Is Implant Coating With Tyrosol- and Antibiotic-loaded Hydrogel Effective in Reducing Cutibacterium (Propionibacterium) acnes Biofilm Formation? A Preliminary In Vitro Study
Authors: Tsikopoulos Konstantinos , Bidossi Alessandro , Drago Lorenzo , Petrenyov Daniil R. , Givissis Panagiotis , Mavridis Dimitris , Papaioannidou Paraskevi 
Published: May 2019

Abstract

Background
Studies have suggested that Cutibacterium acnes (formerly known as Propionibacterium) is the most frequently isolated pathogen after shoulder arthroplasty. To address the burden of periprosthetic joint infections associated with this pathogen, new prevention methods are needed. Tyrosol has a promising record of effectiveness in the field of biofilm-associated infections; however, to our knowledge, it has not been tested against C. acnes thus far.

Questions/purposes
In this in vitro study, we asked: (1) Is tyrosol effective in inhibiting and eradicating C. acnes planktonic growth? (2) Is there synergy between tyrosol and rifampicin? (3) Is supplementation of hydrogel with tyrosol at the minimum inhibitory and subinhibitory concentrations efficacious in reducing free-floating C. acnes growth? (4) Is implant hydrogel coating (either alone or combined with tyrosol, rifampicin, or vancomycin) beneficial in reducing C. acnes biofilm formation? (5) Is the administration of soluble tyrosol an effective measure against C. acnes biofilm formation?

Methods
We assessed C. acnes planktonic growth and eradication by inspecting visually the results of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. We also evaluated macroscopically the presence of synergy among tyrosol and rifampicin by means of the MIC checkerboard testing. Thereafter, we addressed colorimetrically the efficacy of tyrosol-loaded Defensive Antibacterial Coating (DAC®) hydrogel against the C. acnes free-floating form by means of the XTT cell proliferation reduction assay. Then, we explored photometrically the effect of hydrogel and soluble tyrosol at reducing C. acnes biofilm formation on titanium alloy disks that simulated orthopaedic implants by using the minimum biofilm inhibition concentration assay. In particular, 16 disks were sequentially allocated to each of the following testing conditions: (1) hydrogel alone; (2) tyrosol-loaded hydrogel; (3) rifampicin-supplemented hydrogel; (4) vancomycin-loaded hydrogel; and (5) soluble tyrosol. Subsequently, implants were sonicated and cell viability was evaluated in terms of the XTT assay.

Results
Tyrosol was effective in inhibiting C. acnes planktonic (free-floating) growth demonstrating MIC values of 63 mM (9 mg/mL) and MBC values of 250 mM (35 mg/mL). Concerning synergy assessment, the checkerboard testing revealed additivity among tyrosol and rifampicin with a fractional inhibitory concentration index of 0.56. In addition, a hydrogel coating with tyrosol at the MIC showed no difference in the inhibition of free-floating C. Acnes form over control (median absorbance [MA] for tyrosol-supplemented hydrogel versus control groups were 0.21 [interquartile range {IQR}, 0.19–0.24] versus 0.26 [IQR, 0.23–0.31], p = 0.066). Furthermore, loaded hydrogel with tyrosol at 597 mg/mL (1 M) was no more effective than control in reducing C. acnes biofilm formation (MAs for tyrosol versus control were 0.12 [IQR, 0.11–0.13] versus 0.14 [IQR, 0.12–0.16], respectively; p = 0.076). This was also the case when we considered hydrogel in conjunction with vancomycin and rifampicin (MAs for vancomycin at 2% and 5% and rifampicin at 1% versus biofilm control were 0.139 [IQR, 0.133–0.143] and 0.141 [IQR, 0.133–0.143] and 0.135 [IQR, 0.128–0.146] versus 0.142 [IQR, 0.136–0.144], correspondingly). In contrast, soluble tyrosol at 597 mg/mL (1 M) inhibited biofilm formation compared to control (MAs for tyrosol and control groups were 0.11 [IQR, 0.09–0.13] versus 0.13 [IQR, 0.12–0.14], p = 0.007).

Conclusions
Although the implant coating with hydrogel (either pure or supplemented with antimicrobial agents) did not diminish C. acnes biofilm development in vitro, soluble tyrosol at 597 mg/mL (1 M) exceeded the meaningful biofilm inhibition threshold of 80%.

Clinical Relevance
The results of the current preclinical investigation did not support the use of a fast, bioresorbable hydrogel as a coating method against C. acnes biofilms. Instead, direct local administration of soluble tyrosol at high concentrations should be further tested in future animal studies.