Update on the Role of Infection and Biofilms in Wound Healing: Pathophysiology and Treatment.

The presence of biofilms in chronic non-healing wounds, has been identified through in vitro model and in vivo animal data. However, human chronic wound studies are under-represented and generally report low sample sizes. For this reason we sought to ascertain the prevalence of biofilms in human chronic wounds by undertaking a systematic review and meta-analysis. Our initial search identified 554 studies from the literature databases (Cochrane Library, Embase, Medline). After removal of duplicates, and those not meeting the requirements of inclusion, nine studies involving 185 chronic wounds met the inclusion criteria. Prevalence of biofilms in chronic wounds was 78.2 % (confidence interval [CI 61.6-89, p<0.002]). The results of our meta-analysis support our clinical assumptions that biofilms are ubiquitous in human chronic non-healing wounds.

Following confirmation of the presence of biofilms in chronic wounds, the term biofilm became a buzzword within the wound healing community. For more than a century pathogens have been successfully isolated and identified from wound specimens using techniques that were devised in the nineteenth century by Louis Pasteur and Robert Koch. Although this approach still provides valuable information with which to help diagnose acute infections and to select appropriate antibiotic therapies, it is evident that those organisms isolated from clinical specimens with the conditions normally used in diagnostic laboratories are mainly in a planktonic form that is unrepresentative of the way in which most microbial species exist naturally. Usually microbial species adhere to each other, as well as to living and non-living surfaces, where they form complex communities surrounded by collectively secreted extracellular polymeric substances (EPS). Cells within such aggregations (or biofilms) display varying physiological and metabolic properties that are distinct from those of planktonic cells, and which contribute to their persistence. There are many factors that influence healing in wounds and the discovery of biofilms in chronic wounds has provided new insight into the reasons why. Increased tolerance of biofilms to antimicrobial agents explains the limited efficacy of antimicrobial agents in chronic wounds and illustrates the need to develop new management strategies. This review aims to explain the nature of biofilms, with a view to explaining their impact on wounds.

Bacterial biofilms infection is one of the major factors causing delayed wound healing. Therefore, early diagnosis of bacterial biofilms is critical for successful cure of chronic wounds. Although many techniques have been developed to diagnose the free pathogens in infectious wounds, there is no technique which can quickly and accurately identify bacterial biofilms in wounds. In this paper, the mechanism of bacterial biofilm formation is briefly reviewed, and several potential diagnostic techniques for bacterial biofilms are introduced, which are expected to be a means of rapid diagnosis and monitoring of bacterial biofilms in wounds.

Introduction: Prevention and management of biofilm in acute and chronic wounds in ICU settings is critical, as biofilm significantly impedes wound healing. Approximately 90% of all wounds are affected by biofilm. It is not feasible to eliminate biofilm using a single wound-cleaning technology. Early intervention and the combined use of multiple technologies for biofilm removal are essential to promote wound healing. Rapid and effective treatment is vital to prevent severe complications such as infections or amputations in intensive care units. Drawing on extensive knowledge of available technologies and their mechanisms of action, we adopted a multimodal approach to treat wounds with biofilm. The primary objective was to prevent and manage biofilm in both acute and chronic wounds. Methods: Five patients with infected acute and traumatic wounds—including chronic osteomyelitis tunnel wound, gunshot injuries, shrapnel wounds, surgical incisions, and fasciotomies—underwent Three-Step Multimodal Biofilm-Focused Protocol consisting of three sequential steps: Step 1: Cleansing and moistening wounds with a solution containing active pure HOCl (pH 2.5) for at least 30 minutes. Step 2: Cleansing and moistening wounds with a polyhexamethylene biguanide (PHMB) and betaine solution, left in place for 30 minutes, followed by cleaning with a debridement pad. Step 3: Application of a synergistic ionic silver (0.01%)–menthol liquid dressing with unique properties (cleansing, disinfecting, removing waste and slough, destroying biofilm). This dressing offers broad-spectrum antibacterial activity and accelerates healing. Wet pads were left in the wounds for the remainder of the day. Results / Discussion: All wounds demonstrated improvement, with the formation of new granulation tissue. Subsequent treatments included negative pressure therapy, secondary surgical closure, or skin grafting. Gunshot and shrapnel wounds healed successfully, limb amputation was avoided, and the chronic osteomyelitis tunnel wound contracted. This Three-Step Multimodal Biofilm-Focused Protocol enhanced wound healing, prevented amputations, and accelerated recovery. The method was later applied to other complex wounds in ICU patients.

Wound infections involving Candida albicans can be challenging to treat because of the fungus' ability to penetrate wound tissue and form biofilms. The goal of this study was to assess the activity of a hypochlorous acid (HOCl)-generating electrochemical scaffold (e-scaffold) against C. albicans biofilms in vitro and on porcine dermal explants (ex vivo). C. albicans biofilms were grown either on acrylic-bottom six-well plates (in vitro) or on skin tissue excised from porcine ears (ex vivo), and the polarized e-scaffold was used to generate a continuous supply of low concentration HOCl near biofilm surfaces. C. albicans biofilms grown in vitro were reduced to undetectable amounts within 24h of e-scaffold exposure, unlike control biofilms (5·28±0·034 log10 (CFU cm- 2 ); P<0·0001). C. albicans biofilms grown on porcine dermal explants were also reduced to undetectable amounts in 24h, unlike control explant biofilms (4·29±0·057 log10 (CFU cm- 2 ); P<0·0001). There was a decrease in the number of viable mammalian cells (35·6±6·4%) in uninfected porcine dermal explants exposed to continuous HOCl-generating e-scaffolds for 24h compared to explants exposed to nonpolarized e-scaffolds (not generating HOCl) (P<0·05). Our HOCl-generating e-scaffold is a potential antifungal-free strategy to treat C. albicans biofilms in chronic wounds. Wound infections caused by C. albicans are difficult to treat due to presence of biofilms in wound beds. Our HOCl producing e-scaffold provides a promising novel approach to treat wound infections caused by C. albicans.

The potential impact of biofilm on healing in acute and chronic wounds is one of the most controversial current issues in wound care. A significant amount of laboratory-based research has been carried out on this topic, however, in 2013 the European Wound Management Association (EWMA) pointed out the lack of guidance for managing biofilms in clinical practice and solicited the need for guidelines and further clinical research. In response to this challenge, the Italian Nursing Wound Healing Society (AISLeC) initiated a project which aimed to achieve consensus among a multidisciplinary and multiprofessional international panel of experts to identify what could be considered part of 'good clinical practice' with respect to the recognition and management of biofilms in acute and chronic wounds. The group followed a systematic approach, developed by the GRADE working group, to define relevant questions and clinical recommendations raised in clinical practice. An independent librarian retrieved and screened approximately 2000 pertinent published papers to produce tables of levels of evidence. After a smaller focus group had a multistep structured discussion, and a formal voting process had been completed, ten therapeutic interventions were identified as being strongly recommendable for clinical practice, while another four recommendations were graded as being 'weak'. The panel subsequently formulated a preliminary statement (although with a weak grade of agreement): 'provided that other causes that prevent optimal wound healing have been ruled out, chronic wounds are chronically infected'. All members of the panel agreed that there is a paucity of reliable, well-conducted clinical trials which have produced clear evidence related to the effects of biofilm presence. In the meantime it was agreed that expert-based guidelines were needed to be developed for the recognition and management of biofilms in wounds and for the best design of future clinical trials. This is a fundamental and urgent task for both laboratory-based scientists and clinicians.

Chapter 17 - Removal and control of biofilms in wounds

The incidence, cost, morbidity, and mortality associated with non-healing of chronic skin wounds are dramatic. With the increasing numbers of people with obesity, chronic medical conditions, and an increasing life expectancy, the healthcare cost of non-healing ulcers has recently been estimated at $25 billion annually in the United States. The role played by bacterial biofilm in chronic wounds has been emphasized in recent years, particularly in the context of the prolongation of the inflammatory phase of repair. Rapid high-throughput genomic approaches have revolutionized the ability to identify and quantify microbial organisms from wounds. Defining bacterial genomes and using genetic approaches to knock out specific bacterial functions, then studying bacterial survival on cutaneous wounds is a promising strategy for understanding which genes are essential for pathogenicity. When an animal sustains a cutaneous wound, understanding mechanisms involved in adaptations by bacteria and adaptations by the host in the struggle for survival is central to development of interventions that favor the host. Characterization of microbiomes of clinically well characterized chronic human wounds is now under way. The use of in vivo models of biofilm-infected cutaneous wounds will permit the study of the mechanisms needed for biofilm formation, persistence, and potential synergistic interactions among bacteria. A more complete understanding of bacterial survival mechanisms and how microbes influence host repair mechanisms are likely to provide targets for chronic wound therapy.

Biofilms probably induce a chronic and/or 'quiet' inflammation in the chronic wound and so delay healing. This paper reviews current strategies that can be used to suppress biofilms in chronic wounds until better options are available.

We have characterized the microbiome of infected chronic diabetic wounds (CDWs), exploring associations with antibiotic use and wound severity in a Sri Lankan cohort. Fifty CDW patients were enrolled, 38 of whom received antibiotics. Tissue biopsies were analysed by microbiome profiling, and wounds were graded using the University of Texas Wound Grading System. Biofilm presence was assessed in 20 wounds. The microbiome was largely dominated by Enterobacteriaceae, Pseudomonadaceae, Streptococcaceae, and Corynebacteriaceae. Proteobacteria levels were significantly higher in antibiotic-treated wounds (P=.019), with increased Pseudomonas abundance. Wounds were categorized as grade 1 (10), grade 2 (29), and grade 3 (11). Alpha diversity varied by wound grade (P=.015), with grade 2 wounds showing the highest diversity and grade 3 the lowest. All 20 tested wounds were biofilm-positive, and community composition varied more in antibiotic-treated wounds (P=.004). CDW microbiomes were dominated by Enterobacteriaceae and Pseudomonadaceae, with elevated Proteobacteria in antibiotic-treated wounds. Alpha diversity correlated with wound severity, peaking in grade 2 wounds. The high prevalence of biofilms in wounds underscores the need for management of CDWs that address microbial complexity.

ABSTRACTThis study aimed to gain clinician consensus on which signs/symptoms reported to be indicative of biofilm in chronic wounds are likely to be so. An international, two‐round eDelphi process including wound care clinicians ran from December 2023 to February 2024. Participants rated 26 items on a 9‐point Likert scale. Consensus to include: ≥ 70% of respondents rate an item 7–9, ≤ 15% rate it 1–3. Consensus to exclude: ≥ 70% of respondents rate an item 1–3, ≤ 15% rate it 7–9. Eleven items (visual indicators [a shiny, slimy, persistent layer, easily removed, returns quickly without frequent intervention]; failure to respond to antimicrobials; infection > 30 days duration; poor quality granulation tissue; stalled wound despite optimal management; persistent/prolonged inflammation; wound > 6 weeks duration; soft tissue deterioration despite antimicrobials/debridement; signs of local infection; tunnelling/undermining; presence of slough) achieved consensus to include status. To our knowledge, consensus work on this topic has not previously been performed on such a wide scale. When examined alongside similar work, clinical opinion on the matter lacks coherence. We hope that these findings will help direct us toward greater cohesiveness. The work supports a need for research to quantify the predictive abilities of signs and symptoms reported to be indicative of biofilm in chronic wounds.

Biofilms in Chronic Wounds: Pathogenesis and Diagnosis

Introduction: Wound healing is characterised by haemostatic, inflammatory, proliferative and remodelling phases. In the presence of comorbidities such as diabetes, healing can stall and chronic wounds may result. Infection is detrimental to these wounds and associated with poor outcomes. Wounds are contaminated with microbes and debris, and factors such as host resistance, bacterial virulence, species synergy and bioburden determine whether a wound will deteriorate to critically colonised/infected states. Biofilms are sessile microbial communities, exhibiting high-level antibiotic tolerance and resistance to host defences. Biofilm in critically colonised wounds can contribute to delayed healing. Little is known about clinical presentation and diagnosis of wound biofilms. Objective: To identify from the literature clinical signs, symptoms and biomarkers that may indicate biofilm in chronic wounds. Methods: This review will be guided by the Preferred Reporting Items for Systematic Reviews extension for Scoping Reviews (PRISMA-ScR), and the Joanna Briggs Institute Manual for Evidence Synthesis. Studies of any design in any language recruiting adult patients with venous, diabetic, pressure or mixed arterial-venous ulcers and reporting data on clinical signs/symptoms of biofilm are eligible. Searches of Medline, Embase, CINAHL, Cochrane Central, Scopus, Web of Science, Google scholar and BASE will be conducted from inception to present. Reference scanning and contact with content experts will be employed. Title/abstract screening and full text selection will be executed by two reviewers independently. Discrepancies will be resolved by discussion between reviewers or through third party intervention. Data will be extracted by a single reviewer and verified by a second. Clinical signs and symptoms data will be presented in terms of study design, setting and participant demographic data. Discussion: Understanding biofilm impact on chronic wounds is inconsistent and based largely on in vitro research. This work will consolidate clinical signs, symptoms and biomarkers of biofilm in chronic wounds reported in the literature.

Despite the extensive arsenal of antibacterial drugs and the diversity of new wound treatment technologies, it is fair to say that the problem of wound infection has not lost its relevance. As a rule, in patients with concomitant diseases (obesity, diabetes, sensory neuropathies, autoimmune diseases, etc.), the healing of wound defects is prolonged, which makes them especially vulnerable to infections. Wound infection is one of the decisive factors in the pathogenesis of chronic wounds associated with an increase in the number of multiresistant bacterial strains. Two most common wound pathogens are Pseudomonas aeruginosa and Staphylococcus aureus. Wound healing is mediated not only by complex coordinated cellular mechanisms, but also by the impact of the wound microbiome. Objective. Analysis of the mechanisms of pathogenicity and intermicrobial interactions of P. aeruginosa and S. aureus as a significant “instrument” of wound infection to identify priority strategies for antimicrobial therapy (combination antibacterial drugs, phage therapy, use of antimicrobial peptides etc.). Material and methods. A search and analysis of scientific literature for 2018­2025 was performed in the information resources PubMed, eLIBRARY.RU, Europe PMC, Web of Science, CyberLeninka. The search queries included the following combinations of words: for Russian­language publications chronic wound infection; biofilms in chronic wounds, pathophysiological mechanisms of wound healing; for English­language publications chronic wound infection, biofilms in chronic wounds, pathophysiological mechanisms of wound healing, chronic wound infection bacteria, acute and chronic wounds, P. aeruginosa, S. aureus, mechanisms of pathogenicity, virulent properties. Results and discussion. The review summarizes and presents the mechanisms of initiation of wound infection, determinants of virulence, pathogenicity, antibiotic resistance of P. aeruginosa and S. aureus, immune evasion strategies and features of intermicrobial interactions. Conclusions. Wound infections pose a significant global threat due to high rates of morbidity and mortality. P. aeruginosa and S. aureus remain the most common pathogens causing wound infections and form mixed biofilms that hamper their susceptibility to both antimicrobials and the host immune system. Both types of bacteria secrete a broad spectrum of virulence factors, including toxins and enzymes that facilitate their attachment to the wound surface and suppress the host immune response, leading to further tissue damage. Moreover, the spatial organization formed by these pathogens can influence their virulence properties and is key to understanding bacterial interactions within polymicrobial biofilms. Currently, combination antibacterial drugs, phage therapy, antimicrobial peptides, etc. are used to solve the problem of the growth of multidrug­resistant strains.

Treatment of bacterial biofilm in the wound is complicated by the mechanisms underlying biofilm growth. Many clinically relevant biofilms are polymicrobial. Furthermore, mixed species biofilms have complementary metabolic strategies for obtaining nutrients and degrading host immune molecules. Therefore, there is an urgent need to introduce novel or reemerging effective approaches to combat bacterial biofilms in chronic wounds.