Universal antibacterial activity of silver against clinical MRSA isolates: relationship between biofilm characteristics and antimicrobial efficacy

Universal antibacterial activity of silver against clinical MRSA isolates: relationship between biofilm characteristics and antimicrobial efficacy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Universal antibacterial activity of silver against clinical MRSA isolates: relationship between biofilm characteristics and antimicrobial efficacy Keigo Yonemoto, Keiichiro Hara, Taku Ikegami, Hiroki Wakiya, Hiroko Takizawa, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8247298/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: This study investigated the major biofilm components of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from periprosthetic joint infections (PJI) and evaluated the antibacterial activity of Kyocera’s silver-containing hydroxyapatite (Ag-HA) discs against strains with distinct biofilm characteristics. Methods: The MRSA strains examined were USA300, two PJI isolates (No.47 and No.87), and one catheter-related bloodstream infection isolate (MR10). Biofilm composition was assessed using enzymatic degradation with proteinase, DNase, or a polysaccharide-degrading enzyme, followed by quantification of the remaining biomass. The antibacterial effects of Ag-HA discs and their influence on biofilm architecture were analyzed using fluorescence imaging and confocal laser scanning microscopy after ICBiofilm clearing. Results: Strain No.47 produced a biofilm primarily composed of proteins and extracellular DNA, while USA300 and No.87 formed protein-dominant matrices. MR10 exhibited a polysaccharide-rich biofilm. Ag-HA discs suppressed bacterial attachment in all strains and altered their three-dimensional biofilm structures. The application of Ag-HA resulted in a rougher biofilm architecture and reduced surface coverage across all strains. Conclusions: MRSA biofilm composition varies widely among clinical isolates. Ag-HA demonstrates broad antibacterial activity and modifies biofilm architecture, indicating a potential contribution to its antimicrobial efficacy. Three to ten keywords representing the main content of the manuscript Figures Figure 1 Figure 2 Introduction Periprosthetic joint infection (PJI) is a serious complication following joint arthroplasty ( 1 ). Once infection occurs, conservative treatment such as systemic antibiotic administration is rarely curative, and surgical procedures including debridement or implant removal are often required. To prevent and manage PJI, prosthetic materials with inherent antibacterial properties or antibiotic-releasing coatings have been developed( 2 ). Silver (Ag) possesses a broad antibacterial spectrum and exerts potent antimicrobial activity with limited development of bacterial resistance ( 3 ). However, Ag is cytotoxic to osteoblasts and suppresses bone formation, which restricts its direct application within bone tissue ( 4 ). Hydroxyapatite (HA), by contrast, promotes osteoconduction and facilitates early bone integration. To combine the antibacterial benefit of Ag with the osteoconductive properties of HA, silver-containing hydroxyapatite (Ag-HA) coatings have been developed ( 5 ). These coatings are produced by thermally spraying an Ag-HA layer onto titanium substrates ( 6 ). Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen responsible for PJI( 1 ) and is characterized by its robust biofilm-forming ability. Biofilm development proceeds through initial adhesion, maturation driven by adhesion factors, and subsequent dispersion regulated by quorum-sensing systems ( 7 ). MRSA biofilms consist of various extracellular components, including proteins ( 8 ), nucleic acids, and polysaccharides ( 9 ). Extracellular DNA and RNA( 10 ) as well as PNAG polysaccharides( 11 ) are recognized as key structural elements. Importantly, biofilm characteristics differ among clinical isolates and are strongly influenced by culture conditions( 12 ). Although Ag-HA has demonstrated antimicrobial activity against laboratory MRSA strains, its efficacy against clinical isolates derived specifically from PJIs remains insufficiently understood. Therefore, this study aimed to evaluate the antibacterial effects of Ag-HA on MRSA strains exhibiting distinct biofilm compositions and to assess how Ag-HA influences their three-dimensional (3D) biofilm architecture. Materials and Methods Bacterial strains Clinical MRSA isolates were collected with approval from the Pathogen Control Committee and Ethics Committee of Jikei University School of Medicine. The strains used were USA300(13)), No.47 and No.87 (isolated from PJIs), and MR10 (isolated from a catheter-related bloodstream infection)(12) (Table 1). table. Bacterial strains used in this study.The bacterial strains used in this study are listed below. Table1. MRSA strains used in this study Table legends Table1 . MRSA strains used in this study. USA300 and MR10 were provided by the Department of Bacteriology, Jikei University School of Medicine. The MRSA isolates from periprosthetic joint infections (PJI) were collected in the present study. Ag-HA coating Ag-HA was coated on one side of pure titanium discs (14 mm diameter, 1 mm thickness; Kobe Steel, Japan) using a previously described thermal-spray method (6). Additional details of the coating process are available in (5). Biofilm culture and quantification Each MRSA strain was grown on BHI agar plates, and a single colony was inoculated into BHI broth (Becton Dickinson, USA) and incubated at 37 °C overnight. The culture (~1 × 10⁸ CFU/mL) was diluted 1:1000 in TSB containing 0.25% glucose (TSBG). For biofilm quantification, 200 µL of diluted culture was added to 96-well polystyrene plates (Thermo Fisher Scientific, USA) and incubated for 24 h at 37 °C. Wells were washed with PBS, stained with 0.5% crystal violet, washed again, and absorbance was measured at 595 nm(12). Biofilm component analysis Biofilms were cultured under the same conditions, then treated with DNase I (100 U/mL; Roche), proteinase K (100 µg/mL; Wako, Japan), or Dispersin B (20 µg/mL; Kane Biotech, Canada) at 37 °C for 2 h. The remaining biofilm biomass was quantified as above. Ag-HA disc assay Overnight cultures were diluted 1:1000 in TSBG and inoculated onto HA or Ag-HA discs, followed by static incubation at 37 °C overnight. Discs were washed with PBS, fixed with 1% glutaraldehyde for 30 min, quenched with ammonium chloride, then stained with FM1-43 (Thermo Fisher) at 4 °C overnight. ICBiofilm clearing reagent (Tokyo Medical Industry) was applied for 2 h at room temperature (14). Three-dimensional biofilm structures were observed using an all-in-one fluorescence microscope. Statistical analysis Differences in biofilm formation and component susceptibility were evaluated using the Bonferroni–Dunn test. Biofilm coverage on HA vs. Ag-HA discs was analyzed using paired t-tests. A p-value < 0.05 was considered significant. Results Biofilm formation and matrix composition All MRSA strains (USA300, No.47, No.87, MR10) formed biofilms under TSBG conditions (Fig. 1).Enzymatic degradation revealed distinct differences in matrix composition.No.47 biofilms were disrupted by proteinase K and DNase I, indicating protein and extracellular DNA as major components.USA300 and No.87 were degraded by proteinase K only, suggesting predominantly protein-based biofilms.MR10 was specifically disrupted by Dispersin B, indicating polysaccharide-rich biofilm architecture.These results demonstrate substantial heterogeneity in extracellular matrix composition among clinical MRSA isolates. Antibacterial effect of Ag-HA discs Both HA and Ag-HA discs were inoculated with each MRSA strain. The Ag-HA discs manufactured by Kyocera Medical Corporation exhibited clear antibacterial activity. After ICBiofilm clearing and fluorescence imaging (Fig. 2), bacterial coverage on Ag-HA discs was significantly lower than that observed on HA discs for all strains. Ag-HA discs consistently suppressed bacterial attachment and altered the three-dimensional biofilm structures of all strains. The application of Ag-HA resulted in a rougher biofilm architecture and reduced surface coverage across all strains. Three-dimensional biofilm structure Fluorescence imaging revealed thick, multilayered three-dimensional biofilms on HA discs, whereas biofilms on Ag-HA discs appeared noticeably flatter with reduced surface coverage (Fig. 2).Although the strains differed in matrix composition, all exhibited similarly decreased biofilm height and a roughened three-dimensional architecture on Ag-HA substrates. The reduction in surface coverage suggests an inhibitory effect on biofilm formation, while the roughened architecture may contribute to increased susceptibility to antimicrobial agents. Discussion This study compared biofilm formation and extracellular matrix composition among several MRSA isolates, including PJI-associated strains, and evaluated the antibacterial properties of Ag-HA surfaces. MRSA is a frequent cause of implant-related infection due to its strong capacity for biofilm formation and persistence on orthopedic materials ( 1 ). Biofilm formation is strongly influenced by environmental conditions; glucose supplementation, for example, suppresses agr quorum sensing, lowers culture pH, reduces extracellular protease activity, and promotes accumulation of surface-associated proteins ( 15 ). In this study, the two PJI isolates produced predominantly protein-based biofilms. Protein-rich matrices may be susceptible to proteolytic enzymes, including those already used clinically for wound care( 16 ), suggesting possible therapeutic applications for orthopedic infections. Silver is widely utilized as an antimicrobial agent due to its broad antibacterial spectrum and low propensity for resistance( 2 ). Ag ions disrupt bacterial cell wall synthesis, inactivate intracellular proteins, and damage membranes ( 3 ). When incorporated into HA, Ag exhibits synergistic bactericidal effects with antibiotics( 6 ), and silver-coated prostheses have already been used clinically for infection control. In the present study, Ag-HA discs demonstrated antibacterial activity against all MRSA strains tested, irrespective of differences in their biofilm compositions. This is an important finding, as the extracellular matrix contributes substantially to antibiotic tolerance and mechanical stability in MRSA biofilms. Three-dimensional imaging further revealed that Ag-HA not only reduced bacterial burden but also altered biofilm morphology, resulting in flatter and thinner structures. Biofilm thickness is closely associated with reduced metabolic activity and impaired antibiotic penetration ( 17 ). Conversely, mutants forming simplified biofilm architecture display increased antibiotic susceptibility and reduced virulence ( 8 ). Thus, the structural modification induced by Ag-HA may enhance antimicrobial penetration and contribute to its overall antibacterial effect. Overall, our findings indicate that Ag-HA exhibits robust antibacterial activity across diverse MRSA biofilm types and additionally disrupts biofilm 3D architecture. These effects appear independent of matrix composition and may contribute to improved infection prevention around implants. Conclusion MRSA strains isolated from PJI and other infections exhibited diverse biofilm compositions, including protein-rich, DNA-rich, and polysaccharide-rich matrices. Silver-containing HA demonstrated antibacterial activity against all strains and consistently reduced biofilm coverage and thickness. The structural simplification observed on Ag-HA surfaces suggests that modification of biofilm architecture contributes to its antibacterial efficacy. Limitations This study analyzed a limited number of MRSA isolates, which may not capture the full clinical diversity of PJI pathogens. Additionally, all assays were performed under in vitro conditions that do not fully replicate the physiological environment surrounding orthopedic implants. Further studies using additional antimicrobial agents, in vivo models, and time-lapse 3D imaging will be necessary to clarify the mechanisms underlying the antibacterial effects of Ag-HA. Abbreviations Ag: Silver Ag-HA: Silver-containing hydroxyapatite ANOVA: Analysis of variance BHI: Brain heart infusion CFU: Colony-forming unit CLSM: Confocal laser scanning microscopy CV: Crystal violet DNA: Deoxyribonucleic acid eDNA: Extracellular DNA ECM: Extracellular matrix HA: Hydroxyapatite ICBiofilm: Imaging reagent for biofilm clearing (Tokyo Medical Industry) MRSA: Methicillin-resistant Staphylococcus aureus PBS: Phosphate-buffered saline PJI: Periprosthetic joint infection PNAG: Poly-N-acetylglucosamine RNA: Ribonucleic acid 3D: Three-dimensional TSB: Tryptic soy broth TSBG: Tryptic soy broth supplemented with 0.25% glucose Declarations Acknowledgements The authors express their sincere gratitude to Associate Professor Sugimoto of the Department of Bacteriology, Jikei University School of Medicine, for his invaluable advice, provision of experimental reagents and materials, and extensive support throughout this study. The authors also thank Kyocera Medical Corporation for providing the Ag-HA discs used in this research. In addition, we thank the technical staff of the Department of Orthopaedic Surgery, Jikei University School of Medicine, for their assistance with the biofilm analyses. Ethics approval and consent to participate Clinical MRSA isolates were collected with approval from the Pathogen Control Committee and the Ethics Committee of Jikei University School of Medicine (Approval No. 33-021(10631)). Written informed consent was obtained from all patients from whom the bacterial isolates were derived. Consent for publication Not applicable. Availability of data and materials The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by JSPS KAKENHI (Grant Numbers 21K16717 and 25K19987) and by the Japan Orthopaedics and Traumatology Foundation (Grant Number 538). Authors’ contributions K.Y. designed the study, performed the experiments, analyzed the data, and drafted the manuscript. M.S. supervised the project; K.H. contributed to data interpretation; and T.I., H.W., and H.T. assisted with the biofilm experiments. All authors read and approved the final manuscript. References Kapadia BH, Berg RA, Daley JA, Fritz J, Bhave A, Mont MA. Periprosthetic joint infection. Vol. 387, The Lancet. Lancet Publishing Group; 2016. p. 386–94. Schmidt-Braekling T, Streitbuerger A, Gosheger G, Boettner F, Nottrott M, Ahrens H, et al. Silver-coated megaprostheses: review of the literature. Vol. 27, European Journal of Orthopaedic Surgery and Traumatology. Springer-Verlag France; 2017. p. 483–9. Rodrigues AS, Batista JGS, Rodrigues MÁV, Thipe VC, Minarini LAR, Lopes PS, et al. Advances in silver nanoparticles: a comprehensive review on their potential as antimicrobial agents and their mechanisms of action elucidated by proteomics. Vol. 15, Frontiers in Microbiology. Frontiers Media SA; 2024. Albers CE, Hofstetter W, Siebenrock KA, Landmann R, Klenke FM. In vitro cytotoxicity of silver nanoparticles on osteoblasts and osteoclasts at antibacterial concentrations. Nanotoxicology. 2013 Feb;7(1):30–6. Hashimoto A, Miyamoto H, Kii S, Kobatake T, Shobuike T, Noda I, et al. Time-dependent efficacy of combination of silver-containing hydroxyapatite coating and vancomycin on methicillin-resistant Staphylococcus aureus biofilm formation in vitro. BMC Res Notes. 2021 Dec 1;14(1). Hashimoto A, Miyamoto H, Ueno M, noda I, Sonohata M, Mawatari M. The combination of silver-containing hydroxyapatite coating and vancomycin has a synergistic antibacterial effect on methicillin-resistant Staphylococcus aureus biofilm formationCite this article. Bone Joint Res. 2020;9(5):211–8. Moormeier DE, Bayles KW. Staphylococcus aureus biofilm: a complex developmental organism. Vol. 104, Molecular Microbiology. Blackwell Publishing Ltd; 2017. p. 365–76. Yonemoto K, Chiba A, Sugimoto S, Sato C, Saito M, Kinjo Y, et al. Redundant and Distinct Roles of Secreted Protein Eap and Cell Wall-Anchored Protein SasG in Biofilm Formation and Pathogenicity of Staphylococcus aureus. 2019; Available from: https://doi.org/10.1128/IAI Foster TJ, Geoghegan JA, Ganesh VK, Höök M. Adhesion, invasion and evasion: The many functions of the surface proteins of Staphylococcus aureus. Vol. 12, Nature Reviews Microbiology. 2014. p. 49–62. Chiba A, Seki M, Suzuki Y, Kinjo Y, Mizunoe Y, Sugimoto S. Staphylococcus aureus utilizes environmental RNA as a building material in specific polysaccharide-dependent biofilms. NPJ Biofilms Microbiomes. 2022 Dec 1;8(1). Kropec A, Maira-Litran T, Jefferson KK, Grout M, Cramton SE, Götz F, et al. Poly-N-acetylglucosamine production in Staphylococcus aureus is essential for virulence in murine models of systemic infection. Infect Immun. 2005 Oct;73(10):6868–76. Sugimoto S, Sato F, Miyakawa R, Chiba A, Onodera S, Hori S, et al. Broad impact of extracellular DNA on biofilm formation by clinically isolated Methicillin-resistant and -sensitive strains of Staphylococcus aureus. Sci Rep. 2018 Dec 1;8(1). Kennedy AD, Otto M, Braughton KR, Whitney AR, Chen L, Mathema B, et al. Epidemic community-associated methicillin-resistant Staphylococcus aureus: Recent clonal expansion and diversification [Internet]. 2008. Available from: www.pnas.org/cgi/content/full/ Sugimoto S, Kinjo Y. Instantaneous Clearing of Biofilm (iCBiofilm): an optical approach to revisit bacterial and fungal biofilm imaging. Commun Biol. 2023 Dec 1;6(1). Le KY, Otto M. Quorum-sensing regulation in staphylococci-an overview. Vol. 6, Frontiers in Microbiology. Frontiers Media S.A.; 2015. De Francesco F, De Francesco M, Riccio M. Hyaluronic Acid/Collagenase Ointment in the Treatment of Chronic Hard-to-Heal Wounds: An Observational and Retrospective Study. J Clin Med. 2022 Jan 21;11(3):537. Pabst B, Pitts B, Lauchnor E, Stewart PS. Gel-entrapped Staphylococcus aureus bacteria as models of biofilm infection exhibit growth in dense aggregates, oxygen limitation, antibiotic tolerance, and heterogeneous gene expression. Antimicrob Agents Chemother. 2016 Oct 1;60(10):6294–301. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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16:35:43","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":59785,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8247298/v1/40713a071c60d3796dcf2bff.html"},{"id":99223974,"identity":"2a929734-fe8a-4223-8b6f-2a0fb632a0b3","added_by":"auto","created_at":"2025-12-30 10:02:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":169827,"visible":true,"origin":"","legend":"\u003cp\u003eBiofilm formation of the bacterial strains used in this study. Biofilm biomass of each strain is shown. Each experiment was performed in triplicate. Multiple comparison analysis was conducted using one-way ANOVA followed by Bonferroni’s post hoc test.\u003c/p\u003e\n\u003cp\u003e** indicates P \u0026lt; 0.01; NS indicates no significant difference.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8247298/v1/4d9f7245d72b1cef6c67ab86.png"},{"id":99223970,"identity":"c4bdfc67-6bc6-41ee-8424-319b71de634a","added_by":"auto","created_at":"2025-12-30 10:02:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":376809,"visible":true,"origin":"","legend":"\u003cp\u003eThree-dimensional structure of biofilms formed by the bacterial strains.The left column indicates the bacterial strains, and the top row shows the types of discs used in this experiment. Biofilms were stained with fluorescent dyes and visualized using fluorescence microscopy.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8247298/v1/0491ca5201957e86f8f7b491.png"},{"id":102509400,"identity":"beef5a33-2be5-403c-9a92-1601fe0c3871","added_by":"auto","created_at":"2026-02-12 12:11:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1047993,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8247298/v1/7924256a-9529-4e3b-a79d-680fc3c37a4d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eUniversal antibacterial activity of silver against clinical MRSA isolates: relationship between biofilm characteristics and antimicrobial efficacy\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePeriprosthetic joint infection (PJI) is a serious complication following joint arthroplasty (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Once infection occurs, conservative treatment such as systemic antibiotic administration is rarely curative, and surgical procedures including debridement or implant removal are often required. To prevent and manage PJI, prosthetic materials with inherent antibacterial properties or antibiotic-releasing coatings have been developed(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSilver (Ag) possesses a broad antibacterial spectrum and exerts potent antimicrobial activity with limited development of bacterial resistance (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). However, Ag is cytotoxic to osteoblasts and suppresses bone formation, which restricts its direct application within bone tissue (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Hydroxyapatite (HA), by contrast, promotes osteoconduction and facilitates early bone integration. To combine the antibacterial benefit of Ag with the osteoconductive properties of HA, silver-containing hydroxyapatite (Ag-HA) coatings have been developed (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). These coatings are produced by thermally spraying an Ag-HA layer onto titanium substrates (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMethicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) is a major pathogen responsible for PJI(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) and is characterized by its robust biofilm-forming ability. Biofilm development proceeds through initial adhesion, maturation driven by adhesion factors, and subsequent dispersion regulated by quorum-sensing systems (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). MRSA biofilms consist of various extracellular components, including proteins (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), nucleic acids, and polysaccharides (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Extracellular DNA and RNA(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) as well as PNAG polysaccharides(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) are recognized as key structural elements. Importantly, biofilm characteristics differ among clinical isolates and are strongly influenced by culture conditions(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough Ag-HA has demonstrated antimicrobial activity against laboratory MRSA strains, its efficacy against clinical isolates derived specifically from PJIs remains insufficiently understood. Therefore, this study aimed to evaluate the antibacterial effects of Ag-HA on MRSA strains exhibiting distinct biofilm compositions and to assess how Ag-HA influences their three-dimensional (3D) biofilm architecture.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eBacterial strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical MRSA isolates were collected with approval from the Pathogen Control Committee and Ethics Committee of Jikei University School of Medicine. The strains used were USA300(13)), No.47 and No.87 (isolated from PJIs), and MR10 (isolated from a catheter-related bloodstream infection)(12) \u0026nbsp; (Table 1).\u003c/p\u003e\n\u003cp\u003etable. Bacterial strains used in this study.The bacterial strains used in this study are listed below.\u003c/p\u003e\n\u003cp\u003eTable1. MRSA strains used in this study\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"529\" src=\"https://myfiles.space/user_files/58895_8739fc6c57c1c19a/58895_custom_files/img1766999524.png\" align=\"\" alt=\"image\" height=\"157\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable legends\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable1\u003c/strong\u003e. MRSA strains used in this study. USA300 and MR10 were provided by the Department of Bacteriology, Jikei University School of Medicine. The MRSA isolates from periprosthetic joint infections (PJI) were collected in the present study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAg-HA coating\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAg-HA was coated on one side of pure titanium discs (14 mm diameter, 1 mm thickness; Kobe Steel, Japan) using a previously described thermal-spray method (6). Additional details of the coating process are available in (5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiofilm culture and quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach MRSA strain was grown on BHI agar plates, and a single colony was inoculated into BHI broth (Becton Dickinson, USA) and incubated at 37 \u0026deg;C overnight. The culture (~1 \u0026times; 10⁸ CFU/mL) was diluted 1:1000 in TSB containing 0.25% glucose (TSBG).\u003cbr\u003eFor biofilm quantification, 200 \u0026micro;L of diluted culture was added to 96-well polystyrene plates (Thermo Fisher Scientific, USA) and incubated for 24 h at 37 \u0026deg;C. Wells were washed with PBS, stained with 0.5% crystal violet, washed again, and absorbance was measured at 595 nm(12).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiofilm component analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiofilms were cultured under the same conditions, then treated with DNase I (100 U/mL; Roche), proteinase K (100 \u0026micro;g/mL; Wako, Japan), or Dispersin B (20 \u0026micro;g/mL; Kane Biotech, Canada) at 37 \u0026deg;C for 2 h. The remaining biofilm biomass was quantified as above.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAg-HA disc assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvernight cultures were diluted 1:1000 in TSBG and inoculated onto HA or Ag-HA discs, followed by static incubation at 37 \u0026deg;C overnight. Discs were washed with PBS, fixed with 1% glutaraldehyde for 30 min, quenched with ammonium chloride, then stained with FM1-43 (Thermo Fisher) at 4 \u0026deg;C overnight.\u003cbr\u003eICBiofilm clearing reagent (Tokyo Medical Industry) was applied for 2 h at room temperature (14). Three-dimensional biofilm structures were observed using an all-in-one fluorescence microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferences in biofilm formation and component susceptibility were evaluated using the Bonferroni\u0026ndash;Dunn test. Biofilm coverage on HA vs. Ag-HA discs was analyzed using paired t-tests. A p-value \u0026lt; 0.05 was considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eBiofilm formation and matrix composition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll MRSA strains (USA300, No.47, No.87, MR10) formed biofilms under TSBG conditions (Fig. 1).Enzymatic degradation revealed distinct differences in matrix composition.No.47 biofilms were disrupted by proteinase K and DNase I, indicating protein and extracellular DNA as major components.USA300 and No.87 were degraded by proteinase K only, suggesting predominantly protein-based biofilms.MR10 was specifically disrupted by Dispersin B, indicating polysaccharide-rich biofilm architecture.These results demonstrate substantial heterogeneity in extracellular matrix composition among clinical MRSA isolates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibacterial effect of Ag-HA discs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth HA and Ag-HA discs were inoculated with each MRSA strain. The Ag-HA discs manufactured by Kyocera Medical Corporation exhibited clear antibacterial activity. After ICBiofilm clearing and fluorescence imaging (Fig. 2), bacterial coverage on Ag-HA discs was significantly lower than that observed on HA discs for all strains. Ag-HA discs consistently suppressed bacterial attachment and altered the three-dimensional biofilm structures of all strains. The application of Ag-HA resulted in a rougher biofilm architecture and reduced surface coverage across all strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThree-dimensional biofilm structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluorescence imaging revealed thick, multilayered three-dimensional biofilms on HA discs, whereas biofilms on Ag-HA discs appeared noticeably flatter with reduced surface coverage (Fig. 2).Although the strains differed in matrix composition, all exhibited similarly decreased biofilm height and a roughened three-dimensional architecture on Ag-HA substrates. The reduction in surface coverage suggests an inhibitory effect on biofilm formation, while the roughened architecture may contribute to increased susceptibility to antimicrobial agents.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study compared biofilm formation and extracellular matrix composition among several MRSA isolates, including PJI-associated strains, and evaluated the antibacterial properties of Ag-HA surfaces. MRSA is a frequent cause of implant-related infection due to its strong capacity for biofilm formation and persistence on orthopedic materials (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Biofilm formation is strongly influenced by environmental conditions; glucose supplementation, for example, suppresses agr quorum sensing, lowers culture pH, reduces extracellular protease activity, and promotes accumulation of surface-associated proteins (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, the two PJI isolates produced predominantly protein-based biofilms. Protein-rich matrices may be susceptible to proteolytic enzymes, including those already used clinically for wound care(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), suggesting possible therapeutic applications for orthopedic infections.\u003c/p\u003e \u003cp\u003eSilver is widely utilized as an antimicrobial agent due to its broad antibacterial spectrum and low propensity for resistance(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Ag ions disrupt bacterial cell wall synthesis, inactivate intracellular proteins, and damage membranes (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). When incorporated into HA, Ag exhibits synergistic bactericidal effects with antibiotics(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), and silver-coated prostheses have already been used clinically for infection control.\u003c/p\u003e \u003cp\u003eIn the present study, Ag-HA discs demonstrated antibacterial activity against all MRSA strains tested, irrespective of differences in their biofilm compositions. This is an important finding, as the extracellular matrix contributes substantially to antibiotic tolerance and mechanical stability in MRSA biofilms.\u003c/p\u003e \u003cp\u003eThree-dimensional imaging further revealed that Ag-HA not only reduced bacterial burden but also altered biofilm morphology, resulting in flatter and thinner structures. Biofilm thickness is closely associated with reduced metabolic activity and impaired antibiotic penetration (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Conversely, mutants forming simplified biofilm architecture display increased antibiotic susceptibility and reduced virulence (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Thus, the structural modification induced by Ag-HA may enhance antimicrobial penetration and contribute to its overall antibacterial effect.\u003c/p\u003e \u003cp\u003eOverall, our findings indicate that Ag-HA exhibits robust antibacterial activity across diverse MRSA biofilm types and additionally disrupts biofilm 3D architecture. These effects appear independent of matrix composition and may contribute to improved infection prevention around implants.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eMRSA strains isolated from PJI and other infections exhibited diverse biofilm compositions, including protein-rich, DNA-rich, and polysaccharide-rich matrices. Silver-containing HA demonstrated antibacterial activity against all strains and consistently reduced biofilm coverage and thickness. The structural simplification observed on Ag-HA surfaces suggests that modification of biofilm architecture contributes to its antibacterial efficacy.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eThis study analyzed a limited number of MRSA isolates, which may not capture the full clinical diversity of PJI pathogens. Additionally, all assays were performed under in vitro conditions that do not fully replicate the physiological environment surrounding orthopedic implants. Further studies using additional antimicrobial agents, in vivo models, and time-lapse 3D imaging will be necessary to clarify the mechanisms underlying the antibacterial effects of Ag-HA.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAg: Silver\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Ag-HA: Silver-containing hydroxyapatite\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;ANOVA: Analysis of variance\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;BHI: Brain heart infusion\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;CFU: Colony-forming unit\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;CLSM: Confocal laser scanning microscopy\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;CV: Crystal violet\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;DNA: Deoxyribonucleic acid\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;eDNA: Extracellular DNA\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;ECM: Extracellular matrix\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;HA: Hydroxyapatite\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;ICBiofilm: Imaging reagent for biofilm clearing (Tokyo Medical Industry)\u003c/p\u003e\n\u003cp\u003eMRSA: Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;PBS: Phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;PJI: Periprosthetic joint infection\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;PNAG: Poly-N-acetylglucosamine\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;RNA: Ribonucleic acid\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;3D: Three-dimensional\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;TSB: Tryptic soy broth\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;TSBG: Tryptic soy broth supplemented with 0.25% glucose\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their sincere gratitude to Associate Professor Sugimoto of the Department of Bacteriology, Jikei University School of Medicine, for his invaluable advice, provision of experimental reagents and materials, and extensive support throughout this study. The authors also thank Kyocera Medical Corporation for providing the Ag-HA discs used in this research. In addition, we thank the technical staff of the Department of Orthopaedic Surgery, Jikei University School of Medicine, for their assistance with the biofilm analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical MRSA isolates were collected with approval from the Pathogen Control Committee and the Ethics Committee of Jikei University School of Medicine (Approval No. 33-021(10631)). Written informed consent was obtained from all patients from whom the bacterial isolates were derived.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI (Grant Numbers 21K16717 and 25K19987) and by the Japan Orthopaedics and Traumatology Foundation (Grant Number 538).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.Y. designed the study, performed the experiments, analyzed the data, and drafted the manuscript. M.S. supervised the project; K.H. contributed to data interpretation; and T.I., H.W., and H.T. assisted with the biofilm experiments. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKapadia BH, Berg RA, Daley JA, Fritz J, Bhave A, Mont MA. Periprosthetic joint infection. Vol. 387, The Lancet. Lancet Publishing Group; 2016. p. 386\u0026ndash;94. \u003c/li\u003e\n\u003cli\u003eSchmidt-Braekling T, Streitbuerger A, Gosheger G, Boettner F, Nottrott M, Ahrens H, et al. Silver-coated megaprostheses: review of the literature. Vol. 27, European Journal of Orthopaedic Surgery and Traumatology. Springer-Verlag France; 2017. p. 483\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eRodrigues AS, Batista JGS, Rodrigues M\u0026Aacute;V, Thipe VC, Minarini LAR, Lopes PS, et al. Advances in silver nanoparticles: a comprehensive review on their potential as antimicrobial agents and their mechanisms of action elucidated by proteomics. Vol. 15, Frontiers in Microbiology. Frontiers Media SA; 2024. \u003c/li\u003e\n\u003cli\u003eAlbers CE, Hofstetter W, Siebenrock KA, Landmann R, Klenke FM. In vitro cytotoxicity of silver nanoparticles on osteoblasts and osteoclasts at antibacterial concentrations. Nanotoxicology. 2013 Feb;7(1):30\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eHashimoto A, Miyamoto H, Kii S, Kobatake T, Shobuike T, Noda I, et al. Time-dependent efficacy of combination of silver-containing hydroxyapatite coating and vancomycin on methicillin-resistant Staphylococcus aureus biofilm formation in vitro. BMC Res Notes. 2021 Dec 1;14(1). \u003c/li\u003e\n\u003cli\u003eHashimoto A, Miyamoto H, Ueno M, noda I, Sonohata M, Mawatari M. The combination of silver-containing hydroxyapatite coating and vancomycin has a synergistic antibacterial effect on methicillin-resistant Staphylococcus aureus biofilm formationCite this article. Bone Joint Res. 2020;9(5):211\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eMoormeier DE, Bayles KW. Staphylococcus aureus biofilm: a complex developmental organism. Vol. 104, Molecular Microbiology. Blackwell Publishing Ltd; 2017. p. 365\u0026ndash;76. \u003c/li\u003e\n\u003cli\u003eYonemoto K, Chiba A, Sugimoto S, Sato C, Saito M, Kinjo Y, et al. Redundant and Distinct Roles of Secreted Protein Eap and Cell Wall-Anchored Protein SasG in Biofilm Formation and Pathogenicity of Staphylococcus aureus. 2019; Available from: https://doi.org/10.1128/IAI\u003c/li\u003e\n\u003cli\u003eFoster TJ, Geoghegan JA, Ganesh VK, H\u0026ouml;\u0026ouml;k M. Adhesion, invasion and evasion: The many functions of the surface proteins of Staphylococcus aureus. Vol. 12, Nature Reviews Microbiology. 2014. p. 49\u0026ndash;62. \u003c/li\u003e\n\u003cli\u003eChiba A, Seki M, Suzuki Y, Kinjo Y, Mizunoe Y, Sugimoto S. Staphylococcus aureus utilizes environmental RNA as a building material in specific polysaccharide-dependent biofilms. NPJ Biofilms Microbiomes. 2022 Dec 1;8(1). \u003c/li\u003e\n\u003cli\u003eKropec A, Maira-Litran T, Jefferson KK, Grout M, Cramton SE, G\u0026ouml;tz F, et al. Poly-N-acetylglucosamine production in Staphylococcus aureus is essential for virulence in murine models of systemic infection. Infect Immun. 2005 Oct;73(10):6868\u0026ndash;76. \u003c/li\u003e\n\u003cli\u003eSugimoto S, Sato F, Miyakawa R, Chiba A, Onodera S, Hori S, et al. Broad impact of extracellular DNA on biofilm formation by clinically isolated Methicillin-resistant and -sensitive strains of Staphylococcus aureus. Sci Rep. 2018 Dec 1;8(1). \u003c/li\u003e\n\u003cli\u003eKennedy AD, Otto M, Braughton KR, Whitney AR, Chen L, Mathema B, et al. Epidemic community-associated methicillin-resistant Staphylococcus aureus: Recent clonal expansion and diversification [Internet]. 2008. Available from: www.pnas.org/cgi/content/full/\u003c/li\u003e\n\u003cli\u003eSugimoto S, Kinjo Y. Instantaneous Clearing of Biofilm (iCBiofilm): an optical approach to revisit bacterial and fungal biofilm imaging. Commun Biol. 2023 Dec 1;6(1). \u003c/li\u003e\n\u003cli\u003eLe KY, Otto M. Quorum-sensing regulation in staphylococci-an overview. Vol. 6, Frontiers in Microbiology. Frontiers Media S.A.; 2015. \u003c/li\u003e\n\u003cli\u003eDe Francesco F, De Francesco M, Riccio M. Hyaluronic Acid/Collagenase Ointment in the Treatment of Chronic Hard-to-Heal Wounds: An Observational and Retrospective Study. J Clin Med. 2022 Jan 21;11(3):537. \u003c/li\u003e\n\u003cli\u003ePabst B, Pitts B, Lauchnor E, Stewart PS. Gel-entrapped Staphylococcus aureus bacteria as models of biofilm infection exhibit growth in dense aggregates, oxygen limitation, antibiotic tolerance, and heterogeneous gene expression. Antimicrob Agents Chemother. 2016 Oct 1;60(10):6294\u0026ndash;301. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Three to ten keywords representing the main content of the manuscript","lastPublishedDoi":"10.21203/rs.3.rs-8247298/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8247298/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003eThis study investigated the major biofilm components of methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) strains isolated from periprosthetic joint infections (PJI) and evaluated the antibacterial activity of Kyocera’s silver-containing hydroxyapatite (Ag-HA) discs against strains with distinct biofilm characteristics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003eThe MRSA strains examined were USA300, two PJI isolates (No.47 and No.87), and one catheter-related bloodstream infection isolate (MR10). Biofilm composition was assessed using enzymatic degradation with proteinase, DNase, or a polysaccharide-degrading enzyme, followed by quantification of the remaining biomass. The antibacterial effects of Ag-HA discs and their influence on biofilm architecture were analyzed using fluorescence imaging and confocal laser scanning microscopy after ICBiofilm clearing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003eStrain No.47 produced a biofilm primarily composed of proteins and extracellular DNA, while USA300 and No.87 formed protein-dominant matrices. MR10 exhibited a polysaccharide-rich biofilm. Ag-HA discs suppressed bacterial attachment in all strains and altered their three-dimensional biofilm structures. The application of Ag-HA resulted in a rougher biofilm architecture and reduced surface coverage across all strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003eMRSA biofilm composition varies widely among clinical isolates. Ag-HA demonstrates broad antibacterial activity and modifies biofilm architecture, indicating a potential contribution to its antimicrobial efficacy.\u003c/p\u003e","manuscriptTitle":"Universal antibacterial activity of silver against clinical MRSA isolates: relationship between biofilm characteristics and antimicrobial efficacy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 10:02:51","doi":"10.21203/rs.3.rs-8247298/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"52d9c2fa-a9b1-4cad-bd92-2dde1f829adc","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-12T12:10:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-30 10:02:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8247298","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8247298","identity":"rs-8247298","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}