Background: Approximately 40% of osteomyelitis (bone infection) patients experience relapse of disease and chronic infection due to bacterial persistence. Staphylococcus aureus, the predominant pathogen, is known to persist by phenotypic adaptation as small colony variants (SCVs) and the formation of reservoirs inside the most abundant bone cell type, the osteocyte. However, the role of the viable-but-non-culturable (VBNC) phenotype in persistence of S. aureus in bone cells is largely unexplored since validated methodologies are lacking.
Aim: To establish a model for the intracellular formation of the VBNC phenotype in osteocyte-like cells by modifying the host cell type and multiplicity of infection (MOI).
Method: Differentiated osteocyte-like cells (SAOS-2-OCy or normal human bone cells (NHBC-OCy)) were infected with S. aureus strains from the wound (SAw) or the bone (SAb) of a diabetic foot infection patient for 14-22 days at MOIs between 0.05-100. At various times post-infection, the intracellular number and phenotype of recovered colony forming units (CFUs) were determined and bacterial sigB gene levels measured by real-time PCR. Expression of sigB in the absence of colony growth was defined as a VBNC state.
Results: The VBNC phenotype formed after 7d at MOI of 0.5 and after 14d at MOI 100 in NHBC-OCy. In SAOS-2-OCy, VBNC only formed at MOI 0.5 and 50 with SAb. Both SCV and wild-type colonies were present except at the lowest MOI in both host cell types. The total intracellular CFU number was lower in NHBC-OCy than in SAOS-2-OCy cells.
Conclusion: MOI and host-cell type influence the formation of intracellular VBNC S. aureus in osteocytes. Therefore, a model with NHBC-OCy infected with a MOI of 100 over 14d appears suitable for further investigations. This model could be used to explore VBNC resuscitation to a planktonic state and investigate new treatments for osteomyelitis.