Biofilms represent a major challenge, especially in chronic wound care and one of the main factors for wound chronicity and impaired healing (1, 7, 14–16). Adequately addressing this challenge by advancing understanding and developing precise, comprehensive and new therapeutic strategies is one of the most important research goals in modern wound management (7, 17, 18).
Due to the extensive resilience of microorganisms residing within such polymicrobial communities embedded in extracellular polymeric substance (EPS), rigorous and highly efficient antiseptic regimen are necessary. While a form of repetitive, effective debridement represents the fundamental base of every anti-biofilm strategy (1, 7, 19), debridement alone is insufficient for complete biofilm eradication (7, 20), needing a combination with highly efficient antimicrobial and antiseptic agents (7, 21, 22). However, reported efficacy of available antimicrobial and antiseptic agents varies greatly depending on various factors and to date no agents can be recommended over another as being best suited for the treatment of chronic wound biofilms (7, 17). Foremost, heterogeneity in study design/experimental setup and lacking translation from in-vitro to in-vivo studies have been identified as main limitations in current research (17, 18). Another major concern is the liberal extrapolation of results in certain test scenarios to supposedly similar situations. This is the case for experimental setups as demonstrated by the significant differences in efficacy between a standard planktonic (DIN EN 13727 (13)) and biofilm in-vitro assay in this study (Figs. 1a-c) as well as previous works comparing different biofilm models, calling for more comprehensive testing. The results emphasize the direct dependency of an agent’s efficacy on the environment it acts in.
Most static as well as liquid-flow-based in-vitro models are limited to factors such as growth on plastic surfaces and lack of adequate chronic wound reflecting organic conditions, let alone the heterogenous, individual conditions in human chronic wound biofilms (9). The development of our human plasma biofilm model (hpBIOM; Fig. 4a-c) (10, 11), aims to narrow the gap between in-vitro and in-vivo biofilm research and provide a translational approach. The use of a complex biofilm model based on human material, including plasma and active immune cells, addresses the interactions of microbial biofilms with the human wound environment (3, 14) as well as the relevant efficacy loss of antimicrobials under challenge (23, 24), providing a ‘close-to-reality’ test scenario.
The presented results on commercially available hypochlorous wound irrigation solutions compared to high-potency antiseptics highlight the necessity for such models and the careful distinction within agent classes. In a previous publication, we demonstrated and discussed the differences between commercially available hypochlorous wound irrigation solutions depending on agent concentration, pH-value and test setup against planktonic bacteria (12): while LVX, KSL and especially AMF, showed a high short-term efficacy on planktonic bacteria, no efficacy of either agent could be detected in the complex human biofilm model over the course of 72 h (Fig. 2). OCT/PE and PHMB, on the contrary, achieved significant reductions of microorganisms with increasing exposure time (Fig. 1a-c) and visual breaking open of biofilm structures (Fig. 5a-c). Compared to other approaches (11), no re-application of test-substances within 72 h was performed, demonstrating a certain remanence effect of OCT/PE and PHMB. In contrast to our earlier publication (12), hypochlorous agents not only failed to reduce microbial counts in biofilms but also in the here used planktonic method (QSM; Fig. 2). This is most likely attributed to the experimental setup with a deliberately chosen lower ‘agent to microbial test suspension’-ratio in the planktonic QSM (1:5 compared to 8:1 in standards), to unify the ratio in both models (QSM and hpBIOM). OCT/PE and PHMB however, still achieved complete eradication of all tested planktonic microorganisms within 6 hours (Fig. 1a-c) even in low ratio setups. In a planktonic setup, higher ratios for hypochlorous irrigation solutions also resulted in higher reduction rates ((12) and unpublished data).
To investigate whether an increase in substance volume would yield anti-biofilm effects of hypochlorous solutions, the volume capabilities of the hpBIOM were exhausted to administer as much agent as possible (1.0 instead of 0.3 mL): while OCT/PE and PHMB showed a significant increase in anti-biofilm efficacy, even partially achieving complete eradication, the only hypochlorous solution, demonstrating a (small) increase in efficacy compared to the lower volume was AMF (Fig. 3a & b). LVX and KSL showed no reduction (Suppl. Tbl. 2). This underlines the dose-dependency of antiseptics and antimicrobials as well as the relevance of additional aspects such as dilution, mechanical detachment/debridement and reduction in surface tension to the overall antimicrobial and cleansing effects of especially wound irrigation solutions reported in other studies(25, 26). The discrepancies in studies evaluating different forms of chlorine-releasing solutions, reporting higher efficacies, than reported in this study, mainly derive from the vast heterogeneity of study designs. Many in-vitro studies used stationary biofilms on plastic surfaces without organic challenge (27–29), even though the relevance of such challenge has been widely described (9, 30). Also, the exact composition of solutions needs to be considered, differentiating between solutions containing mainly sodium-hypochlorite, hypochlorous acid or both, as well as the concentration and pH of solution and environment, whereby more acidic and alkaline conditions and solutions can be more effective, as described before (12, 27, 29, 31, 32) and shown in this work, as the only hypochlorous irrigation solution showing any anti-biofilm effect was the more alkaline, higher-concentrated (0.2% NaClO) AMF (Fig. 3b).