Test parameter selection affects the perceived antibacterial efficacy of a contact-killing coating developed to prevent prosthetic joint infection

Abstract

Introduction Prosthetic joint infections occur in 1% -4% of total hip and knee arthroplasties. Many new treatment approaches to prevent bacterial adhesion and biofilm formation are under development. This study highlights the importance of methodological parameter selection and data processing in in vitro antibacterial testing by evaluating a contact-killing coating composed of poly-ε-lysine and hyaluronic acid (PEL-10/HA-1). Methods Two broth compositions, inoculum volumes, and agitation speeds were evaluated to test bacterial adhesion on PEL-10/HA-1-coated and uncoated titanium alloy Ti6Al4V (Ti) samples. The samples were inoculated with 1 × 107 colony-forming units (CFU)/mL of Staphylococcus aureus (ATCC 25923). The inoculum was prepared in tryptic soy broth (TSB) or Mueller-Hinton broth (MHB) in volumes of 0.5 or 1 mL. The inoculated samples were incubated for 24 h at 60 or 120 RPM (0.05 or 0.2 g, respectively). After incubation, the attached CFU per sample were counted. Results In MHB, a >5 log reduction in CFU/sample was observed for the coated samples compared to the uncoated ones. In TSB, reductions ranged from 0.4 to 3.5 log. A volume of 0.5 mL and an agitation speed of 120 RPM resulted in higher reductions in CFU/sample compared to 1 mL and 60 RPM, respectively. The main source of variation was the type of broth, followed by agitation speed and inoculum volume. Discussion In conclusion, methodological choices in seemingly standard antibacterial efficacy testing protocols significantly influence the outcomes. These results underscore the critical need to carefully select and thoroughly document parameters to ensure the effectivity and reliability of testing antibacterial approaches.