A semi-automated cell tracking protocol for quantitative analyses of neutrophil swarming to sterile and S. aureus contaminated bone implants in a mouse femur model.

Implant-associated osteomyelitis remains a major orthopaedic problem. As neutrophil swarming to the surgical site is a critical host response to prevent infection, visualization and quantification of this dynamic behavior at the native microenvironment of infection will elucidate previously unrecognized mechanisms central to understanding the host response. We recently developed longitudinal intravital imaging of the bone marrow (LIMB) to visualize host cells and fluorescent S. aureus on a contaminated transfemoral implant in live mice, which allows for direct visualization of bacteria colonization of the implant and host cellular responses using two-photon laser scanning microscopy. To the end of rigorous and reproducible quantitative outcomes of neutrophil swarming kinetics in this model, we developed a protocol for robust segmentation, tracking, and quantifications of neutrophil dynamics adapted from Trainable Weka Segmentation and TrackMate, two readily available Fiji/ImageJ plugins. In this work, Catchup mice with tdTomato expressing neutrophils received a transfemoral pin with or without ECFP/EGFP-expressing USA300 methicillin-resistant Staphylococcus aureus (MRSA) to obtain 30-minute LIMB videos at 2-, 4-, and 6-hours post-implantation. The developed semi-automated neutrophil tracking protocol was executed independently by two users to quantify the distance, displacement, speed, velocity, and directionality of the target cells. The results revealed high inter-user reliability for all outcomes (ICC > 0.96; p > 0.05). Consistent with the established paradigm on increased neutrophil swarming during active infection, the results also demonstrated increased neutrophil speed and velocity at all measured time points, and increased displacement at later time points (6 hours) in infected versus uninfected mice (p < 0.05). Neutrophils and bacteria also exhibit directionality during migration in the infected mice. The semi-automated cell tracking protocol provides a streamlined approach to robustly identify and track individual cells across diverse experimental settings and eliminates inter-observer variability.

In vitro models for studying implant-associated biofilms - A review from the perspective of bioengineering 3D microenvironments.

Biofilm research has grown exponentially over the last decades, arguably due to their contribution to hospital acquired infections when they form on foreign body surfaces such as catheters and implants. Yet, translation of the knowledge acquired in the laboratory to the clinic has been slow and/or often it is not attempted by research teams to walk the talk of what is defined as 'bench to bedside'. We therefore reviewed the biofilm literature to better understand this gap. Our search revealed substantial development with respect to adapting surfaces and media used in models to mimic the clinical settings, however many of the in vitro models were too simplistic, often discounting the composition and properties of the host microenvironment and overlooking the biofilm-implant-host interactions. Failure to capture the physiological growth conditions of biofilms in vivo results in major differences between lab-grown- and clinically-relevant biofilms, particularly with respect to phenotypic profiles, virulence, and antimicrobial resistance, and they essentially impede bench-to-bedside translatability. In this review, we describe the complexity of the biological processes at the biofilm-implant-host interfaces, discuss the prerequisite for the development and characterization of biofilm models that better mimic the clinical scenario, and propose an interdisciplinary outlook of how to bioengineer biofilms in vitro by converging tissue engineering concepts and tools.

Sonication protocols and their contributions to the microbiological diagnosis of implant-associated infections: a review of the current scenario.

Addressing the existing problem in the microbiological diagnosis of infections associated with implants and the current debate about the real power of precision of sonicated fluid culture (SFC), the objective of this review is to describe the methodology and analyze and compare the results obtained in current studies on the subject. Furthermore, the present study also discusses and suggests the best parameters for performing sonication. A search was carried out for recent studies in the literature (2019-2023) that addressed this research topic. As a result, different sonication protocols were adopted in the studies analyzed, as expected, and consequently, there was significant variability between the results obtained regarding the sensitivity and specificity of the technique in relation to the traditional culture method (periprosthetic tissue culture - PTC). Coagulase-negative Staphylococcus (CoNS) and Staphylococcus aureus were identified as the main etiological agents by SFC and PTC, with SFC being important for the identification of pathogens of low virulence that are difficult to detect. Compared to chemical biofilm displacement methods, EDTA and DTT, SFC also produced variable results. In this context, this review provided an overview of the most current scenarios on the topic and theoretical support to improve sonication performance, especially with regard to sensitivity and specificity, by scoring the best parameters from various aspects, including sample collection, storage conditions, cultivation methods, microorganism identification techniques (both phenotypic and molecular) and the cutoff point for colony forming unit (CFU) counts. This study demonstrated the need for standardization of the technique and provided a theoretical basis for a sonication protocol that aims to achieve the highest levels of sensitivity and specificity for the reliable microbiological diagnosis of infections associated with implants and prosthetic devices, such as prosthetic joint infections (PJIs). However, practical application and additional complementary studies are still needed.

A polypeptide coating for preventing biofilm on implants by inhibiting antibiotic resistance genes.

Aging population has been boosting the need for orthopedic implants. However, biofilm has been a major obstacle for orthopedic implants due to its insensitivity to antibiotics and tendency to drive antimicrobial resistance. Herein, an antibacterial polypeptide coating with excellent in vivo adhesive capacity was prepared to prevent implants from forming biofilms and inducing acquired antibiotic resistance. A peptide-based copolymer, poly[phenylalanine10-stat-lysine12]-block-3,4-dihydroxy-l-phenylalanine [Poly(Phe10-stat-Lys12)-DOPA] was modularly designed, where poly(Phe10-stat-Lys12) is antibacterial polypeptide with high antibacterial activity, and DOPA provides strong adhesion in both wet and dry microenvironments. Meanwhile, compared to traditional "graft-onto" methods, this antibacterial coating can be facilely achieved by immersing Titanium substrates into antibacterial polypeptide solution for 5 min at room temperature. The poly(Phe10-stat-Lys12)-DOPA polymer showed good antibacterial activity with minimum inhibitory concentrations against S. aureus and E. coli of 32 and 400 µg/mL, respectively. Compared to obvious antimicrobial resistance of S. aureus after continuous treatment with vancomycin, this antibacterial coating doesn't drive antimicrobial resistance upon long-term utilization. Transcriptome sequencing and qPCR tests further confirmed that the antibacterial coating was able to inhibit the expression of multiple peptide resistance factor (mprF) and lipoteichoic acid modification D-alanylation genes (dltB and dltC) that can increase the net positive charge of bacterial cell wall to induce the resistance to cationic antimicrobial peptides. In vivo experiments confirmed that this poly(Phe10-stat-Lys12)-DOPA coating can both effectively prevent biofilm formation through surface contact sterilization and avoid local and systemic infections. Overall, we proposed a facile method for preparing antibacterial orthopedic implants with longer indwelling time and without inducing antimicrobial resistance by coating a polypeptide-based polymer on the implants.

Detection of Microorganisms in Clinical Sonicated Orthopedic Devices Using Conventional Culture and qPCR / Detecção de microrganismos em dispositivos ortopédicos sonicados clínicos usando cultura convencional e qPCR

Abstract Objective To evaluate the sensitivity and specificity of the quantitative real-time polymerase chain reaction (qPCR) for 16S rDNA gene screening using sonicated fluid from orthopedic implants. Methods A retrospective study was conducted on 73 sonicated fluids obtained from patients with infection associated with orthopedic implants. The samples were subjected to conventional culture and molecular testing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and qPCR for 16S rDNA. The cycle threshold values were used to define a cut-off of the qPCR of the 16S rDNA for negative and positive cultures. Results No statistical differences were observed between the positive and negative culture groups based on the time from the first surgery to infection (p= 0.958), age (p =0.269), or general comorbidities. Nevertheless, a statistical difference was found between the mean duration of antibiotic use before device removal (3.41 versus 0.94; p =0.016). Bacterial DNA was identified in every sample from the sonicated fluids. The median cycle thresholds of the positive and negative cultures were of 25.6 and 27.3 respectively (p< 0.001). As a diagnostic tool, a cycle threshold cut-off of 26.89 demonstrated an area under the curve of the receiver operating characteristic of 0.877 (p≤ 0.001). Conclusion The presence of antimicrobial agents for more than 72 hours decreased culture positivity, but did not influence the qPCR results. Despite this, amplification of the 16S rDNA may overestimate infection diagnosis.

Resumo Objetivo Avaliar a sensibilidade e a especificidade da reação em cadeia de polimerase em tempo real quantitativa (quantitative real-time polymerase chain reaction, qPCR, em inglês) para a triagem do gene rDNA 16S, com a utilização do fluido sonicado de implantes ortopédicos. Métodos Um estudo retrospectivo foi realizado em 73 fluidos sonicados obtidos de pacientes com infecção associada aos implantes ortopédicos. As amostras foram submetidas a cultura convencional e a teste molecular utilizando ionização e dessorção a laser assistida por matriz com espectrometria de massa por tempo de voo (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS, em inglês) e qPCR para o gene rDNA 16S. Os valores limiares do ciclo foram usados para definir um ponto de corte para a qPCR do gene rDNA 16S para culturas negativas e positivas. Resultados Não foram observadas diferenças estatísticas entre os grupos de cultura positiva e negativa com base no tempo desde a primeira cirurgia até a infecção (p= 0,958), na idade (p= 0,269), ou nas comorbidades em geral. No entanto, uma diferença estatística foi encontrada entre a duração média do uso de antibióticos antes da remoção do dispositivo (3,41 versus 0,94; p= 0,016). O DNA bacteriano foi identificado em todas as amostras dos fluidos sonicados. Os limiares do ciclo médio de culturas positivas e negativas foram de 25,6 e 27,3, respectivamente (p< 0,001). Como uma ferramenta de diagnóstico, um corte do limite do ciclo de 26,89 demonstrou uma área sob a curva da característica de operação do receptor de 0,877 (p ≤ 0,001). Conclusão A presença de agentes antimicrobianos por mais de 72 horas diminuiu a positividade da cultura, mas não influenciou os resultados da qPCR. Apesar disso, a amplificação do rDNA 16S pode sobrestimar o diagnóstico de infecção.

Effect of Tranexamic Acid against Staphylococcus spp. and Cutibacterium acnes Associated with Peri-Implant Infection: Results from an In Vitro Study.

Tranexamic acid (TXA) is extensively used in orthopedic surgery and traumatology as an antifibrinolytic agent to control intra- and postoperative bleeding and, therefore, indirectly, to reduce postsurgery infection rates. The hypothesis of an additional antibiotic effect against microorganisms associated with periprosthetic joint infection needs to be further evaluated. We aimed to assess whether TXA could reduce bacterial growth using an in vitro model. ATCC and clinical strains of staphylococci and Cutibacterium acnes were tested against TXA in both planktonic and sessile forms. We recorded the percent reduction in the following variables: log CFU/mL by microbiological culture, percentage of live cells by confocal laser scanning microscopy, and, additionally in sessile cells, metabolic activity by the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) assay. Variables were compared between groups using the Kruskal-Wallis test, and the results were reported as median (interquartile range [IQR]). Statistical significance was set at a P value of <0.05. Clinical significance was defined as a reduction of ≥25%. TXA at 50 mg/mL led to a slight reduction in CFU counts (4.5%). However, it was at 10 mg/mL that the reduction reached 27.2% and 33.0% for log CFU/mL counts and percentage of live cells, respectively. TXA was not efficacious for reducing preformed 24-h mature staphylococci and 48-h mature C. acnes biofilms, regardless of its concentration. TXA did not exert an antimicrobial effect against bacterial biofilms. However, when bacteria were in the planktonic form, it led to a clinically and statistically significant reduction in bacterial growth at 10 mg/mL. IMPORTANCE The possible use of TXA as an antibiotic agent in addition to its antifibrinolytic effect may play an important role in the prevention of prosthetic joint infection.

Heterogeneity of Staphylococcus epidermidis in prosthetic joint infections: time to reevaluate microbiological criteria?

Prosthetic joint infection (PJI) is a feared and challenging to diagnose complication after arthroplasty, with Staphylococcus epidermidis as the major pathogen. One important criteria to define PJI is the detection of phenotypically indistinguishable microorganisms with identical antibiotic susceptibility pattern in at least two different samples. However, owing to phenotypical variation within genetic clones and clonal variation within a phenotype, the criteria may be ambiguous. We investigated the extent of diversity among coagulase-negative staphylococci (CoNS) in PJI and characterised S. epidermidis isolates from PJI samples, specifically multiple S. epidermidis isolates identified in individual PJI patients. We performed a retrospective cohort study on 62 consecutive patients with PJI caused by CoNS from two hospitals in Northern Sweden. In 16/62 (26%) PJIs, multiple S. epidermidis isolates were available for whole-genome analyses. Hospital-adapted multidrug-resistant genetic clones of S. epidermidis were identified in samples from 40/62 (65%) of the patients using a combination of pulsed-field gel electrophoresis and multilocus sequence typing. Whole-genome sequencing showed the presence of multiple sequence types (STs) in 7/16 (44%) PJIs where multiple S. epidermidis isolates were available. Within-patient phenotypical variation in the antibiotic susceptibility and/or whole-genome antibiotic resistance gene content was frequent (11/16, 69%) among isolates with the same ST. The results highlight the ambiguity of S. epidermidis phenotypic characterisation as a diagnostic method in PJI and call for larger systematic studies for determining the frequency of CoNS diversity in PJIs, the implications of such diversity for microbiological diagnostics, and the therapeutic outcomes in patients.

Antibiotic Susceptibility of Staphylococcus aureus Plankton and Biofilm Forms Isolated in Implant-Associated Infection.

Comparison of activity of antibiotics against Staphylococcus aureus strains in their plankton form and in biofilms of varying maturity showed that the concentrations of antibiotics causing death of 90% S. aureus strains (MIC90) in their plankton form was 0.07-2.80 µg/ml and significantly (p<0.05) differed from MIC90 concentrations (3-245 µg/ml) for the strains in 24 and 48 h biofilms. MIC90 value was affected by the degree of biofilm maturity: microbial resistance in 48-h biofilms for all analyzed antibiotics was significantly (p<0.05) higher than in 24-h biofilms.

Recent Strategies to Combat Infections from Biofilm-Forming Bacteria on Orthopaedic Implants.

Biofilm-related implant infections (BRII) are a disastrous complication of both elective and trauma orthopaedic surgery and occur when an implant becomes colonised by bacteria. The definitive treatment to eradicate the infections once a biofilm has established is surgical excision of the implant and thorough local debridement, but this carries a significant socioeconomic cost, the outcomes for the patient are often poor, and there is a significant risk of recurrence. Due to the large volumes of surgical procedures performed annually involving medical device implantation, both in orthopaedic surgery and healthcare in general, and with the incidence of implant-related infection being as high as 5%, interventions to prevent and treat BRII are a major focus of research. As such, innovation is progressing at a very fast pace; the aim of this study is to review the latest interventions for the prevention and treatment of BRII, with a particular focus on implant-related approaches.

Point-of-care antimicrobial coating protects orthopaedic implants from bacterial challenge.

Implant related infections are the most common cause of joint arthroplasty failure, requiring revision surgeries and a new implant, resulting in a cost of $8.6 billion annually. To address this problem, we created a class of coating technology that is applied in the operating room, in a procedure that takes less than 10 min, and can incorporate any desired antibiotic. Our coating technology uses an in situ coupling reaction of branched poly(ethylene glycol) and poly(allyl mercaptan) (PEG-PAM) polymers to generate an amphiphilic polymeric coating. We show in vivo efficacy in preventing implant infection in both post-arthroplasty infection and post-spinal surgery infection mouse models. Our technology displays efficacy with or without systemic antibiotics, the standard of care. Our coating technology is applied in a clinically relevant time frame, does not require modification of implant manufacturing process, and does not change the implant shelf life.

Alternating magnetic fields and antibiotics eradicate biofilm on metal in a synergistic fashion.

Hundreds of thousands of human implant procedures require surgical revision each year due to infection. Infections are difficult to treat with conventional antibiotics due to the formation of biofilm on the implant surface. We have developed a noninvasive method to eliminate biofilm on metal implants using heat generated by intermittent alternating magnetic fields (iAMF). Here, we demonstrate that heat and antibiotics are synergistic in biofilm elimination. For Pseudomonas aeruginosa biofilm, bacterial burden was reduced >3 log with iAMF and ciprofloxacin after 24 h compared with either treatment alone (p < 0.0001). This effect was not limited by pathogen or antibiotic as similar biofilm reductions were seen with iAMF and either linezolid or ceftriaxone in Staphylococcus aureus. iAMF and antibiotic efficacy was seen across various iAMF settings, including different iAMF target temperatures, dose durations, and dosing intervals. Initial mechanistic studies revealed membrane disruption as one factor important for AMF enhanced antibacterial activity in the biofilm setting. This study demonstrates the potential of utilizing a noninvasive approach to reduce biofilm off of metallic implants.

A Rat Model of Orthopedic Implant-Associated Infection for Identification of Staphylococcal Biofilm Proteins.

Secreted bacterial proteins are difficult to identify directly from an infection site due to a limited amount of bacteria and presence of a large quantity of host proteins. Here we describe a rat model of orthopedic implant that allows us to harvest bacterial biofilm materials sufficient for identification of bacterial proteins in the biofilm matrix by liquid chromatography-tandem MS (GeLC-MS/MS) analysis.

Diclofenac Resensitizes Methicillin-Resistant Staphylococcus aureus to ß-Lactams and Prevents Implant Infections.

Implant infections caused by methicillin-resistant Staphylococcus aureus (MRSA) can cause major complications during the perioperative period. Diclofenac, one of the most widely used nonsteroidal anti-inflammatory drugs, is often used to relieve pain and inflammation. In this study, it is found that high-dose diclofenac can inhibit the growth of MRSA, and does not easily induce drug-resistant mutations after continuous passage. However, low-doses diclofenac can resensitize bacteria to ß-lactams, which help to circumvent drug resistance and improve the antibacterial efficacy of conventional antibiotics. Further, low-dose diclofenac in combination with ß-lactams inhibit MRSA associated biofilm formation in implants. Transcriptomic and proteomic analyses indicate that diclofenac can reduce the expression of genes and proteins associated with ß-lactam resistance: mecA, mecR, and blaZ; peptidoglycan biosynthesis: murA, murC, femA, and femB; and biofilm formation: altE and fnbP. Murine implant infection models indicate that diclofenac combined with ß-lactams, can substantially alleviate MRSA infections in vivo. In addition, it is investigated that low dose diclofenac can inhibit MRSA antibiotic resistance via the mecA/blaZ pathway and related biofilms in implants. The synergistic effect of diclofenac and ß-lactams might have promising applications for preventing perioperative infection, considering its multitarget effects against MRSA.

Establishment of a localized acute implant-associated Staphylococcus aureus bone infection model in sheep.

Orthopedic implant-associated bacterial infections with Staphylococcus aureus constitute a major clinical problem, and large pre-clinical animal models remain scarce. The aim of this study was to establish a standardized method of a localized, acute S. aureus bone infection in the presence of complex implanted devices in a sheep model. Four sheep underwent surgery receiving a complex implanted metallic device with a component stabilizing a bone defect created in the left tibial metaphysis, and an attached component placed in adjacent soft tissue. The bone defect was inoculated with S. aureus strain ATCC25293 (1 × 104 CFU). Twenty one days later, the surgery site was macroscopically evaluated, tissue samples and implants harvested for bacterial cell count quantification and tissue samples histologically analyzed. The animals exhibited clinical signs of localized infection (e.g. swelling, lameness, pain) but did not develop symptoms of sepsis. After euthanasia, macroscopic assessment revealed a localized bone and soft tissue infection at the surgery site. Histologically, an acute inflammation with neutrophils but also signs of bone destruction with necrosis was noted. An ovine model of a localized, acute S. aureus bone infection with complex implants was successfully established and could be used to test novel treatments against orthopedic implant-associated infections.

High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity.

Peptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

Bioactive Functional Nanolayers of Chitosan-Lysine Surfactant with Single- and Mixed-Protein-Repellent and Antibiofilm Properties for Medical Implants.

Medical implant-associated infections resulting from biofilm formation triggered by unspecific protein adsorption are the prevailing cause of implant failure. However, implant surfaces rendered with multifunctional bioactive nanocoatings offer a promising alternative to prevent the initial attachment of bacteria and effectively interrupt biofilm formation. The need to research and develop novel and stable bioactive nanocoatings for medical implants and a comprehensive understanding of their properties in contact with the complex biological environment are crucial. In this study, we developed an aqueous stable and crosslinker-free polyelectrolyte-surfactant complex (PESC) composed of a renewable cationic polysaccharide, chitosan, a lysine-based anionic surfactant (77KS), and an amphoteric antibiotic, amoxicillin, which is widely used to treat a number of infections caused by bacteria. We successfully introduced the PESC as bioactive functional nanolayers on the "model" and "real" polydimethylsiloxane (PDMS) surfaces under dynamic and ambient conditions. Besides their high stability and improved wettability, these uniformly deposited nanolayers (thickness: 44-61 nm) with mixed charges exhibited strong repulsion toward three model blood proteins (serum albumin, fibrinogen, and γ-globulin) and their competitive interactions in the mixture in real-time, as demonstrated using a quartz crystal microbalance with dissipation (QCM-D). The functional nanolayers with a maximum negative zeta potential (ζ: -19 to -30 mV at pH 7.4), water content (1628-1810 ng cm-2), and hydration (low viscosity and elastic shear modulus) correlated with the mass, conformation, and interaction nature of proteins. In vitro antimicrobial activity testing under dynamic conditions showed that the charged nanolayers actively inhibited the growth of both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria compared to unmodified PDMS. Given the ease of fabrication of multifunctional and charged biobased coatings with simultaneous protein-repellent and antimicrobial activities, the limitations of individual approaches could be overcome leading to a better and advanced design of various medical devices (e.g., catheters, prosthetics, and stents).

Microbiological diagnosis of polymicrobial periprosthetic joint infection revealed superiority of investigated tissue samples compared to sonicate fluid generated from the implant surface.

OBJECTIVES: In the microbiological diagnosis of periprosthetic joint infection (PJI), there is much discussion about the methodology of obtaining proper specimens, the processing technique, and suitable culture media. This retrospective study was conducted to analyse the accuracy of our culture techniques. METHODS: Tissue samples and components from 258 patients after revision arthroplasty of the hip, knee, and shoulder were investigated, and the results of tissue cultures (TC) were compared to those of sonicate fluid cultures (SFC). Furthermore, an evaluation was performed of the influence of different culture media on the detection rate. RESULTS: PJI was confirmed in 186 patients. The overall sensitivity of TC was no different to that of SFC (91.3% vs 90.8%, P = 1). In 153 cases (82.3%), TC and SFC showed concordant positive results. Results were discordant in 33 cases (17.7%). When differentiated according to the type of infection, TC showed significantly better results than SFC in detecting polymicrobial infections (97.0% vs 67.0%, P = 0.004). There were also significant differences between the culture media regarding the yield of microorganisms. CONCLUSIONS: TC was more effective in detecting co-infections. The best results were obtained using both TC and SFC. The choice of culture media has a significant influence on the quality of results.

Antibacterial Activity in Iodine-coated Implants Under Conditions of Iodine Loss: Study in a Rat Model Plus In Vitro Analysis.

BACKGROUND: We developed iodine-coated titanium implants to suppress microbial activity and prevent periprosthetic joint infection (PJI); their efficacy was demonstrated in animal and in vitro models. The iodine content in iodine-coated implants naturally decreases in vivo. However, to our knowledge, the effect of reduced iodine content on the implant's antimicrobial activity has not been evaluated to date. QUESTIONS/PURPOSES: (1) How much does the iodine content on the implant surface decrease after 4 and 8 weeks in vivo in a rat model? (2) What effect does the reduced iodine content have on the antimicrobial effect of the implant against multiple bacteria in an in vitro model? METHODS: This experiment was performed in two parts: an in vivo experiment to determine attenuation of iodine levels over time in rats, and an in vitro experiment in which we sought to assess whether the reduced iodine content observed in the in vivo experiment was still sufficient to deliver antimicrobial activity against common pathogens seen in PJI. For the in vivo experiment, three types of titanium alloy washers were implanted in rats: untreated (Ti), surface-anodized to produce an oxide film (Ti-O), and with an iodine layer on the oxidation film (Ti-I). The attenuation of iodine levels in rats was measured over time using inductively coupled plasma-mass spectrometry. Herein, only the Ti-I washer was used, with five implanted in each rat that were removed after 4 or 8 weeks. For the 4- and 8-week models, two rats and 15 washers were used. For the in vitro study, to determine the antibacterial effect, three types of washers (Ti, Ti-O, and Ti-I) (nine washers in total) were implanted in each rat. Then, the washers were removed and the antibacterial effect of each washer was examined on multiple bacterial species using the spread plate method and fluorescence microscopy. For the spread plate method, six rats were used, and five rats were used for the observation using fluorescence microscopy; further, 4- and 8-week models were made for each method. Thus, a total of 22 rats and 198 washers were used. Live and dead bacteria in the biofilm were stained, and the biofilm coverage percentage for quantitative analysis was determined using fluorescence microscopy in a nonblinded manner. Ti-I was used as the experimental group, and Ti and Ti-O were used as control groups. The total number of rats and washers used throughout this study was 24 and 213, respectively. RESULTS: Iodine content in rats implanted with Ti-I samples decreased to 72% and 65% after the in vivo period of 4 and 8 weeks, respectively (p = 0.001 and p < 0.001, respectively). In the in vitro experiment, the Ti-I implants demonstrated a stronger antimicrobial activity than Ti and Ti-O implants in the 4- and 8-week models. Both the median number of bacterial colonies and the median biofilm coverage percentage with live bacteria on Ti-I were lower than those on Ti or Ti-O implants for each bacterial species in the 4- and 8-week models. There was no difference in the median biofilm coverage percentage of dead bacteria. In the 8-week model, the antibacterial activity using the spread plate method had median (interquartile range) numbers of bacteria on the Ti, Ti-O, and Ti-I implants of 112 (104 to 165) × 105, 147 (111 to 162) × 105, and 55 (37 to 67) × 105 of methicillin-sensitive Staphylococcus aureus (Ti-I versus Ti, p = 0.026; Ti-I versus Ti-O, p = 0.009); 71 (39 to 111) × 105, 50 (44 to 62) × 105, and 26 (9 to 31)× 105 CFU of methicillin-resistant S. aureus (Ti-I versus Ti, p = 0.026; Ti-I versus Ti-O, p = 0.034); and 77 (74 to 83) × 106, 111 (95 to 117) × 106, and 30 (21 to 45) × 106 CFU of Pseudomonas aeruginosa (Ti-I versus Ti, p = 0.004; Ti-I versus Ti-O, p = 0.009). Despite the decrease in the iodine content of Ti-I after 8 weeks, it demonstrated better antibacterial activity against all tested bacteria than the Ti and Ti-O implants. CONCLUSION: Iodine-coated implants retained their iodine content and antibacterial activity against methicillin-sensitive S. aureus, methicillin-resistant S. aureus, and P. aeruginosa for 8 weeks in vivo in rats. To evaluate the longer-lasting antibacterial efficacy, further research using larger infected animal PJI models with implants in the joints of both males and females is desirable. CLINICAL RELEVANCE: Iodine-coated titanium implants displayed an antibacterial activity for 8 weeks in rats in vivo. Although the findings in a rat model do not guarantee efficacy in humans, they represent an important step toward clinical application.