{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T11:46:27Z","timestamp":1785843987309,"version":"3.56.0"},"reference-count":33,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T00:00:00Z","timestamp":1785801600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Biocomposites Limited, Keele, UK"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibiotics"],"abstract":"<jats:p>Background: Bacterial biofilms play a key role in causing periprosthetic joint infection (PJI). Current PJI management strategies commonly combine systemic antibiotic therapy with localized delivery such as antibiotic-loaded cement or beads to achieve effective tissue penetration and high antimicrobial concentrations at the implant site. We hypothesized that antibiotic-loaded calcium sulfate beads would effectively eradicate early Staphylococcus aureus biofilms but exhibit reduced efficacy against mature biofilms formed in synovial fluid (SF). This study evaluated the efficacy of vancomycin combined with gentamicin (V+G) or tobramycin (V+T) against GFP-expressing S. aureus biofilms grown in the presence of SF. Methods: Biofilms were established on clinically relevant orthopedic implant materials such as titanium (Ti), stainless steel (316L), and polyethylene (PE). Early (1-day) and mature (3-day) biofilms were treated with calcium sulfate beads loaded with V+G or V+T. Antimicrobial efficacy was quantified by colony-forming unit (CFU) enumeration. Minimum inhibitory concentration (MIC) testing was performed on surviving populations to assess potential development of antibiotic resistance. Results: Both antibiotic combinations resulted in no detectable viable bacteria in the 1-day biofilm across all tested materials following treatment, demonstrating strong reduction in early biofilm burden below the detection limit. Treatment of mature biofilms resulted in a 4\u20135 log reduction. MIC analysis of representative bacteria surviving post-treatment indicated no substantial increase in resistance. Conclusions: These findings show that while locally delivered antibiotic combinations eliminate early biofilms, mature biofilms demonstrate significant tolerance. MIC analysis showed little or no change in vancomycin, gentamicin, and tobramycin susceptibility after treatment, indicating no evident increase in planktonic antibiotic resistance among the surviving isolates. Together, these results highlight the importance of early intervention and the need for strategies that disrupt biofilm structure or improve antibiotic penetration to enhance PJI treatment outcomes.<\/jats:p>","DOI":"10.3390\/antibiotics15080752","type":"journal-article","created":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T10:38:08Z","timestamp":1785839888000},"page":"752","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Efficacy of Antibiotic-Loaded Stimulan Beads Against Biofilms on Clinically Relevant Orthopedic Implant Surfaces"],"prefix":"10.3390","volume":"15","author":[{"given":"Tripti Thapa","family":"Gupta","sequence":"first","affiliation":[{"name":"Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathan","family":"Sacaria","sequence":"additional","affiliation":[{"name":"Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8832-8497","authenticated-orcid":false,"given":"Phillip A.","family":"Laycock","sequence":"additional","affiliation":[{"name":"Biocomposites Ltd., Keele Science Park, Keele, Staffordshire ST5 5NL, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sean S.","family":"Aiken","sequence":"additional","affiliation":[{"name":"Biocomposites Ltd., Keele Science Park, Keele, Staffordshire ST5 5NL, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6069-273X","authenticated-orcid":false,"given":"Paul","family":"Stoodley","sequence":"additional","affiliation":[{"name":"Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210, USA"},{"name":"Department of Orthopedics, The Ohio State University, Columbus, OH 43210, USA"},{"name":"National Centre for Advanced Tribology at Southampton, Department of Mechanical Engineering, University of Southampton, Southampton SO17 1BJ, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel J.","family":"Wozniak","sequence":"additional","affiliation":[{"name":"Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210, USA"},{"name":"Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1016\/j.arth.2012.02.022","article-title":"Economic burden of periprosthetic joint infection in the United States","volume":"27","author":"Kurtz","year":"2012","journal-title":"J. Arthroplast."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e158","DOI":"10.2106\/JBJS.M.01363","article-title":"Risk factors for surgical site infection following total joint arthroplasty","volume":"96","author":"Rasouli","year":"2014","journal-title":"J. Bone Jt. Surg."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1007\/s11999-009-0945-0","article-title":"The epidemiology of revision total knee arthroplasty in the United States","volume":"468","author":"Bozic","year":"2010","journal-title":"Clin. Orthop. Relat. Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1302\/2633-1462.31.BJO-2021-0051.R1","article-title":"Cefazolin remains the linchpin for preventing acute periprosthetic joint infection following primary total knee arthroplasty","volume":"3","author":"Buchalter","year":"2022","journal-title":"Bone Jt. Open"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"859","DOI":"10.1302\/2633-1462.411.BJO-2023-0078.R1","article-title":"The effect of the surgical helmet system on intraoperative contamination in arthroplasty surgery: A fluorescent quantitative simulation","volume":"4","author":"Chen","year":"2023","journal-title":"Bone Jt. Open"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1302\/2046-3758.111.BJR-2021-0499","article-title":"Periprosthetic joint infection: Patients benefit from a multidisciplinary team approach","volume":"11","author":"Walter","year":"2022","journal-title":"Bone Jt. Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1696","DOI":"10.1016\/j.arth.2020.01.080","article-title":"Is treatment of periprosthetic joint infection improving over time?","volume":"35","author":"Xu","year":"2020","journal-title":"J. Arthroplast."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Gutowski, C.J., Chen, A.F., and Parvizi, J. (2016). The incidence and socioeconomic impact of periprosthetic joint infection: United States perspective. Periprosthetic Joint Infections: Changing Paradigms, Springer.","DOI":"10.1007\/978-3-319-30091-7_2"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"521","DOI":"10.1016\/j.arth.2017.09.021","article-title":"Revision total knee arthroplasty for periprosthetic joint infection is associated with increased postoperative morbidity and mortality relative to noninfectious revisions","volume":"33","author":"Boddapati","year":"2018","journal-title":"J. Arthroplast."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"196515","DOI":"10.1100\/2012\/196515","article-title":"Economic impact of infected total knee arthroplasty","volume":"2012","author":"Haenle","year":"2012","journal-title":"Sci. World J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"302","DOI":"10.1128\/CMR.00111-13","article-title":"Prosthetic joint infection","volume":"27","author":"Tande","year":"2014","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"ofz452","DOI":"10.1093\/ofid\/ofz452","article-title":"Predictors of treatment failure for hip and knee prosthetic joint infections in the setting of 1-and 2-stage exchange arthroplasty: A multicenter retrospective cohort","volume":"6","author":"Kandel","year":"2019","journal-title":"Open Forum Infect. Dis."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1038\/s41579-022-00682-4","article-title":"Tolerance and resistance of microbial biofilms","volume":"20","author":"Ciofu","year":"2022","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1111\/joim.12233","article-title":"Clinical presentation and treatment of orthopaedic implant-associated infection","volume":"276","author":"Zimmerli","year":"2014","journal-title":"J. Intern. Med."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1111\/apm.12923","article-title":"Mapping bacterial biofilms on recovered orthopaedic implants by a novel agar candle dip method","volume":"127","author":"Moley","year":"2019","journal-title":"Apmis"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"11302","DOI":"10.1039\/D4TB01563K","article-title":"Surface engineering of orthopedic implants for better clinical adoption","volume":"12","author":"Tripathi","year":"2024","journal-title":"J. Mater. Chem. B"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"147","DOI":"10.3233\/BMR-210319","article-title":"Microbiological profiles and antibiotic resistance of periprosthetic joint infection after hip replacement in patients with fracture or non-fracture: A comparative study","volume":"36","author":"Hu","year":"2023","journal-title":"J. Back Musculoskelet. Rehabil."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"015002","DOI":"10.1088\/1748-605X\/12\/1\/015002","article-title":"Biofilm prevention of gram-negative bacterial pathogens involved in periprosthetic infection by antibiotic-loaded calcium sulfate beads in vitro","volume":"12","author":"Howlin","year":"2017","journal-title":"Biomed. Mater."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"297","DOI":"10.3928\/01477447-20140430-03","article-title":"What do we (not) know about antibiotic-loaded hip spacers?","volume":"37","author":"Anagnostakos","year":"2014","journal-title":"Orthopedics"},{"key":"ref_20","first-page":"32","article-title":"Dissolvable antibiotic beads in treatment of periprosthetic joint infection and revision arthroplasty-the use of synthetic pure calcium sulfate (Stimulan\u00ae) impregnated with vancomycin & tobramycin","volume":"3","author":"McPherson","year":"2013","journal-title":"Reconstr. Rev."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1318","DOI":"10.1126\/science.284.5418.1318","article-title":"Bacterial biofilms: A common cause of persistent infections","volume":"284","author":"Costerton","year":"1999","journal-title":"Science"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1143","DOI":"10.1002\/jor.25135","article-title":"Identifying alternative antibiotics that elute from calcium sulfate beads for treatment of orthopedic infections","volume":"40","author":"Levack","year":"2022","journal-title":"J. Orthop. Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2349","DOI":"10.1002\/jor.23903","article-title":"Antibiotic loaded calcium sulfate bead and pulse lavage eradicates biofilms on metal implant materials in vitro","volume":"36","author":"Knecht","year":"2018","journal-title":"J. Orthop. Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1038\/nature12479","article-title":"Bacteria activate sensory neurons that modulate pain and inflammation","volume":"501","author":"Chiu","year":"2013","journal-title":"Nature"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e03081-19","DOI":"10.1128\/mBio.03081-19","article-title":"Stochastic expression of Sae-dependent virulence genes during Staphylococcus aureus biofilm development is dependent on SaeS","volume":"11","author":"DelMain","year":"2020","journal-title":"mBio"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"632","DOI":"10.1302\/0301-620X.106B6.BJJ-2023-1175.R1","article-title":"Antibiotic calcium sulphate beads lower the bacterial burden and prevent infection in a mouse model of periprosthetic joint infection","volume":"106","author":"Hart","year":"2024","journal-title":"Bone Jt. J."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Laycock, P.A., Cooper, J.J., Howlin, R.P., Delury, C., Aiken, S., and Stoodley, P. (2018). In vitro efficacy of antibiotics released from calcium sulfate bone void filler beads. Materials, 11.","DOI":"10.3390\/ma11112265"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Pestrak, M.J., Gupta, T.T., Dusane, D.H., Guzior, D.V., Staats, A., Harro, J., Horswill, A.R., and Stoodley, P. (2020). Investigation of synovial fluid induced Staphylococcus aureus aggregate development and its impact on surface attachment and biofilm formation. PLoS ONE, 15.","DOI":"10.1371\/journal.pone.0231791"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e0023622","DOI":"10.1128\/mbio.00236-22","article-title":"Rapid aggregation of Staphylococcus aureus in synovial fluid is influenced by synovial fluid concentration, viscosity, and fluid dynamics, with evidence of polymer bridging","volume":"13","author":"Staats","year":"2022","journal-title":"mBio"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"655873","DOI":"10.3389\/fmicb.2021.655873","article-title":"Staphylococcus aureus floating biofilm formation and phenotype in synovial fluid depends on albumin, fibrinogen, and hyaluronic acid","volume":"12","author":"Knott","year":"2021","journal-title":"Front. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"176","DOI":"10.4161\/biom.22905","article-title":"Infection of orthopedic implants with emphasis on bacterial adhesion process and techniques used in studying bacterial-material interactions","volume":"2","author":"Ribeiro","year":"2012","journal-title":"Biomatter"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Stoodley, P., Brooks, J., Peters, C.W., Jiang, N., Delury, C.P., Laycock, P.A., Aiken, S.S., and Dusane, D.H. (2020). Prevention and Killing Efficacy of Carbapenem Resistant Enterobacteriaceae (CRE) and Vancomycin Resistant Enterococci (VRE) Biofilms by Antibiotic-Loaded Calcium Sulfate Beads. Materials, 13.","DOI":"10.3390\/ma13153258"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3160","DOI":"10.1128\/AEM.66.8.3160-3165.2000","article-title":"Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506","volume":"66","author":"Lowder","year":"2000","journal-title":"Appl. Environ. Microbiol."}],"container-title":["Antibiotics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6382\/15\/8\/752\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T11:24:21Z","timestamp":1785842661000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6382\/15\/8\/752"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,4]]},"references-count":33,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["antibiotics15080752"],"URL":"https:\/\/doi.org\/10.3390\/antibiotics15080752","relation":{},"ISSN":["2079-6382"],"issn-type":[{"value":"2079-6382","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,4]]}}}