Wound Biofilm Research Articles

N-Acetyl-cysteine and Mechanisms Involved in Resolution of Chronic Wound Biofilm

Chronic wounds are a major global health problem with the presence of biofilm significantly contributing to wound chronicity. Current treatments are ineffective in resolving biofilm and simultaneously killing the bacteria; therefore, effective biofilm-resolving drugs are needed. We have previously shown that, together with α-tocopherol, N-acetyl-cysteine (NAC) significantly improves the healing of biofilm-containing chronic wounds, in a diabetic mouse model we developed, by causing disappearance of the bacteria and breakdown of the extracellular polymeric substance (EPS). We hypothesize that NAC creates a microenvironment that affects bacterial survival and EPS integrity. To test this hypothesis, we developed an in vitro biofilm system using microbiome taken directly from diabetic mouse chronic wounds. For these studies, we chose mice in which chronic wound microbiome was rich in Pseudomonas aeruginosa (97%). We show that NAC at concentrations with pH < pKa causes bacterial cell death and breakdown of EPS. When used before biofilm is formed, NAC leads to bacterial cell death whereas treatment after the biofilm is established NAC causes biofilm dismantling accompanied by bacterial cell death. Mechanistically, we show that NAC can penetrate the bacterial membrane, increase oxidative stress, and halt protein synthesis. We also show that low pH is important for the actions of NAC and that bacterial death occurs independently of the presence of biofilm. In addition, we show that both the acetyl and carboxylic groups play key roles in NAC functions. The results presented here provide insight into the mechanisms by which NAC dismantles biofilm and how it could be used to treat chronic wounds after debridement (NAC applied at the start of culture) or without debridement (NAC applied when biofilm is already formed). This approach can be taken to develop biofilm from microbiome taken directly from human chronic wounds to test molecules that could be effective for the treatment of specific biofilm compositions.

The efficacy of topical agents used in wounds for managing chronic biofilm infections: A systematic review

ObjectivesClinicians have increasingly adopted the widespread use of topical agents to manage chronic wound infections, despite limited data on their effectiveness in vivo. This study sought to evaluate the evidence for commonly employed topical agents used in wounds for the purpose of treating chronic infections caused by biofilm. MethodWe included in vitro, animal and human in vivo studies where topical agents were tested for their efficacy against biofilms, for use in wound care. For human studies, we only included those which utilised appropriate identification techniques for visualising and confirming the presence of biofilms. ResultA total of 640 articles were identified, with 43 included after meeting eligibility. In vitro testing accounted for 90% (n = 39) of all included studies, five studies using animal models and three human in vivo studies. Sixteen different laboratory models were utilised, with the most frequent being the minimum biofilm eradication concentration (MBEC™) / well plate assay (38%, n = 15 of 39). A total of 44 commercially available topical agents were grouped into twelve categories with the most commonly tested agents being silver, iodine and polyhexamethylene biguanide (PHMB). In vitro results on efficacy demonstrated iodine as having the highest mean log10 reductions of all agents (4.81, ±3.14). ConclusionThere is large disparity in the translation of laboratory studies to researchers undertaking human trials relating to the effectiveness of commercially available topical agents. There is insufficient human in vivo evidence to definitively recommend any commercially available topical agent over another for the treatment of chronic wound biofilms. The heterogeneity identified between study designs (in vitro to in vivo) further limits the generalisability of results.

Honey can inhibit and eliminate biofilms produced by Pseudomonas aeruginosa

Chronic wound treatment is becoming increasingly difficult and costly, further exacerbated when wounds become infected. Bacterial biofilms cause most chronic wound infections and are notoriously resistant to antibiotic treatments. The need for new approaches to combat polymicrobial biofilms in chronic wounds combined with the growing antimicrobial resistance crisis means that honey is being revisited as a treatment option due to its broad-spectrum antimicrobial activity and low propensity for bacterial resistance. We assessed four well-characterised New Zealand honeys, quantified for their key antibacterial components, methylglyoxal, hydrogen peroxide and sugar, for their capacity to prevent and eradicate biofilms produced by the common wound pathogen Pseudomonas aeruginosa. We demonstrate that: (1) honey used at substantially lower concentrations compared to those found in honey-based wound dressings inhibited P. aeruginosa biofilm formation and significantly reduced established biofilms; (2) the anti-biofilm effect of honey was largely driven by its sugar component; (3) cells recovered from biofilms treated with sub-inhibitory honey concentrations had slightly increased tolerance to honey; and (4) honey used at clinically obtainable concentrations completely eradicated established P. aeruginosa biofilms. These results, together with their broad antimicrobial spectrum, demonstrate that manuka honey-based wound dressings are a promising treatment for infected chronic wounds, including those with P. aeruginosa biofilms.

An in vitro collagen perfusion wound biofilm model; with applications for antimicrobial studies and microbial metabolomics

BackgroundThe majority of in vitro studies of medically relevant biofilms involve the development of biofilm on an inanimate solid surface. However, infection in vivo consists of biofilm growth on, or suspended within, the semi-solid matrix of the tissue, whereby current models do not effectively simulate the nature of the in vivo environment. This paper describes development of an in vitro method for culturing wound associated microorganisms in a system that combines a semi-solid collagen gel matrix with continuous flow of simulated wound fluid. This enables culture of wound associated reproducible steady state biofilms under conditions that more closely simulate the dynamic wound environment. To demonstrate the use of this model the antimicrobial kinetics of ceftazidime, against both mature and developing Pseudomonas aeruginosa biofilms, was assessed. In addition, we have shown the potential application of this model system for investigating microbial metabolomics by employing selected ion flow tube mass spectrometry (SIFT-MS) to monitor ammonia and hydrogen cyanide production by Pseudomonas aeruginosa biofilms in real-time.ResultsThe collagen wound biofilm model facilitates growth of steady-state reproducible Pseudomonas aeruginosa biofilms under wound like conditions. A maximum biofilm density of 1010 cfu slide− 1 was achieved by 30 h of continuous culture and maintained throughout the remainder of the experiment. Treatment with ceftazidime at a clinically relevant dose resulted in a 1.2–1.6 log reduction in biofilm density at 72 h compared to untreated controls. Treatment resulted in loss of complex biofilm architecture and morphological changes to bacterial cells, visualised using confocal microscopy. When monitoring the biofilms using SIFT-MS, ammonia and hydrogen cyanide levels peaked at 12 h at 2273 ppb (±826.4) and 138 ppb (±49.1) respectively and were detectable throughout experimentation.ConclusionsThe collagen wound biofilm model has been developed to facilitate growth of reproducible biofilms under wound-like conditions. We have successfully used this method to: (1) evaluate antimicrobial efficacy and kinetics, clearly demonstrating the development of antimicrobial tolerance in biofilm cultures; (2) characterise volatile metabolite production by P. aeruginosa biofilms, demonstrating the potential use of this method in metabolomics studies.

Chronic wound biofilms: diagnosis and therapeutic strategies.

Objective:To review the diagnosis of chronic wound biofilms and discuss current treatment approaches.Data sources:Articles included in this review were obtained from the following databases: Wanfang, China National Knowledge Infrastructure, PubMed, and the Web of Science. We focused on research published before August 2019 with keywords including chronic wound, biofilm, bacterial biofilms, and chronic wound infection.Study selection:Relevant articles were selected by carefully reading the titles and abstracts. Further, different diagnosis and clinical treatment methods for chronic wound biofilm were compared and summarized from the selected published articles.Results:Recent guidelines on medical biofilms stated that approaches such as the use of scanning electron microscopy and confocal laser scanning microscopy are the most reliable types of diagnostic techniques. Further, therapeutic strategies include debridement, negative pressure wound therapy, ultrasound, antibiotic, silver-containing dressing, hyperbaric oxygen therapy, and others.Conclusion:This review provides the identification and management of biofilms, and it can be used as a tool by clinicians for a better understanding of biofilms and translating research to develop best clinical practices.

Optimal environmental and culture conditions allow the in vitro coexistence of Pseudomonas aeruginosa and Staphylococcus aureus in stable biofilms

The coexistence between species that occurs in some infections remains hard to achieve in vitro since bacterial fitness differences eventually lead to a single organism dominating the mixed culture. Pseudomonas aeruginosa and Staphylococcus aureus are major pathogens found growing together in biofilms in disease-affected lungs or wounds. Herein, we tested and analyzed different culture media, additives and environmental conditions to support P. aeruginosa and S. aureus coexistence in vitro. We have unraveled the potential of DMEM to support the growth of these two organisms in mature cocultured biofilms (three days old) in an environment that dampens the pH rise. Our conditions use equal initial inoculation ratios of both strains and allow the stable formation of separate S. aureus microcolonies that grow embedded in a P. aeruginosa biofilm, as well as S. aureus biofilm overgrowth when bovine serum albumin is added to the system. Remarkably, we also found that S. aureus survival is strictly dependent on a well-characterized phenomenon of oxygen stratification present in the coculture biofilm. An analysis of differential tolerance to gentamicin and ciprofloxacin treatment, depending on whether P. aeruginosa and S. aureus were growing in mono- or coculture biofilms, was used to validate our in vitro coculture conditions.

Delivery and quantification of hydrogen peroxide generated via cold atmospheric pressure plasma through biological material

The ability of plasma-generated hydrogen peroxide (H2O2) to traverse bacterial biofilms and the subsequent fate of the generated H2O2 has been investigated. An in vitro model, comprising a nanoporous membrane impregnated with artificial wound fluid and biofilms of varying maturity was treated with a helium-driven, cold atmospheric pressure plasma (CAP) jet. The concentration of H2O2 generated below the biofilms was quantified. The results showed that the plasma-generated H2O2 interacted significantly with the biofilm, thus exhibiting a reduction in concentration across the underlying nanoporous membrane. Biofilm maturity exhibited a significant effect on the penetration depth of H2O2, suggesting that well established, multilayer biofilms are likely to offer a shielding effect with respect to cells located in the lower layers of the biofilm, thus rendering them less susceptible to plasma disinfection. This may prove clinically significant in the plasma treatment of chronic, deep tissue infections such as diabetic and venous leg ulcers. Our results are discussed in the context of plasma-biofilm interactions, with respect to the fate of the longer lived reactive species generated by CAP, such as H2O2.

The Effect of Biofilm-Forming Bacteria on Inflammasome Activity

Background and Hypothesis: Over 75% of chronic wounds have biofilm infection. Biofilm infection derails the overall process of wound healing. Biofilm bacteria have developed strategies to subvert macrophage inflammatory response. The underlying mechanisms of such subversion are currently unknown. Inflammasomes are multiprotein, intracellular complexes that contain caspase-1. Caspase-1 is responsible for cleavage and activation of cytokines IL-1β, a major pro-inflammatory cytokine in chronic wounds. We hypothesized that biofilm bacteria subvert macrophage inflammatory response by attenuating caspase-1 activity and thereby reducing IL-1β. The primary objective of the study was to determine Caspase-1 activity and IL-1β production by blood monocytes derived from patient blood samples exposed to biofilm infection ex vivo and clinical wound macrophages.&#x0D; Methods: Wound macrophages were isolated from chronic patients seen at Indiana University Health Comprehensive Wound Center (CWC). Blood monocyte derived macrophages (BMDMs) were exposed to conditioning media from isogenic mutant strains SA300ΔsarA or SA300ΔrexB (derived from Staphylococcus aureus USA300LAC) as hypo- and hyper-biofilm forming mutants. Wound macrophages were isolated from wound fluid with known bacterial content. The IL-β release and caspase-1 activity from macrophages were determined using ELISA, PCR, and a colorimetric assay.&#x0D; Results: Both RT-PCR and ELISA independently exhibited a significant reduction in IL-1β production in macrophages exposed to conditioned media from hyper-biofilm forming SA300ΔrexB as compared to hypo-biofilm forming SA300ΔsarA mutant.The Caspase-1 activity was significantly reduced in the macrophages challenged with hyper-biofilm forming bacteria as compared to hypo-biofilm forming mutant.&#x0D; Conclusion and Potential Impact: Biofilm forming bacteria leads to an attenuation in production of IL-1β from macrophages indicating a subversion of inflammatory response. The caspase-1 activity data exhibited a significant role of caspase-1 in biofilm mediated subversion of the inflammatory response indicating a reduction in inflammasome activity. A clear understanding of the mechanisms of biofilm mediated suppression of host response will help improve therapeutic strategies against wound biofilm infection.

Chronic wound healing: A specific antibiofilm protein-asymmetric release system

Chronic infection is a major cause of delayed wound-healing. It is recognized to be associated with infectious bacterial communities called biofilms. Currently used conventional antibiotics alone often reveal themselves ineffective, since they do not specifically target the wound biofilm. Here, we report a new conceptual tool aimed at overcoming this drawback: an antibiofilm drug delivery system targeting the bacterial biofilm as a whole, by inhibiting its formation and/or disrupting it once it is formed. The system consists of a micro/nanostructured poly(butylene-succinate-co-adipate) (PBSA)-based asymmetric membrane (AM) with controlled porosity. By the incorporation of hydrophilic porogen agents, polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG), we were able to obtain AMs with high levels of porosity, exhibiting interconnections between pores. The PBSA-PEG membrane presented a dense upper layer with pores small enough to block bacteria penetration. Upon using such porogen agents, under dry and wet conditions, membrane's integrity and mechanical properties were maintained. Using bovine serum albumin (BSA) as a model protein, we demonstrated that protein loading and release from PBSA membranes were affected by the membrane structure (porosity) and the presence of residual porogen. Furthermore, the release curve profile consisted of a fast initial slope followed by a second slow phase approaching a plateau within 24 h. This can be highly beneficial for the promotion of wound healing. Cross-sectional confocal laser scanning microscopy (CLSM) images revealed a heterogeneous distribution of fluorescein isothiocyanate (FITC) labeled BSA throughout the entire membrane. PBSA membranes were loaded with dispersin B (DB), a specific antibiofilm matrix enzyme. Studies using a Staphylococcus epidermidis model, indicate significant efficiency in both inhibiting or dispersing preformed biofilm (up to 80 % eradication). The asymmetric PBSA membrane prepared with the PVP porogen (PBSA-PVP) displayed highest antibiofilm activity. Moreover, in vitro cytotoxicity assays using HaCaT and reconstructed human epidermis (RHE) models revealed that unloaded and DB-loaded PBSA-PVP membranes had excellent biocompatibility suitable for wound dressing applications.

Ultrasound-mediated therapies for the treatment of biofilms in chronic wounds: a review of present knowledge.

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.

Laser-induced vapor nanobubbles improve diffusion in biofilms of antimicrobial agents for wound care

Being responsible for delayed wound healing, the presence of biofilms in infected wounds leads to chronic, and difficult to treat infections. One of the reasons why antimicrobial treatment often fails to cure biofilm infections is the reduced penetration rate of antibiotics through dense biofilms. Strategies that have the ability to somehow interfere with the integrity of biofilms and allowing a better penetration of drugs are highly sought after. A promising new approach is the use of laser-induced vapor nanobubbles (VNB), of which it was recently demonstrated that it can substantially enhance the penetration of antibiotics into biofilms, resulting in a marked improvement of the killing efficiency. In this study, we examined if treatment of biofilms with laser-induced vapor nanobubbles (VNB) can enhance the potency of antimicrobials which are commonly used to treat wound infections, including povidone-iodine, chlorhexidine, benzalkonium chloride, cetrimonium bromide and mupirocin. Our investigations were performed on Pseudomonas aeruginosa and Staphylococcus aureus biofilms, which are often implicated in chronic wound infections. Pre-treatment of biofilms with laser-induced VNB did enhance the killing efficiency of those antimicrobials which experience a diffusion barrier in the biofilms, while this was not the case for those compounds for which there is no diffusion barrier. The magnitude of the enhanced potency was in most cases similar to the enhancement that was obtained when the biofilms were completely disrupted by vortexing and sonication. These results show that laser-induced VNB are indeed a very efficient way to enhance drug penetration deep into biofilms, and pave the way towards clinical translation of this novel approach for treatment of wound infections.

Evaluation of the Antimicrobial Peptide, RP557, for the Broad-Spectrum Treatment of Wound Pathogens and Biofilm.

The relentless growth of multidrug resistance and generation of recalcitrant biofilm are major obstacles in treating wounds, particularly in austere military environments where broad-spectrum pathogen coverage is needed. Designed antimicrobial peptides (dAMPs) are constructed analogs of naturally occurring AMPs that provide the first line of defense in many organisms. RP557 is a dAMP resulting from iterative rational chemical structural analoging with endogenous AMPs, human cathelicidin LL-37 and Tachyplesin 1 and the synthetic D2A21 used as structural benchmarks. RP557 possesses broad spectrum activity against Gram-positive and Gram-negative bacteria and fungi, including recalcitrant biofilm with substantial selective killing over bacterial cells compared to mammalian cells. RP557 did not induce resistance following chronic passages of Pseudomonas aeruginosa and Staphylococcus aureus at subinhibitory concentrations, whereas concurrently run conventional antibiotics, gentamycin, and clindamycin, did. Furthermore, RP557 was able to subsequently eliminate the generated gentamycin resistant P. aeruginosa and clindamycin resistant S. aureus strains without requiring an increase in minimum inhibitory concentration (MIC) concentrations. RP557 was evaluated further in a MRSA murine wound abrasion infection model with a topical application of 0.2% RP557, completely eliminating infection. If these preclinical results are translated into the clinical setting, RP557 may become crucial for the empirical broad-spectrum treatment of wound pathogens, so that infections can be reduced to a preventable complication of combat-related injuries.

Evaluation of Platensimycin and Platensimycin-Inspired Thioether Analogues against Methicillin-Resistant Staphylococcus aureus in Topical and Systemic Infection Mouse Models.

Staphylococcus aureus is one of the most common pathogens causing hospital-acquired and community-acquired infections. Methicillin-resistant S. aureus (MRSA)-formed biofilms in wounds are difficult to treat with conventional antibiotics. By targeting FabB/FabF of bacterial fatty acid synthases, platensimycin (PTM) was discovered to act as a promising natural antibiotic against MRSA infections. In this study, PTM and its previously synthesized sulfur-Michael derivative PTM-2t could reduce over 95% biofilm formation by S. aureus ATCC 29213 when used at 2 μg/mL in vitro. Topical application of ointments containing PTM or PTM-2t (2 × 4 mg/day/mouse) was successfully used to treat MRSA infections in a BABL/c mouse burn wound model. As a potential prodrug lead, PTM-2t showed improved in vivo efficacy in a mouse peritonitis model compared with PTM. Our study suggests that PTM and its analogue may be used topically or locally to treat bacterial infections. In addition, the use of prodrug strategies might be instrumental to improve the poor pharmacokinetic properties of PTM.

Treatment with the Pseudomonas aeruginosa Glycoside Hydrolase PslG Combats Wound Infection by Improving Antibiotic Efficacy and Host Innate Immune Activity.

Pseudomonas aeruginosa is an opportunistic, nosocomial bacterial pathogen that forms persistent infections due to the formation of protective communities, known as biofilms. Once the biofilm is formed, the bacteria embedded within it are recalcitrant to antimicrobial treatment and host immune defenses. Moreover, the presence of biofilms in wounds is correlated with chronic infection and delayed healing. The current standard of care for chronic wound infections typically involves physical disruption of the biofilm via debridement and subsequent antimicrobial treatment. The glycoside hydrolases PelAh and PslGh have been demonstrated in vitro to disrupt biofilm integrity through degradation of the key biofilm matrix exopolysaccharides Pel and Psl, respectively. Herein, we demonstrate that PslGh hydrolase therapy is a promising strategy for controlling P. aeruginosa wound infections. Hydrolase treatment of P. aeruginosa biofilms resulted in increased antibiotic efficacy and penetration into the biofilm. PslGh treatment of P. aeruginosa biofilms also improved innate immune activity leading to greater complement deposition, neutrophil phagocytosis, and neutrophil reactive oxygen species production. Furthermore, when P. aeruginosa-infected wounds were treated with a combination of PslGh and tobramycin, we observed an additive effect leading to greater bacterial clearance than treatments of tobramycin or PslGh alone. This study demonstrates that PelAh and PslGh have promising therapeutic potential and that PslGh may aid in the treatment of P. aeruginosa wound infections.

The Use of Lab on a Chip Devices to Evaluate Infectious Biofilm Formation and Assess Antibiotics and Nano Drugs Treatments

The microfluidics devices are identified as Lab On a Chip (LOC) and they consist of a network of microchannels that are compose by different sections, chambers, columns and reservoirs [1]. The LOC application to study organisms is an emerging field where the benefits of miniaturization of the system offer new opportunities to study and sense different bioprocesses. In addition, microorganisms form surface-adherent community structures called biofilms and these biofilms play a critical role in wound infections. Microbial biofilms have inherent defense and survival mechanisms including: avoidance of host inflammatory cells, resistance to antibiotics, and dynamic cell-cell communication pathways with gene exchange (Quorum Sensing), which inhibit wound healing [2]. Also nanoparticles targeting anti-biofilm therapy have gained recognition due to their unique features. In this work we propose LOC as a platform for the formation of infectious biofilm in skin wounds and assess new treatments with antibiotics and nano drugs and these drugs have antimicrobial and anti-biofilm properties. The microdevices were built with glass base and polydimethylsiloxane (PDMS) cover. PDMS was mixed with curing agent in a 10:1 ratio and then the mixture was placed under vacuum to remove air bubbles, poured onto the SU8-mold and cured in an oven at 80 °C overnight. The microchip design microchannels (Figure 1) allows for higher throughput with multiple channels are located in one chip (parallelized measurements). Each microchannel has 11 culture chambers with a height of 100 µm and an internal volume of 5.36 µL with. The microchannels were washed with 70% ethanol and were disinfected using NaOH (0.5 mol L-1) for 30 minutes. Pseudomonas aeruginosa and Propionibacterium acnes were used in a continuous culture either as a single culture or as co-culture in the microchip as a bioreactor for biofilm formation in the microchannels. After 3 days of culture good cell adhesions to the substrate and biofilm formation inside the microchannels were observed. Different concentrations of nanoparticles of silver, gold and antibiotics were evaluated for the inhibition of the infections biofilm. After 48 h of each anti-biofilm treatment condition the physiological characteristics were analyzed by transcriptomics (RNAm sequencing) and the morphological characterization of biofilm architecture was evaluated by Scanning Electron Microscopy (SEM) and Confocal Scanning Laser Microscopy (CSLM).The results of this study show that the use of microchip as a culture system allows a good biofilm formation and is a valuable tool for quickly screening of antimicrobial activity of nano drugs in biofilms. This microchip can be used in drug testing and biomedical applications. 1- Whitesides, G.M., (2006). The origins and the future of microfluidics. Nature. 442(7101):368-373 2- Alvarado-Gomez, E., Martínez-Castañon, G., Sanchez-Sanchez, R., Ganem-Rondero, A., Yacaman, M. J., & Martinez-Gutierrez, F. (2018). Evaluation of anti-biofilm and cytotoxic effect of a gel formulation with Pluronic F-127 and silver nanoparticles as a potential treatment for skin wounds. Materials Science and Engineering: C, 92:621-630. Figure 1

Current Status of In Vitro Models and Assays for Susceptibility Testing for Wound Biofilm Infections.

Biofilm infections have gained recognition as an important therapeutic challenge in the last several decades due to their relationship with the chronicity of infectious diseases. Studies of novel therapeutic treatments targeting infections require the development and use of models to mimic the formation and characteristics of biofilms within host tissues. Due to the diversity of reported in vitro models and lack of consensus, this review aims to provide a summary of in vitro models currently used in research. In particular, we review the various reported in vitro models of Pseudomonas aeruginosa biofilms due to its high clinical impact in chronic wounds and in other chronic infections. We assess advances in in vitro models that incorporate relevant multispecies biofilms found in infected wounds, such as P. aeruginosa with Staphylococcus aureus, and additional elements such as mammalian cells, simulating fluids, and tissue explants in an attempt to better represent the physiological conditions found at an infection site. It is hoped this review will aid researchers in the field to make appropriate choices in their proposed studies with regards to in vitro models and methods.

Recent Advances in Non-Conventional Antimicrobial Approaches for Chronic Wound Biofilms: Have We Found the 'Chink in the Armor'?

Chronic wounds are a major healthcare burden, with huge public health and economic impact. Microbial infections are the single most important cause of chronic, non-healing wounds. Chronic wound infections typically form biofilms, which are notoriously recalcitrant to conventional antibiotics. This prompts the need for alternative or adjunct ‘anti-biofilm’ approaches, notably those that account for the unique chronic wound biofilm microenvironment. In this review, we discuss the recent advances in non-conventional antimicrobial approaches for chronic wound biofilms, looking beyond standard antibiotic therapies. These non-conventional strategies are discussed under three groups. The first group focuses on treatment approaches that directly kill or inhibit microbes in chronic wound biofilms, using mechanisms or delivery strategies distinct from antibiotics. The second group discusses antimicrobial approaches that modify the biological, chemical or biophysical parameters in the chronic wound microenvironment, which in turn enables the disruption and removal of biofilms. Finally, therapeutic approaches that affect both, biofilm bacteria and microenvironment factors, are discussed. Understanding the advantages and limitations of these recent approaches, their stage of development and role in biofilm management, could lead to new treatment paradigms for chronic wound infections. Towards this end, we discuss the possibility that non-conventional antimicrobial therapeutics and targets could expose the ‘chink in the armor’ of chronic wound biofilms, thereby providing much-needed alternative or adjunct strategies for wound infection management.

Efficacy and safety assessment of two enterococci phages in an in vitro biofilm wound model

Chronic wounds affect thousands of people worldwide, causing pain and discomfort to patients and represent significant economical burdens to health care systems. The treatment of chronic wounds is very difficult and complex, particularly when wounds are colonized by bacterial biofilms which are highly tolerant to antibiotics. Enterococcus faecium and Enterococcus faecalis are within the most frequent bacteria present in chronic wounds. Bacteriophages (phages) have been proposed as an efficient and alternative against antibiotic-resistant infections, as those found in chronic wounds. We have isolated and characterized two novel enterococci phages, the siphovirus vB_EfaS-Zip (Zip) and the podovirus vB_EfaP-Max (Max) to be applied during wound treatment. Both phages demonstrated lytic behavior against E. faecalis and E. faecium. Genome analysis of both phages suggests the absence of genes associated with lysogeny. A phage cocktail containing both phages was tested against biofilms formed in wound simulated conditions at a multiplicity of infection of 1.0 and a 2.5 log CFU.mL−1 reduction in the bacterial load after at 3 h of treatment was observed. Phages were also tested in epithelial cells colonized by these bacterial species and a 3 log CFU.mL−1 reduction was observed using both phages. The high efficacy of these new isolated phages against multi-species biofilms, their stability at different temperatures and pH ranges, short latent periods and non-cytotoxicity to epithelial cells suggest their therapeutic use to control infectious biofilms present in chronic wounds.

Preclinical evaluation of a novel silver gelling fiber dressing on Pseudomonas aeruginosa in a porcine wound infection model.

The wound environment is a fertile ground for biofilm forming pathogens. Once biofilms form within the wound, they can be very challenging to eradicate. The purpose of this study was to examine the effect of a gelling fiber dressing with silver using a well-established porcine wound biofilm model. Deep partial thickness wounds were inoculated with Pseudomonas aeruginosa ATCC 27312 and covered with a polyurethane film dressing to promote biofilm formation. Wounds were then divided into treatment groups: gelling fiber dressing with silver, gelling fiber dressing without silver, hydrofiber dressing with silver, benzethonium chloride and ethylenediaminetetraacetic acid and compared to untreated control. Microbiological, biofilm, and histological wound assessments were performed on days 3, 5, and 7 postinfection. Treatment with gelling fiber dressing with silver resulted in significant reduction of P. aeruginosa biofilm when compared to all other treatment groups on every assessment time point. In addition, gelling fiber dressing with silver treatment resulted in detachment of biofilm from the wound, while wounds treated with gelling fiber dressing with and without silver showed more granulation tissue formation on day 3. Our data show that a new gelling fiber dressing with silver was effective in reducing biofilm associated P. aeruginosa in vivo. This study may have important clinical implications especially for wounds heavily colonized with gram-negative biofilm-forming bacteria.

Diabetic foot syndrome. Replacement for inpatient treatment technique based on the fast track surgery

One of the main problems of modern surgery is treatment of trophic skin and soft tissues defects due to the ambiguity of the pathogenesis, tendency to recurrence, deterioration in the patients’ quality of life, complexity and duration of treatment, large economic costs of treatment and rehabilitation. The article describes an algorithm for outpatient treatment of patients with trophic skin and soft tissues defects in diabetic foot syndrome. Debridement the wound from necrosis, purulent-fibrinous pellicles, wound biofilms is produced by a chemical necrectomy. A method of closing the wound defects by the means of high-tech biomaterials based on collagen type 1 is described.