Figure Legends
Figure 1. Effect of temperature on biofilm formation in differentBurkholderia strains (B. pseudomallei 1026b Δasd , Bp82, JW270 and B. thailandensis E264). a. Biofilm formation life cycle in B. pseudomallei JW270 b. Pellicle images ofBurkholderia strains. c. Image of a B. pseudomallei JW270 pellicle post processing vs micrograph image of the pellicle. Red lines lead to an SEM image which shows that the pellicles are made up the extracellular materials (eDNA, proteins and polysaccharides) and bacterial cells. There was no difference in pellicle morphology on the SEM. Bar 2µM. d. Crystal violet quantification of Burkholderiabiofilms under different temperatures shows that there is no significant difference in biofilm formation at the end of the experiment -72 hours (P > 0.05). e. Biofilm formation of all 4 strains at 37°C after 72 hours shows that there is no significant difference between the biofilms formed by B. pseudomallei strains or by B. thailandensis , (P >0.05 for each comparison).
Figure 2. Microscopic images of Burkholderia biofilm. a. 10x Microscopic images of 72 hours Burkholderia biofilms which shows the biofilms are composed of eDNA (Dapi), proteins (Sypro ruby) and polysaccharides (WGA) White bar - 200µm. Inserts are 100x Microscopic images showing the cells within a biofilm. Red bar - 100µm. b. Live/dead image of cells 0-48 hours showing that the cells are viable throughout the biofilm process (Bar- 10µm).
Figure 3 shows that there is a significant difference between the amounts exported when comparing between growth modes (p ˂ 0.05). As expected, biofilm cells export higher amount of all constituents compared to planktonic cells. In figures 3a -3c, we observe that 1026bΔasd and Bp82 export high levels of (a) protein and (b) polysaccharide and low levels of (c) eDNA. JW270 biofilm on the other hand show higher levels of eDNA and minimal protein and polysaccharide export while B . thailandensis exports high protein and eDNA and lower polysaccharide levels when compared with B. pseudomallei 1026b Δasd and Bp82.
Figure 4. Effects of enzymatic treatments on 72 hoursBurkholderia biofilms. Data shows that there is a significant difference between untreated biofilms and enzyme treated biofilm. (* = p ˂0.05, ** = p = 0.01-0.05, *** = p =0.01- 0.007).
Figure 5. Morphology and composition of Burkholderia pellicles. a. Fluorescent images of a Burkholderia pellicles showing eDNA (blue), Polysaccharides (green), Sypro ruby (red). b. Polysaccharide (black bars) and protein (red line) compositions extracted from 72 hour pellicles. c. Quantification of eDNA extracted from a 72 hour pellicle and read using propidium iodide and a spectrophotometer. Bars represent average of three independent experiment and Error bars represent SEM.
Figure 6. Comparison between 1026b, DD503, and JW270 biofilms. Graphs a-c show concentrations of protein and glucose and eDNA levels respectively. There is no significant difference between the protein and eDNA of 1026b and DD503 at the end of the experiment, 72 hours (p > 0.05) whereas DD503 and JW270 differed significantly at each time point except the 48 hours eDNA concentration (p < 0.05). There is also a significant difference between the glucose concentrations for all three strains.