Bacteriophage-Mediated Reduction of Pseudomonas aeruginosa Biofilm on Titanium Surfaces for Biomedical Applications

Bacteriophage-Mediated Reduction of Pseudomonas aeruginosa Biofilm on Titanium Surfaces for Biomedical Applications | 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 Research Article Bacteriophage-Mediated Reduction of Pseudomonas aeruginosa Biofilm on Titanium Surfaces for Biomedical Applications Amanda Kelly Ferreira Sousa, Viviani Tadioto, Anderson Giehl, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8376090/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Apr, 2026 Read the published version in European Journal of Clinical Microbiology & Infectious Diseases → Version 1 posted 9 You are reading this latest preprint version Abstract Biofilm-associated infections on biomedical devices are a major clinical challenge. These infections are resistant to antimicrobial treatments and often cause chronic and recurrent problems. Pseudomonas aeruginosa is a significant biofilm-forming pathogen, particularly on titanium-based implants used in medical and dental applications. Bacteriophages have emerged as a promising alternative for preventing and eradicating biofilms. This study evaluated the effectiveness of bacteriophages in controlling P. aeruginosa biofilms on titanium. It tested their ability to eradicate mature biofilms and prevent the formation of new ones. Microbiological assays, efficacy analyses, and scanning electron microscopy (SEM) were used. Bacteriophages significantly reduced biofilm biomass, with reductions over 90% in most cases. For the ATCC strain, efficiencies ranged from 50.53% to 99.78%. For the clinical strain, reductions varied between 0.01% and 99.94%. Among the tested phages, PA4 and PA5 were the most effective, consistently achieving reductions of over 99%. Prophylactic treatment completely inhibited biofilm formation, regardless of phage concentration or bacterial strain. Although variations in efficacy were observed depending on strain, phage type, and multiplicity of infection, complicating the establishment of a clear dose-response relationship, the results confirm the strong potential of bacteriophages as both therapeutic and preventive agents against biofilm-related infections on titanium biomedical devices. These findings provide a crucial foundation for future clinical and industrial applications, contributing to the development of safer and more reliable titanium-based implants. Biocontrol Bacterial Bacteriophages Infection control Medical devices Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Biofilms, complex microbial conglomerates that adhere to solid surfaces and are encased in a matrix of extracellular substances, represent a particularly challenging and prevalent form of infection in clinical and dental settings. In recent years, understanding of the formation and clinical impact of biofilms on medical, dental devices, and biomaterials has expanded substantially, as these multicellular structures continue to be a significant source of morbidity and associated healthcare costs (Flemming et al., 2023; Taner et al., 2024; Zhao, Sun, Liu, 2023). Bacterial biofilms have the ability to colonize a wide range of surfaces, including intravascular catheters, orthopedic implants, dental restorations, and dental prostheses. Once established, these biofilms not only demonstrate remarkable resistance to traditional antimicrobial treatments but also promote the development of chronic and recurrent infections, often requiring repeated clinical interventions and resulting in considerable financial and health burdens (Alves et al., 2022; Li et al., 2023). The use of implanted medical devices has become an integral part of modern medicine; however, these devices also carry a heightened risk of biofilm-associated infections (Yu et al., 2024a). The processes governing biofilm formation are rather complex, involving several steps, and almost all surfaces are susceptible to being colonised. (Li et al., 2023). Effects of material/surface properties—such as surface charge, hydrophobicity, roughness, topography, and chemistry may influence biofilm formation (Khan; Shakoor, 2023). Despite efforts to maintain sterility, implantable and prosthetic medical devices can easily become contaminated, when placed in the human body, it becomes a site where host cells and microbes compete to reach while it is carrying out its intended purpose of salvaging the normal functions of vital organs (Yu et al., 2024; Li et al., 2023; Khan; Shankoor, 2023; Li et al., 2024). Several infections are characterized by biofilm formation and include, among others, infective endocarditis, osteomyelitis, chronic non-healing wounds, and chronic obstructive pulmonary disease. These infections compromise quality of life, can be fatal, and are often traced to bacterial species in water, air, soil, or on human skin. The mortality, morbidity, and costs associated with these infections affect a large and increasing proportion of the population. Therefore, the prevention of bacterial biofilm-associated infections is highly important, necessitating a better understanding of the agents involved. New studies are necessary to develop safe, effective, practical, and economically viable strategies for combating medical device-related biofilm infections (Malviya et al., 2023; Bouhrour et al., 2024; Kaushik et al., 2024). The growing understanding of the complexity of biofilms on medical and dental devices has prompted multidisciplinary efforts to investigate the most effective prevention and control strategies. Furthermore, advances in nanotechnology and biomaterials have fueled the development of innovative approaches to inhibit the formation and promote the eradication of biofilms (Yu et al., 2024b). In this context, the study aimed to evaluate the effectiveness of bacteriophages in reducing and preventing Pseudomonas aeruginosa biofilm formation, with the goal of applying this approach to titanium surfaces. The research contributes to advancing strategies for preventing and treating infections associated with biomedical devices, ultimately enhancing patient quality of life and improving the safety of medical and dental procedures. MATERIALS AND METHODS Pseudomonas aeruginosa and Bacteriophages Pseudomonas aeruginosa strain ATCC® 27853™ and Pseudomonas aeruginosa clinical strains were provided by the Polydoro Ernani de São Thiago University Hospital, Federal University of Santa Catarina (HU/UFSC). Native lytic bacteriophages designated Phage PA1, PA2, PA3, PA4, and PA5 were isolated from clinical P. aeruginosa strains and are maintained at the Laboratory of Applied Virology (LVA/UFSC). These five phages were tested for their lytic activity against P. aeruginosa using the double agar layer method, and demonstrated effective lytic profiles (Adams, 1959; Kropinski et al., 2009). P. aeruginosa and Biofilm Control on Titanium Surfaces Mediated by Bacteriophages Assays To induce biofilm formation on titanium surfaces, individual assays were performed using Pseudomonas aeruginosa . For each test, 20 µL of bacterial inoculum (5 Log CFU/mL) was added to a 24-well microplate containing 980 µL of Brain Heart Infusion (BHI) broth. Titanium elements were immersed in the bacterial suspension using sterile forceps and incubated at 37°C for 4 days, following a protocol adapted from Freitas, Sand, and Simonetti (2010). A negative control consisting of titanium elements immersed in sterile BHI broth without bacterial inoculation was incubated under identical conditions. Every two days, the titanium fragments were aseptically transferred to fresh BHI broth (1 mL) after being washed with sterile saline to remove planktonic cells. All experiments were conducted in triplicate for each bacterial strain tested. Post-treatment titanium disks were incubated for 4 days in a 24-well microplate containing 1 mL of Brain Heart Infusion (BHI) broth. Every two days, the disks were transferred to fresh BHI medium (1 mL) to assess the prophylactic activity of the bacteriophages. Both the disruption and prophylactic experiments were conducted using three different bacteriophage concentrations, expressed as multiplicity of infection (MOI): low (MOI 0.01), intermediate (MOI 0.1), and high (MOI 1). Viable P. aeruginosa viability count To quantify viable bacteria adhered to treated and untreated specimens, each fragment was removed from the nutrient medium and gently rinsed with sterile saline solution. The biofilm remaining on the metal surface was collected using a sterile swab, which was then immersed in a tube containing 2 mL of sterile distilled water. The swab was vortexed for 2 minutes to detach bacterial cells. After discarding the swab, the tube containing the specimen was vortexed for an additional 1 minute to ensure thorough cell detachment. Subsequently, 10 µL of the resulting suspension was serially diluted in phosphate-buffered saline (PBS) using base-10 dilutions in 96-well microplates, ranging from 10⁻² to 10⁻¹⁰, to obtain countable colony numbers (between 3 and 30 colonies). Aliquots of 10 µL from each dilution were then vertically dripped onto BHI agar plates. The plates were left open in a laminar flow cabinet to dry the inoculum droplets, followed by incubation at 37°C for 24 hours. All procedures were performed in triplicate. Colony-forming units (CFU) were calculated as described by Chaves (2004). Assessment by Scanning Electron Microscopy (SEM) The evaluation of titanium specimens, both with and without biofilms and with or without bacteriophage treatment, was performed using Scanning Electron Microscopy (SEM) at the Central Electronic Microscopy Laboratory (LCME/UFSC). Samples were prepared following biofilm cultivation and bacteriophage treatment protocols prior to analysis. Sample preparation for SEM was based on the method recommended by Castro (2002), with modifications. After removal from the suspension, fragments were washed in sterile saline for 1 minute and then fixed in 2.5% glutaraldehyde for at least 12 hours. Following fixation, samples were dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, and 100%), with each step lasting 30 minutes. After dehydration, the specimens were immersed in 1% osmium tetroxide for 1 hour. Chemical drying was performed using hexamethyldisilazane (HMDS). Samples were then mounted on aluminum stubs using double-sided conductive adhesive tape and sputter-coated with a 10 nm layer of gold using a Bal-Tec SDC 050 sputter coater for 2 minutes. Finally, specimens were observed under the SEM at magnifications of 100×, 1,000×, and 10,000× to capture detailed images. For quantitative analysis of biofilm reduction on the surfaces of titanium elements, the ImageJ software version 1.49, written in Java, was used. This software is available on the website http://rsweb.nih.gov/ij . For each experimental group, 5 digital images were used, totaling 50 images, referring to the five bacteriophages used, in the three doses applied, and the two strains of P. aeruginosa . The images were standardized to maintain an adequate scale, with the intention of reducing uncertainties and increasing the precision of the data obtained. Estimates of the percentage of total area occupied by the biofilm (in black) and the exposed disc (in white) were determined. The images were processed and saved in the TIFF format (Tagged Image File Format), a format commonly used in image binarization and analysis processes. The percentage values obtained were used to calculate the percentage reduction, which is the difference between the initial value (untreated element) and the new value (treated element), a-b, divided by the initial absolute value (untreated element). STATISTICAL ANALYSIS The obtained results were subjected to Two-Way ANOVA, a two-way analysis of variance. The statistical significance level was set at p < 0.05. Tukey's test was employed for group comparisons. The analyses were conducted using GraphPad Prism 8.0 software. Results that did not support the inferential procedure were presented descriptively. RESULTS P. aeruginosa biofilm in titanium The SEM images (Figs. 1 and 2) illustrate the surface morphology of titanium elements covered by biofilms formed by P. aeruginosa (positive control) compared to titanium discs without biofilm formation (negative control). The titanium surface exhibits notable irregularities, with its rough texture clearly visible. In the positive control images, a dense accumulation of cellular biomass is evident, highlighting extensive biofilm development on the surface. Bacteriophage-Mediated Biofilm Control: Proof of Concept Table 1 presents the survival rates of viable bacterial cells following treatment with bacteriophages for each tested infection. Phages demonstrated the ability to infect and reduce the number of viable cells; however, complete eradication was not achieved. Additionally, the results did not exhibit a consistent dose-dependent pattern or clear variation between host strains, preventing the establishment of a definitive cause-and-effect relationship. Table 1 . Viable cells recovered from Pseudomonas aeruginosa biofilm after treatment with bacteriophages in mature biofilm on a titanium surface. Where 0.01, 0.1 and 1 correspond to the viral infection multiplicity indices (PFU/CFU). CFU = Colony Forming Unit per mL; PFU = Plaque Forming Unit per mL. Rates were calculated considering the averages of three independent experiments. Pseudomonas aeruginosa Bacteriophages Concentration (PFU/CFU) Survival Rate (%) ATCC® 27853™ PA1 0.01 46.70 PA1 0.1 26.15 PA1 1 35.06 PA2 0.01 28.90 PA2 0.1 55.42 PA2 1 8.62 PA3 0.01 20.30 PA3 0.1 62.31 PA3 1 8.84 PA4 0.01 27.83 PA4 0.1 37.10 PA4 1 18.15 PA5 0.01 61.63 PA5 0.1 8.84 PA5 1 26.53 CLINIC PA1 0.01 3.25 PA1 0.1 3.20 PA1 1 7.84 PA2 0.01 8.81 PA2 0.1 46.29 PA2 1 0.22 PA3 0.01 69.23 PA3 0.1 76.95 PA3 1 81.20 PA4 0.01 31.63 PA4 0.1 34.27 PA4 1 53.79 PA5 0.01 12.41 PA5 0.1 61.53 PA5 1 33.82 The micrographs presented in Fig. 3 show the reduction in biofilm of the P. aeruginosa clinical strain on the surface of the titanium elements after treatment with the 5 bacteriophages tested, compared to the control. Regarding bacteriophages PA1, PA2, and PA3, the intermediate dose was less efficient in eradicating biofilm compared to the other concentrations applied. Among these, PA3 was the least efficient at the lowest dose. The PA4, on the other hand, was considered the most efficient, as there was no presence of post-treatment biofilm at either concentration, as was the case with PA5; however, traces of biofilm were observed at the highest concentration tested. The same general observation regarding the disaggregation capacity was noted for P. aeruginosa ATCC strain (Fig. 4). However, there was a difference regarding the profile. The PA3 and PA4 phages had the lowest efficiency, allowing for the visualization of biofilm and a high bacterial population density, respectively. The intermediate and high doses were effective in both, except for PA2, where planktonic cells were observed in abundance. However, when comparing the intermediate and high dosages of PA1 bacteriophage, the high dosage was more efficient. Bacteriophage-Mediated Biofilm Control Quantify Table 2 presents the reduction of P. aeruginosa biofilm per total surface area (mm2) of the titanium piece. For the ATCC strain, the PA1 phage showed a biofilm removal efficiency range of 50.53% to 99.78%; PA2, greater than 99%; PA3, between 71.60% and 99.16%; PA4, 87.55% to 99.70%; and PA5, also higher than 99%. Considering clinical strain, PA1 showed a reduction rate between 0.01% and 99.82%; PA2, between 92.61% and 99.78%; PA3, between 75.31% and 99.94%; PA4, greater than 99%; and finally, PA5, greater than 90%. Therefore, there is efficiency in the application of bacteriophages for the destruction and disintegration of P. aeruginosa biofilm. Table 2. Reduction of Pseudomonas aeruginosa biofilm on the surface of titanium elements by Scanning Electron Microscopy (SEM). Software for editing and analyzing micrographs, ImageJ© version 1.49, using the Java language. Pseudomonas aeruginosa Bacteriophage (MOI) MOI Micrograph area (%) Disc biofilm area mm 2 (control) Disc biofilm area mm 2 (treatment) Reduction rate (%) ATCC® 27853™ PA1 0.01 0.202 17.69 0.040 98.78 PA1 0.1 44.557 8.751 50.53 PA1 1.0 31.012 6.091 65.57 PA2 0.01 0.004 0.001 100 PA2 0.1 0.519 0.116 99.34 PA2 1.0 0.077 0.015 99.91 PA3 0.01 25.579 5.024 71.60 PA3 0.1 0.761 0.149 99.16 PA3 1.0 3.143 0.617 96.51 PA4 0.01 11.216 2.203 87.55 PA4 0.1 0.563 0.111 99.37 PA4 1.0 0.273 0.054 99.70 PA5 0.01 0.147 0.029 99.84 PA5 0.1 0.45 0.088 99.50 PA5 1.0 0.162 0.032 99.82 CLINIC PA1 0.01 0.196 16.66 0.038 99.77 PA1 0.1 84.81 16.657 0.01 PA1 1.0 0.188 0.037 99.78 PA2 0.01 0.253 0.050 99.70 PA2 0.1 6.269 1.231 92.61 PA2 1.0 0.125 0.025 99.85 PA3 0.01 2.084 0.409 97.54 PA3 0.1 20.942 4.113 75.31 PA3 1.0 0.048 0.009 99.94 PA4 0.01 0.209 0.041 99.75 PA4 0.1 0.104 0.020 99.88 PA4 1.0 0.052 0.010 99.94 PA5 0.01 6.839 1.347 91.91 PA5 0.1 0.01 0.002 99.99 PA5 1.0 0.188 0.37 99.78 Bacteriophage-Mediated Biofilm Control: Prophylaxis Assay Bacteria viable count Table 3 displays the bacterial survival after biofilm propagation and post-prophylactic treatment for the clinical and reference strains. It is evident that both bacteriophages successfully infected and reduced the number of viable bacterial cells, achieving eradication rates greater than 97%. Therefore, the results did not reveal a clear relationship between the administered dose and variation between the host and the infectious agent, which again made it difficult to establish a cause-and-effect relationship. Table 3. Viable cells recovered from Pseudomonas aeruginosa biofilm on a titanium surface after prophylactic treatment with bacteriophages at different concentrations. Where 0.01, 0.1, and 1 correspond to the viral infection multiplicity indices (UFP/UFC). UFC = Colony Forming Unit per mL; UFP Unit of plaque per mL. Pseudomonas aeruginosa Bacteriophages MOI Survival Rate (%) ATCC® 27853™ PA1 0.01 0.19 PA1 0.1 0.07 PA1 1 0.14 PA2 0.01 0.15 PA2 0.1 0.52 PA2 1 0.3 PA3 0.01 0.09 PA3 0.1 0.08 PA3 1 0.61 PA4 0.01 0.15 PA4 0.1 0.40 PA4 1 0.10 PA5 0.01 0.67 PA5 0.1 2.51 PA5 1 1.69 CLINIC PA1 0.01 0.05 PA1 0.1 0.15 PA1 1 0.04 PA2 0.01 2.92 PA2 0.1 0.41 PA2 1 0.01 PA3 0.01 0.23 PA3 0.1 0.16 PA3 1 0.26 PA4 0.01 1.39 PA4 0.1 0.45 PA4 1 0.11 PA5 0.01 0.07 PA5 0.1 0.21 PA5 1 0.34 In Fig. 5, micrographs of the titanium specimen surface obtained through SEM are presented. The SEM images show prophylactic activity with 100% inhibition of biofilm formation in the observed area, without variation between the study variables (concentration and host-pathogen interaction). The same can be found in Fig. 6 , for tests with the clinical strain. Figure 5. Assessment of the prophylactic activity of bacteriophages in the formation of Pseudomonas aeruginosa ATCC® 27853™ biofilm on the surface of titanium elements, in increasing order of multiplicity of infection. Where, the columns correspond to (A, D, G, J and M) = MOI 0.01; (B, E, H, K and N) = MOI 0.1 and (C, F, I, L and O) = MOI 1. Organized by line, with (A, B and C) = PA1; (D, E and F) = PA2; (G, H and I) = PA5; (J, K and L) = PA3 and (M, N and O) = PA4. All micrographs were captured at the central region of the titanium specimen surface, at a magnification of 1,000×. Considering the percentage reduction of P. aeruginosa biofilm on the surface of titanium elements measured by Scanning Electron Microscopy (SEM) after prophylactic treatment and calculated per surface area (mm²), an efficiency of 100% was observed for all bacteriophages and concentrations tested. These results indicate complete inhibition of biofilm formation, demonstrating the total efficacy of bacteriophage application as a preventive strategy against P. aeruginosa biofilm development on titanium surfaces. DISCUSSION This study demonstrated the effective use of bacteriophages in controlling Pseudomonas aeruginosa biofilms on titanium surfaces. There are several notable findings in the study: (i) The possible multifage collections used were able to infect and reduce viable bacterial cells, making the pathogen more susceptible; (ii) the efficacy of bacteriophages in reducing viable cells in the biofilm is highly dependent on the specificity of the bacteriophage, bacterial strain, and treatment concentration; (iii) analysis of micrographs showed that bacteriophages are capable of reducing P. aeruginosa biofilms on titanium discs by over 90%; (iv) When used prophylactically, bacteriophages inhibited 100% of biofilm formation. Strategies for combating bacterial biofilms are divided into two segments: eradicating already formed biofilms or inhibiting their formation. Among the strategies for biofilm inhibition, inhibiting bacterial growth using bactericidal or bacteriostatic compounds, as well as blocking bacterial adhesion, are considered. Antivirulence therapies are also seen as an alternative, as they make the pathogen more susceptible to the immune system and antimicrobials (Blackledge; Worthington; Melander, 2013; Sharahi et al., 2019) The findings in this study also demonstrate the efficacy of bacteriophages in inhibiting biofilm formation and disintegration of already formed biofilm, as well as their activity in reducing the growth rate of P. aeruginosa . The phage can also indirectly destroy biofilms either through its enzymatic activity or by causing bacterial lysis before or after colonization of the surface (Chegini et al., 2020; Wiguna; Waturangi; Yogiara, 2022). The use of bacteriophage cocktails containing two or more phage mixtures with different host ranges in a single suspension has been shown to be more effective in inhibiting bacterial infections. As in the study by Kovacs et al. (2024), the multifage collection resulted in better reduction of bacterial density in vitro, enhancing the efficacy of bacteriophages. Unlike antibiotics used for such purposes, bacteriophages have greater ease of penetration into P. aeruginosa biofilm; the enzymatic activity of bacteriophages, as portrayed in various studies, aids in the adsorption, invasion, and disintegration of the host bacteria. Additionally, bacteriophages produce endolysins capable of degrading peptidoglycans and assisting in the formation of new progeny phages for release and initiation of a new cycle of viral infection (Kim et al., 2024; Kovacs et al., 2024; Kunisch et al., 2024; Kunz Coyne et al., 2022) Therefore, the advantages of using bacteriophages compared to antibiotics in inhibiting infections caused by bacterial biofilms are evident due to their high specificity and unique mode of action against sessile cells, such as their ability to infect and destroy persistent cells upon reactivation. Another important mechanism is their ability to dissolve the extracellular polymeric matrix through the production of their own enzymes or induction of enzymatic production by the host organism, as well as through interruption of quorum-sensing communication (Abdelghafar et al., 2023; Kovacs et al., 2024). Several studies have highlighted the advances in phage therapy against clinically important bacteria in the clinical environment, such as P. aeruginosa , which causes diseases like chronic otitis, cystic fibrosis, and burns. However, these studies have been limited by preclinical evaluations. Despite the absence of legislation in Brazil authorizing and regulating the use of phage therapy, it should be noted that phage therapy is not a new approach, having been widely used in Brazil in 1920 by José da Costa Cruz of the Oswaldo Cruz Institute (Rio de Janeiro, Brazil) in the control and treatment of bacillary dysentery and staphylococcal infections (Almeida; Sundberg, 2020). In vitro, in silico, and in vivo studies have been conducted, indicating the safety and efficacy of phages in controlling major clinical gram-negative and gram-positive pathogens, especially those microorganisms with high resistance to antibiotics, high morbidity and mortality, and high biofilm production. Kutateladze and Adamia (2010) suggested that phage therapy can be used in biocontrol in medical and dental devices, hospital environments, and mainly in the treatment of nosocomial infections in humans and animals (Hampton, Watson, Fineran, 2020; Kutateladze and Adamia, 2010; Węgrzyn, 2023; Zagaliotis et al., 2022) CONCLUSION The inefficiency of therapeutic strategies currently commonly used to eradicate and inhibit biofilm formation has caused extensive damage in the field of public health, thus necessitating the search for new, efficient strategies capable of combating it. Therefore, the present study identifies phage therapy as one of the effective methods for preventing and eradicating the formation of P. aeruginosa biofilms, specifically on surfaces composed of titanium, an important metallic element used in the manufacture of medical and dental devices. The specificity of the bacteriophage, the bacterial strain, and the concentration of the treatment emerge as limiting factors for the widespread use of phage therapy. Additionally, the need for clinical studies in real-world situations is imperative, providing crucial insights for personalization, further studies, and future developments based on phage therapy as a safe and effective strategy. The proven effectiveness of bacteriophages represents a significant advancement in the fight against biofilm-associated infections, paving the way for further research, technological development, and the biotechnological refinement of clinical products based on phage therapy. Declarations Author Contribution Conceptualization: Gislaine Fongaro, Maria Célia da Silva Lanna. Methodology: Amanda Kelly Ferreira Sousa, Viviani Tadioto, Anderson Giehl, Helena Yurevna Caio. Investigation: Amanda Kelly Ferreira Sousa, Estevão Brasiliense de Souza, Evelin Padilha Corrêa, Mariana Alves Elois, Estevan Rodrigues dos Santos Neto. Data curation: Amanda Kelly Ferreira Sousa, Viviani Tadioto. Formal analysis: Anderson Giehl, Sérgio Luiz Alves Junior. Resources: Ariadne Cristiane Cabral da Cruz, Mara Cristina Scheffer. Writing – original draft: Amanda Kelly Ferreira Sousa. Writing – review & editing: Viviani Tadioto, Anderson Giehl, Gislaine Fongaro. Supervision: Gislaine Fongaro, Maria Célia da Silva Lanna. Project administration: Gislaine Fongaro. Funding acquisition: Gislaine Fongaro.All authors read and approved the final manuscript. Acknowledgement We thank the Division of Clinical Analyses at the University Hospital of the Federal University of Santa Catarina (HU/UFSC) and the Multiuser Laboratory for Biological Studies (LAMEB/CCB) for their technical support, infrastructure, and essential collaboration in conducting this work. 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Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 08 Apr, 2026 Read the published version in European Journal of Clinical Microbiology & Infectious Diseases → Version 1 posted Editorial decision: Revision requested 23 Jan, 2026 Reviews received at journal 23 Jan, 2026 Reviewers agreed at journal 14 Jan, 2026 Reviews received at journal 07 Jan, 2026 Reviewers agreed at journal 05 Jan, 2026 Reviewers invited by journal 22 Dec, 2025 Editor assigned by journal 17 Dec, 2025 Submission checks completed at journal 16 Dec, 2025 First submitted to journal 16 Dec, 2025 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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09:06:05","extension":"xml","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89848,"visible":true,"origin":"","legend":"","description":"","filename":"2285cff713f74240a59200c4704e87441structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8376090/v1/76b325c29584f59af2f1d112.xml"},{"id":99216172,"identity":"7daf3286-bb74-4aac-b513-bcfcc9411b2e","added_by":"auto","created_at":"2025-12-30 09:06:05","extension":"html","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":106083,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8376090/v1/e259dc5a72dd8eaba9990652.html"},{"id":99216146,"identity":"44800ba1-f5e3-4763-8a39-f17bcded4335","added_by":"auto","created_at":"2025-12-30 09:06:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":273684,"visible":true,"origin":"","legend":"\u003cp\u003eMicrograph of experimental controls: Clinical Pseudomonas aeruginosa. Where A, B and C = Positive control, bacterial biofilm adhered to the surface of the specimens; D, E, and F= Negative control, surface of the titanium discs subjected to the same cultivation conditions, but without bacterial inoculum. Both micrographs were taken at the same location, considered the central area of the surface of the specimen and at a size of X1,000. Further information can be found at the bottom of the micrographs.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8376090/v1/0ccd06eaf47167f98ee69c0b.png"},{"id":99216142,"identity":"5ea9f69c-1d79-4ea4-8333-e692fe43d125","added_by":"auto","created_at":"2025-12-30 09:06:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":66702,"visible":true,"origin":"","legend":"\u003cp\u003eMicrograph of experimental controls: \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC® 27853™. Where, A, Band C = Positive control, bacterial biofilm adhered to the surface of the specimens; D, E and F= Negative control, surface of the titanium discs subjected to the same cultivation conditions, but without bacterial inoculum. Both micrographs were taken at the same location considered the central area of the surface of the specimen and at a size of X1,000. Further information can be found at the bottom of the micrographs.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8376090/v1/9e8a5bff19f5e35fbe06f796.jpg"},{"id":99318427,"identity":"acfa9215-71fb-4a53-83f4-5f7fcc0224ca","added_by":"auto","created_at":"2025-12-31 16:33:10","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1287360,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of the disaggregation and destructive activity of clinical \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003ebiofilms on titanium surfaces after treatment with bacteriophages, shown in increasing order of multiplicity of infection (MOI). SEM micrographs illustrate the surface of titanium specimens post-treatment. Columns represent different MOIs: (A, D, G, J, M) = MOI 0.01; (B, E, H, K, N) = MOI 0.1; and (C, F, I, L, O) = MOI 1. Rows correspond to the specific bacteriophages used: (A–C) = PA1; (D–F) = PA2; (G–I) = PA5; (J–L) = PA3; and (M–O) = PA4. All images were captured from the central area of the titanium surface at a magnification of 1,000×.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8376090/v1/9fdd77bbf9fa5ba9ec68a0e0.jpg"},{"id":99317066,"identity":"cb7d9d5a-0844-459e-bba8-6c604765786a","added_by":"auto","created_at":"2025-12-31 16:29:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1468056,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of the disaggregation and destruction activity of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC® 27853™ biofilm on titanium surfaces, following bacteriophage treatment at increasing multiplicities of infection (MOI). Columns represent MOI levels: (A, D, G, J, M) = MOI 0.01; (B, E, H, K, N) = MOI 0.1; and (C, F, I, L, O) = MOI 1. Rows correspond to the different bacteriophages applied: (A–C) = PA1; (D–F) = PA2; (G–I) = PA5; (J–L) = PA3; and (M–O) = PA4. All micrographs were captured at the central region of the titanium specimen surface, at a magnification of 1,000×.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8376090/v1/6018ca749ed9c262dfd709e0.jpg"},{"id":99317139,"identity":"efe26706-d176-4dbc-b118-cee790716fba","added_by":"auto","created_at":"2025-12-31 16:29:41","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1778606,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of the prophylactic activity of bacteriophages in the formation of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003eATCC® 27853™ biofilm on the surface of titanium elements, in increasing order of multiplicity of infection. Where, the columns correspond to (A, D, G, J and M) = MOI 0.01; (B, E, H, K and N) = MOI 0.1 and (C, F, I, L and O) = MOI 1. Organized by line, with (A, B and C) = PA1; (D, E and F) = PA2; (G, H and I) = PA5; (J, K and L) = PA3 and (M, N and O) = PA4. All micrographs were captured at the central region of the titanium specimen surface, at a magnification of 1,000×.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8376090/v1/811c85879d056bcd38193a77.jpg"},{"id":99317387,"identity":"1e8ac0cb-45c1-42f6-8877-c6035d5c7f99","added_by":"auto","created_at":"2025-12-31 16:30:06","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":208753,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the prophylactic activity of bacteriophages in the formation of clinical \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e biofilm on the surface of titanium elements, in increasing order of multiplicity of infection. Where, the columns correspond to (A, D, G, J and M) = MOI 0.01; (B, E, H, K and N) = MOI 0.1 and (C, F, I, L and O) = MOI 1. Organized by line, with (A, B and C) = PA1; (D, E and F) = PA2; (G, H and I) = PA5; (J, K and L) = PA3 and (M, N and O) = PA4. All micrographs were captured at the central region of the titanium specimen surface, at a magnification of 1,000×.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8376090/v1/090b7d031a1a0b4ad0e22df2.jpg"},{"id":106810692,"identity":"55cfcee5-02fa-40dd-9a8a-a353b50b532f","added_by":"auto","created_at":"2026-04-13 16:16:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6545567,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8376090/v1/0cd2cf72-bb2d-4908-9b77-197f47c3aaa4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bacteriophage-Mediated Reduction of Pseudomonas aeruginosa Biofilm on Titanium Surfaces for Biomedical Applications","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eBiofilms, complex microbial conglomerates that adhere to solid surfaces and are encased in a matrix of extracellular substances, represent a particularly challenging and prevalent form of infection in clinical and dental settings. In recent years, understanding of the formation and clinical impact of biofilms on medical, dental devices, and biomaterials has expanded substantially, as these multicellular structures continue to be a significant source of morbidity and associated healthcare costs (Flemming et al., 2023; Taner et al., 2024; Zhao, Sun, Liu, 2023).\u003c/p\u003e \u003cp\u003eBacterial biofilms have the ability to colonize a wide range of surfaces, including intravascular catheters, orthopedic implants, dental restorations, and dental prostheses. Once established, these biofilms not only demonstrate remarkable resistance to traditional antimicrobial treatments but also promote the development of chronic and recurrent infections, often requiring repeated clinical interventions and resulting in considerable financial and health burdens (Alves et al., 2022; Li et al., 2023).\u003c/p\u003e \u003cp\u003eThe use of implanted medical devices has become an integral part of modern medicine; however, these devices also carry a heightened risk of biofilm-associated infections (Yu et al., 2024a). The processes governing biofilm formation are rather complex, involving several steps, and almost all surfaces are susceptible to being colonised. (Li et al., 2023). Effects of material/surface properties\u0026mdash;such as surface charge, hydrophobicity, roughness, topography, and chemistry may influence biofilm formation (Khan; Shakoor, 2023). Despite efforts to maintain sterility, implantable and prosthetic medical devices can easily become contaminated, when placed in the human body, it becomes a site where host cells and microbes compete to reach while it is carrying out its intended purpose of salvaging the normal functions of vital organs (Yu et al., 2024; Li et al., 2023; Khan; Shankoor, 2023; Li et al., 2024).\u003c/p\u003e \u003cp\u003eSeveral infections are characterized by biofilm formation and include, among others, infective endocarditis, osteomyelitis, chronic non-healing wounds, and chronic obstructive pulmonary disease. These infections compromise quality of life, can be fatal, and are often traced to bacterial species in water, air, soil, or on human skin. The mortality, morbidity, and costs associated with these infections affect a large and increasing proportion of the population. Therefore, the prevention of bacterial biofilm-associated infections is highly important, necessitating a better understanding of the agents involved. New studies are necessary to develop safe, effective, practical, and economically viable strategies for combating medical device-related biofilm infections (Malviya et al., 2023; Bouhrour et al., 2024; Kaushik et al., 2024).\u003c/p\u003e \u003cp\u003eThe growing understanding of the complexity of biofilms on medical and dental devices has prompted multidisciplinary efforts to investigate the most effective prevention and control strategies. Furthermore, advances in nanotechnology and biomaterials have fueled the development of innovative approaches to inhibit the formation and promote the eradication of biofilms (Yu et al., 2024b).\u003c/p\u003e \u003cp\u003eIn this context, the study aimed to evaluate the effectiveness of bacteriophages in reducing and preventing \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e biofilm formation, with the goal of applying this approach to titanium surfaces. The research contributes to advancing strategies for preventing and treating infections associated with biomedical devices, ultimately enhancing patient quality of life and improving the safety of medical and dental procedures.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cb\u003ePseudomonas aeruginosa\u003c/b\u003e \u003cb\u003eand Bacteriophages\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e strain ATCC\u0026reg; 27853\u0026trade; and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e clinical strains were provided by the Polydoro Ernani de S\u0026atilde;o Thiago University Hospital, Federal University of Santa Catarina (HU/UFSC). Native lytic bacteriophages designated Phage PA1, PA2, PA3, PA4, and PA5 were isolated from clinical \u003cem\u003eP. aeruginosa\u003c/em\u003e strains and are maintained at the Laboratory of Applied Virology (LVA/UFSC). These five phages were tested for their lytic activity against \u003cem\u003eP. aeruginosa\u003c/em\u003e using the double agar layer method, and demonstrated effective lytic profiles (Adams, 1959; Kropinski et al., 2009).\u003c/p\u003e \u003cp\u003e \u003cb\u003eP. aeruginosa\u003c/b\u003e \u003cb\u003eand Biofilm Control on Titanium Surfaces Mediated by Bacteriophages Assays\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo induce biofilm formation on titanium surfaces, individual assays were performed using \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. For each test, 20 \u0026micro;L of bacterial inoculum (5 Log CFU/mL) was added to a 24-well microplate containing 980 \u0026micro;L of Brain Heart Infusion (BHI) broth. Titanium elements were immersed in the bacterial suspension using sterile forceps and incubated at 37\u0026deg;C for 4 days, following a protocol adapted from Freitas, Sand, and Simonetti (2010). A negative control consisting of titanium elements immersed in sterile BHI broth without bacterial inoculation was incubated under identical conditions. Every two days, the titanium fragments were aseptically transferred to fresh BHI broth (1 mL) after being washed with sterile saline to remove planktonic cells. All experiments were conducted in triplicate for each bacterial strain tested. Post-treatment titanium disks were incubated for 4 days in a 24-well microplate containing 1 mL of Brain Heart Infusion (BHI) broth. Every two days, the disks were transferred to fresh BHI medium (1 mL) to assess the prophylactic activity of the bacteriophages. Both the disruption and prophylactic experiments were conducted using three different bacteriophage concentrations, expressed as multiplicity of infection (MOI): low (MOI 0.01), intermediate (MOI 0.1), and high (MOI 1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eViable\u003c/b\u003e \u003cb\u003eP. aeruginosa\u003c/b\u003e \u003cb\u003eviability count\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo quantify viable bacteria adhered to treated and untreated specimens, each fragment was removed from the nutrient medium and gently rinsed with sterile saline solution. The biofilm remaining on the metal surface was collected using a sterile swab, which was then immersed in a tube containing 2 mL of sterile distilled water. The swab was vortexed for 2 minutes to detach bacterial cells. After discarding the swab, the tube containing the specimen was vortexed for an additional 1 minute to ensure thorough cell detachment.\u003c/p\u003e \u003cp\u003eSubsequently, 10 \u0026micro;L of the resulting suspension was serially diluted in phosphate-buffered saline (PBS) using base-10 dilutions in 96-well microplates, ranging from 10⁻\u0026sup2; to 10⁻\u0026sup1;⁰, to obtain countable colony numbers (between 3 and 30 colonies). Aliquots of 10 \u0026micro;L from each dilution were then vertically dripped onto BHI agar plates. The plates were left open in a laminar flow cabinet to dry the inoculum droplets, followed by incubation at 37\u0026deg;C for 24 hours. All procedures were performed in triplicate. Colony-forming units (CFU) were calculated as described by Chaves (2004).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAssessment by Scanning Electron Microscopy (SEM)\u003c/h2\u003e \u003cp\u003eThe evaluation of titanium specimens, both with and without biofilms and with or without bacteriophage treatment, was performed using Scanning Electron Microscopy (SEM) at the Central Electronic Microscopy Laboratory (LCME/UFSC). Samples were prepared following biofilm cultivation and bacteriophage treatment protocols prior to analysis.\u003c/p\u003e \u003cp\u003eSample preparation for SEM was based on the method recommended by Castro (2002), with modifications. After removal from the suspension, fragments were washed in sterile saline for 1 minute and then fixed in 2.5% glutaraldehyde for at least 12 hours. Following fixation, samples were dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, and 100%), with each step lasting 30 minutes. After dehydration, the specimens were immersed in 1% osmium tetroxide for 1 hour.\u003c/p\u003e \u003cp\u003eChemical drying was performed using hexamethyldisilazane (HMDS). Samples were then mounted on aluminum stubs using double-sided conductive adhesive tape and sputter-coated with a 10 nm layer of gold using a Bal-Tec SDC 050 sputter coater for 2 minutes. Finally, specimens were observed under the SEM at magnifications of 100\u0026times;, 1,000\u0026times;, and 10,000\u0026times; to capture detailed images.\u003c/p\u003e \u003cp\u003eFor quantitative analysis of biofilm reduction on the surfaces of titanium elements, the ImageJ software version 1.49, written in Java, was used. This software is available on the website \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rsweb.nih.gov/ij\u003c/span\u003e\u003cspan address=\"http://rsweb.nih.gov/ij\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. For each experimental group, 5 digital images were used, totaling 50 images, referring to the five bacteriophages used, in the three doses applied, and the two strains of \u003cem\u003eP. aeruginosa\u003c/em\u003e. The images were standardized to maintain an adequate scale, with the intention of reducing uncertainties and increasing the precision of the data obtained.\u003c/p\u003e \u003cp\u003eEstimates of the percentage of total area occupied by the biofilm (in black) and the exposed disc (in white) were determined. The images were processed and saved in the TIFF format (Tagged Image File Format), a format commonly used in image binarization and analysis processes. The percentage values obtained were used to calculate the percentage reduction, which is the difference between the initial value (untreated element) and the new value (treated element), a-b, divided by the initial absolute value (untreated element).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSTATISTICAL ANALYSIS\u003c/h2\u003e \u003cp\u003eThe obtained results were subjected to Two-Way ANOVA, a two-way analysis of variance. The statistical significance level was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Tukey's test was employed for group comparisons. The analyses were conducted using GraphPad Prism 8.0 software. Results that did not support the inferential procedure were presented descriptively.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eP. aeruginosa\u003c/strong\u003e \u003cstrong\u003ebiofilm in titanium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SEM images (Figs. 1 and 2) illustrate the surface morphology of titanium elements covered by biofilms formed by \u003cem\u003eP. aeruginosa\u003c/em\u003e (positive control) compared to titanium discs without biofilm formation (negative control). The titanium surface exhibits notable irregularities, with its rough texture clearly visible. In the positive control images, a dense accumulation of cellular biomass is evident, highlighting extensive biofilm development on the surface.\u003c/p\u003e\n\u003ch3\u003eBacteriophage-Mediated Biofilm Control: Proof of Concept\u003c/h3\u003e\n\u003cp\u003eTable\u0026nbsp;1 presents the survival rates of viable bacterial cells following treatment with bacteriophages for each tested infection. Phages demonstrated the ability to infect and reduce the number of viable cells; however, complete eradication was not achieved. Additionally, the results did not exhibit a consistent dose-dependent pattern or clear variation between host strains, preventing the establishment of a definitive cause-and-effect relationship.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;1\u003c/strong\u003e. Viable cells recovered from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e biofilm after treatment with bacteriophages in mature biofilm on a titanium surface. Where 0.01, 0.1 and 1 correspond to the viral infection multiplicity indices (PFU/CFU). CFU\u0026thinsp;=\u0026thinsp;Colony Forming Unit per mL; PFU\u0026thinsp;=\u0026thinsp;Plaque Forming Unit per mL. Rates were calculated considering the averages of three independent experiments.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"509\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacteriophages\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentration (PFU/CFU)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSurvival Rate (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"15\" valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eATCC\u0026reg; 27853\u0026trade;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e46.70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e26.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e35.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e28.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e55.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e8.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e20.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e62.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e8.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e27.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e37.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e18.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e61.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e8.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e26.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"15\" valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCLINIC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e3.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e3.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e7.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e8.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e46.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e69.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e76.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e81.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e31.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e34.27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e53.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e12.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e61.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e33.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe micrographs presented in Fig. 3 show the reduction in biofilm of the \u003cem\u003eP. aeruginosa\u003c/em\u003e clinical strain on the surface of the titanium elements after treatment with the 5 bacteriophages tested, compared to the control. Regarding bacteriophages PA1, PA2, and PA3, the intermediate dose was less efficient in eradicating biofilm compared to the other concentrations applied. Among these, PA3 was the least efficient at the lowest dose. The PA4, on the other hand, was considered the most efficient, as there was no presence of post-treatment biofilm at either concentration, as was the case with PA5; however, traces of biofilm were observed at the highest concentration tested.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThe same general observation regarding the disaggregation capacity was noted for \u003cem\u003eP. aeruginosa\u003c/em\u003e ATCC strain (Fig. 4). However, there was a difference regarding the profile. The PA3 and PA4 phages had the lowest efficiency, allowing for the visualization of biofilm and a high bacterial population density, respectively. The intermediate and high doses were effective in both, except for PA2, where planktonic cells were observed in abundance. However, when comparing the intermediate and high dosages of PA1 bacteriophage, the high dosage was more efficient.\u003c/p\u003e\n\u003ch3\u003eBacteriophage-Mediated Biofilm Control Quantify\u003c/h3\u003e\n\u003cp\u003eTable 2 presents the reduction of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm per total surface area (mm2) of the titanium piece. For the ATCC strain, the PA1 phage showed a biofilm removal efficiency range of 50.53% to 99.78%; PA2, greater than 99%; PA3, between 71.60% and 99.16%; PA4, 87.55% to 99.70%; and PA5, also higher than 99%. Considering clinical strain, PA1 showed a reduction rate between 0.01% and 99.82%; PA2, between 92.61% and 99.78%; PA3, between 75.31% and 99.94%; PA4, greater than 99%; and finally, PA5, greater than 90%. Therefore, there is efficiency in the application of bacteriophages for the destruction and disintegration of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;2.\u003c/strong\u003e Reduction of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e biofilm on the surface of titanium elements by Scanning Electron Microscopy (SEM). Software for editing and analyzing micrographs, ImageJ\u0026copy; version 1.49, using the Java language.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"764\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacteriophage (MOI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMOI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMicrograph area (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDisc biofilm area mm\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e (control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDisc biofilm area mm\u003csup\u003e2\u003c/sup\u003e (treatment)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReduction rate (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"15\" valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eATCC\u0026reg; 27853\u0026trade;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"15\" valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e17.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e98.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e44.557\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e8.751\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e50.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e31.012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e6.091\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e65.57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.519\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.077\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e25.579\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e5.024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e71.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.761\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.149\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e3.143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.617\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e96.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e11.216\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e2.203\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e87.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.563\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.054\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.147\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.088\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.162\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.032\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"15\" valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCLINIC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.196\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"15\" valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e16.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.038\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e84.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e16.657\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.188\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.253\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.050\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e6.269\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e1.231\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e92.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e2.084\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.409\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e97.54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e20.942\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e4.113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e75.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.209\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.041\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.104\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e6.839\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e1.347\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e91.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.188\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e99.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eBacteriophage-Mediated Biofilm Control: Prophylaxis Assay\u003c/h2\u003e\n \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n \u003ch2\u003eBacteria viable count\u003c/h2\u003e\n \u003cp\u003eTable\u0026nbsp;3 displays the bacterial survival after biofilm propagation and post-prophylactic treatment for the clinical and reference strains. It is evident that both bacteriophages successfully infected and reduced the number of viable bacterial cells, achieving eradication rates greater than 97%. Therefore, the results did not reveal a clear relationship between the administered dose and variation between the host and the infectious agent, which again made it difficult to establish a cause-and-effect relationship.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;3.\u003c/strong\u003e Viable cells recovered from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e biofilm on a titanium surface after prophylactic treatment with bacteriophages at different concentrations. Where 0.01, 0.1, and 1 correspond to the viral infection multiplicity indices (UFP/UFC). UFC\u0026thinsp;=\u0026thinsp;Colony Forming Unit per mL; UFP Unit of plaque per mL.\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"509\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacteriophages\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMOI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSurvival Rate (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"15\" valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eATCC\u0026reg; 27853\u0026trade;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e2.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e1.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"15\" valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCLINIC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e2.92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e1.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ePA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eIn Fig. 5, micrographs of the titanium specimen surface obtained through SEM are presented. The SEM images show prophylactic activity with 100% inhibition of biofilm formation in the observed area, without variation between the study variables (concentration and host-pathogen interaction). The same can be found in \u003cstrong\u003eFig.\u0026nbsp;6\u003c/strong\u003e, for tests with the clinical strain.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure 5.\u003c/strong\u003e Assessment of the prophylactic activity of bacteriophages in the formation of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC\u0026reg; 27853\u0026trade; biofilm on the surface of titanium elements, in increasing order of multiplicity of infection. Where, the columns correspond to (A, D, G, J and M)\u0026thinsp;=\u0026thinsp;MOI 0.01; (B, E, H, K and N)\u0026thinsp;=\u0026thinsp;MOI 0.1 and (C, F, I, L and O)\u0026thinsp;=\u0026thinsp;MOI 1. Organized by line, with (A, B and C)\u0026thinsp;=\u0026thinsp;PA1; (D, E and F)\u0026thinsp;=\u0026thinsp;PA2; (G, H and I)\u0026thinsp;=\u0026thinsp;PA5; (J, K and L)\u0026thinsp;=\u0026thinsp;PA3 and (M, N and O)\u0026thinsp;=\u0026thinsp;PA4. All micrographs were captured at the central region of the titanium specimen surface, at a magnification of 1,000\u0026times;.\u003c/p\u003e\n \u003cp\u003eConsidering the percentage reduction of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm on the surface of titanium elements measured by Scanning Electron Microscopy (SEM) after prophylactic treatment and calculated per surface area (mm\u0026sup2;), an efficiency of 100% was observed for all bacteriophages and concentrations tested. These results indicate complete inhibition of biofilm formation, demonstrating the total efficacy of bacteriophage application as a preventive strategy against \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm development on titanium surfaces.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study demonstrated the effective use of bacteriophages in controlling \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e biofilms on titanium surfaces. There are several notable findings in the study: (i) The possible multifage collections used were able to infect and reduce viable bacterial cells, making the pathogen more susceptible; (ii) the efficacy of bacteriophages in reducing viable cells in the biofilm is highly dependent on the specificity of the bacteriophage, bacterial strain, and treatment concentration; (iii) analysis of micrographs showed that bacteriophages are capable of reducing \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms on titanium discs by over 90%; (iv) When used prophylactically, bacteriophages inhibited 100% of biofilm formation.\u003c/p\u003e \u003cp\u003eStrategies for combating bacterial biofilms are divided into two segments: eradicating already formed biofilms or inhibiting their formation. Among the strategies for biofilm inhibition, inhibiting bacterial growth using bactericidal or bacteriostatic compounds, as well as blocking bacterial adhesion, are considered. Antivirulence therapies are also seen as an alternative, as they make the pathogen more susceptible to the immune system and antimicrobials (Blackledge; Worthington; Melander, 2013; Sharahi et al., 2019)\u003c/p\u003e \u003cp\u003eThe findings in this study also demonstrate the efficacy of bacteriophages in inhibiting biofilm formation and disintegration of already formed biofilm, as well as their activity in reducing the growth rate of \u003cem\u003eP. aeruginosa\u003c/em\u003e. The phage can also indirectly destroy biofilms either through its enzymatic activity or by causing bacterial lysis before or after colonization of the surface (Chegini et al., 2020; Wiguna; Waturangi; Yogiara, 2022).\u003c/p\u003e \u003cp\u003eThe use of bacteriophage cocktails containing two or more phage mixtures with different host ranges in a single suspension has been shown to be more effective in inhibiting bacterial infections. As in the study by Kovacs et al. (2024), the multifage collection resulted in better reduction of bacterial density in vitro, enhancing the efficacy of bacteriophages. Unlike antibiotics used for such purposes, bacteriophages have greater ease of penetration into \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm; the enzymatic activity of bacteriophages, as portrayed in various studies, aids in the adsorption, invasion, and disintegration of the host bacteria. Additionally, bacteriophages produce endolysins capable of degrading peptidoglycans and assisting in the formation of new progeny phages for release and initiation of a new cycle of viral infection (Kim et al., 2024; Kovacs et al., 2024; Kunisch et al., 2024; Kunz Coyne et al., 2022)\u003c/p\u003e \u003cp\u003eTherefore, the advantages of using bacteriophages compared to antibiotics in inhibiting infections caused by bacterial biofilms are evident due to their high specificity and unique mode of action against sessile cells, such as their ability to infect and destroy persistent cells upon reactivation. Another important mechanism is their ability to dissolve the extracellular polymeric matrix through the production of their own enzymes or induction of enzymatic production by the host organism, as well as through interruption of quorum-sensing communication (Abdelghafar et al., 2023; Kovacs et al., 2024).\u003c/p\u003e \u003cp\u003eSeveral studies have highlighted the advances in phage therapy against clinically important bacteria in the clinical environment, such as \u003cem\u003eP. aeruginosa\u003c/em\u003e, which causes diseases like chronic otitis, cystic fibrosis, and burns. However, these studies have been limited by preclinical evaluations. Despite the absence of legislation in Brazil authorizing and regulating the use of phage therapy, it should be noted that phage therapy is not a new approach, having been widely used in Brazil in 1920 by Jos\u0026eacute; da Costa Cruz of the Oswaldo Cruz Institute (Rio de Janeiro, Brazil) in the control and treatment of bacillary dysentery and staphylococcal infections (Almeida; Sundberg, 2020).\u003c/p\u003e \u003cp\u003eIn vitro, in silico, and in vivo studies have been conducted, indicating the safety and efficacy of phages in controlling major clinical gram-negative and gram-positive pathogens, especially those microorganisms with high resistance to antibiotics, high morbidity and mortality, and high biofilm production. Kutateladze and Adamia (2010) suggested that phage therapy can be used in biocontrol in medical and dental devices, hospital environments, and mainly in the treatment of nosocomial infections in humans and animals (Hampton, Watson, Fineran, 2020; Kutateladze and Adamia, 2010; Węgrzyn, 2023; Zagaliotis et al., 2022)\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe inefficiency of therapeutic strategies currently commonly used to eradicate and inhibit biofilm formation has caused extensive damage in the field of public health, thus necessitating the search for new, efficient strategies capable of combating it. Therefore, the present study identifies phage therapy as one of the effective methods for preventing and eradicating the formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms, specifically on surfaces composed of titanium, an important metallic element used in the manufacture of medical and dental devices. The specificity of the bacteriophage, the bacterial strain, and the concentration of the treatment emerge as limiting factors for the widespread use of phage therapy. Additionally, the need for clinical studies in real-world situations is imperative, providing crucial insights for personalization, further studies, and future developments based on phage therapy as a safe and effective strategy. The proven effectiveness of bacteriophages represents a significant advancement in the fight against biofilm-associated infections, paving the way for further research, technological development, and the biotechnological refinement of clinical products based on phage therapy.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: Gislaine Fongaro, Maria C\u0026eacute;lia da Silva Lanna. Methodology: Amanda Kelly Ferreira Sousa, Viviani Tadioto, Anderson Giehl, Helena Yurevna Caio. Investigation: Amanda Kelly Ferreira Sousa, Estev\u0026atilde;o Brasiliense de Souza, Evelin Padilha Corr\u0026ecirc;a, Mariana Alves Elois, Estevan Rodrigues dos Santos Neto. Data curation: Amanda Kelly Ferreira Sousa, Viviani Tadioto. Formal analysis: Anderson Giehl, S\u0026eacute;rgio Luiz Alves Junior. Resources: Ariadne Cristiane Cabral da Cruz, Mara Cristina Scheffer. Writing \u0026ndash; original draft: Amanda Kelly Ferreira Sousa. Writing \u0026ndash; review \u0026amp; editing: Viviani Tadioto, Anderson Giehl, Gislaine Fongaro. Supervision: Gislaine Fongaro, Maria C\u0026eacute;lia da Silva Lanna. Project administration: Gislaine Fongaro. Funding acquisition: Gislaine Fongaro.All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank the Division of Clinical Analyses at the University Hospital of the Federal University of Santa Catarina (HU/UFSC) and the Multiuser Laboratory for Biological Studies (LAMEB/CCB) for their technical support, infrastructure, and essential collaboration in conducting this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFINANCIAL SUPPORT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Council for Scientific and Technological Development (CNPq - n° 304434/2023-0 and n° - 408073/2023-3) and the Coordination for the Improvement of Higher Education Personnel (CAPES).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eABDELGHAFAR, Aliaa \u003cem\u003eet al.\u003c/em\u003e Isolation of a bacteriophage targeting Pseudomonas aeruginosa and exhibits a promising in vivo efficacy. \u003cstrong\u003eAMB Express\u003c/strong\u003e, v. 13, n. 1, p. 79, 26 jul. 2023. 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Accessed on August 2, 2025.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-clinical-microbiology-and-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejcm","sideBox":"Learn more about [European Journal of Clinical Microbiology \u0026 Infectious Diseases](https://www.springer.com/journal/10096)","snPcode":"10096","submissionUrl":"https://submission.nature.com/new-submission/10096/3","title":"European Journal of Clinical Microbiology \u0026 Infectious Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Biocontrol, Bacterial, Bacteriophages, Infection control, Medical devices","lastPublishedDoi":"10.21203/rs.3.rs-8376090/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8376090/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBiofilm-associated infections on biomedical devices are a major clinical challenge. These infections are resistant to antimicrobial treatments and often cause chronic and recurrent problems. \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e is a significant biofilm-forming pathogen, particularly on titanium-based implants used in medical and dental applications. Bacteriophages have emerged as a promising alternative for preventing and eradicating biofilms. This study evaluated the effectiveness of bacteriophages in controlling \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms on titanium. It tested their ability to eradicate mature biofilms and prevent the formation of new ones. Microbiological assays, efficacy analyses, and scanning electron microscopy (SEM) were used. Bacteriophages significantly reduced biofilm biomass, with reductions over 90% in most cases. For the ATCC strain, efficiencies ranged from 50.53% to 99.78%. For the clinical strain, reductions varied between 0.01% and 99.94%. Among the tested phages, PA4 and PA5 were the most effective, consistently achieving reductions of over 99%. Prophylactic treatment completely inhibited biofilm formation, regardless of phage concentration or bacterial strain. Although variations in efficacy were observed depending on strain, phage type, and multiplicity of infection, complicating the establishment of a clear dose-response relationship, the results confirm the strong potential of bacteriophages as both therapeutic and preventive agents against biofilm-related infections on titanium biomedical devices. These findings provide a crucial foundation for future clinical and industrial applications, contributing to the development of safer and more reliable titanium-based implants.\u003c/p\u003e","manuscriptTitle":"Bacteriophage-Mediated Reduction of Pseudomonas aeruginosa Biofilm on Titanium Surfaces for Biomedical Applications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 09:05:53","doi":"10.21203/rs.3.rs-8376090/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-23T16:54:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-23T14:37:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147294012655081949383749389204954591434","date":"2026-01-14T12:47:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-07T09:53:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"202405382578127291908522815878858652421","date":"2026-01-05T08:52:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-22T08:11:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-17T15:01:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-17T00:41:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Clinical Microbiology \u0026 Infectious Diseases","date":"2025-12-16T12:16:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-clinical-microbiology-and-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejcm","sideBox":"Learn more about [European Journal of Clinical Microbiology \u0026 Infectious Diseases](https://www.springer.com/journal/10096)","snPcode":"10096","submissionUrl":"https://submission.nature.com/new-submission/10096/3","title":"European Journal of Clinical Microbiology \u0026 Infectious Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0454df52-0808-4355-afd0-4fa99907b4d7","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:14:40+00:00","versionOfRecord":{"articleIdentity":"rs-8376090","link":"https://doi.org/10.1007/s10096-026-05462-z","journal":{"identity":"european-journal-of-clinical-microbiology-and-infectious-diseases","isVorOnly":false,"title":"European Journal of Clinical Microbiology \u0026 Infectious Diseases"},"publishedOn":"2026-04-08 15:59:02","publishedOnDateReadable":"April 8th, 2026"},"versionCreatedAt":"2025-12-30 09:05:53","video":"","vorDoi":"10.1007/s10096-026-05462-z","vorDoiUrl":"https://doi.org/10.1007/s10096-026-05462-z","workflowStages":[]},"version":"v1","identity":"rs-8376090","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8376090","identity":"rs-8376090","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}