Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix.

We developed a new approach that couples Southwestern blotting and mass spectrometry to discover proteins that bind extracellular DNA (eDNA) in bacterial biofilms. Using Staphylococcus aureus as a model pathogen, we identified proteins with known DNA-binding activity and uncovered a series of lipoproteins with previously unrecognized DNA-binding activity. We demonstrated that expression of these lipoproteins results in an eDNA-dependent biofilm enhancement. Additionally, we found that while deletion of lipoproteins had a minimal impact on biofilm accumulation, these lipoprotein mutations increased biofilm porosity, suggesting that lipoproteins and their associated interactions contribute to biofilm structure. For one of the lipoproteins, SaeP, we showed that the biofilm phenotype requires the lipoprotein to be anchored to the outside of the cellular membrane, and we further showed that increased SaeP expression correlates with more retention of high-molecular-weight DNA on the bacterial cell surface. SaeP is a known auxiliary protein of the SaeRS system, and we also demonstrated that the levels of SaeP correlate with nuclease production, which can further impact biofilm development. It has been reported that S. aureus biofilms are stabilized by positively charged cytoplasmic proteins that are released into the extracellular environment, where they make favorable electrostatic interactions with the negatively charged cell surface and eDNA. In this work we extend this electrostatic net model to include secreted eDNA-binding proteins and membrane-attached lipoproteins that can function as anchor points between eDNA in the biofilm matrix and the bacterial cell surface.IMPORTANCE Many bacteria are capable of forming biofilms encased in a matrix of self-produced extracellular polymeric substances (EPS) that protects them from chemotherapies and the host defenses. As a result of these inherent resistance mechanisms, bacterial biofilms are extremely difficult to eradicate and are associated with chronic wounds, orthopedic and surgical wound infections, and invasive infections, such as infective endocarditis and osteomyelitis. It is therefore important to understand the nature of the interactions between the bacterial cell surface and EPS that stabilize biofilms. Extracellular DNA (eDNA) has been recognized as an EPS constituent for many bacterial species and has been shown to be important in promoting biofilm formation. Using Staphylococcus aureus biofilms, we show that membrane-attached lipoproteins can interact with the eDNA in the biofilm matrix and promote biofilm formation, which suggests that lipoproteins are potential targets for novel therapies aimed at disrupting bacterial biofilms.

Thermal shock susceptibility and regrowth of Pseudomonas aeruginosa biofilms.

Biofilms on implanted medical devices cause thousands of patients each year to undergo multiple surgeries to remove and replace the implant, driving billions of dollars in increased health care costs due to the lack of viable treatment options for in situ biofilm eradication. Remotely activated localised heating is under investigation to mitigate these biofilms; however, little is known about the temperatures required to kill the biofilms. To better understand the required parameters this study investigated the thermal susceptibility of biofilms as a function of their fluidic and chemical environment during growth, as well as their propensity for regrowth following thermal shock. Pseudomonas aeruginosa biofilms were cultured in shaker plate fluidic conditions in four different growth media, then thermally shocked at various temperatures and exposure times. Biofilms were re-incubated to determine their regrowth potential following thermal shocks of various intensities. Results indicate that growth media has little impact on thermal susceptibility, while fluidic conditions strongly influence susceptibility to modest thermal shocks. This effect disappears, however, with increasingly aggressive shocks, reducing biofilm populations by up to 5 orders of magnitude. Regrowth studies indicate a critical post-shock bacterial loading (∼103 CFU/cm2) below which the biofilms were no longer viable, while biofilms above that loading slowly regrew to their previous population density.

Synergistic effects of heat and antibiotics on Pseudomonas aeruginosa biofilms.

Upon formation of a biofilm, bacteria undergo several changes that prevent eradication with antimicrobials alone. Due to this resistance, the standard of care for infected medical implants is explantation of the infected implant and surrounding tissue, followed by eventual reimplantation of a replacement device. Recent studies have demonstrated the efficacy of heat shock for biofilm eradication. To minimize the heat required for in situ biofilm eradication, this study investigated the hypothesis that antibiotics, while ineffective by themselves, may substantially increase heat shock efficacy. The combined effect of heat and antibiotics on Pseudomonas aeruginosa biofilms was quantified via heat shock in combination with ciprofloxacin, tobramycin, or erythromycin at multiple concentrations. Combined treatments had synergistic effects for all antibiotics for heat shock conditions of 60°C for 5 min to 70°C for 1 min, indicating an alternative to surgical explantation.

Thermal mitigation of Pseudomonas aeruginosa biofilms.

Bacterial biofilms infect 2-4% of medical devices upon implantation, resulting in multiple surgeries and increased recovery time due to the very great increase in antibiotic resistance in the biofilm phenotype. This work investigates the feasibility of thermal mitigation of biofilms at physiologically accessible temperatures. Pseudomonas aeruginosa biofilms were cultured to high bacterial density (1.7 × 10(9) CFU cm(-2)) and subjected to thermal shocks ranging from 50°C to 80°C for durations of 1-30 min. The decrease in viable bacteria was closely correlated with an Arrhenius temperature dependence and Weibull-style time dependence, demonstrating up to six orders of magnitude reduction in bacterial load. The bacterial load for films with more conventional initial bacterial densities dropped below quantifiable levels, indicating thermal mitigation as a viable approach to biofilm control.