Effect of Bacterial Biofilms on Topical Antimicrobial Therapy

Chronic non-healing wounds represent a major global health burden, affecting millions of patients and generating significant socioeconomic costs exceeding 98 billion USD annually in the United States alone (Khan, 2024). The persistence of these wounds is often associated with bacterial colonization and infection, especially when microorganisms organize into complex biofilm structures. Biofilms are now recognized as a fundamental barrier to wound healing and one of the main reasons for the failure of both systemic and topical antimicrobial therapies (Schneider, 2025). A biofilm is defined as a structured community of microorganisms enclosed in a self-produced extracellular polymeric substance (EPS) composed of polysaccharides, proteins, lipids, and nucleic acids (Kirketerp-Møller, 2011 and Fonseca, 2011). Biofilms are polymicrobial ecosystems, often involving Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus spp., and Candida spp., whose synergistic interactions further enhance pathogenicity and persistence (Chegini, 2025). It is estimated that more than 70% of hard-to-heal wounds contain biofilm-associated bacteria (Khan, 2024), and biofilm-associated infections display up to a 1,000-fold increase in antimicrobial resistance compared to planktonic bacteria (Chegini, 2025). The alarming rise in multidrug-resistant (MDR) pathogens, notably P. aeruginosa and S. aureus, underscores the need for localized strategies that circumvent systemic antibiotic resistance (Chegini, 2025). Topical antiseptics and antimicrobial dressings provide the advantage of achieving high local concentrations with minimal systemic toxicity, yet their efficacy is frequently limited by the biofilm barrier (Khan, 2024). Local therapy strategies include well-known agents like silver or Polyhexamethylene biguanide (Muslim 2025, Watson, 2024). Other emerging systems include biopolymer-based composite films that combine natural matrices such as hydroxyethyl cellulose with inorganic nanoparticles and essential oils. For instance, films embedding zinc oxide nanoparticles and mesoporous silica loaded with cinnamon essential oil have demonstrated synergistic antimicrobial and antibiofilm effects against S. aureus and E. coli (Motelica, 2024). Additionally, natural compounds such as asiatic acid and ursolic acid derived from Eucalyptus globulus have exhibited promising antibiofilm activity against Gram-positive and Gram-negative bacteria (Mezzasalma, 2025). Given the multifactorial nature of chronic wound infection, optimal therapy requires a multimodal approach that combines mechanical or enzymatic debridement, effective antibiofilm and antimicrobial topical agents, and management of systemic risk factors. Clinical evidence increasingly supports the concept of “wound hygiene,” involving regular cleansing, biofilm disruption, and re-application of topical antiseptics within a structured regimen (Khan, 2024). However, despite growing preclinical and early clinical data, further research is essential to standardize testing methods, establish optimal application protocols, and integrate these advanced topical therapies into evidence-based wound care algorithms (Schneider, 2025).

opical antimicrobials play an important role in complex wound care, especially in non-healing wounds, which we encounter with increasing frequency in our daily clinical practice. For this reason, and because we currently lack a standardized microbiological testing method that would inform us about the effectiveness of various topical antimicrobial agents in individual patients, we decided to conduct a laboratory study to verify the effect of local treatment on biofilm-forming bacteria. Multidrug-resistant bacterial strains were isolated from non-healing wounds of patients treated at the University Hospital Bratislava and used to create an appropriate bacterial collection for laboratory research. Clinically important wound pathogens (P. aeruginosa – two strains, S. aureus, E. coli, E. faecalis, and A. baumannii) were selected from the bacterial collection and tested for biofilm production according to Stepanović et al. (2007). These five strains—biofilm producers—were used to evaluate the efficacy of topical antimicrobials commonly available for wound treatment. This evaluation was performed in the modified in vitro wound model according to Hammond et al. (2011).

1. Biofilm formation testing according to Stepanović et al.

Triplicates of wells on a polystyrene 96-well microtiter plate were inoculated with 200 μL of bacterial suspension of each tested strain in LB (Luria–Bertani) broth with a density of 1×10⁶ CFU/mL (colony-forming units per milliliter). In parallel, three microtiter plates were inoculated and incubated at 37 °C for 4, 6, and 24 hours, respectively. After incubation, the medium was aspirated, and the wells were washed three times with PBS (phosphate-buffered saline) to remove non-adherent cells. Subsequently, the biofilm was fixed with methanol and stained with 0.5% crystal violet. After decanting the dye, gently rinsing the wells, and drying, the bound dye was eluted with 99% ethanol. The optical density, corresponding to the amount of biofilm formed, was measured spectrophotometrically at 570 nm (MRX Microplate Reader, DYNEX Technologies, USA). The intensity of biofilm formation was evaluated semi-quantitatively according to the criteria described by Stepanović et al. (2007).

2. Testing the antimicrobial efficacy of topical agents using an in vitro burn wound model according to Hammond et al. (2011)

Three monobacterial contaminated burn wound models were prepared:

• Model A: wound 4 hours after bacterial inoculation

• Model B: wound 6 hours after bacterial inoculation

• Model C: wound 24 hours after bacterial inoculation

On all three models, the semi-quantitative efficacy of 11 topical antimicrobial agents commonly used in burn surgery was evaluated in various pharmaceutical forms.

The results of our study confirm that the timing of topical antimicrobial application plays a crucial role in the effective suppression of bacterial growth in contaminated wounds. A clear pattern emerged across all experiments: the longer the bacterial growth was allowed to proceed before treatment, the weaker the antimicrobial effect observed. This finding correlates strongly with the dynamic process of biofilm maturation, which fundamentally alters microbial susceptibility to antimicrobial agents. In our biofilm formation assay, all strains were capable of producing biofilm, but the intensity distinctly increased over time, with P. aeruginosa and A. baumannii demonstrating strong biofilm formation already at 24 hours. This reflects clinical observations that these pathogens are among the most problematic in chronic wounds due to their ability to rapidly generate structured, protective biofilm communities (Khan, 2024; Schneider, 2025).

In the in vitro wound model, this phenomenon resulted in a marked decline in antimicrobial efficacy between Models A and C. While several agents—including Octenisept^®, Betadine^®, silver-based dressings, and silver sulfadiazine—showed excellent performance when applied 4–6 hours after inoculation, nearly all preparations lost activity in the 24-hour model, with results approaching those of sterile gauze application. A similar time-dependent loss of efficacy was reported by Roche et al., who demonstrated significantly reduced activity of silver-containing therapies when applied 24 hours after MRSA contamination compared with early (4 h) treatment (Roche, 2012). The convergence of our findings with published data further supports the interpretation that biofilm maturation fundamentally limits the capacity of topical antimicrobial agents to eradicate bacteria, even when these agents are known to be potent in planktonic conditions. This phenomenon aligns closely with current understanding of biofilm biology. Biofilm formation begins within minutes to hours after bacterial attachment, and as the extracellular polymeric substance (EPS) develops, diffusion barriers, altered metabolic states, and quorum sensing all contribute to dramatic increases in antimicrobial tolerance—up to 1000-fold compared with planktonic bacteria (Chegini, 2025). Findings from the available literature further demonstrate that once biofilm is established, topical antimicrobials rarely achieve complete eradication. For example, Mezzasalma et al. showed that although plant-derived compounds such as asiatic and ursolic acid exhibit notable antibiofilm effects, none were able to fully inhibit biofilm formation across all tested strains (Mezzasalma, 2025). Similarly, Thomas et al. observed that an N-chlorotaurine gel—despite stability and activity against S. aureus—was ineffective against P. aeruginosa biofilms in a chronic wound model (Thomas, 2025). These findings reiterate that biofilms represent a robust defensive phenotype, particularly in Gram-negative pathogens, and that complete eradication is rarely achieved using topical agents alone.

Our results also highlight differences in the spectrum of activity of tested agents, with Octenisept^® and Betadine^® retaining partial efficacy even in Model C against select bacteria (S. aureus, E. coli). This is consistent with known characteristics of iodine-based and octenidine-based preparations, which exhibit rapid, non-specific mechanisms of action able to partially overcome biofilm-associated tolerance (Kessler, 2025). However, even these agents were unable to fully inhibit growth across all strains in the mature biofilm environment, further emphasizing the resilience of biofilm-mediated infections.

Recent research has increasingly focused on novel strategies to disrupt biofilm structures or potentiate the effects of antimicrobials. Several studies from our dataset demonstrate promising avenues: hydrogels enriched with plant extracts (Lethongkam, 2025), composite films incorporating zinc oxide nanoparticles and essential oils (Motelica, 2024), and enzyme-based debridement systems targeting matrix polysaccharides (Schneider et al., 2025). These interventions showed significant reductions of biofilm biomass—often 3–7 log units—in ex vivo or in vitro chronic wound models, particularly when applied in combination with physical disruption methods such as mechanical or enzymatic debridement. The study by Schneider et al. is especially relevant, demonstrating that biofilm dispersal enzymes dramatically improve the effect of subsequent antimicrobial therapy and surgical debridement, providing a compelling model for integrated wound care.

Collectively, these insights suggest that topical antimicrobials alone are insufficient when applied to wounds with established biofilms, whether in vitro, ex vivo, or in vivo. Instead, successful management of chronic or heavily contaminated wounds requires a multimodal approach: early and repeated cleansing, timely antimicrobial application, mechanical or enzymatic debridement, and ongoing biofilm management within a structured wound hygiene protocol. Our data strongly support this approach and further indicate that timing of intervention is one of the most critical determinants of treatment success.

Translating our experimental results into clinical practice, it is reasonable to assume that delayed application of topical antimicrobials—for example, 24 hours after microbial contamination of a burn wound—is unlikely to sufficiently suppress bacterial proliferation or prevent infection. By this stage, biofilm maturation likely impedes diffusion and neutralizes active agents. These findings underscore the necessity of early and proactive wound management, particularly in burn surgery, where rapid colonization by biofilm-forming pathogens is well documented.

Our study demonstrates that the timing of topical antimicrobial application is a critical determinant of its efficacy in contaminated wound models. The results clearly show that early intervention—within the first hours following bacterial contamination—results in significantly higher antimicrobial activity, whereas delayed application, particularly after 24 hours, is associated with a substantial decline in efficacy. This reduction strongly correlates with the process of biofilm maturation, which represents a key barrier to successful antimicrobial therapy.

All tested bacterial strains could form biofilm, with a marked increase in biofilm intensity over time. This finding supports the concept that even initially planktonic bacterial populations rapidly transition into structured biofilm communities, which exhibit significantly increased tolerance to antimicrobial agents. In the in vitro burn wound model, this was reflected by the progressive loss of effectiveness of most tested topical agents, with several preparations showing activity comparable to sterile gauze in the mature biofilm stage.

Among the evaluated agents, octenidine- and iodine-based preparations demonstrated the most consistent antimicrobial activity across different time points, although even these agents were unable to fully eradicate bacteria in the established biofilm environment. These findings highlight the inherent limitations of topical antimicrobial therapy when used as a standalone intervention in wounds with developed biofilm.

From a clinical perspective, our results emphasize the necessity of early and proactive wound management. Delayed initiation of topical antimicrobial therapy may be insufficient to prevent bacterial proliferation and infection due to the protective nature of biofilm. Therefore, effective treatment strategies should incorporate timely antimicrobial application combined with regular wound cleansing, biofilm disruption, and, when necessary, mechanical or enzymatic debridement as part of a structured wound hygiene approach. In conclusion, the successful management of contaminated and chronic wounds requires not only the appropriate selection of antimicrobial agents but also the precise timing of their application and integration into a multimodal therapeutic strategy targeting both planktonic bacteria and biofilm-associated communities.