Biofilm Producing Staphylococcus epidermidis (RP62A Strain) Inhibits Osseous Integration Without Osteolysis and Histopathology in a Murine Septic Implant Model

Abstract

Despite its presence in orthopaedic infections, Staphylococcus epidermidis's ability to directly induce inflammation and bone destruction is unknown. Thus, we compared a clinical strain of methicillin‐resistant biofilm‐producing S. epidermidis (RP62A) to a highly virulent and osteolytic strain of methicillin‐resistant Staphylococcus aureus (USA300) in an established murine implant‐associated osteomyelitis model. Bacterial burden was assessed by colony forming units (CFUs), tissue damage was assessed by histology and micro‐computed tomography, biofilm was assessed by scanning electron microscopy (SEM), host gene expression was assessed by quantitative polymerase chain reaction, and osseous integration was assessed via biomechanical push‐out test. While CFUs were recovered from RP62A‐contaminated implants and surrounding tissues after 14 days, the bacterial burden was significantly less than USA300‐infected tibiae (p < 0.001). In addition, RP62A failed to produce any of the gross pathologies induced by USA300 (osteolysis, reactive bone formation, Staphylococcus abscess communities, marrow necrosis, and biofilm). However, fibrous tissue was present at the implant‐host interface, and rigorous SEM confirmed the rare presence of cocci on RP62A‐contaminated implants. Gene expression studies revealed that IL‐1β, IL‐6, RANKL, and TLR‐2 mRNA levels in RP62A‐infected bone were increased versus Sterile controls. Ex vivo push‐out testing showed that RP62A‐infected implants required significantly less force compared with the Sterile group (7.5 ± 3.4 vs. 17.3 ± 4.1 N; p < 0.001), but required 10‐fold greater force than USA300‐infected implants (0.7 ± 0.3 N; p < 0.001). Taken together, these findings demonstrate that S. epidermidis is a commensal pathogen whose mechanisms to inhibit osseous integration are limited to minimal biofilm formation on the implant, and low‐grade inflammation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:852‐860, 2020

Distinct vasculotropic versus osteotropic features ofS. agalactiaeversusS. aureusimplant‐associated bone infection in mice

“…Finally, this is the first work to describe direct S. agalactiae‐ mediated bone degradation, as evidenced by scalloping of bone. Previously described in S. aureus , but not in S. epidermidis infection, 51 the degradation of bone may release a nutrient source to the pathogen as well as expose extracellular matrix proteins for bacterial cell attachment and colonization 56 …”

“…For S. aureus , it is understood that biofilm formation is a key survival mechanism in PJIs, as the biofilm provides protection from immune cell attack as well as resistance to antibiotic therapies 14,36,49,50 . In this model of osteomyelitis, both S. aureus and the less virulent Staphylococcus epidermidis 51 colonize the implant with approximately 10 4 CFUs at Day 14 post‐infection. Importantly, this study revealed that S. agalactiae does not persistently colonize the implant hardware, despite their known ability to form biofilms 52 .…”

“…Previous studies investigating the lesser pathogenic organism, S. epidermidis , infection in osteomyelitis showed that its infection phenotype differs from both S. agalactiae and S. aureus . In the same murine model of implant associated osteomyelitis, S. epidermidis infection does not form bone marrow abscesses and does not colonize necrotic bone fragments, but successfully forms robust biofilms on the implant surface 51 . Taken together, it is clear that infection severity and disease pathology can be dependent on the infecting organism.…”

The limitations of mono‐ and combination antibiotic therapies on immature biofilms in a murine model of implant‐associated osteomyelitis

“…A constant 12‐h light/dark cycle was maintained. The murine implant‐associated tibial osteomyelitis model, which does not display sexual dimorphism, was used as previously described 10,21 . Briefly, a methicillin‐susceptible S. aureus (MSSA) strain (UAMS‐1) was cultured in tryptic soy broth (TSB) and used to contaminate a transtibial stainless‐steal implant with 2.5 × 10 5 colony‐forming unit (CFU) 22 .…”

“…The biofilm on the implant was assessed with SEM as a primary outcome (SEM: S‐4700; Hitachi Ltd.). The percent of the biofilm present on the 1 mm 2 region of interest (%biofilm area) was quantified with ImageJ (NIH) ( n = 5 in each group), as previously described 10,21 …”