM) observation of the CellTracker™ Orange-B. pseudomallei stained cells. The results demonstrated the ability of P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 to graze B. pseudomallei. However, the number of internalized B. pseudomallei substantially decreased and the bacterial cells disappeared during the observation period, suggesting they had been digested. We found that B. pseudomallei promoted the growth of Acanthamoeba sp. and isolate A-ST39-E1 in co-cultures at MOI 100 at 30°C, 24 h. These findings indicated that P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 may prey upon B. pseudomallei rather than representing potential environmental reservoirs in which the bacteria can persist.

Fig 1. Bright field microscopic images demonstrate trophozoite and cyst morphology (A and B) Paravahlkampfia ustiana, (C and D) Acanthamoeba sp., (E and F) Naegleria pagei and (G and H) isolate A-ST39-E1.

P. ustiana, N. pagei and isolate A-ST39-E1 were stained with trypan blue while Acanthamoeba sp. was stained with crystal violet. The letter in images indicate the following: Uf = Uroidal filaments, N = Nucleus, Ac = Acanthopodia, Cv = Contractile vacuole, Fv = Food vacuole, O = Outer-cyst wall, I = Inner-cyst wall, F = Flagella, S = Sub-pseudopodium and U = Uroid.

Fig 2. Intracellular survival through time of B. pseudomallei in P. ustiana (A), Acanthamoeba sp. (B) and isolate A-ST39-E1 (C).

Time zero represents 3 hours after B. pseudomallei feeding. Bars represent the standard errors of the means of duplicate, three times independent experiments, * p < 0. 0001 using ANOVA.

Fig 3. B. pseudomallei is internalized into amoebae but could not resist digestion.

CLSM micrographs show the internalized B. pseudomallei in P. ustiana (A-C), Acanthamoeba sp. (D-F) and isolate A-ST39-E1 (G-I) at 0, 3 and 6 h after kanamycin treatment. Orange fluorescence represents CellTracker^™ Orange-B. pseudomallei and green fluorescence indicates the amoebae stained with FITC-ConA for visualization.

Fig 4. Numbers of Acanthamoeba sp. and isolate A-ST39-E1 over time (A-B and C-D respectively) after feeding with B. pseudomallei (▲) or E. coli (positive control) (■) or deprived of bacteria as a negative control (●).

Graphs and figures show no significant differences between amoebae fed on B. pseudomallei and E. coli. However, numbers of amoebae in the negative control group were significantly lower than in the pother groups (p ≤ 0.0001). Data are mean ± SD from duplicates of the three independent experiments.