Test Parameter Selection Severely Affects in Vitro Antibacterial Efficacy of Contact-Killing Coatings Developed to Prevent Prosthetic Joint Infection

Prosthetic joint infections occur in 1-4% of total hip and knee arthroplasties. Many new treatment technologies to prevent bacterial adhesion and biofilm formation are under development. In addition to the required standard testing methods (ISO 22196, ASTM E2180-18, and JIS Z 2801), additional testing methods are necessary to validate and fully understand the antibacterial activity of new technologies. This study highlights the importance of selecting methodological parameters and processing data in antibacterial testing by evaluating a contact-killing coating composed of poly-ε-lysine and hyaluronic acid (PEL-10/HA-1). Two broth compositions, inoculum volumes, and agitation speeds were evaluated to assess bacterial adhesion to PEL-10/HA-1-coated and uncoated titanium alloy Ti6Al4V (Ti) samples. Samples were inoculated with 1 × 107 CFU/mL of S. aureus (ATCC 25923). The inoculum was prepared in tryptic soy broth (TSB) or Mueller-Hinton broth (MHB) at volumes of 0.5 or 1 mL. Inoculated samples were incubated for 24 hours at 60 or 120 RPM (0.05 or 0.2 g, respectively). After incubation, the attached colony-forming units (CFU) to the samples were counted. In MHB, PEL-10/HA-1-coated samples showed a >5 log reduction in CFU/sample compared with uncoated samples. In TSB, reductions ranged from 0.4 to 3.5 log. A volume of 0.5 mL and an agitation speed of 120 RPM produced higher reductions in CFU/sample than 1 mL and 60 RPM, respectively. The main source of variation was the type of broth, followed by agitation speed and inoculum volume. In conclusion, methodological choices in seemingly standard antibacterial efficacy testing protocols significantly influence outcomes and perceived antibacterial efficacy. These results underscore the critical need to carefully select and thoroughly document parameters to ensure the effectiveness and reliability of testing antibacterial technologies. Without clear parameters, comparisons between studies become unreliable, and the true antibacterial efficacy remains unknown. ISO and adhesion tests are suitable initial steps in evaluating antibacterial technologies. However, a comprehensive evaluation using multiple antibacterial tests is essential to thoroughly assess and characterize antibacterial technology.