Lysosomal alkalization to potentiate eradication of intraosteoblastic Staphylococcus aureus in the bone and joint infection setting

Abstract Objectives Beyond intracellular penetration, acidic lysosomal pH might affect the intracellular activity of some antimicrobials. This study evaluated the ability of lysosomotropic alkalizing agents to potentiate antimicrobial eradication of intraosteoblastic S. aureus reservoir in the bone and joint infection (BJI) setting. Methods MICs of sixteen anti-staphylococcal molecules against MSSA were evaluated at pH 5 and 7. Additionally, lysosomal alkalinizing potential (spectrofluorometry) and cytotoxicity (MTT assay) of hydroxychloroquine, amantadine and NH4Cl were assessed. Results led to further investigate clindamycin, cotrimoxazole, daptomycin and levofloxacin in an in vitro model of osteoblast infection, alone or in combination with hydroxychloroquine. The impact of hydroxychloroquine on autophagy was finally investigated, using Western blot detection of two autophagic flux indicators, the LC3 membrane protein and the SQSTM1 cargo protein. Results Daptomycin, cotrimoxazole, clindamycin and levofloxacin alone decreased significantly the intracellular staphylococcal reservoir (5.12 log10CFU/100,000 cells) by 0.14 (95%CI, 0.01-0.34), 0.25 (95%CI, 0.12-0.43), 0.16 (95%CI, 0.004-0.39) and 1.18 (95%CI,1.04-1.38) log10CFU/100,000 cells, respectively (p Conclusion At high concentrations, hydroxychloroquine used as an adjuvant to antimicrobials improves eradication of S. aureus intraosteoblastic reservoir in our in vitro cell infection model. These findings advocate for further in vivo evaluation of alkalization efficacy and tolerance in S. aureus BJI.