Understanding biofilm in practice: a global survey of health professionals.

OBJECTIVE: The aim of this survey was to examine health professionals' views and practices relating to biofilm in chronic wounds. METHOD: A global online survey was conducted to assess the current understanding of biofilm and wound management practices. The survey consisted of 20 questions designed to evaluate health professional knowledge of biofilm, perception and understanding of biofilm behaviour, detection and diagnosis, and treatment. Respondents were classified as 'specialists' if wounds were their primary focus and they developed protocols and determined formularies. Respondents were classified as 'generalists' if wounds were part of multiple indications they treat and they were able to choose wound care products from a restricted list of products. The Pearson's chi-square or Fisher's exact test was used to assess whether the responses were independent of the clinician role, health-care setting and country. RESULTS: Overall, 3011 health professionals took part in the survey, of which 397 were excluded or disqualified. Of the remaining 2614 respondents, 1223 (46.8%) completed the entire survey. Although the majority of health professionals were aware of biofilm, knowledge gaps regarding its prevalence in chronic wounds were evident. In general, the majority indicated that they understood that biofilm is detrimental to wound healing. With the exception of wound stalling, there was a lack of consensus on other clinical signs in the detection and diagnosis of biofilm. Knowledge gaps were also evident over the treatment of biofilm and the efficacy of antimicrobial treatments, debridement and wound dressing. CONCLUSION: Our results show that though there is a broad recognition of biofilm and its possible role in chronic wounds, there is still a need to educate and increase knowledge on recognition and treatment of biofilm.

The prevalence of biofilms in chronic wounds: a systematic review and meta-analysis of published data.

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.

The presence of biofilm structures in atherosclerotic plaques of arteries from legs amputated as a complication of diabetic foot ulcers.

OBJECTIVE: Atherosclerosis, rather than microcirculatory impairment caused by endothelial cell dysfunction, is the main driver of circulatory compromise in patients with diabetic limbs. The presence of atherosclerotic plaque at the trifurcation is a significant contributor to amputation of diabetic legs. The presence of bacteria and other microorganisms in atherosclerotic plaque has long been known, however, the cause of chronic inflammation and the role of bacteria/viruses in atherosclerosis have not been studied in detail. The objective of this study was to clarify the cause of the chronic inflammation within atherosclerotic plaques, and determine if any bacteria and/or viruses are involved in the inflammatory pathway. METHOD: This study uses fluorescence microscopy and fluorescence in-situ hybridisation (FISH) to identify components of biofilm in atherosclerotic arteries. These tools are also used to identify individual bacteria, and determine the architectural spatial location within the atherosclerotic plaque where the bacteria can be found. RESULTS: The results indicate that the presence of biofilms in grossly involved arteries may be an important factor in chronic inflammatory pathways of atherosclerotic progression, in the amputated limbs of patients with diabetic foot ulcers and vascular disease. CONCLUSION: While the presence of bacterial biofilm structures in atherosclerotic plaque does not prove that biofilm is the proximate cause of atherosclerosis, it could contribute to the persistent inflammation associated with it. Second, the synergistic relationship between the atherosclerotic infection and the diabetic foot ulcer may ultimately contribute to higher amputation rates in diabetics. DECLARATION OF INTEREST: RAW and RDW have equity interest in PathoGenius, a clinical laboratory using DNA to identify microbes.

Microbiota is a primary cause of pathogenesis of chronic wounds.

OBJECTIVE: Diverse microorganisms present on the surface of chronic wounds have been established to constitute wound microbiota. The aims of this study were to quantify the viability of wound microbiota, classify dispersal of viable microbes from the wound, and determine if human wound microbiota can produce a chronic wound in an animal model. METHOD: Wound microbiotas as units (multiple microbial species acting as one infectious agent) were obtained from well-defined human chronic wounds and seeded onto mouse surgical excision wounds to produce chronically infected wounds that closely resembled the chronic wounds observed in the original hosts. RESULTS: We found the wound microbiota harvested from 35 out of 43 (81%) patients could produce similar chronic wounds (producing slough and exudate) in a murine chronic wound model. The top 30 species present in patient wounds were identified in the mouse wounds by molecular sequencing. Koch's postulates could therefore be applied to establish wound microbiota as the cause of the original human chronic wound infections. Evidence-based medicine criteria such as Hill's criteria for causation can all be satisfied by what is currently known about wound microbiota. CONCLUSION: This study demonstrates that wound microbiota actively disseminates from the chronic wound by forces and mechanisms intrinsic to the wound. Koch's postulates and Hill's criteria for causation together suggest chronic wound microbiota to be the main cause underlying the pathogenesis of chronic wounds. DECLARATION OF INTEREST: RW has an equity interest in PathoGenius Labs. No funding was received for this study.

Recommendations for the management of biofilm: a consensus document.

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.

Economic aspects of biofilm-based wound care in diabetic foot ulcers.

OBJECTIVE: There has been a dramatic rise in the number of chronic wounds globally, which is placing an increased demand on decreasing health-care resources. With significant cuts in health-care budgets, wound care, providers will have to achieve better outcomes quicker and with fewer resources. By using new molecular methods to fully identify wound microbiota, commercially available antimicrobials can be used more efficiently, thereby improving outcomes and decreasing cost. METHOD: This study is a retrospective analysis of patients treated for diabetic foot ulcers (DFU); one group healed DFUs in 2005, the other in 2013. The 2005 patients were treated with standard of care methods common today. The second cohort from 2013 included patients treated using biofilm-based wound management anchored by molecular diagnostics. DNA methods were used to identify individual wound microbiota. Then personalised gels with commercially available antibiotics were applied topically to manage the microorganisms identified. RESULTS: For the 2013 cohort, total charges per patient for the entire course of treatment was $4,756 (total payments $3,060; £1,987). For the 2005 cohort, each patient required treatments that culminated in total charges of $14,690 (total payments $11,444; £7,429). The economic difference per patient from 2013 compared to 2005 was a reduction in total charges of 68% (reduction in total reimbursement of 73%). CONCLUSION: In conjunction with other cohort analysis we previously reported, we feel this economic data demonstrates the benefits not only in wounds healed faster but also more wounds healed at a greatly reduced total cost.

Disrupting the biofilm matrix improves wound healing outcomes.

OBJECTIVE: The most unyielding molecular component of biofilm communities is the matrix structure that it can create around the individual microbes that constitute the biofilm. The type of polymeric substances (polymeric sugars, bacterial proteins, bacterial DNA and even co-opted host substances) are dependent on the microbial species present within the biofilm. The extracellular polymeric substances that make up the matrix give the wound biofilm incredible colony defences against host immunity, host healing and wound care treatments. This polymeric slime layer, which is secreted by bacteria, encases the population of microbes, creating a physical barrier that limits the ingress of treatment agents to the bacteria. The aim of this study was to determine if degrading the wound biofilm matrix would improve wound healing outcomes and if so, if there was a synergy between treating agents that disrupted biofilm defenses with Next Science Wound Gel (wound gel) and cidal agents (topical antibiotics). METHOD: A three-armed randomised controlled trial was designed to determine if standard of care (SOC) was superior to SOC plus wound gel (SOC + gel) and wound gel alone. The wound gel used in this study contains components that directly attack the biofilm extracellular polymeric substance. The gel was applied directly to the wound bed on a Monday-Wednesday-Friday interval, either alone or with SOC topical antibiotics. RESULTS: Using a surrogate endpoint of 50% reduction in wound volume, the results showed that SOC healed at 53%, wound gel healed at 80%, while SOC plus wound gel showed 93% of wounds being successfully treated. CONCLUSION: By directly targeting the wound biofilm matrix, wound healing outcomes are improved.

More effective cell-based therapy through biofilm suppression.

OBJECTIVE: To determine if targeted biofilm suppression of a specific wound improves the efficacy of cell-based therapy (CBT). METHOD: A retrospective study designed to compare a cohort of patients who received CBT in conjunction with biofilm-based wound management with published outcomes from randomised controlled trials in the literature, utilising standard care and CBT. Biofilm-based wound management is the use of multiple simultaneous strategies to suppress wound biofilm, specifically targeting the individual wound's bioburden with agents and methods to suppress the wound biofilm and improve wound-bed preparation.

Molecular diagnostics and personalised medicine in wound care: assessment of outcomes.

OBJECTIVE: This large, level A, retrospective cohort study set out to compare healing outcomes in three large cohorts of wound patients managed universally for bioburden: standard of care group, who were prescribed systemic antibiotics on the basis of empiric and traditional culture-based methodologies; treatment group 1, who were prescribed an improved selection of systemic antibiotics based on the results of molecular diagnostics; treatment group 2 who received personalised topical therapeutics (including antibiotics) based on the results of molecular diagnostics. METHOD: Apart from the differences in diagnostic methods and antibiotic treatments described above, all three cohorts were subjected to the same biofilm-based wound care protocol, which included evaluation of the host and bioburden, frequent sharp debridement, use of wound dressings and comprehensive standard care (reperfusion therapy, nutritional support, offloading, compression and management of comorbidities). RESULTS: In all, 1378 patients were recruited into the study. In the standard of care group 48.5% of patients (244/503) healed completely during the 7-month study period. This increased to 62.4% (298/479) in treatment group 1 and 90.4% (358/396) in treatment group 2. Cox proportional hazards analysis revealed the time to complete closure decreased by 26% in treatment group 1 (p<0.001) and 45.9% in treatment group 2 (p<0.001) compared with the standard of care group. Patients in treatment group 2 had >200% better odds of healing at any given time point compared with the other cohorts. CONCLUSION: Implementation of personalised topical therapeutics guided by molecular diagnosis resulted in statistically and clinically significant improvements in outcome. The integration of molecular diagnostics and personalised medicine provides a directed and targeted approach to wound care. CONFLICT OF INTEREST: SED and RDW are owners of PathoGenius Laboratories, a clinical diagnostic laboratory. SED and RDW are owners of Research and Testing Laboratory, which develops molecular diagnostics. CJ and JK are clinical advisors for PathoGenius. CJ and JK are owners of Southeastern Medical Compounding, Savannah, GA and Southeastern Medical Technologies, Savannah, GA.

Survey of fungi and yeast in polymicrobial infections in chronic wounds.

OBJECTIVE: To assess the incidence, abundance and species diversity of fungi in chronic wounds, as well as to describe the associations of major fungi populations. METHOD: Comprehensive molecular diagnostic reports were evaluated from a total of 915 chronic wounds in a retrospective study. RESULTS: Of the 915 clinical specimens, 208 (23%) were positive for fungal species. These samples were further compared in a compiled dataset, and sub-classified among the four major chronic wound types (decubitus ulcer, diabetic foot ulcer, non-healing surgical wound, and venous leg ulcer). The most abundant fungi were yeasts in the genus Candida; however, Curvularia, Malessezia, Aureobasidium, Cladosporium, Ulocladium, Engodontium and Trichtophyton were also found to be prevalent components of these polymicrobial infections. A notable bacterial/fungal negative correlation was found to be apparent between Staphylococcus and Candida. There were also significant relationships between both bacterial and fungal genera and patient metadata including gender, diabetes status and cardiovascular comorbidities. CONCLUSION: This microbial survey shows that fungi are more important wound pathogens and opportunistic pathogens than previously reported, exemplifying the impact of these under-reported pathogens. With the application of modern cost-effective and comprehensive molecular diagnostics, clinicians can now identify and address this significant component of chronic wound bioburden with targeted therapies, thereby improving healing trajectories.

Production of cell-cell signalling molecules by bacteria isolated from human chronic wounds.

AIM: To (i) identify chronic wound bacteria and to test their ability to produce acyl-homoserine-lactones (AHLs) and autoinducer-2 (AI-2) cell-cell signalling molecules and (ii) determine whether chronic wound debridement samples might contain these molecules. METHODS AND RESULTS: Partial 16S rRNA gene sequencing revealed the identity of 46 chronic wound strains belonging to nine genera. Using bio-reporter assays, 69.6% of the chronic wound strains were inferred to produce AI-2, while 19.6% were inferred to produce AHL molecules. At least one strain from every genus, except those belonging to the genera Acinetobacter and Pseudomonas, were indicated to produce AI-2. Production of AI-2 in batch cultures was growth-phase dependent. Cross-feeding assays demonstrated that AHLs were produced by Acinetobacter spp., Pseudomonas aeruginosa and Serratia marcescens. Independent from studies of the bacterial species isolated from wounds, AHL and/or AI-2 signalling molecules were detected in 21 of 30 debridement samples of unknown microbial composition. CONCLUSION: Chronic wound bacteria produce cell-cell signalling molecules. Based on our findings, we hypothesize that resident species generally produce AI-2 molecules, and aggressive transient species associated with chronic wounds typically produce AHLs. Both these classes of cell-cell signals are indicated to be present in human chronic wounds. SIGNIFICANCE AND IMPACT OF THE STUDY: Interbacterial cell-cell signalling may be an important factor influencing wound development and if this is the case, the presence of AHLs and AI-2 could be used as a predictor of wound severity. Manipulation of cell-cell signalling may provide a novel strategy for improving wound healing.

Molecular diagnosis of Raoultella planticola infection of a surgical site.

Raoultella planticola has been rarely diagnosed in clinical specimens. A case of a polymicrobial surgical site infection primarily caused by R. planticola in a 66-year-old Caucasian male with a fractured left tibia after an open reduction internal fixation of his left ankle is described and confirms this organism to be an opportunistic human pathogen. This pathogen was diagnosed with rapid clinical molecular pathogen diagnostic methods, which allowed an appropriate therapy to be implemented, thereby improving prognosis.

Healing and healing rates of chronic wounds in the age of molecular pathogen diagnostics.

OBJECTIVE: To compare healing outcomes at a wound healing centre both before and after the introduction of molecular pathogen diagnostics. METHOD: An IT consultant was recruited to analyse the medical records of patients at a wound healing centre, comparing patient groups from 2007 and 2009 - before and after the introduction of comprehensive molecular pathogen diagnostic methods. RESULTS: Before the implementation of molecular diagnostics, 244/503 patients (48.5%) healed completely, while after implementation 298/479 patients (62.4%) healed. Furthermore, based on survival analysis and after controlling for potential confounding factors, time to healing was significantly shorter in 2009 than 2007 (p<0.05). Specifically, biofilm-based wound care, along with the implementation of comprehensive molecular diagnostics for venous leg ulcers, pressure ulcers and diabetic foot ulcers and all wounds combined showed, respectively, 21%, 23%, 25% and 22% reductions in the time to healing. In addition, after implementing molecular diagnostics, the use of expensive fi rst-line antibiotics also declined in 2009, while a broader range of targeted antibiotics was used. CONCLUSION: The results of modern molecular pathogen diagnostic applications allow comprehensive evaluation of the microbial bioburden in chronic wounds. This comprehensive diagnostic in turn has led to a more precise and targeted therapeutic approach to wound care. With the comprehensive nature of molecular diagnostics future advances in topical patient specific therapeutics are now possible.

Chronic wounds and the medical biofilm paradigm.

There is a growing recognition that biofilms are the principal cause of wound chronicity. The development of treatments for wound biofilms raises the prospect that chronic wounds can be treated, potentially saving many patients' lives.

Biofilm maturity studies indicate sharp debridement opens a time- dependent therapeutic window.

OBJECTIVE: To investigate the hypothesis that newly formed wound biofilms (or bioburdens) are more susceptible to antimicrobial treatment. METHOD: Four separate and distinct models were performed by four separate biofilm research laboratories to evaluate the resistance of biofilms to antimicrobial treatments over time. These included a drip-flow biofilm model along with a hydrodebridement study, a porcine skin punch biopsy ex vivo model, a mouse chronic wound model and clinical longitudinal debridement study. RESULTS: All four models showed that, within the first 24 hours, the biofilm community was more susceptible to the selected antibiotics, and after maturing for up to 48 hours became increasingly tolerant. In each model, there was at least a 24-hour period in which the biofilms were more resistant to antibiotics. Each of the models utilised showed a significant decrease in the resistance of the biofilm/ burden to gentamicin for up to 24 hours with a confidence interval of at least 95%. The resistance increased in each of the models by 48 hours and reached original resistance levels by 72 hours. CONCLUSION: These data suggest the principles of biofilm-based wound care, along with the use of serial debridement to continually remove mature biofilm, followed by biofilm wound management strategies, including topical antibiotics while the bioburden is still immature and more susceptible, are valid.

Ethanol exhibits specificity in its effects on differentiation of hematopoietic progenitors.

Ethanol is a known teratogen but the mechanisms by which this simple compound affects fetal development remain unresolved. The goal of the current study was to determine the mechanism by which ethanol affects lymphoid differentiation using an in vitro model of ethanol exposure. Primitive hematopoietic oligoclonal-neonatal-progenitor cells (ONP), with the phenotype Lin(-)HSA(lo)CD43(lo)Sca-1(-)c-Kit(+) that are present in neonatal but not adult bone marrow were sorted from the bone marrow of 2-week-old C57BL/6J mice and cultured under conditions that favor either B cell or myeloid cell differentiation with or without addition of ethanol. The overall growth of the ONP cells was not significantly affected by inclusion of up to 100mM ethanol in the culture medium. However, the differentiation of the progenitor cells along the B-cell pathway was significantly impaired by ethanol in a dose-dependent manner. Exposure of ONP cells to 100mM ethanol resulted in greater than 95% inhibition of B cell differentiation. Conversely, ethanol concentrations up to and including 100mM had no significant effect on differentiation along the myeloid pathway. The effect of ethanol on transcription factor expression was consistent with the effects on differentiation. ONP cells grown in 100mM ethanol failed to upregulate Pax5 and EBF, transcriptional regulators that are necessary for B cell development. However, ethanol had no significant effect on the upregulation of PU.1, a transcription factor that, when expressed in high concentration, favors myeloid cell development. Taken together, these results suggest that ethanol has specificity in its effects on differentiation of hematopoietic progenitors.

Propagation of anaerobic bacteria within an aerobic multi-species chronic wound biofilm model.

OBJECTIVE: Most chronic wound biofilms have been shown to have significant populations of anaerobes. In order to better screen antimicrobial and antibiofilm therapeutics, we evaluated the ability of key anaerobes to incorporate and propagate within our aerobic chronic wound biofilm. METHOD: We had previously developed a rapid model to simulate polymicrobial chronic wound biofilms. This model incorporated meticillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE) and Pseudomonas aeruginosa. The model was used along with a variety of anaerobes to determine whether this biofilm model would support propagation of anaerobes similar to that we have identified in chronic wounds. RESULTS: Using our previously defined Lubbock Chronic Wound Biofilm (LCWB) model combined with quantitative PCR, anaerobic bacteria were shown to proliferate through integration into the biofilm under aerobic conditions. Using electron microscopy we show close association between aerobes and anaerobes within the biofilm suggesting a synergistic relationship. CONCLUSION: We have expanded the utility of the LCBW to show the ability of clinically significant anaerobic bacteria to thrive in aerobic conditions. The expansion of this model can further simulate the functional characteristics of chronic wound pathogenic biofilms and the species that dwell within them allowing improved ability to evaluate therapeutics that target anaerobes.

Regular debridement is the main tool for maintaining a healthy wound bed in most chronic wounds.

Sharp debridement is the most clinically and cost-effective way of physically removing and suppressing a biofilm. Continued debridement, as part of a multifaceted treatment strategy, will keep the biofilm in a weakened state.

Identification of yeast in chronic wounds using new pathogen-detection technologies.

OBJECTIVE: To evaluate the ability of two new diagnostic methods to detect and accurately identify yeast associated with chronic wound infections. METHOD: Fungal tag-encoded FLX amplicon pyrosequencing (fTEFAP), a universal fungal identification method, bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP), a universal bacterial identification method, and a new quantitative polymerase chain reaction (qPCR) wound pathogen panel were used to evaluate three chronic wounds suspected to contain yeast. RESULTS: Forty wound samples were analysed in addition to the three samples suspected of containing yeast. The qPCR panel, which targets Candida albicans, detected this yeast in two of the three wound samples. In contrast, fTEFAP detected yeast in each of the three samples: two showed Candida albicans and the third Candida parapsilosis. fTEFAP also identified a lower level of Candida tropicalis in one of the wounds that was positive for Candida albicans. The qPCR wound panel results were returned within two hours, while the fTEFAP results were returned within 24 hours. CONCLUSION: Two new molecular methods have been developed to aid wound pathogen diagnostics. The quantitative PCR wound panel is rapid but is limited to major wound-associated bacteria and yeasts. The universal fTEFAP and bTEFAP methods take 24 hours to return results but are able to detect the relative contribution of any bacteria of yeast in a chronic wound diagnostic sample. DECLARATION OF INTEREST: Southwest Regional Wound Care Center is a clinical wound-care provider seeking to improve the ability of wound care practitioners to help patients. The Research and Testing Laboratory develops molecular methods including fTEFAP, bTEFAP and the quantitative PCR wound panel.

Bacterial diversity in surgical site infections: not just aerobic cocci any more.

OBJECTIVE: To evaluate the microbial diversity in chronic surgical site infections (SSIs). METHOD: Bacterial populations in 23 chronic SSIs were identified using bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP),which is an universal bacterial identification method.These results were then validated using quantitative polymerase chain reaction (qPCR). RESULTS: bTEFAP identified two previously uncharacterised Bacteroidales in all of the SSIs and showed that it was the predominant population in the majority of these chronic wounds. Other bacteria identified included Corynebacterium spp., Peptoniphilus spp., Staphylococcus spp., Staphylococcus aureus, Serratia marcescens, Prevotella spp. and Pseudomonas aeruginosa. Rarefaction analysis of the data indicated that, on average, six genera occurred in any given SSI, suggesting that such infections are multispecies. On average, over 60% of the bacteria evaluated in the SSIs were anaerobic bacilli. The previous literature indicates that aerobic cocci predominate in such wounds. CONCLUSION: This modern molecular survey indicates that our previous understanding of which bacteria cause SSIs may be faulty. The high prevalence of anaerobic bacilli and the overwhelming predominance of two previously uncharacterised Bacteroidales suggest that such bacteria may be a leading contributor to such infections. Further research on the identification and treatment of such bacteria are warranted.