Burkholderia pseudomallei biofilm resists Acanthamoeba sp. grazing and produces 8-O-4′-diferulic acid, a superoxide scavenging metabolite after passage through the amoeba

Abstract Burkholderia pseudomallei, an etiological agent of melioidosis is an environmental bacterium that can survive as an intracellular pathogen. The biofilm produced by B. pseudomallei is crucial for cellular pathogenesis of melioidosis. The purpose of this investigation is to explore the role of biofilm in survival of B. pseudomallei during encounters with Acanthamoeba sp. using B. pseudomallei H777 (a biofilm wild type), M10 (a biofilm defect mutant) and C17 (a biofilm-complemented strain). The results demonstrated similar adhesion to amoebae by both the biofilm wild type and biofilm mutant strains. There was higher initial internalisation, but the difference diminished after longer encounter with the amoeba. Interestingly, confocal laser scanning microscopy demonstrated that pre-formed biofilm of B. pseudomallei H777 and C17 were markedly more persistent in the face of Acanthamoeba sp. grazing than that of M10. Metabolomic analysis revealed a significant increased level of 8-O-4′-diferulic acid, a superoxide scavenger metabolite, in B. pseudomallei H777 serially passaged in Acanthamoeba sp. The interaction between B. pseudomallei with a free-living amoeba may indicate the evolutionary pathway that enables the bacterium to withstand superoxide radicals in intracellular environments. This study supports the hypothesis that B. pseudomallei biofilm persists under grazing by amoebae and suggests a strategy of metabolite production that turns this bacterium from saprophyte to intracellular pathogen

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

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

Intracellular survival through time of <i>B</i>. <i>pseudomallei</i> in <i>P</i>. <i>ustiana</i> (A), <i>Acanthamoeba</i> sp. (B) and isolate A-ST39-E1 (C).

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

<i>B</i>. <i>pseudomallei</i> is internalized into amoebae but could not resist digestion.

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

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

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

Specific primers for the 18s rRNA gene of amoebae.

<p>Specific primers for the 18s rRNA gene of amoebae.</p