{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T23:03:20Z","timestamp":1790895800443,"version":"4.1.0"},"reference-count":37,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,6,24]],"date-time":"2022-06-24T00:00:00Z","timestamp":1656028800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:p><jats:italic>S. aureus<\/jats:italic> infection of bone is difficult to eradicate due to its ability to colonize the osteocyte-lacuno-canalicular network (OLCN), rendering it resistant to standard-of-care (SOC) antibiotics. To overcome this, we proposed two bone-targeted bisphosphonate-conjugated antibiotics (BCA): bisphosphonate-conjugated sitafloxacin (BCS) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS). Initial studies demonstrated that the BCA kills <jats:italic>S. aureus in vitro<\/jats:italic>. Here we demonstrate the <jats:italic>in vivo<\/jats:italic> efficacy of BCS and HBCS versus bisphosphonate, sitafloxacin, and vancomycin in mice with implant-associated osteomyelitis. Longitudinal bioluminescent imaging (BLI) confirmed the hypothesized \u201ctarget and release\u201d-type kinetics of BCS and HBCS. Micro-CT of the infected tibiae demonstrated that HBCS significantly inhibited peri-implant osteolysis versus placebo and free sitafloxacin (p &amp;lt; 0.05), which was not seen with the corresponding non-antibiotic-conjugated bisphosphonate control. TRAP-stained histology confirmed that HBCS significantly reduced peri-implant osteoclast numbers versus placebo and free sitafloxacin controls (p &amp;lt; 0.05). To confirm <jats:italic>S. aureus<\/jats:italic> killing, we compared the morphology of <jats:italic>S. aureus<\/jats:italic> autolysis within <jats:italic>in vitro<\/jats:italic> biofilm and infected tibiae <jats:italic>via<\/jats:italic> transmission electron microscopy (TEM). Live bacteria <jats:italic>in vitro<\/jats:italic> and <jats:italic>in vivo<\/jats:italic> presented as dense cocci ~1 \u03bcm in diameter. <jats:italic>In vitro<\/jats:italic> evidence of autolysis presented remnant cell walls of dead bacteria or \u201cghosts\u201d and degenerating (non-dense) bacteria. These features of autolyzed bacteria were also present among the colonizing <jats:italic>S. aureus<\/jats:italic> within OLCN of infected tibiae from placebo-, vancomycin-, and sitafloxacin-treated mice, similar to placebo. However, most of the bacteria within OLCN of infected tibiae from BCA-treated mice were less dense and contained small vacuoles and holes &amp;gt;100 nm. Histomorphometry of the bacteria within the OLCN demonstrated that BCA significantly increased their diameter versus placebo and free antibiotic controls (p &amp;lt; 0.05). As these abnormal features are consistent with antibiotic-induced vacuolization, bacterial swelling, and necrotic phenotype, we interpret these findings to be the initial evidence of BCA-induced killing of <jats:italic>S. aureus<\/jats:italic> within the OLCN of infected bone. Collectively, these results support the bone targeting strategy of BCA to overcome the biodistribution limits of SOC antibiotics and warrant future studies to confirm the novel TEM phenotypes of bacteria within OLCN of <jats:italic>S. aureus<\/jats:italic>-infected bone of animals treated with BCS and HBCS.<\/jats:p>","DOI":"10.3389\/fcimb.2022.910970","type":"journal-article","created":{"date-parts":[[2022,6,24]],"date-time":"2022-06-24T07:45:14Z","timestamp":1656056714000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":24,"title":["Efficacy of Bisphosphonate-Conjugated Sitafloxacin in a Murine Model of S. aureus Osteomyelitis: Evidence of \u201cTarget &amp; Release\u201d Kinetics and Killing of Bacteria Within Canaliculi"],"prefix":"10.3389","volume":"12","author":[{"given":"Youliang","family":"Ren","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Xue","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joshua","family":"Rainbolt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karen L. de Mesy","family":"Bentley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chad A.","family":"Galloway","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuting","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philip","family":"Cherian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeffrey","family":"Neighbors","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marloes I.","family":"Hofstee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Frank H.","family":"Ebetino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas Fintan","family":"Moriarty","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuting","family":"Sun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edward M.","family":"Schwarz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chao","family":"Xie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2022,6,24]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"732","DOI":"10.3390\/antibiotics10060732","article-title":"Development of Bisphosphonate-Conjugated Antibiotics to Overcome Pharmacodynamic Limitations of Local Therapy: Initial Results With Carbamate Linked Sitafloxacin and Tedizolid","volume":"10","author":"Adjei-Sowah","year":"2021","journal-title":"Antibiotics (Basel)"},{"key":"B2","doi-asserted-by":"publisher","first-page":"43","DOI":"10.1093\/jac\/44.1.43","article-title":"Antibiotic Susceptibility Assay for Staphylococcus Aureus in Biofilms Developed In Vitro","volume":"44","author":"Amorena","year":"1999","journal-title":"J. Antimicrob. Chemother."},{"key":"B3","doi-asserted-by":"publisher","first-page":"96","DOI":"10.1097\/00003086-198411000-00014","article-title":"Antibiotic-Loaded Acrylic Cement: Current Concepts","author":"Buchholz","year":"1984","journal-title":"Clin. Orthop Relat. Res."},{"key":"B4","doi-asserted-by":"publisher","first-page":"845","DOI":"10.1002\/jor.23080","article-title":"Antioxidant Impregnated Ultra-High Molecular Weight Polyethylene Wear Debris Particles Display Increased Bone Remodeling and a Superior Osteogenic:Osteolytic Profile vs. Conventional UHMWPE Particles in a Murine Calvaria Model","volume":"34","author":"Chen","year":"2016","journal-title":"J. Orthop Res."},{"key":"B5","doi-asserted-by":"publisher","first-page":"627","DOI":"10.1006\/jmbi.1996.0274","article-title":"DNA Gyrase and Topoisomerase IV on the Bacterial Chromosome: Quinolone-Induced DNA Cleavage","volume":"258","author":"Chen","year":"1996","journal-title":"J. Mol. Biol."},{"key":"B6","doi-asserted-by":"publisher","first-page":"376","DOI":"10.1002\/jor.24968","article-title":"Emerging Electron Microscopy and 3D Methodologies to Interrogate Staphylococcus Aureus Osteomyelitis in Murine Models","volume":"39","author":"de Mesy Bentley","year":"2021","journal-title":"J. Orthop Res."},{"key":"B7","doi-asserted-by":"publisher","first-page":"e8","DOI":"10.2106\/JBJS.CC.17.00154","article-title":"Chronic Osteomyelitis With Staphylococcus Aureus Deformation in Submicron Canaliculi of Osteocytes: A Case Report","volume":"8","author":"de Mesy Bentley","year":"2018","journal-title":"JBJS Case Connect"},{"key":"B8","doi-asserted-by":"publisher","first-page":"985","DOI":"10.1002\/jbmr.3055","article-title":"Evidence of Staphylococcus Aureus Deformation, Proliferation, and Migration in Canaliculi of Live Cortical Bone in Murine Models of Osteomyelitis","volume":"32","author":"de Mesy Bentley","year":"2017","journal-title":"J. Bone Miner Res."},{"key":"B9","doi-asserted-by":"publisher","first-page":"51","DOI":"10.3109\/01913129209074550","article-title":"Large Block Embedding and \"Pop-Off\" Technique for Immunoelectron Microscopy","volume":"16","author":"de Mesy Jensen","year":"1992","journal-title":"Ultrastruct Pathol."},{"key":"B10","doi-asserted-by":"publisher","first-page":"51","DOI":"10.3109\/01913129209074550","article-title":"Large Block Embedding and \u201cPop-Off\u201d Technique for Immunoelectron Microscopy","volume":"16","author":"de Mesy Jensen","year":"1992","journal-title":"Ultrastruct. Pathol."},{"key":"B11","volume-title":"Bisphosphonate Quinolone Conjugates and Uses Thereof","author":"Ebetino","year":"2020"},{"key":"B12","doi-asserted-by":"publisher","first-page":"47","DOI":"10.1016\/j.bonr.2018.06.007","article-title":"Bisphosphonate Conjugation for Bone Specific Drug Targeting","volume":"9","author":"Farrell","year":"2018","journal-title":"Bone Rep."},{"key":"B13","doi-asserted-by":"publisher","first-page":"855","DOI":"10.1128\/AAC.01003-12","article-title":"In Vivo Bioluminescence Imaging to Evaluate Systemic and Topical Antibiotics Against Community-Acquired Methicillin-Resistant Staphylococcus Aureus-Infected Skin Wounds in Mice","volume":"57","author":"Guo","year":"2013","journal-title":"Antimicrob. Agents Chemother."},{"key":"B14","doi-asserted-by":"publisher","first-page":"1446","DOI":"10.3390\/pathogens10111446","article-title":"In Vitro 3d Staphylococcus Aureus Abscess Communities Induce Bone Marrow Cells to Expand Into Myeloid-Derived Suppressor Cells","volume":"10","author":"Hofstee","year":"2021","journal-title":"Pathogens"},{"key":"B15","doi-asserted-by":"publisher","first-page":"385","DOI":"10.1016\/j.ajic.2005.06.006","article-title":"Emergence of Community-Associated Methicillin-Resistant Staphylococcus Aureus USA 300 Clone as a Cause of Health Care-Associated Infections Among Patients With Prosthetic Joint Infections","volume":"33","author":"Kourbatova","year":"2005","journal-title":"Am. J. Infect. Control"},{"key":"B16","doi-asserted-by":"publisher","first-page":"84","DOI":"10.1111\/j.1749-6632.2009.05210.x","article-title":"Effects of Antiresorptive Agents on Osteomyelitis: Novel Insights Into the Pathogenesis of Osteonecrosis of the Jaw","volume":"1192","author":"Li","year":"2010","journal-title":"Ann. N Y Acad. Sci."},{"key":"B17","doi-asserted-by":"publisher","first-page":"96","DOI":"10.1002\/jor.20452","article-title":"Quantitative Mouse Model of Implant-Associated Osteomyelitis and the Kinetics of Microbial Growth, Osteolysis, and Humoral Immunity","volume":"26","author":"Li","year":"2008","journal-title":"J. Orthop Res."},{"key":"B18","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41579-022-00686-0","article-title":"Skeletal Infections: Microbial Pathogenesis, Immunity and Clinical Management","author":"Masters","year":"2022","journal-title":"Nat. Rev. Microbiol"},{"key":"B19","doi-asserted-by":"publisher","first-page":"20","DOI":"10.1038\/s41413-019-0061-z","article-title":"Evolving Concepts in Bone Infection: Redefining \"Biofilm\", \"Acute vs. Chronic Osteomyelitis\", \"the Immune Proteome\" and \"Local Antibiotic Therapy\"","volume":"7","author":"Masters","year":"2019","journal-title":"Bone Res."},{"key":"B20","doi-asserted-by":"publisher","first-page":"326","DOI":"10.1111\/j.1439-0450.2006.00962.x","article-title":"Comparative Assessment of the Antimicrobial Susceptibility of Staphylococcus Aureus Isolates From Bovine Mastitis in Biofilm Versus Planktonic Culture","volume":"53","author":"Melchior","year":"2006","journal-title":"J. Vet. Med. B Infect. Dis. Vet. Public Health"},{"key":"B21","doi-asserted-by":"publisher","first-page":"136","DOI":"10.1002\/jor.24804","article-title":"The AO Trauma CPP Bone Infection Registry: Epidemiology and Outcomes of Staphylococcus Aureus Bone Infection","volume":"39","author":"Morgenstern","year":"2021","journal-title":"J. Orthop Res."},{"key":"B22","doi-asserted-by":"publisher","first-page":"1311","DOI":"10.1002\/jor.22907","article-title":"Quantifying the Natural History of Biofilm Formation In Vivo During the Establishment of Chronic Implant-Associated Staphylococcus Aureus Osteomyelitis in Mice to Identify Critical Pathogen and Host Factors","volume":"33","author":"Nishitani","year":"2015","journal-title":"J. Orthop Res."},{"key":"B23","doi-asserted-by":"publisher","first-page":"S1","DOI":"10.1016\/j.arth.2018.09.038","article-title":"Introduction: Proceedings of International Consensus on Orthopedic Infections","volume":"34","author":"Parvizi","year":"2018","journal-title":"J. Arthroplasty"},{"key":"B24","doi-asserted-by":"publisher","first-page":"1007","DOI":"10.1002\/jor.24229","article-title":"The 2018 International Consensus Meeting on Musculoskeletal Infection: Summary From the Biofilm Workgroup and Consensus on Biofilm Related Musculoskeletal Infections","volume":"37","author":"Saeed","year":"2019","journal-title":"J. Orthop Res"},{"key":"B25","doi-asserted-by":"publisher","first-page":"227","DOI":"10.1002\/jor.24616","article-title":"Adjuvant Antibiotic-Loaded Bone Cement: Concerns With Current Use and Research to Make it Work","volume":"39","author":"Schwarz","year":"2021","journal-title":"J. Orthop Res."},{"key":"B26","doi-asserted-by":"publisher","first-page":"997","DOI":"10.1002\/jor.24293","article-title":"The 2018 International Consensus Meeting on Musculoskeletal Infection: Research Priorities From the General Assembly Questions","volume":"37","author":"Schwarz","year":"2019","journal-title":"J. Orthopaedic Research\u00ae"},{"key":"B27","doi-asserted-by":"publisher","first-page":"997","DOI":"10.1002\/jor.24293","article-title":"2018 International Consensus Meeting on Musculoskeletal Infection: Research Priorities From the General Assembly Questions","volume":"37","author":"Schwarz","year":"2019","journal-title":"J. Orthop Res."},{"key":"B28","doi-asserted-by":"publisher","first-page":"2326","DOI":"10.1021\/acs.jmedchem.6b01615","article-title":"Design, Synthesis, and Antimicrobial Evaluation of a Novel Bone-Targeting Bisphosphonate-Ciprofloxacin Conjugate for the Treatment of Osteomyelitis Biofilms","volume":"60","author":"Sedghizadeh","year":"2017","journal-title":"J. Med. Chem."},{"key":"B29","doi-asserted-by":"publisher","first-page":"2008","DOI":"10.1111\/bph.15251","article-title":"Bisphosphonates for Delivering Drugs to Bone","volume":"178","author":"Sun","year":"2021","journal-title":"Br. J. Pharmacol."},{"key":"B30","doi-asserted-by":"publisher","first-page":"6585","DOI":"10.4049\/jimmunol.1002794","article-title":"Staphylococcus Aureus Biofilms Prevent Macrophage Phagocytosis and Attenuate Inflammation In Vivo","volume":"186","author":"Thurlow","year":"2011","journal-title":"J. Immunol."},{"key":"B31","doi-asserted-by":"publisher","first-page":"94","DOI":"10.1128\/mSphere.00422-18","article-title":"A High-Throughput Screening Approach To Repurpose FDA-Approved Drugs for Bactericidal Applications Against Staphylococcus Aureus Small-Colony Variants","volume":"3","author":"Trombetta","year":"2018","journal-title":"mSphere"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.3390\/pharmaceutics11020094","article-title":"Calcium Phosphate Spacers for the Local Delivery of Sitafloxacin and Rifampin to Treat Orthopedic Infections: Efficacy and Proof of Concept in a Mouse Model of Single-Stage Revision of Device-Associated Osteomyelitis","volume":"11","author":"Trombetta","year":"2019","journal-title":"Pharmaceutics"},{"key":"B33","doi-asserted-by":"publisher","first-page":"154","DOI":"10.3390\/pharmaceutics10030154","article-title":"Synthesis of a Bone-Targeted Bortezomib With In Vivo Anti-Myeloma Effects in Mice","volume":"10","author":"Wang","year":"2018","journal-title":"Pharmaceutics"},{"key":"B34","doi-asserted-by":"publisher","first-page":"343","DOI":"10.1002\/jbmr.3889","article-title":"Targeting Bortezomib to Bone Increases Its Bone Anabolic Activity and Reduces Systemic Adverse Effects in Mice","volume":"35","author":"Wang","year":"2020","journal-title":"J. Bone Miner Res."},{"key":"B35","doi-asserted-by":"publisher","first-page":"325","DOI":"10.1002\/jor.21500","article-title":"Unique Angiogenic and Vasculogenic Properties of Renal Cell Carcinoma in a Xenograft Model of Bone Metastasis are Associated With High Levels of Vegf-a and Decreased Ang-1 Expression","volume":"30","author":"Xie","year":"2012","journal-title":"J. Orthop Res."},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.1016\/j.bone.2020.115492","article-title":"Targeting Anti-Cancer Agents to Bone Using Bisphosphonates","volume":"138","author":"Xing","year":"2020","journal-title":"Bone"},{"key":"B37","doi-asserted-by":"publisher","first-page":"2124","DOI":"10.1359\/JBMR.050806","article-title":"Periosteal Progenitor Cell Fate in Segmental Cortical Bone Graft Transplantations: Implications for Functional Tissue Engineering","volume":"20","author":"Zhang","year":"2005","journal-title":"J. Bone Miner Res."}],"container-title":["Frontiers in Cellular and Infection Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2022.910970\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,6,24]],"date-time":"2022-06-24T07:45:16Z","timestamp":1656056716000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2022.910970\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,6,24]]},"references-count":37,"alternative-id":["10.3389\/fcimb.2022.910970"],"URL":"https:\/\/doi.org\/10.3389\/fcimb.2022.910970","relation":{},"ISSN":["2235-2988"],"issn-type":[{"value":"2235-2988","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,6,24]]},"article-number":"910970"}}