Biofilm formation by staphylococci on fresh, fresh-frozen and processed human and bovine bone grafts

Abstract

Biofilm formation is a multi-step process influenced by surface properties. We investigated early and mature biofilm of Staphylococcus aureus on 4 different biological calcium phosphate (CaP) bone grafts used for filling bone defects. We investigated standardised cylinders of fresh and fresh-frozen human bone grafts were harvested from femoral heads; processed human and bovine bone grafts were obtained preformed. Biofilm formation was done in tryptic soy broth (TSB) using S. aureus (ATCC 29213) with static conditions. Biofilm density after 3 h (early biofilm) and 24 h (mature biofilm) was investigated by sonication and microcalorimetry. After 3 h, bacterial density was highest on fresh-frozen and fresh bone grafts. After 24 h, biofilm density was lowest on fresh bone grafts (p < 0.001) compared to the other 3 materials, which did not differ quantitatively (p > 0.05). The lowest increase in bacterial density was detected on fresh bone grafts (p < 0.001). Despite normal shaped colonies, we found additional small colonies on the surface of the fresh and fresh-frozen samples by sonication. This was also apparent in microcalorimetric heat-flow curves. The four investigated CaP bone grafts showed minor structural differences in architecture but marked differences concerning serum coverage and the content of bone marrow, fibrous tissue and bone cells. These variations resulted in a decreased biofilm density on fresh and fresh-frozen bone grafts after 24 h, despite an increased early biofilm formation and might also be responsible for the variations in colony morphology (small colonies). Detection of small colony variants by microcalorimetry might be a new approach to improve the understanding of biofilm formation.

Staphylococcus aureus Infections: Epidemiology, Pathophysiology, Clinical Manifestations, and Management

“…Strains of S. aureus lacking genes that encode certain MSCRAMMs are less likely to cause osteoarticular infections in animal models (417,419,420). S. aureus is also able to form biofilms on foreign materials that act as sanctuary sites, where it is relatively protected from antimicrobial agents and the host immune response (421). Finally, S. aureus can invade osteoblasts (422) and form small-colony variants (SCVs) (423) in the intracellular compartment, where they are able to survive in a metabolically inactive state while preserving the integrity of the host cell.…”

Microcalorimetric detection of staphylococcal biofilm growth on various prosthetic biomaterials after exposure to daptomycin

“…The biofilm evaluations were performed according to a well‐established protocol including sonication, culture, and microcalorimetric heat flow measurement previously used to analyze the influence of physicochemical biomaterial properties, as well as studies of antimicrobial susceptibility of biofilm bacteria …”

“…We investigated two well‐characterized reference strains of S. aureus (ATCC 29213, methicillin‐susceptible) and S. epidermidis (ATCC 35984, methicillin‐resistant) . These ATCC‐strains, capable of biofilm production through adherence to and aggregation on biomaterial surfaces, have been used in numerous experimental studies, and were considered representative of staphylococcal species commonly causing PJI. The susceptibility of these strains has been determined in a previous experimental study analyzing the inhibitory effect of daptomycin against a 24 h biofilm compared to the minimal inhibitory concentration (MIC) of planktonic bacteria .…”

“…Isothermal microcalorimetry and sonication were recently used to analyze the influence of bone graft material properties on the initial adhesion and formation of biofilm under in vitro and in vivo settings . Microcalorimetry is a highly sensitive and accurate method for the detection of slow‐growing microorganisms and reduced bacterial numbers after antibiotic pre‐exposure .…”

◾ Calorimetric Approaches to Studying Complex Protein Structure–Function–Stability Relationships in Conformational Diseases: The Case of Cystathionine β-Synthase

“…For instance, Clauss et al (2013) monitored biofilms of S. aureus growing on human and bovine bone grafts, while Astasov-Frauenhoffer et al (2012) investigated the variability and dynamics of a triple-species biofilm and determined the efficacy of amoxicillin and metronidazole combinations against biofilms of various species (Astasov-Frauenhoffer et al 2014). Said et al (2014a) determined the efficacy and mechanism of action of an antibiofilm wound dressing.…”