Polymicrobial interactions between Staphylococcus aureus and Pseudomonas aeruginosa promote biofilm formation and persistence in chronic wound infections

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.

Chronic wound infections are a global health concern, affecting up to 4% of diabetic populations. Biofilm-producing bacteria significantly delay healing and increase multidrug resistance (MDR. This study assessed the prevalence of biofilm-forming bacteria in chronic wounds and their antibiotic resistance patterns. A total of 100 chronic wound samples were collected. Bacterial identification and biofilm quantification were performed using standard microbiological techniques, while antibiotic susceptibility testing followed CLSI guidelines. Data were analyzed using SPSS v26, with p < 0.05 considered significant. Of 100 samples, 85 (85%) showed bacterial growth. Gram-negative isolates predominated (70/85, 82.4%) over Gram-positives (15/85, 17.6%). The most frequent pathogens were Pseudomonas aeruginosa (25, mean 0.29 ± 0.06), Staphylococcus aureus (20, 0.24 ± 0.05), Escherichia coli (15, 0.18 ± 0.04), and Klebsiella pneumoniae (10, 0.12 ± 0.03). Biofilm production was detected in 62 isolates (72.9%): strong (18, 0.29 ± 0.07), moderate (25, 0.40 ± 0.09), and weak (19, 0.31 ± 0.08). Strong biofilm formation was most common in P. aeruginosa (48%, 0.83 ± 0.08) and S. aureus (20%, 0.50 ± 0.08). Antibiotic resistance was high: S. aureus showed 80% penicillin and 60% erythromycin resistance, with 35% MRSA. E. coli (40%) and K. pneumoniae (50%) were ESBL producers. Overall, 45/85 isolates (52.9%, mean 0.53 ± 0.15) were MDR, significantly higher in biofilm producers (67.7%, mean 0.68 ± 0.10) than non-producers (13%, 0.13 ± 0.05; p < 0.01). Polymicrobial infections showed greater biofilm prevalence (83.3%) and MDR (70%) than monomicrobial (45.5%). Chronic wound infections are dominated by biofilm-forming MDR bacteria, particularly P. aeruginosa and S. aureus. Biofilm formation correlates strongly with MDR, wound duration, and polymicrobial infections, emphasizing the need for anti-biofilm therapies in wound management.

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.

Biofilms in Chronic Wounds: Pathogenesis and Diagnosis

While much is known about acute infection pathogenesis, the understanding of chronic infections has lagged. Here we sought to identify the genes and functions that mediate fitness of the pathogen Pseudomonas aeruginosa in chronic wound infections, and to better understand the selective environment in wounds. We found that clinical isolates from chronic human wounds were frequently defective in virulence functions and biofilm formation, and that many virulence and biofilm formation genes were not required for bacterial fitness in experimental mouse wounds. In contrast, genes involved in anaerobic growth, some metabolic and energy pathways, and membrane integrity were critical. Consistent with these findings, the fitness characteristics of some wound impaired-mutants could be represented by anaerobic, oxidative, and membrane-stress conditions ex vivo, and more comprehensively by high-density bacterial growth conditions, in the absence of a host. These data shed light on the bacterial functions needed in chronic wound infections, the nature of stresses applied to bacteria at chronic infection sites, and suggest therapeutic targets that might compromise wound infection pathogenesis.

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.

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.

The foodborne pathogen Listeria monocytogenes has the ability to develop biofilm in food-processing environment, which becomes a major concern for the food safety. The biofilm formation is strongly influenced by the availability of nutrients and environmental conditions, and particularly enhanced in poor minimal essential medium (MEM) containing glucose rather than in rich brain heart infusion (BHI) broth. To gain better insight into the conserved protein expression profile in these biofilms, the proteomes from biofilm- and planktonic-grown cells from MEM with 50mM glucose or BHI were compared using two-dimensional polyacrylamide gel electrophoresis followed by MALDI-TOF/TOF analysis. 47 proteins were successfully identified to be either up (19 proteins) or down (28 proteins) regulated in the biofilm states. Most (30 proteins) of them were assigned to the metabolism functional category in cluster of orthologous groups of proteins. Among them, up-regulated proteins were mainly associated with the pentose phosphate pathway and glycolysis, whereas a key enzyme CitC involved in tricarboxylic acid cycle was down-regulated in biofilms compared to the planktonic states. These data implicate the importance of carbon catabolite control for L. monocytogenes biofilm formation in response to nutrient availability.

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.

BackgroundChronic wounds are frequently colonized or infected with multiple bacterial or fungal species, which can both promote or inhibit each other. Network analyses are helpful to understand the interplay of these species in polymicrobial infections. Our aim was to analyse the network of bacterial and fungal species in chronic wounds.MethodsSwabs (n = 163) from chronic wound infections (Masanga, Sierra Leone, 2019–2020) were screened for bacterial and fungal species using non-selective agars. Some of these wounds were suspected but not confirmed Buruli ulcer. Species identification was done with MALDI-TOF mass spectrometry. Network analysis was performed to investigate co-occurrence of different species within one patient. All species with n ≥ 10 isolates were taken into account.ResultsOf the 163 patients, 156 had a positive wound culture (median of three different species per patient; range 1–7). Pseudomonas aeruginosa (n = 75) was the dominating species with frequent co-detections of Klebsiella pneumoniae (21 cases; OR = 1.36, 95%CI: 0.63–2.96, p = 0.47), Staphylococcus aureus (14 cases; OR = 1.06, 95%CI: 0.44–2.55, p = 1) and Proteus mirabilis (13 cases; OR = 0.84, 95%CI: 0.35–1.99, p = 0.69).ConclusionThe culturome of chronic wounds in Sierra Leonean patients is highly diverse and characterized by the co-occurrence of P. aeruginosa, K. pneumoniae and S. aureus.

Chronic wound infections are an important cause of delayed wound healing, posing a significant healthcare burden with consequences that include hospitalization, amputation, and death. These infections most often take the form of three-dimensional biofilm communities, which are notoriously recalcitrant to antibiotics and immune clearance, contributing to the chronic wound state. In the chronic wound microenvironment, microbial biofilms interact closely with other key components, including host cellular and matrix elements, immune cells, inflammatory factors, signaling components, and mechanical cues. Intricate relationships between these contributing factors not only orchestrate the development and progression of wound infections but also influence the therapeutic outcome. Current medical treatment for chronic wound infections relies heavily on long-term usage of antibiotics; however, their efficacy and reasons for failure remain uncertain. To develop effective therapeutic approaches, it is essential to better understand the complex pathophysiology of the chronic wound infection microenvironment, including dynamic interactions between various key factors. For this, it is critical to develop bioengineered platforms or model systems that not only include key components of the chronic wound infection microenvironment but also recapitulate interactions between these factors, thereby simulating the infection state. In doing so, these platforms will enable the testing of novel therapeutics, alone and in combinations, providing insights toward composite treatment strategies. In the first section of this review, we discuss the key components and interactions in the chronic wound infection microenvironment, which would be critical to recapitulate in a bioengineered platform. In the next section, we summarize the key features and relevance of current bioengineered chronic wound infection platforms. These are categorized and discussed based on the microenvironmental components included and their ability to recapitulate the architecture, interactions, and outcomes of the infection microenvironment. While these platforms have advanced our understanding of the underlying pathophysiology of chronic wound infections and provided insights into therapeutics, they possess certain insufficiencies that limit their clinical relevance. In the final section, we propose approaches that can be incorporated into these existing model systems or developed into future platforms developed, thus enhancing their biomimetic and translational capabilities, and thereby their human-relevance.

Laboratory models provide tractable, reproducible systems that have long served as foundational tools in microbiology. However, the extent to which these models accurately mimic the biological environments they represent remains poorly understood. A quantitative framework was recently introduced to assess how well laboratory models capture microbial physiology in situ. However, applications of this framework have been limited to characterizing the physiology of a single species in human infections, leaving a gap in our understanding of overall microbial community physiology in polymicrobial contexts. Here, we extended this framework to evaluate the accuracy of laboratory model systems in capturing community-level functions in polymicrobial infection. As a proof of concept, we applied the extended framework to a polymicrobial model of human chronic wound (CW) infection. CWs harbor metabolically diverse bacterial species that engage in a range of microbe-microbe interactions, ultimately impacting community dynamics and disease progression. However, studies on the mechanistic drivers of chronic wound infection have relied on single species or pairwise approaches. Here, we demonstrate that our adapted framework can be used to develop accurate polymicrobial models. Further, we demonstrate that this extended framework can evaluate the occurrence of known microbe-microbe interactions. Building on our prior work in large-scale metagenomic and metatranscriptomic analysis, we propose a highly accurate 6-member synthetic bacterial community model i.e. representative of the taxonomic and functional complexity of human CW infections. This approach will support the development of ecologically relevant polymicrobial models and better treatment strategies.

Chronic wounds are a drain on global health services and remain a major area of unmet clinical need. Chronic wounds are characterised by a stable and stubborn bacterial biofilm which hinders innate immune response and delays or prevents wound healing. Bioactive glass (BG) fibres offer a promising novel treatment for chronic wounds by targeting the wound-associated biofilm. In this study, the antimicrobial properties of silver-doped BG fibres were tested against Pseudomonas aeruginosa biofilms, which are commonly found in chronic wound infections. Results showed that BG fibres doped with silver resulted in a 5log10 reduction in biofilm formation whereas silver-free fibres only reduced formation by log10, therefore silver-doped fibres possess stronger antimicrobial effects. Moreover, there appeared to be a synergistic effect between the fibres and the silver as the application of the silver-doped fibres placed directly in contact with the forming biofilm resulted in a higher reduction in biofilm formation compared to treatments either: using the dissolution ions, using BG powder, or when the fibres were placed in an insert above the biofilm, inhibiting physical contact, instead. This suggests that the physical properties of the fibres, as well as silver, influence biofilm formation. Finally, results demonstrated that silver chloride, which is not antimicrobial, forms and the concentrations of antimicrobial silver species, namely silver ions and nanoparticles, reduce over time when fibres are soaked in cell culture media, which partially explains why the silver-doped dissolution ions contained lower antimicrobial activity compared to the fibres. As silver chloride is more likely to form with increased temperature and time, the antimicrobial activity of silver-containing dissolution ions is highly dependent on the length of ageing and storage conditions. Many studies investigate the antimicrobial and cytotoxic properties of biomaterials through their dissolution products. However, instability of antimicrobial silver species due to silver chloride formation and its effect on antimicrobial properties of silver-based biomaterials has not been reported before and could influence past and future dissolution-based assays as results showed that the antimicrobial activity of silver-based dissolution ions can vary greatly depending on post processing steps and can therefore produce misleading data.

Extracellular DNA-dependent biofilm formation by Staphylococcus epidermidis RP62A in response to subminimal inhibitory concentrations of antibiotics

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.