From Biofilms to Bubbles: Dispersion of Bacteria from Biofilms Using Histotripsy

Abstract

Orthopedic infections are difficult to treat and diagnose because bacteria often persist within biofilms, fibrin networks, host tissue, and other protective infection-associated matrices. These structures limit antimicrobial penetration, reduce bacterial susceptibility, and make viable bacteria difficult to recover for culture. This thesis investigated whether focused ultrasound technologies could mechanically disrupt these protective bacterial environments to improve both antibiotic-mediated bacterial killing and culture-based diagnostic recovery. The first objective was to evaluate histotripsy as an adjunctive strategy to improve antibiotic killing of biofilm-associated bacteria in equine synovial environments. Staphylococcus aureus ATCC™ 25923 and an agrC knockout strain were grown as biofilm-associated aggregates in either biofilm media or pooled equine synovial fluid and treated with histotripsy, proteinase K (200 µg/ml), amikacin sulfate, or combination treatments. Histotripsy consistently disrupted biofilm structure and produced greater dispersal than untreated controls and proteinase K (200 µg/ml), as measured by optical density. Amikacin alone had limited activity against protected biofilm-associated bacteria, while combination treatment with histotripsy and amikacin produced greater bacterial killing than amikacin alone. In biofilm media, combination treatment reduced viable bacterial counts by up to approximately 6 log₁₀ CFU/mL compared with untreated controls. Bacterial killing was less pronounced in synovial fluid, where viable bacteria persisted despite dispersal and antibiotic exposure. These findings suggest that mechanical disruption of biofilm-associated aggregates can improve antibiotic access and enhance bacterial killing, while also demonstrating the protective effect of synovial fluid and infection-associated matrices. The second objective was to evaluate Focused Ultrasound Extraction (FUSE), as a rapid dispersal method to improve bacterial recovery from biofilms and infected tissue. Biofilms of Staphylococcus aureus ATCC™ 25923 and Pseudomonas aeruginosa PAO1 were grown for 24 hours in RPMI containing 20% equine plasma and treated with proteinase K (200 µg/ml), sonication, or FUSE at increasing total pulse doses. For S. aureus ATCC™ 25923, FUSE at 40 cycles, 200 Hz pulse repetition frequency, and 20,000 total pulses produced the greatest mean recovery of culturable bacteria, while higher pulse doses reduced recoverability. For P. aeruginosa PAO1, lower FUSE doses did not significantly increase recovery, and higher pulse doses significantly reduced recoverability. These findings demonstrate that FUSE dose must be optimized to favor bacterial release while preserving viability, and that the effective dose window differs by organism. FUSE was then evaluated using equine-derived clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. FUSE significantly improved recovery from K. pneumoniae M26-1142 at 10,000 pulses and from S. aureus M22-2451 at 20,000 pulses, while no significant treatment effect was detected for E. coli M25-0692 under the conditions tested. These isolate-dependent differences likely reflect differences in biofilm structure, density, and matrix composition. S. aureus formed a visibly more robust and structured biofilm than K. pneumoniae or E. coli, creating a greater opportunity for dispersal-based methods to improve bacterial recovery. In contrast, relatively high bacterial recovery from untreated controls for some Gram-negative isolates suggests that these organisms may have been more readily recoverable without active dispersal in this model. Finally, FUSE was evaluated using human periprosthetic joint infection tissue samples and compared with an enzymatic dispersal agent. FUSE rapidly disrupted infected tissue and released culturable bacteria within minutes. In the majority of samples, FUSE produced greater colony recovery per gram of tissue than enzymatic dispersal, while requiring only a brief treatment period. These findings support the potential use of FUSE as a rapid culture-preparation strategy for infected tissue. Together, these studies support focused ultrasound as a versatile mechanical disruption platform for orthopedic infections. In the therapeutic setting, histotripsy improved antibiotic-mediated bacterial killing by disrupting protected bacterial aggregates. In the diagnostic setting, FUSE improved recovery of viable bacteria from biofilms and infected tissue when delivered within an organism-appropriate dose range. These findings address a shared clinical problem: bacteria embedded within protective matrices are difficult to kill and difficult to culture. Further optimization and validation using larger clinical sample sets may support future development of focused ultrasound strategies for both treatment and diagnosis of biofilm-associated orthopedic infections.