Pseudomonas aeruginosa biofilms react with and precipitate toxic soluble gold

The interaction between biofilms of Pseudomonas aeruginosa PAO1 and 0.01-5 mM gold chloride was investigated using flow-cells. Scanning confocal laser microscopy (SCLM) of these biofilms revealed the formation of two distinct structural features: (i) confluent areas of uniform thickness and (ii) cell clusters which often emerged as 30-40 micro m, tall narrow pillars (or pedestals) of cells and exopolymeric substance (EPS). When 5-day-old, quasi-steady state biofilms (as indicated by the stability of film thickness and overall structure) were exposed to relatively high AuCl3 (i.e. 0.5-5 mM) for 30 min at 20 degrees C, reduction of the auric ion resulted in the formation of both extracellular and intracellular metallic gold colloids, as revealed by transmission electron microscopy (TEM). Most mineralization occurred on cell surfaces with lesser amounts within cells and little throughout the EPS. Little to no mineralization of gold was seen at 0.01-0.1 mM concentrations. As initial AuCl3 concentrations approached 0.5 mM or greater, more gold particles were seen and cell viability, as determined by a BacLight live/dead viability probe, approached zero. At an intermediate concentration of 0.1 mM, the live:dead ratio increased to 4:1. However, when planktonic cells were exposed to this same 0.1 mM concentration, it resulted in a 4-log reduction in viable counts as determined by plating. The higher resistance of biofilm cells to 0.1 mM gold can be attributed to its binding to the EPS and cell surfaces of the biofilm which ensured a (presumably) low effective cytoplasmic concentration of gold (i.e. no gold crystals were seen in cells by TEM). In addition, SCLM revealed the formation of larger extracellular gold crystals at the substratum (coverslip) level of the biofilms, with a higher proportion of crystals detected beneath pillars (cell cluster structures), suggesting the possibility of unique cell types, more reduced microenvironments at the base of each cluster, or a combination of both. These results suggest that the biomineralization of gold is impacted by biofilm structure.