Early clinical performance of an adaptive self-assembling barrier scaffold in nonhealing chronic wounds: a review of six cases

INTRODUCTION: Chronic nonhealing wounds pose a serious concern for patient health and the health care system. Management of chronic wounds becomes especially challenging in the setting of systemic comorbidities and patient nonadherence. OBJECTIVE: Authors evaluated the performance of a proprietary adaptive self-assembling barrier scaffold (aSABS) in the management and healing of complex chronic wounds. MATERIALS AND METHODS: Six patients with anatomically and etiologically diverse chronic wounds were considered for treatment with aSABS, which is for prescription use under the supervision of a licensed health care professional. The wounds had been unresponsive to various treatment regimens for 8 weeks to more than 20 years. The adaptive self-assembling barrier scaffold was applied in the clinic weekly, with the exception of 1 case in which it was applied every 2 weeks. Institutional Review Board approval was not required because use of aSABS was in accordance with the US Food and Drug Administration-cleared indications for use. RESULTS: After only 3 to 6 applications of aSABS, these wounds showed notable improvement in healing, accompanied by suppression of both inflammation and infection, granulation tissue formation, and reepithelialization. The adaptive self-assembling barrier scaffold also facilitated aggressive debridement to remove inflamed, infected, and necrotic tissues, providing effective wound management and bleeding control while functioning as a protective barrier. Furthermore, use of aSABS reduced the at-home burden of wound care for patients and caretakers. Additionally, use of this aSABS may offer clinicians an alternative to high acuity operating rooms by facilitating debridement and management of some complex wounds in a low acuity outpatient clinic setting-a particularly crucial product attribute during the COVID-19 pandemic that helped ensure timely and effective treatment. CONCLUSIONS: In this study, aSABS demonstrated clinical benefit in a short period of time in patients with significant comorbidities and nonhealing wounds. Use of aSABS may offer clinicians an alternative to high-acuity operating rooms by facilitating debridement and management of some complex wounds in a low-acuity outpatient clinic setting. These outcomes can be used to make a compelling argument for use of aSABS as a central aspect of treatment at the onset of wound care and as a rescue product for wounds for which prior standard and advanced treatment protocols were unsuccessful.

Urethral Catheter Biofilms Reveal Plasticity in Bacterial Composition and Metabolism and Withstand Host Immune Defenses in Hypoxic Environment.

Biofilms composed of multiple microorganisms colonize the surfaces of indwelling urethral catheters that are used serially by neurogenic bladder patients and cause chronic infections. Well-adapted pathogens in this niche are Escherichia coli, Proteus, and Enterococcus spp., species that cycle through adhesion and multilayered cell growth, trigger host immune responses, are starved off nutrients, and then disperse. Viable microbial foci retained in the urinary tract recolonize catheter surfaces. The molecular adaptations of bacteria in catheter biofilms (CBs) are not well-understood, promising new insights into this pathology based on host and microbial meta-omics analyses from clinical specimens. We examined catheters from nine neurogenic bladder patients longitudinally over up to 6 months. Taxonomic analyses from 16S rRNA gene sequencing and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics revealed that 95% of all catheter and corresponding urinary pellet (UP) samples contained bacteria. CB biomasses were dominated by Enterobacteriaceae spp. and often accompanied by lactic acid and anaerobic bacteria. Systemic antibiotic drug treatments of patients resulted in either transient or lasting microbial community perturbations. Neutrophil effector proteins were abundant not only in UP but also CB samples, indicating their penetration of biofilm surfaces. In the context of one patient who advanced to a kidney infection, Proteus mirabilis proteomic data suggested a combination of factors associated with this disease complication: CB biomasses were high; the bacteria produced urease alkalinizing the pH and triggering urinary salt deposition on luminal catheter surfaces; P. mirabilis utilized energy-producing respiratory systems more than in CBs from other patients. The NADH:quinone oxidoreductase II (Nqr), a Na+ translocating enzyme not operating as a proton pump, and the nitrate reductase A (Nar) equipped the pathogen with electron transport chains promoting growth under hypoxic conditions. Both P. mirabilis and E. coli featured repertoires of transition metal ion acquisition systems in response to human host-mediated iron and zinc sequestration. We discovered a new drug target, the Nqr respiratory system, whose deactivation may compromise P. mirabilis growth in a basic pH milieu. Animal models would not allow such molecular-level insights into polymicrobial biofilm metabolism and interactions because the complexity cannot be replicated.

Biofilm exacerbates antibiotic resistance: Is this a current oversight in antimicrobial stewardship?

OBJECTIVE: To raise awareness of the role of environmental biofilm in the emergence and spread of antibiotic resistance and its consideration in antimicrobial stewardship. BACKGROUND: Antibiotic resistance is a major threat to public health. Overuse of antibiotics, increased international travel, and genetic promiscuity amongst bacteria have contributed to antibiotic resistance, and global containment efforts have so far met with limited success. Antibiotic resistance is a natural mechanism by which bacteria have adapted to environmental threats over billions of years and is caused either by genetic mutations or by horizontal gene transfer. Another ancient survival strategy involves bacteria existing within a self-produced polymeric matrix, which today is termed biofilm. Biofilm similarly enables bacterial tolerance to environmental threats, and also encourages the transfer of antibiotic resistance genes between bacterial species. This natural and ubiquitous mode of bacterial life has not been considered amongst strategies to tackle antibiotic resistance in healthcare facilities, despite its ability to significantly enhance bacterial survival and persistence, and to encourage antibiotic resistance. CONCLUSION: Biofilm must be considered synonymously with antibiotic resistance because of its proficiency in transferring resistance genes as well as its innate phenotypic tolerance to antibiotics. Although biofilm falls outside of the current definition of antimicrobial stewardship, greater awareness of the existence, ubiquity, and consequences of environmental biofilm amongst healthcare practitioners is crucial to improving hygiene practices and controlling the emergence and spread of antibiotic resistance in healthcare facilities.

A review of iodine-based compounds, with a focus on biofilms: results of an expert panel.

Biofilms play a central role in the chronicity of non-healing lesions such as venous leg ulcers and diabetic foot ulcers. Therefore, biofilm management and treatment is now considered an essential part of wound care. Many antimicrobial treatments, whether topical or systemic, have been shown to have limited efficacy in the treatment of biofilm phenotypes. The antimicrobial properties of iodine compounds rely on multiple and diverse interactions to exert their effects on microorganisms. An expert panel, held in Las Vegas during the autumn Symposium on Advanced Wound Care meeting in 2018, discussed these properties, with the focus on iodine and iodophors and their effects on biofilm prevention and treatment.

Patient genetics is linked to chronic wound microbiome composition and healing.

The clinical importance of microbiomes to the chronicity of wounds is widely appreciated, yet little is understood about patient-specific processes shaping wound microbiome composition. Here, a two-cohort microbiome-genome wide association study is presented through which patient genomic loci associated with chronic wound microbiome diversity were identified. Further investigation revealed that alternative TLN2 and ZNF521 genotypes explained significant inter-patient variation in relative abundance of two key pathogens, Pseudomonas aeruginosa and Staphylococcus epidermidis. Wound diversity was lowest in Pseudomonas aeruginosa infected wounds, and decreasing wound diversity had a significant negative linear relationship with healing rate. In addition to microbiome characteristics, age, diabetic status, and genetic ancestry all significantly influenced healing. Using structural equation modeling to identify common variance among SNPs, six loci were sufficient to explain 53% of variation in wound microbiome diversity, which was a 10% increase over traditional multiple regression. Focusing on TLN2, genotype at rs8031916 explained expression differences of alternative transcripts that differ in inclusion of important focal adhesion binding domains. Such differences are hypothesized to relate to wound microbiomes and healing through effects on bacterial exploitation of focal adhesions and/or cellular migration. Related, other associated loci were functionally enriched, often with roles in cytoskeletal dynamics. This study, being the first to identify patient genetic determinants for wound microbiomes and healing, implicates genetic variation determining cellular adhesion phenotypes as important drivers of infection type. The identification of predictive biomarkers for chronic wound microbiomes may serve as risk factors and guide treatment by informing patient-specific tendencies of infection.

Defying hard-to-heal wounds with an early antibiofilm intervention strategy: 'wound hygiene'.

Biofilm has been implicated as a barrier to wound healing and it is widely accepted that the majority of wounds not following a normal healing trajectory contain biofilm. Therefore, strategies that inform and engage clinicians to reduce biofilm and optimise the wound tissue environment to enable wound progression are of interest to wound care providers. In March 2019, an advisory board was convened where experts considered the barriers and opportunities to drive a broader adoption of a biofilm-based approach to wound care. Poor clarity and articulation of wound terminology were identified as likely barriers to clinical adoption of rigorous and proactive microbial decontamination that is supportive of wound healing advancement. A transition to an intuitive term such as 'wound hygiene' was proposed to communicate a comprehensive wound decontamination plan with an associated message of expected habitual routine. 'Wound hygiene', is a relatable concept that supports meticulous wound practice that addresses barriers to wound healing, such as biofilm, while aligning with antimicrobial stewardship programmes.

Aerococcus urinae and Globicatella sanguinis Persist in Polymicrobial Urethral Catheter Biofilms Examined in Longitudinal Profiles at the Proteomic Level.

Aerococcus urinae (Au) and Globicatella sanguinis (Gs) are gram-positive bacteria belonging to the family Aerococcaceae and colonize the human immunocompromised and catheterized urinary tract. We identified both pathogens in polymicrobial urethral catheter biofilms (CBs) with a combination of 16S rDNA sequencing, proteomic analyses, and microbial cultures. Longitudinal sampling of biofilms from serially replaced catheters revealed that each species persisted in the urinary tract of a patient in cohabitation with 1 or more gram-negative uropathogens. The Gs and Au proteomes revealed active glycolytic, heterolactic fermentation, and peptide catabolic energy metabolism pathways in an anaerobic milieu. A few phosphotransferase system (PTS)-based sugar uptake and oligopeptide ABC transport systems were highly expressed, indicating adaptations to the supply of nutrients in urine and from exfoliating squamous epithelial and urothelial cells. Differences in the Au vs Gs metabolisms pertained to citrate lyase and utilization and storage of glycogen (evident only in Gs proteomes) and to the enzyme Xfp that degrades d-xylulose-5'-phosphate and the biosynthetic pathways for 2 protein cofactors, pyridoxal 6'-phosphate and 4'-phosphopantothenate (expressed only in Au proteomes). A predicted ZnuA-like transition metal ion uptake system was identified for Gs while Au expressed 2 LPXTG-anchored surface proteins, one of which had a predicted pilin D adhesion motif. While these proteins may contribute to fitness and virulence in the human host, it cannot be ruled out that Au and Gs fill a niche in polymicrobial biofilms without being the direct cause of injury in urothelial tissues.

Chronic wound microbiome colonization on mouse model following cryogenic preservation.

Chronic wound infections are increasingly recognized to be dynamic and polymicrobial in nature, necessitating the development of wound models which reflect the complexities of infection in a non-healing wound. Wound slough isolated from human chronic wounds and transferred to mice was recently shown to create polymicrobial infection in mice, and there is potential this tool may be improved by cryogenic preservation. The purpose of this study was to investigate the application of cryogenic preservation to transferring polymicrobial communities, specifically by quantifying the effects of cryopreservation and wound microbiome transplantation. Slough from an established murine polymicrobial surgical excision model and five patients were subjected to three preservation strategies: refrigeration until infection, freezing in liquid nitrogen, or freezing in liquid nitrogen with glycerol solution prior to infection in individual mice. Four days following inoculation onto mice, wound microbiota were quantified using either culture isolation or by 16s rRNA gene community profiling and quantitative PCR. Cryogenic preservation did not significantly reduce bacterial viability. Reestablished microbial communities were significantly associated with patient of origin as well as host context (i.e., originally preserved from a patient versus mouse infection). Whereas preservation treatment did not significantly shape community composition, the transfers of microbiomes from human to mouse were characterized by reduced diversity and compositional changes. These findings indicated that changes should be expected to occur to community structure after colonization, and that compositional change is likely due to the rapid change in infection context as opposed to preservation strategy. Furthermore, species that were present in higher relative abundance in wound inoculate were more likely to colonize subsequent wounds, and wound inoculate with higher bacterial load established wound communities that were more compositionally similar. Results inform expectations for the complementation of chronic wound in vivo modeling with cryogenic preservation archives.

Fungal Diversity and Onychomycosis An Analysis of 8,816 Toenail Samples Using Quantitative PCR and Next-Generation Sequencing.

BACKGROUND: Onychomycosis is a fungal infection of the nail that is often recalcitrant to treatment and prone to relapse. Traditional potassium hydroxide and culture diagnosis is costly and time-consuming. Therefore, molecular methods were investigated to demonstrate effectiveness in diagnosis and to quantify the microbial flora present that may be contributing to disease. METHODS: A total of 8,816 clinically suspicious toenail samples were collected by podiatric physicians across the United States from patients aged 0 to 103 years and compared with a control population (N = 20). Next-generation sequencing and quantitative polymerase chain reaction were used to identify and quantify dermatophytes, nondermatophyte molds, and bacteria. RESULTS: Approximately 50% of suspicious toenails contained both fungi and bacteria, with the dermatophyte Trichophyton rubrum contributing the highest relative abundance and presence in 40% of these samples. Of the remaining 50% of samples, 34% had bacterial species present and 16% had neither. Fungi only were present in less than 1% of samples. Nondermatophyte molds contributed to 11.0% of occurrences in fungus-positive samples. All of the control samples were negative for fungi, with commensal bacterial species composing most of the flora population. CONCLUSIONS: Molecular methods were successful in efficiently quantifying microbial and mycologic presence in the nail. Contributions from dermatophytes were lower than expected, whereas the opposite was true for nondermatophyte molds. The clinical significance of these results is currently unknown.

Bacterial fitness in chronic wounds appears to be mediated by the capacity for high-density growth, not virulence or biofilm functions.

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.

Improving the quality of clinical research on chronic wound infection treatment: expert-based recommendations.

OBJECTIVE:: To produce recommendations for the design of reliable and informative clinical investigations in chronic wound infection. METHOD:: A multidisciplinary panel of international experts from four countries (Italy, UK, Ireland and the US) were involved in a detailed, semi-structured discussion on how to better select and describe a target population, interventions and outcomes, and which infection-related criteria to apply in order to achieve a high-quality trial. Consent among the experts was measured using the Delphi method and GRADE Working Group suggestions. The project was fully supported by AISLeC 2016 (Italian Nursing Society for Wound Care Study). RESULTS:: In total, 37 recommendations achieved substantial agreement among the experts; 10 concerned the most appropriate description and selection of a target population, four related to interventions and 15 to outcomes. A further eight statements about critical methodological points were approved. CONCLUSION:: Developing recommendations in a systematic manner through a representative group of experts could generate tools for improving the design of clinical trials in this challenging area.

Efficacy of hyperbaric oxygen therapy in bacterial biofilm eradication.

OBJECTIVE: Chronic wounds typically require several concurrent therapies, such as debridement, pressure offloading, and systemic and/or topical antibiotics. The aim of this study was to examine the efficacy of hyperbaric oxygen therapy (HBOT) towards reducing or eliminating bacterial biofilms in vitro and in vivo. METHOD: Efficacy was determined using in vitro grown biofilms subjected directly to HBOT for 30, 60 and 90 minutes, followed by cell viability determination using propidium monoazide-polymerase chain reaction (PMA-PCR). The efficacy of HBOT in vivo was studied by searching our chronic patient wound database and comparing time-to-healing between patients who did and did not receive HBOT as part of their treatment. RESULTS: In vitro data showed small but significant decreases in cell viability at the 30- and 90-minute time points in the HBOT group. The in vivo data showed reductions in bacterial load for patients who underwent HBOT, and ~1 week shorter treatment durations. Additionally, in patients' chronic wounds there was a considerable emergence of anaerobic bacteria and fungi between intermittent HBOT treatments. CONCLUSION: The data demonstrate that HBOT does possess a certain degree of biofilm killing capability. Moreover, as an adjuvant to standard treatment, more favourable patient outcomes are achieved through a quicker time-to-healing which reduces the chance of complications. Furthermore, the data provided insights into biofilm adaptations to challenges presented by this treatment strategy which should be kept in mind when treating chronic wounds. Further studies will be necessary to evaluate the benefits and mechanisms of HBOT, not only for patients with chronic wounds but other chronic infections caused by bacterial biofilms.

Consensus guidelines for the identification and treatment of biofilms in chronic nonhealing wounds.

BACKGROUND: Despite a growing consensus that biofilms contribute to a delay in the healing of chronic wounds, conflicting evidence pertaining to their identification and management can lead to uncertainty regarding treatment. This, in part, has been driven by reliance on in vitro data or animal models, which may not directly correlate to clinical evidence on the importance of biofilms. Limited data presented in human studies have further contributed to the uncertainty. Guidelines for care of chronic wounds with a focus on biofilms are needed to help aid the identification and management of biofilms, providing a clinical focus to support clinicians in improving patient care through evidence-based medicine. METHODS: A Global Wound Biofilm Expert Panel, comprising 10 clinicians and researchers with expertise in laboratory and clinical aspects of biofilms, was identified and convened. A modified Delphi process, based on published scientific data and expert opinion, was used to develop consensus statements that could help identify and treat biofilms as part of the management of chronic nonhealing wounds. Using an electronic survey, panel members rated their agreement with statements about biofilm identification and treatment, and the management of chronic nonhealing wounds. Final consensus statements were agreed on in a face-to-face meeting. RESULTS: Participants reached consensus on 61 statements in the following topic areas: understanding biofilms and the problems they cause clinicians; current diagnostic options; clinical indicators of biofilms; future options for diagnostic tests; treatment strategies; mechanical debridement; topical antiseptics; screening antibiofilm agents; and levels of evidence when choosing antibiofilm treatments. CONCLUSION: This consensus document attempts to clarify misunderstandings about the role of biofilms in clinical practice, and provides a basis for clinicians to recognize biofilms in chronic nonhealing wounds and manage patients optimally. A new paradigm for wound care, based on a stepped-down treatment approach, was derived from the consensus statements.

Planktonic Growth of Pseudomonas aeruginosa around a Dual-Species Biofilm Supports the Growth of Fusobacterium nucleatum within That Biofilm.

PURPOSE: The goal of this study was to understand the potential interaction between Pseudomonas aeruginosa and Fusobacterium nucleatum within the middle ear. METHODS: We examined the microbiota of ear fluid and tympanostomy tubes (TTs) obtained from patients with posttympanostomy tube otorrhea. We also examined biofilms formed by P. aeruginosa and F. nucleatum, singly or together, under aerobic or anaerobic conditions. RESULTS: While the facultative anaerobe P. aeruginosa dominated the bacterial population within the ear fluid, strict anaerobes, including F. nucleatum, dominated bacterial populations within the TTs. F. nucleatum was able to grow under aerobic conditions only in the presence of P. aeruginosa, whose growth reduced the level of dissolved oxygen within the broth to nearly anoxic condition within 4 h after inoculation. The presence of P. aeruginosa allowed F. nucleatum to maintain its growth for 72 h within the dual-species biofilm but not within the planktonic growth. Visualization of the biofilms revealed coaggregation of P. aeruginosa and F. nucleatum. CONCLUSION: Extrapolation of these results suggests that, within the middle ear fluid, the growth of P. aeruginosa produces the anaerobic conditions required for the growth of F. nucleatum, both within effusion and within biofilms.

Temporal dynamics of relative abundances and bacterial succession in chronic wound communities.

Polymicrobial bacterial infection is an important factor contributing to wound chronicity. Consequently, clinicians frequently adopt a biofilm-based wound care approach, in which wounds are treated utilizing DNA sequencing information about microbial communities. While more successful than treatment not using community information, there is little information about temporal dynamics of wound communities and optimal approaches over the course of treatment. To characterize these dynamics, temporal analysis over three sampling points was conducted for 167 chronic wounds. Across sampling intervals, wound communities from the same patients changed in composition, and most commonly shared less than 50% of observed species. There was a significant relationship between community similarity and time between sampling. Classifying wounds into state types, we found that communities frequently transitioned from Pseudomonas or Staphylococcus dominated, into a highly variable state type. Although low abundance microbial species are typically disregarded due to uncertainty of biological importance, we found that 80% of wound microbiomes included common or dominant species at subsequent time points that were in low abundance in earlier samples. Moreover, these species were often those known to frequently infect wounds. Results document compositional shifts through the course of treatment and suggest that routine consideration of low abundance species may improve biofilm-based wound care. Moreover, findings indicate that integrating ecological modeling to understand wound microbiome succession may lead to more informed therapy.

A post-planktonic era of in vitro infectious models: issues and changes addressed by a clinically relevant wound like media.

Medical science is pitted against an ever-increasing rise in antibiotic tolerant microorganisms. Concurrently, during the past decade, biofilms have garnered much attention within research and clinical practice. Although the significance of clinical biofilms is becoming very apparent, current methods for diagnostics and direction of therapy plans in many hospitals do not reflect this knowledge; with many of the present tools proving to be inadequate for accurately mimicking the biofilm phenomenon. Based on current findings, we address some of the fundamental issues overlooked by clinical labs: the paradigm shifts that need to occur in assessing chronic wounds; better simulation of physiological conditions in vitro; and the importance of incorporating polymicrobial populations into biofilm models. In addition, this review considers using a biofilm relevant in vitro model for cultivating and determining the antibiotic tolerance and susceptibility of microorganisms associated with chronic wounds. This model presents itself as a highly rapid and functional tool that can be utilized by hospitals in an aim to improve bedside treatments.