Biofilms in Chronic Wounds: Pathogenesis and Diagnosis

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.

Increased local temperature is a classic sign of wound infection, and its quantitative measurement has the potential to assist with assessment and diagnosis of chronic deep wound and surrounding skin infection at the bedside. Evidence supporting such use in chronic wound care is very limited. This clinical pilot study was conducted in an attempt to quantify the relationship between increased periwound skin temperature and wound infection, as well as validate use of a handheld infrared thermometer for the wound care practitioner. Using a cross-sectional design, 2 groups of participants were recruited from a chronic wound clinic: without wounds (n = 20) and with chronic leg ulcers (n = 40). Participant and wound characteristics were documented. All skin temperatures were documented using a handheld infrared thermometer under consistent environmental conditions within the clinic. Data analysis was based on the difference (Delta) in skin temperature (in degrees Fahrenheit) between a target or wound site and an equivalent contralateral control site. Wound infection was identified using the combination of a validated assessment tool and clinical judgment. Supplemental semiquantitative bacterial swabs were collected from all wounds. Descriptive statistics were analyzed using the chi-squared calculation. A Pearson r calculation of test-retest skin temperature data collected from nonwounded participants initially determined reliability of the infrared thermometer. Correlation of increased periwound skin temperature to wound infection was determined by calculation of a 1-way analysis of variance. The infrared thermometer was found to be reliable (r = 0.939, P = .000 at a 95% confidence interval). A statistically significant relationship between increased periwound skin temperature and wound infection was identified (F = 44.238, P = .000 at a 95% confidence interval). Neither patient nor wound characteristics were significantly different between the participants with noninfected or infected wounds. The results of this study demonstrate that incorporating quantitative skin temperature measurement into routine wound assessment provides a timely and reliable method for a wound care practitioner to quantify the heat associated with deep and surrounding skin infection and to monitor ongoing wound status. Study limitations may reduce transferability of these findings to wound types other than chronic leg ulcers. Further research is needed to support and strengthen these results.

Chronic wounds cause significant morbidity and mortality and cost our health care system millions of dollars each year. A major impediment to wound healing is the formation of bacterial biofilms. Biofilms are communities of bacteria associated with chronic infections. This article reviews the literature on chronic wounds and biofilms. The role of biofilms in chronic wounds is not widely known. The purpose is to increase awareness of their role and to discuss research into novel therapeutic options. PubMed searches were performed to identify publications on chronic wounds and biofilms. Biofilms contribute to chronic wound nonhealing. There is an abundance of research into novel antibiofilm strategies for chronic wounds. Current research is being targeted at antibiofilm strategies needed to restore an optimal wound-healing environment. A combined treatment approach involving aggressive débridement and the addition of antibiofilm agents is needed.

The survey on prevailing medical practices for non-healing chronic wounds and its impact provides valuable insights into the management and treatment strategies adopted in the context of chronic wound care. The discussion highlights key findings related to diabetes as a prevalent cause, wound location, treatment approaches, and the utilization of serological tests. Diabetes emerged as a significant risk factor for chronic wounds, with 90% of the respondents having a family history of diabetes, and 70% currently under medication for diabetes management. These findings emphasize the crucial role of diabetes in contributing to non-healing chronic wounds. Healthcare providers should prioritize diabetic patients for preventive wound care measures and regular assessments to reduce the incidence of chronic wounds. The survey reveals that a substantial proportion of chronic wounds (75%) are diabetes-related, further supporting the link between diabetes and wound development. Infection-related wounds (10%), autoimmune-related wounds (3%), and other causes (5%) also contribute to the overall burden of chronic wounds. Understanding the various causative factors allows healthcare professionals to tailor treatment plans accordingly and address the specific needs of individual patients. The location of wounds showed a gender disparity, with a higher prevalence of chronic wounds in male limbs (74%) compared to female limbs (26%). Similarly, chronic wounds in other sites were more common in males (74%) than females (26%). The reasons for these gender-based differences warrant further investigation and consideration in treatment planning. In terms of treatment and management, nearly all respondents (100%) were on multiple antibiotics for wound care, indicating the widespread use of antimicrobial therapy. Additionally, a significant portion of respondents (57%) reported trying alternative treatment approaches, suggesting the need for a more diverse and holistic approach to chronic wound management. Hospitalization for chronic wound care was common, with 80% of the respondents having been hospitalized multiple times. This suggests that chronic wounds often require extensive medical attention and resources, making the management of chronic wounds a challenging and resource- intensive aspect of healthcare. The utilization of wound-related serological tests was relatively low, with only 3% of respondents undergoing such tests. This indicates a potential gap in healthcare practices, as serological tests can provide valuable diagnostic information for guiding wound management strategies. Increased awareness and promotion of serological tests among healthcare providers could lead to better wound care outcomes. Overall, the survey findings underscore the complexities and challenges associated with non-healing chronic wounds, especially in the context of diabetes-related wounds. The study highlights the need for comprehensive and personalized wound management strategies, increased awareness about wound-related serological tests, and the exploration of alternative treatment approaches to improve the outcomes and quality of life for individuals affected by chronic wounds. Addressing these challenges would lead to better wound healing outcomes, reduced healthcare burdens, and enhanced overall patient well-being Keywords: Non-healing chronic wounds, Wounds Prevalence, Antimicrobial therapy, Alternative treatment approaches, Serological tests.

Polymicrobial biofilms in chronic infected wounds harbour different bacterial species that interact with each other, competing or co-operating to survive. The two most common pathogens co-isolated from chronic wound biofilms are Pseudomonas aeruginosa and Staphylococcus aureus. Evidence from in vitrobiofilms models have shown these two bacteria interactand data suggests that P. aeruginosa inhibits the growth of S. aureusin mixed biofilms. This study aimed to assess the growth of these two species in a complex polymicrobial biofilm in a 3D matrix comprised of either alginate (1.5% w/v) or a collagen scaffold. Using a five-species biofilm (S. aureus, P. aeruginosa, Citrobacter freundii, Enterococcus faecalisand Escherichia coli), with all bacteria inoculated at time zero, it was consistently observed that P. aeruginosa was not recoverable over a 72h period, with sampling every 24h. However, P. aeruginosagrew well if it was added to a pre-formed four-species biofilm. Further, P. aeruginosa was seen to inhibit the growth of S. aureus after 24h subsequent co-culture in the pre-formed biofilm, which resulted in the emergence of small colony variants of S. aureus. Interestingly when P. aeruginosawas co-inoculated in a four species biofilm that did not contain S. aureus,its growth was not inhibited, suggesting a competitive interaction between these two bacteria during establishment of the early biofilm. These data were consistent in alginate beads and collagen scaffolds. In a chronic wound P. aeruginosais regarded as a late coloniser and the phenomena observed in this study might be reflective of this.

The occurrence of chronic wounds is a major global health issue. These wounds are difficult to heal as a result of disordered healing mechanisms. The most common types of chronic wounds are diabetic ulcers, pressure ulcers, arterial/venous ulcers and nonhealing surgical wounds. Although bacteria are an important cause of chronic nonhealing wounds, fungi also play a substantial role in them. The fungal infection rate varies with different chronic wound types, but overall, the prevalence of fungi is extremely underestimated in the clinical treatment and management of chronic wounds. Wounds and ulcers can be colonized by host cutaneous, commensal or environmental fungi and evolve into local infections, causing fungemia as well as invasive fungal disease. Furthermore, the fungi involved in nonhealing wound-related infections help commensal bacteria resist antibiotics and the host immune response, forcing wounds to become reservoirs for multiresistant species, which are considered a potential key factor in the microbial bioburden of wounds and ulcers. Fungi can be recalcitrant to the healing process. Biofilm establishment is the predominant mechanism of fungal resistance or tolerance to antimicrobials in chronic nonhealing wounds. Candida albicans yeast and Trichophyton rubrum filamentous fungi are the main fungi involved in chronic wound infection. Fungal species diversity and drug resistance phenotypes in different chronic nonhealing wound types will be emphasized. In this review, we outline the latest research on fungi in chronic wounds and discuss challenges and future perspectives related to diagnosing and managing chronic wounds.

Biofilms are sessile communities of microorganisms, mainly bacteria, that grow on biotic and abiotic surfaces. These microorganisms are embedded within an extracellular polymeric substance that provides enhanced protection from antimicrobials. Chronic wounds provide an ideal habitat for biofilm formation. Bacteria can easily attach to wound debris and can infect the wound due to an impaired host immune response. This review highlights the mechanism of biofilm formation and the role of biofilms in the pathophysiology of chronic wounds. Our major focus is on various formulation strategies and delivery systems that are employed to eradicate or disperse biofilms, thereby effectively managing acute and chronic wounds. We also discuss clinical research that has studied or is studying the treatment of biofilm-infected chronic wounds.

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 sought to examine whether vacuum-assisted closure (VAC) is associated with fewer surgical site infections (SSIs) or infections of chronic wounds than other management procedures for surgical wounds. The PubMed and Scopus databases were searched systematically. Randomized controlled trials (RCTs) comparing the development of SSIs or infections of chronic wounds between patients treated with VAC for acute or chronic wounds and those whose wounds were treated without VAC were considered eligible for inclusion in the study. Eight RCTs met the inclusion criteria for the study. Four of the studies included chronic or diabetic lower extremity wounds and four included fractures. In three of four studies reporting on fractures, the wounds were not closed post-operatively, whereas in one study primary closure of the wound was performed. With regard to wounds left open after the stabilization of fractures, patients whose wounds were treated with VAC developed fewer SSIs than those whose wounds were treated without VAC ([367 patients (196 with VAC; 171 without VAC) relative risk [RR], 0.47; 95% CI 0.28-0.81]). On the contrary, no difference in the development of SSIs occurred among patients with chronic or diabetic lower-extremity wounds treated with VAC and those whose wounds were treated without VAC ([638 patients (320 with VAC; 318 without VAC) RR 1.67; 95% CI: 0.71-3.94]). The available evidence suggests that the development of infections in wounds treated with VAC depends on the type of wound being treated.

SummaryBacterial biofilms are an ever‐growing concern for public health, featuring both inherited genetic resistance and a conferred innate tolerance to traditional antibiotic therapies. Consequently, there is a growing interest in novel methods of drug delivery, in order to increase the efficacy of antimicrobial agents. One such method is the use of acoustically activated microbubbles, which undergo volumetric oscillations and collapse upon exposure to an ultrasound field. This facilitates physical perturbation of the biofilm and provides the means to control drug delivery both temporally and spatially. In line with current literature in this area, this review offers a rounded argument for why ultrasound‐responsive agents could be an integral part of advancing wound care. To achieve this, we will outline the development and clinical significance of biofilms in the context of chronic infections. We will then discuss current practices used in combating biofilms in chronic wounds and then critically evaluate the use of acoustically activated gas microbubbles as an emerging treatment modality. Moreover, we will introduce the novel concept of microbubbles carrying biologically active gases that may facilitate biofilm dispersal.

Introduction. Chronic wounds are notoriously difficult to treat and are associated with decreased limb function, reduced quality of life and significant morbidity. Their recurrent nature, despite aggressive antibiotic therapy, is due in part to the presence of polymicrobial biofilms. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most frequently co-isolated pathogens in these infections and are known to form complex biofilms that hinder treatment.Hypothesis. We hypothesized that co-existence and competitive dynamics between P. aeruginosa and S. aureus in chronic wound infections are influenced by strain-specific interactions and may not rely solely on well-characterized inhibitory mechanisms such as 2-alkyl-4-quinolone (AQ) production by P. aeruginosa impacting on S. aureus fitness.Aim. To establish a polymicrobial chronic wound infection model and assess the contribution of AQ signalling and strain-specific interactions on co-existence.Methodology. We used a modified chronic wound biofilm model to co-culture matched and mismatched clinical isolate pairs of P. aeruginosa and S. aureus, collected from two different chronic wound patients. Viable bacterial counts (c.f.u.) were quantified over an 8-day period. AQ production by each P. aeruginosa strain was quantified using liquid chromatography-MS.Results. A stable culture of P. aeruginosa strains was achieved, but distinct behaviours between each S. aureus strain were seen. One matched clinical isolate pair maintained stable c.f.u. levels of both species throughout the 8-day model, indicating a compatible co-existence. In contrast, mismatched pairs showed early loss of S. aureus viability and the emergence of small colony variants after 4 days, not seen in matched pair growth. Interestingly, the most competitive P. aeruginosa strain exhibited undetectable levels of all AQs tested, indicating that its dominance was not due to AQ-mediated antagonism, as has previously been described.Conclusion. Our findings demonstrate that stable dual-species biofilm formation in chronic wounds is strain-dependent and that P. aeruginosa can impact on S. aureus fitness through AQ-independent mechanisms. These results highlight the importance of using clinical isolates in biofilm research and caution against generalizing findings from laboratory strains to complex clinical infections.

To identify general competence areas for graduating registered nurses and podiatrists providing chronic wound care. The provision of care for chronic wounds involves a team of multiple professionals, and registered nurses and podiatrists play an important role. However, previous studies have found that registered nurses have limited competence in wound care. In addition, there are no standardised international competence areas for the provision of chronic wound care by registered nurses and podiatrists. A qualitative design was used in this study. The data were collected using six focus-group interviews with the following professionals: (a) registered nurses; (b) authorised wound care nurses; (c) nurse educators; (d) physicians; (e) podiatrists; and (f) podiatry educators (N=23). The data were analysed using inductive and deductive content analysis. COREQ guidelines were followed, See Appendix S1. The competence areas for registered nurses and podiatrists providing care for chronic wounds include knowledge, skills and performance in anatomy and physiology; aetiology, care and prevention of chronic wounds; and wound management and assessment. The competence areas also include a set of attitudes and values relating to chronic wound care and patients with chronic wounds. In nursing and podiatry education and in working life, teaching should focus on these areas of competence in chronic wound care. This would standardise and integrate education on wound care, especially that of chronic wounds. Wound care is an important part of clinical practice in nursing and podiatry. It is suggested that registered nurses and podiatrists need general competence in chronic wound care to carry out evidence-based, safe, high-quality and cost-effective care. These results could be used to define registered nurses' and podiatrists' competence areas and roles in evidence-based wound care as independent healthcare professionals.

The application of antimicrobials in the management of wounds is a complex procedure requiring appropriate clinical decision making, judgment and a thorough understanding of antimicrobial therapies, together with their potential disadvantages. There is considerable direct and indirect evidence for the presence of bacterial biofilms in the chronic wound bed, and it has been demonstrated that bacteria within these biofilms may exhibit both specific and nonspecific antimicrobial tolerance. The antimicrobial tolerance of biofilms is a major concern in the treatment of both infected and nonhealing chronic wounds and an understanding of the mechanisms involved is of fundamental importance in managing wound infections and developing future wound management strategies. The aim of this review is therefore to provide an overview of our current understanding of the mechanisms by which bacteria in wound biofilms can resist conventional antibiotic and antibacterial therapies which is very important to wound healing.

Infection is the likeliest single cause of delayed healing in healing of chronic open wounds by secondary intention. If neglected it can progress from contamination to colonization and local infection through to systemic infection, sepsis and multiple organ dysfunction syndrome, and it can be life-threatening. Infection in chronic wounds is not as easy to define as in acute wounds, and is complicated by the presence of biofilms. There is, as yet, no diagnostic for biofilm presence, but it contributes to excessive inflammation - through excessive and prolonged stimulation of nitric oxide, inflammatory cytokines and free radicals - and activation of immune complexes and complement, leading to a delay in healing. Control of biofilm is a key part of chronic wound management. Maintenance debridement and use of topical antimicrobials (antiseptics) are more effective than antibiotics, which should be reserved for treating spreading local and systemic infection. The continuing rise of antimicrobial resistance to antibiotics should lead us to reserve their use for these indications, as no new effective antibiotics are in the research pipeline. Antiseptics are effective through many mechanisms of action, unlike antibiotics, which makes the development of resistance to them unlikely. There is little evidence to support the theoretical risk that antiseptics select resistant pathogens. However, the use of antiseptic dressings for preventing and managing biofilm and infection progression needs further research involving well-designed, randomized controlled trials.

Opportunistic pathogen Pseudomonas aeruginosa plays a crucial role in chronic wound biofilms, increasing infection's morbidity and mortality. In recent years, the signalling molecule nitric oxide (NO) and chelating agent tetrasodium EDTA (T-EDTA) have been applied therapeutically owing to their multifactorial effects including bacterial killing, biofilm dispersal, and wound healing. However, previous studies assessing NO's antibiofilm efficacy have not considered the variable pH and temperature of the wound environment. Here, pH-dependent NO donors N-diazeniumdiolates (NONOates), PAPA NONOate (PA-NO) and Spermine NONOate (SP-NO), and T-EDTA were applied in wound-relevant pH environments (pH 5.5-8.5) and temperatures (32°C and 37°C) to P. aeruginosa PAO1 biofilms grown for either 24 or 48h. At 32°C and pH 7.5, 250μM PA-NO reduced 24-h biofilm biomass by 35%. At 37°C, 250μM PA-NO and 4% w/v T-EDTA caused 21% and 57% biomass reduction in 24-h biofilms, respectively. In 48-h biofilms, NONOates did not induce significant biomass reduction, while T-EDTA maintained its efficacy with a 64% reduction. A subsequent experiment investigated the impact of NONOates and T-EDTA as pre-treatments before exposure to ciprofloxacin. Unexpectedly, NONOate pre-treatment decreased ciprofloxacin's effectiveness, resulting in approximately 1-log increase in viable planktonic and biofilm-residing cells compared to ciprofloxacin alone. It was hypothesized that this protective effect might stem from NO-induced decreased cellular respiration, which inhibits reactive oxygen species (ROS)-mediated bactericidal mechanisms. These findings highlight both the potential and complexities of developing effective antimicrobial strategies for chronic wound infections, emphasizing the need for further research to optimize treatment approaches.