(191al) Reincubation of Heat Shocked Pseudomonas aeruginosa Biofilm

When bacteria colonize a medical implant surface, they form a biofilm which cannot be eradicated chemically. The current standard of care is surgical explantation of the device and surrounding tissue, with eventual reimplantation of a replacement device with twice the probability of infection. These infections are a $5 billion problem in the U.S. alone, impacting over 100,000 patients annually. By applying a localized thermal shock, we have shown that these biofilms can be eliminated, though the mechanism of elimination has been unclear. In the presence of antibiotics which have little effect on the biofilm by themselves, biofilms are eliminated by heat shocks which are too mild to have any noticeable effect in the absence of antibiotics. One hypothesis for this synergistic efficacy is that the heat shock triggers a change in bacterial phenotype, prompting the bacteria to disperse from the biofilm in the planktonic state, and thereby making them susceptible to antibiotic elimination.

Reincubation studies, however, indicate that biofilm elimination by thermal shock is not instantaneous. Biofilms were cultured to a steady-state population density of 10⁷ colony forming units (CFU) per cm² and subjected to heat shocks of varying intensity (60, 70, or 80 °C for 1, 5, or 30 min). Their population density was quantified immediately following thermal shock, demonstrating populations reductions ranging from none (no statistically significant reduction) after mild heat shock to complete (no viable bacteria detected) after aggressive heat shocks. Reincubation of thermally shocked biofilms for 2, 4, 12, 24 and 96 hours followed by resuspension and plating showed a bimodal response. Following heat shock, biofilms slowly regrew, eventually reaching their original 10⁷ CFU/cm² population density—if the population density immediately following these heat shocks was typically 10³ CFU/cm² or above. However, if the population density immediately following these heat shocks was typically below 10³ CFU/cm² (yet clearly detectable and quantifiable), the biofilms would continue dying off, with no viable CFU after a few hours, despite being able to form colonies when immediately resuspended and plated. This supports an opposing hypothesis that the bacteria do not flee the biofilm, but rather die in place, leaving a matrix full of toxic enzymes that proceed to kill off the surviving bacteria. These hypotheses are further examined by applying thermal shock in a flow cell system in which any fleeing bacteria are collected in the flowing fluid and quantified as a function of time.