Imidazoles and Quaternary Ammonium Compounds as Effective Therapies against (Multidrug-Resistant) Bacterial Wound Infections.

BACKGROUND/OBJECTIVES: The rise and spread of antimicrobial resistance complicates the treatment of bacterial wound pathogens, further increasing the need for newer, effective therapies. Azoles such as miconazole have shown promise as antibacterial compounds; however, they are currently only used as antifungals. Previous research has shown that combining azoles with quaternary ammonium compounds yields synergistic activity against fungal pathogens, but the effect on bacterial pathogens has not been studied yet. METHODS: In this study, the focus was on finding active synergistic combinations of imidazoles and quaternary ammonium compounds against (multidrug-resistant) bacterial pathogens through checkerboard assays. Experimental evolution in liquid culture was used to evaluate the possible emergence of resistance against the most active synergistic combination. RESULTS: Several promising synergistic combinations were identified against an array of Gram-positive pathogens: miconazole/domiphen bromide, ketoconazole/domiphen bromide, clotrimazole/domiphen bromide, fluconazole/domiphen bromide and miconazole/benzalkonium chloride. Especially, miconazole with domiphen bromide exhibits potential, as it has activity at a low concentration against a broad range of pathogens and shows an absence of strong resistance development over 11 cycles of evolution. CONCLUSIONS: This study provides valuable insight into the possible combinations of imidazoles and quaternary ammonium compounds that could be repurposed for (topical) wound treatment. Miconazole with domiphen bromide shows the highest application potential as a possible future wound therapy. However, further research is needed into the mode of action of these compounds and their efficacy and toxicity in vivo.

From sugars to aliphatic amines: as sweet as it sounds? Production and applications of bio-based aliphatic amines.

Aliphatic amines encompass a diverse group of amines that include alkylamines, alkyl polyamines, alkanolamines and aliphatic heterocyclic amines. Their structural diversity and distinctive characteristics position them as indispensable components across multiple industrial domains, ranging from chemistry and technology to agriculture and medicine. Currently, the industrial production of aliphatic amines is facing pressing sustainability, health and safety issues which all arise due to the strong dependency on fossil feedstock. Interestingly, these issues can be fundamentally resolved by shifting toward biomass as the feedstock. In this regard, cellulose and hemicellulose, the carbohydrate fraction of lignocellulose, emerge as promising feedstock for the production of aliphatic amines as they are available in abundance, safe to use and their aliphatic backbone is susceptible to chemical transformations. Consequently, the academic interest in bio-based aliphatic amines via the catalytic reductive amination of (hemi)cellulose-derived substrates has systematically increased over the past years. From an industrial perspective, however, the production of bio-based aliphatic amines will only be the middle part of a larger, ideally circular, value chain. This value chain additionally includes, as the first part, the refinery of the biomass feedstock to suitable substrates and, as the final part, the implementation of these aliphatic amines in various applications. Each part of the bio-based aliphatic amine value chain will be covered in this Review. Applying a holistic perspective enables one to acknowledge the requirements and limitations of each part and to efficiently spot and potentially bridge knowledge gaps between the different parts.

Collateral sensitivity counteracts the evolution of antifungal drug resistance in Candida auris.

Antifungal drug resistance represents a serious global health threat, necessitating new treatment strategies. Here we investigated collateral sensitivity (CS), in which resistance to one drug increases sensitivity to another, and cross-resistance (XR), in which one drug resistance mechanism reduces susceptibility to multiple drugs, since CS and XR dynamics can guide treatment design to impede resistance development, but have not been systematically explored in pathogenic fungi. We used experimental evolution and mathematical modelling of Candida auris population dynamics during cyclic and combined drug exposures and found that especially CS-based drug cycling can effectively prevent the emergence of drug resistance. In addition, we found that a CS-based treatment switch can actively select against or eradicate resistant sub-populations, highlighting the potential to consider CS in therapeutic decision-making upon resistance detection. Furthermore, we show that some CS trends are robust among different strains and resistance mechanisms. Overall, these findings provide a promising direction for improved antifungal treatment approaches.

Salmonella biofilm formation diminishes bacterial proliferation in the C. elegans intestine.

Non-typhoidal Salmonella serovars are a significant global cause of foodborne infections, owing their transmission success to the formation of biofilms. While the role of these biofilms in Salmonella's persistence outside the host is well understood, their significance during infection remains elusive. In this study, we investigated the impact of Salmonella biofilm formation on host colonization and virulence using the nematode model Caenorhabditis elegans. This infection model enables us to isolate the effect of biofilm formation on gut colonization and proliferation, as no gut microbiome is present and Salmonella cannot invade the intestinal tissue of the nematode. We show that a biofilm-deficient ΔcsgD mutant enhances gut proliferation compared to the wild-type strain, while the pathogen's virulence, the host's immune signaling pathways, and host survival remain unaffected. Hence, our work suggests that biofilm formation does not significantly contribute to Salmonella infection in C. elegans. However, complementary assays in higher-order in vivo models are required to further characterize the role of biofilm formation during infection and to take into account the impact of biofilm formation on competition with gut microbiome and epithelial invasion.

Selective pressures for public antibiotic resistance.

The rapid increase of antibiotic-resistant pathogens is severely limiting our current treatment possibilities. An important subset of the resistance mechanisms conferring antibiotic resistance have public effects, allowing otherwise susceptible bacteria to also survive antibiotic treatment. As susceptible bacteria can survive treatment without bearing the metabolic cost of producing the resistance mechanism, there is potential to increase their relative frequency in the population and, as such, select against resistant bacteria. Multiple studies showed that this altered selection for resistance is dependent on various environmental and treatment parameters. In this review, we provide a comprehensive overview of their most important findings and describe the main factors impacting the selection for resistance. In-depth understanding of the driving forces behind selection can aid in the design and implementation of alternative treatments which limit the risk of resistance development.

Fabrication of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)/ZnO Nanocomposite Films for Active Packaging Applications: Impact of ZnO Type on Structure-Property Dynamics.

Bio-based and biodegradable polyhydroxyalkanoates (PHAs) have great potential as sustainable packaging materials. The incorporation of zinc oxide nanoparticles (ZnO NPs) could further improve their functional properties by providing enhanced barrier and antimicrobial properties, although current literature lacks details on how the characteristics of ZnO influence the structure-property relationships in PHA/ZnO nanocomposites. Therefore, commercial ZnO NPs with different morphologies (rod-like, spherical) and silane surface modification are incorporated into poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) via extrusion and compression molding. All ZnO NPs are homogeneously distributed in the PHBHHx matrix at 1, 3 and 5 wt.%, but finer dispersion is achieved with modified ZnO. No chemical interactions between ZnO and PHBHHx are observed due to a lack of hydroxyl groups on ZnO. The fabricated nanocomposite films retain the flexible properties of PHBHHx with minimal impact of ZnO NPs on crystallization kinetics and the degree of crystallinity (53 to 56%). The opacity gradually increases with ZnO loading, while remaining translucent up to 5 wt.% ZnO and providing an effective UV barrier. Improved oxygen barrier and antibacterial effects against S. aureus are dependent on the intrinsic characteristics of ZnO rather than its morphology. We conclude that PHBHHx retains its favorable processing properties while producing nanocomposite films that are suitable as flexible active packaging materials.

Competition quenching strategies reduce antibiotic tolerance in polymicrobial biofilms.

Bacteria typically live in dense communities where they are surrounded by other species and compete for a limited amount of resources. These competitive interactions can induce defensive responses that also protect against antimicrobials, potentially complicating the antimicrobial treatment of pathogens residing in polymicrobial consortia. Therefore, we evaluate the potential of alternative antivirulence strategies that quench this response to competition. We test three competition quenching approaches: (i) interference with the attack mechanism of surrounding competitors, (ii) inhibition of the stress response systems that detect competition, and (iii) reduction of the overall level of competition in the community by lowering the population density. We show that either strategy can prevent the induction of antimicrobial tolerance of Salmonella Typhimurium in response to competitors. Competition quenching strategies can thus reduce tolerance of pathogens residing in polymicrobial communities and could contribute to the improved eradication of these pathogens via traditional methods.

Dynamic social interactions and keystone species shape the diversity and stability of mixed-species biofilms - an example from dairy isolates.

Identifying interspecies interactions in mixed-species biofilms is a key challenge in microbial ecology and is of paramount importance given that interactions govern community functionality and stability. We previously reported a bacterial four-species biofilm model comprising Stenotrophomonas rhizophila, Bacillus licheniformis, Microbacterium lacticum, and Calidifontibacter indicus that were isolated from the surface of a dairy pasteuriser after cleaning and disinfection. These bacteria produced 3.13-fold more biofilm mass compared to the sum of biofilm masses in monoculture. The present study confirms that the observed community synergy results from dynamic social interactions, encompassing commensalism, exploitation, and amensalism. M. lacticum appears to be the keystone species as it increased the growth of all other species that led to the synergy in biofilm mass. Interactions among the other three species (in the absence of M. lacticum) also contributed towards the synergy in biofilm mass. Biofilm inducing effects of bacterial cell-free-supernatants were observed for some combinations, revealing the nature of the observed synergy, and addition of additional species to dual-species combinations confirmed the presence of higher-order interactions within the biofilm community. Our findings provide understanding of bacterial interactions in biofilms which can be used as an interaction-mediated approach for cultivating, engineering, and designing synthetic bacterial communities.

Competitive interactions facilitate resistance development against antimicrobials.

While the evolution of antimicrobial resistance is well studied in free-living bacteria, information on resistance development in dense and diverse biofilm communities is largely lacking. Therefore, we explored how the social interactions in a duo-species biofilm composed of the brewery isolates Pseudomonas rhodesiae and Raoultella terrigena influence the adaptation to the broad-spectrum antimicrobial sulfathiazole. Previously, we showed that the competition between these brewery isolates enhances the antimicrobial tolerance of P. rhodesiae. Here, we found that this enhanced tolerance in duo-species biofilms is associated with a strongly increased antimicrobial resistance development in P. rhodesiae. Whereas P. rhodesiae was not able to evolve resistance against sulfathiazole in monospecies conditions, it rapidly evolved resistance in the majority of the duo-species communities. Although the initial presence of R. terrigena was thus required for P. rhodesiae to acquire resistance, the resistance mechanisms did not depend on the presence of R. terrigena. Whole genome sequencing of resistant P. rhodesiae clones showed no clear mutational hot spots. This indicates that the acquired resistance phenotype depends on complex interactions between low-frequency mutations in the genetic background of the strains. We hypothesize that the increased tolerance in duo-species conditions promotes resistance by enhancing the selection of partially resistant mutants and opening up novel evolutionary trajectories that enable such genetic interactions. This hypothesis is reinforced by experimentally excluding potential effects of increased initial population size, enhanced mutation rate, and horizontal gene transfer. Altogether, our observations suggest that the community mode of life and the social interactions therein strongly affect the accessible evolutionary pathways toward antimicrobial resistance.IMPORTANCEAntimicrobial resistance is one of the most studied bacterial properties due to its enormous clinical and industrial relevance; however, most research focuses on resistance development of a single species in isolation. In the present study, we showed that resistance evolution of brewery isolates can differ greatly between single- and mixed-species conditions. Specifically, we observed that the development of antimicrobial resistance in certain species can be significantly enhanced in co-culture as compared to the single-species conditions. Overall, the current study emphasizes the need of considering the within bacterial interactions in microbial communities when evaluating antimicrobial treatments and resistance evolution.

Anomalous diffusion of nanoparticles in the spatially heterogeneous biofilm environment.

Biofilms contain extracellular polymeric substances (EPS) that provide structural support and restrict penetration of antimicrobial treatment. To overcome limited penetration, functionalized nanoparticles (NPs) have been suggested as carriers for antimicrobial delivery. Using microscopy, we evaluate the diffusion of nanoparticles in function of the structure of Salmonella biofilms. We observe anomalous diffusion and heterogeneous mobility of NPs resulting in distinct NPs distribution that depended on biofilm structure. Through Brownian dynamics modeling with spatially varying viscosity around bacteria, we demonstrated that spatial gradients in diffusivity generate viscous sinks that trap NPs near bacteria. This model replicates the characteristic diffusion signature and vertical distribution of NPs in the biofilm. From a treatment perspective, our work indicates that both biofilm structure and the level of EPS can impact NP drug delivery, where low levels of EPS might benefit delivery by immobilizing NPs closer to bacteria and higher levels hamper delivery due to shielding effects.

Antibiotic Cycling Affects Resistance Evolution Independently of Collateral Sensitivity.

Antibiotic cycling has been proposed as a promising approach to slow down resistance evolution against currently employed antibiotics. It remains unclear, however, to which extent the decreased resistance evolution is the result of collateral sensitivity, an evolutionary trade-off where resistance to one antibiotic enhances the sensitivity to the second, or due to additional effects of the evolved genetic background, in which mutations accumulated during treatment with a first antibiotic alter the emergence and spread of resistance against a second antibiotic via other mechanisms. Also, the influence of antibiotic exposure patterns on the outcome of drug cycling is unknown. Here, we systematically assessed the effects of the evolved genetic background by focusing on the first switch between two antibiotics against Salmonella Typhimurium, with cefotaxime fixed as the first and a broad variety of other drugs as the second antibiotic. By normalizing the antibiotic concentrations to eliminate the effects of collateral sensitivity, we demonstrated a clear contribution of the evolved genetic background beyond collateral sensitivity, which either enhanced or reduced the adaptive potential depending on the specific drug combination. We further demonstrated that the gradient strength with which cefotaxime was applied affected both cefotaxime resistance evolution and adaptation to second antibiotics, an effect that was associated with higher levels of clonal interference and reduced cost of resistance in populations evolved under weaker cefotaxime gradients. Overall, our work highlights that drug cycling can affect resistance evolution independently of collateral sensitivity, in a manner that is contingent on the antibiotic exposure pattern.

Nutrient Availability and Biofilm Polysaccharide Shape the Bacillaene-Dependent Antagonism of Bacillus subtilis against Salmonella Typhimurium.

Salmonella enterica is one of the most common foodborne pathogens and, due to the spread of antibiotic resistance, new antimicrobial strategies are urgently needed to control it. In this study, we explored the probiotic potential of Bacillus subtilis PS-216 and elucidated the mechanisms that underlie the interactions between this soil isolate and the model pathogenic strain S. Typhimurium SL1344. The results reveal that B. subtilis PS-216 inhibits the growth and biofilm formation of S. Typhimurium through the production of the pks cluster-dependent polyketide bacillaene. The presence of S. Typhimurium enhanced the activity of the PpksC promoter that controls bacillaene production, suggesting that B. subtilis senses and responds to Salmonella. The level of Salmonella inhibition, overall PpksC activity, and PpksC induction by Salmonella were all higher in nutrient-rich conditions than in nutrient-depleted conditions. Although eliminating the extracellular polysaccharide production of B. subtilis via deletion of the epsA-O operon had no significant effect on inhibitory activity against Salmonella in nutrient-rich conditions, this deletion mutant showed an enhanced antagonism against Salmonella in nutrient-depleted conditions, revealing an intricate relationship between exopolysaccharide production, nutrient availability, and bacillaene synthesis. Overall, this work provides evidence on the regulatory role of nutrient availability, sensing of the competitor, and EpsA-O polysaccharide in the social outcome of bacillaene-dependent competition between B. subtilis and S. Typhimurium. IMPORTANCE Probiotic bacteria represent an alternative for controlling foodborne disease caused by Salmonella enterica, which constitutes a serious concern during food production due to its antibiotic resistance and resilience to environmental stress. Bacillus subtilis is gaining popularity as a probiotic, but its behavior in biofilms with pathogens such as Salmonella remains to be elucidated. Here, we show that the antagonism of B. subtilis is mediated by the polyketide bacillaene and that the production of bacillaene is a highly dynamic trait which depends on environmental factors such as nutrient availability and the presence of competitors. Moreover, the production of extracellular polysaccharides by B. subtilis further alters the influence of these factors. Hence, this work highlights the inhibitory effect of B. subtilis, which is condition-dependent, and the importance of evaluating probiotic strains under conditions relevant to the intended use.

Origanum vulgare ethanolic extracts as a promising source of compounds with antimicrobial, anti-biofilm, and anti-virulence activity against dental plaque bacteria.

Dental caries and periodontal diseases remain a challenge for oral health, especially given the lack of effective and safe treatment options that are currently available. Against the backdrop of an ongoing antimicrobial resistance crisis, a renewed interest in traditional medicinal plants as a potential source of new bioactive compounds has surfaced. In this context, we systematically screened the antimicrobial and anti-biofilm activities of both ethanolic and aqueous extracts of nine Algerian medicinal plants (Artemisia herba alba, Centaurium erythraea, Juglans regia, Laurus nobilis, Matricaria recutita, Mentha pulegium, Mentha piperita, Origanum vulgare and Taraxacum officinale). To evaluate the activity spectrum of the extracts, the screening was carried out against an extensive collection of Streptococcus, Enterococcus and Lacticaseibacillus isolates recovered from dental plaques of Algerian patients. Broad-spectrum antimicrobial and anti-biofilm properties were observed, especially among ethanolic extracts, which marks them as a promising source for bioactive compounds to control oral biofilms. The ethanolic extract of O. vulgare, which showed the most promising effects in the initial screening, was further characterized. We first verified the biocompatibility of this extract using human oral keratinocytes and selected a range of non-cytotoxic concentrations (0.195-0.781 mg/ml) to further validate its anti-biofilm and anti-virulence potential. At these concentrations, the extract not only prevented biofilm formation (10.04 ± 0.75-87.91 ± 9.08% of reduction) of most dental plaque isolates on a polystyrene surface, but also significantly reduced their adherence to hydroxyapatite (34.58 ± 9.09-62.77 ± 0.95%). Moreover, the extract showed curative potential against mature biofilms grown under conditions mimicking the oral niche. In addition to its anti-biofilm properties, we observed an inhibition of glucosyltransferase activity, a reduction in acidogenesis and a downregulation in the expression of multiple virulence-associated genes for extract-treated samples. Since anti-virulence properties are more robust to the development of resistance, they provide an attractive complementation to the antimicrobial activities of the extract. Thymol was identified as an important active compound of the extract using GC-MS analysis, but synergy with other compounds was also detected, suggesting a potential advantage of using the whole extract over purified thymol. Further research into the bioactive compounds of the O. vulgare ethanolic extract could yield novel products to fight dental caries.

Permissive aggregative group formation favors coexistence between cooperators and defectors in yeast.

In Saccharomyces cerevisiae, the FLO1 gene encodes flocculins that lead to formation of multicellular flocs, that offer protection to the constituent cells. Flo1p was found to preferentially bind to fellow cooperators compared to defectors lacking FLO1 expression, enriching cooperators within the flocs. Given this dual function in cooperation and kin recognition, FLO1 has been termed a "green beard gene". Because of the heterophilic nature of the Flo1p bond however, we hypothesize that kin recognition is permissive and depends on the relative stability of the FLO1+/flo1- versus FLO1+/FLO1+ detachment force F. We combine single-cell measurements of adhesion, individual cell-based simulations of cluster formation, and in vitro flocculation to study the impact of relative bond stability on the evolutionary stability of cooperation. We identify a trade-off between both aspects of the green beard mechanism, with reduced relative bond stability leading to increased kin recognition at the expense of cooperative benefits. We show that the fitness of FLO1 cooperators decreases as their frequency in the population increases, arising from the observed permissive character (F+- = 0.5 F++) of the Flo1p bond. Considering the costs associated with FLO1 expression, this asymmetric selection often results in a stable coexistence between cooperators and defectors.

Evolution-proof inhibitors of public good cooperation: a screening strategy inspired by social evolution theory.

Interference with public good cooperation provides a promising novel antimicrobial strategy since social evolution theory predicts that resistant mutants will be counter-selected if they share the public benefits of their resistance with sensitive cells in the population. Although this hypothesis is supported by a limited number of pioneering studies, an extensive body of more fundamental work on social evolution describes a multitude of mechanisms and conditions that can stabilize public behaviour, thus potentially allowing resistant mutants to thrive. In this paper we theorize on how these different mechanisms can influence the evolution of resistance against public good inhibitors. Based hereon, we propose an innovative 5-step screening strategy to identify novel evolution-proof public good inhibitors, which involves a systematic evaluation of the exploitability of public goods under the most relevant experimental conditions, as well as a careful assessment of the most optimal way to interfere with their action. Overall, this opinion paper is aimed to contribute to long-term solutions to fight bacterial infections.

Microbial Interspecies Interactions and Their Impact on the Emergence and Spread of Antimicrobial Resistance.

Bacteria are social organisms that commonly live in dense communities surrounded by a multitude of other species. The competitive and cooperative interactions between these species not only shape the bacterial communities but also influence their susceptibility to antimicrobials. While several studies have shown that mixed-species communities are more tolerant toward antimicrobials than their monospecies counterparts, only limited empirical data are currently available on how interspecies interactions influence resistance development. We here propose a theoretic framework outlining the potential impact of interspecies social behavior on different aspects of resistance development. We identify factors by which interspecies interactions might influence resistance evolution and distinguish between their effect on (a) the emergence of a resistant mutant and (b) the spread of this resistance throughout the population. Our analysis indicates that considering the social life of bacteria is imperative to the rational design of more effective antibiotic treatment strategies with a minimal hazard for resistance development.

Effect of chemical modifications of tannins on their antimicrobial and antibiofilm effect against Gram-negative and Gram-positive bacteria.

Background: Tannins have demonstrated antibacterial and antibiofilm activity, but there are still unknown aspects on how the chemical properties of tannins affect their biological properties. We are interested in understanding how to modulate the antibiofilm activity of tannins and in delineating the relationship between chemical determinants and antibiofilm activity. Materials and methods: The effect of five different naturally acquired tannins and their chemical derivatives on biofilm formation and planktonic growth of Salmonella Typhimurium, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus was determined in the Calgary biofilm device. Results: Most of the unmodified tannins exhibited specific antibiofilm activity against the assayed bacteria. The chemical modifications were found to alter the antibiofilm activity level and spectrum of the tannins. A positive charge introduced by derivatization with higher amounts of ammonium groups shifted the anti-biofilm spectrum toward Gram-negative bacteria, and derivatization with lower amounts of ammonium groups and acidifying derivatization shifted the spectrum toward Gram-positive bacteria. Furthermore, the quantity of phenolic OH-groups per molecule was found to have a weak impact on the anti-biofilm activity of the tannins. Conclusion: We were able to modulate the antibiofilm activity of several tannins by specific chemical modifications, providing a first approach for fine tuning of their activity and antibacterial spectrum.

Pre-clinical In Vitro Models of Vascular Graft Coating in the Prevention of Vascular Graft Infection: A Systematic Review.

OBJECTIVE: Vascular graft infection (VGI) is a feared complication. Prevention is of the utmost importance and vascular graft coatings (VGCs) could offer a potential to do this, with in vitro research a first crucial step. The aim of this study was to summarise key features of in vitro models investigating coating strategies to prevent VGI in order to provide guidance for the setup of future translational research. DATA SOURCES: A comprehensive search was performed in MEDLINE, Embase, and Web of Science. METHODS: A systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. For each database, a specific search strategy was developed. Quality was assessed with the Toxicological data Reliability Assessment Tool (ToxRTool). In vitro models using a VGC and inoculation of the graft with a pathogen were included. The type of graft, coating, and pathogen were summarised. The outcome assessment in each study was evaluated. RESULTS: In total, 4 667 studies were identified, of which 45 papers met the inclusion criteria. The majority used polyester grafts (68.2%). Thirty-one studies (68.9%) included antibiotics, and nine studies (20%) used a commercial silver graft in their protocol. New antibacterial strategies (e.g., proteolytic enzymes) were investigated. A variety of testing methods was found and focused mainly on bacterial adherence, coating adherence and dilution, biofilm formation, and cytotoxicity. Ninety-three per cent of the studies (n = 41) were considered unreliable. CONCLUSION: Polyester is the preferred type of graft to coat on. The majority of coating studies are based on antibiotics; however, new coating strategies (e.g., antibiofilm coating) are coming. Many in vitro setups are available. In vitro studies have great potential, they can limit the use, but cannot replace in vivo studies completely. This paper can be used as a guidance document for future in vitro research.

An Improved 2-Aminoimidazole Based Anti-Biofilm Coating for Orthopedic Implants: Activity, Stability, and in vivo Biocompatibility.

Orthopedic device-related infections remain a serious challenge to treat. Central to these infections are bacterial biofilms that form on the orthopedic implant itself. These biofilms shield the bacteria from the host immune system and most common antibiotic drugs, which renders them essentially antibiotic-tolerant. There is an urgent clinical need for novel strategies to prevent these serious infections that do not involve conventional antibiotics. Recently, a novel antibiofilm coating for titanium surfaces was developed based on 5-(4-bromophenyl)-N-cyclopentyl-1-octyl-1H-imidazol-2-amine as an active biofilm inhibitor. In the current study we present an optimized coating protocol that allowed for a 5-fold higher load of this active compound, whilst shortening the manufacturing process. When applied to titanium disks, the newly optimized coating was resilient to the most common sterilization procedures and it induced a 1 log reduction in biofilm cells of a clinical Staphylococcus aureus isolate (JAR060131) in vitro, without affecting the planktonic phase. Moreover, the antibiofilm effect of the coating in combination with the antibiotic cefuroxime was higher than cefuroxime treatment alone. Furthermore, the coating was successfully applied to a human-scale fracture fixation device resulting in a loading that was comparable to the titanium disk model. Finally, an in vivo biocompatibility and healing study in a rabbit osteotomy model indicated that these coated implants did not negatively affect fracture healing or osteointegration. These findings put our technology one step closer to clinical trials, confirming its potential in fighting orthopedic infections without compromising healing.