Probe-Based Ultrasonication Removal of Staphylococcal Biofilm: In Vitro and Ex Vivo Proof of Concept

Introduction
Biofilm formation on implant surfaces is a major clinical challenge in periprosthetic joint infections
Ultrasonication has shown promise for disrupting biofilms, but efficacy on orthopaedic implant materials requires investigation
Presented here:
1. Quantification of ultrasonication removal of biofilm grown in vitro on orthopaedic materials
2. Qualitative observations of ex vivo biofilm removal using a novel mammalian model for staphylococcal knee PJI

Methods

The in vitro study:
Staphylococcus aureus cultured on four orthopaedic materials: bone cement (BC), polyethylene (PE), rough metal (RM, titanium alloy), and smooth metal (SM, polished cobalt chromium) (Fig. 1A-H)
Three ultrasonication protocols (pulse, 20-sec continuous, 60-sec continuous) with a submerged piezoelectric probe (Fig. 1I)
Biofilm presence/removal quantified by crystal violet absorbance assay
Colony forming units (CFU) remaining quantified by water bath sonication in culture media, serial dilution, agar plate culture, and colony counting
Statistics: Two-way ANOVAs with Tukey’s multiple comparisons and 95% confidence intervals were performed in GraphPad Prism 11.0.1.
Scanning electron microscopy (SEM) visualized biofilm changes

The in vivo study:
Novel rabbit model for knee PJI with a cementless 3D-printed porous titanium femoral unicondylar arthroplasty (Fig. 1J-L) [PMID: 42393525]
Intraarticular inoculation 2 weeks post-implant surgery with 2*105 CFU S. aureus
Biofilm developed in vivo for 2 weeks, distal end of the femur containing the implant removed (Fig. 1M) and placed in a sterile silicone mold (Fig. 1N), sterile PBS added, submerged piezoelectric probe ultrasonication performed, and SEM acquired
Negative control: no inoculation, no sonication
Positive control: inoculation, no sonication
Ex vivo biofilm removal: inoculation, 60 sec sonication

Results

Quantitative in vitro assays
Ultrasonication reduced biofilm matrix on all materials to negative control levels (p<0.0001, minimum Cohen’s D effect size=3.1) with no difference between sonication protocols (Fig. 2A)
Ultrasonication reduced viable CFU on all materials (p<0.0001, minimum Cohen’s D effect size=2.29) with no difference between sonication protocols (Fig. 2B)
Ultrasonication decreased the bacterial burden on BC discs by 2 orders of magnitude and on the PE, RM, and SM discs by 3 orders of magnitude (Fig. 2B)

Qualitative in vitro assay - SEM imaging
Robust biofilm observed on all materials without sonication, with abundant bacteria and biofilm extracellular matrix (ECM) (Fig. 3)
Biofilm matrices were eliminated on all materials post-treatment, with only sparse bacteria (black arrows) and ECM debris (white arrowheads) remaining (Fig. 3)
Qualitative ex vivo assay - SEM imaging
Uninoculated negative control exhibited host cell adhesion (Fig. 4A-B)
Untreated biofilm positive control showed robust purulent material and the presence of bacteria (black arrows) (Fig. 4C-D)
Ultrasonication demonstrated effective removal of biofilm and purulent material (Fig. 4E-F)
The ultrasonication probe was stationary, with differences in effect observed based on distance from the probe
Center of ultrasonic field: full biofilm and purulent material removal (Fig. 4G)
Further from probe: a layer of ECM (Fig. 4H-I)
~8 mm off-center: dense purulent material (Fig. 4J)

Conclusions

Submerged probe ultrasonication effectively disrupted S. aureus biofilms in vitro and ex vivo.