In vitro Activity of Antimicrobials Alone and in Combination Therapy against Methicillin-resistant Staphylococcus pseudintermedius Biofilm Formation

Abstract

Biofilms are defined as complex, surface-attached communities of microorganisms, the formation of which allows bacteria to become recalcitrant to antibiotic therapy and host immune response. Surgical site infections (SSIs) caused by biofilm formation on medical devices have a tremendous impact on the management of patient health in veterinary medicine often leading to morbidity, prolonged hospitalization, and increased treatment costs. Currently, methicillin-resistant Staphylococcus pseudintermedius (MRSP) has emerged as the leading cause of SSIs in canines in veterinary hospitals across North America. Because of the importance of SSIs in companion animals and the increasing evidence that biofilms play a key role in hospital-acquired infections, ways to prevent or eliminate biofilm formation are needed. To test the efficacy of therapeutic eradication treatments of chosen antimicrobials, crystal violet assays had 20 MRSP isolates sub-cultured and inoculated into a broth dilution before addition to microtiter plates. Absorbance readings, OD570, were then taken after removal of planktonic bacteria followed by staining, heat fixing, and finally with elution of biofilm-embedded bacteria. Quantitative assay results (P = 0.512) suggest that the influence of clarithromycin in the remediation of MRSP biofilm formation was insignificant between 4 and 24 h time points, indicating that MRSP biofilms exhibit higher resistance to clarithromycin in therapeutic doses than other staphylococci. Adhesion was characterized through growth of MRSP isolates on stainless-steel orthopaedic screws exposed to antimicrobials at various time points using Scanning Electron microscopy (SEM). Qualitative analysis of SEM images revealed the ability of the MRSP isolate to form biofilm across the surface of the orthopaedic screws and in-between threads treated with Clarithromycin. Disk diffusion and microtiter plate assay studies along with Scanning Electron Imaging investigations reveals that Clartithromycin does not inhibit MRSP adherence and biofilm formation.