Burn Wound Infection Model Research Articles

Amino Acid-Conjugated Polymer-Silver Bromide Nanocomposites for Eradicating Polymicrobial Biofilms and Treating Burn Wound Infections.

The rise in antimicrobial resistance, the increasing occurrence of bacterial, and fungal infections, and the challenges posed by polymicrobial biofilms necessitate the exploration of innovative therapeutic strategies. Silver-based antimicrobials have garnered attention for their broad-spectrum activity and multimodal mechanisms of action. However, their effectiveness against single-species or polymicrobial biofilms remains limited. In this study, we present the fabrication of polymer-silver bromide nanocomposites using amino acid conjugated polymers (ACPs) through a green and water-based in situ technique. The nanocomposite architecture facilitated prolonged and controlled release of the active components. Remarkably, the nanocomposites exhibited broad-spectrum activity against multidrug-resistant (MDR) human pathogenic bacteria (MIC = 2-16 μg/mL) and fungi (MIC = 1-8 μg/mL), while displaying no detectable toxicity to human erythrocytes (HC50 > 1024 μg/mL). In contrast to existing antimicrobials and silver-based therapies, the nanocomposite effectively eradicated bacterial, fungal, and polymicrobial biofilms, and prevented the development of microbial resistance due to their membrane-active properties. Furthermore, the lead polymer-silver bromide nanocomposite demonstrated a 99% reduction in the drug-resistant Pseudomonas aeruginosa burden in a murine model of burn wound infection, along with excellent in vivo biocompatibility.

Neem-hypericum-bacterial cellulose wound care paste characterized in vitro and in Galleria mellonella in vivo model

Surgical wound infections represent a global health emergency because of antibiotic-resistant bacteria. Commensal and pathogenic bacteria can grow in the form of biofilms on damaged skin, and often exhibit an antibiotic-resistance profile. For this reason, the development of new strategies is necessary for the management of skin wounds, which are based not only on antimicrobial activity but also on the inhibition of bacterial biofilm formation or biofilm removal. In this study, we show the development and the biofilm-detaching properties of a novel bacterial cellulose formulation for wound healing. This paste is made by mixing bacterial cellulose pulp with neem-hypericum oil/water emulsion. Once its biocompatibility on murine fibroblasts has been determined, its efficacy was evaluated in vitro against Pseudomonas aeruginosa and Staphylococcus aureus biofilms and in vivo by using Galleria mellonella larvae as a burn wound infection model. Unlike pure BC, the paste showed promising biofilm detachment properties in vitro and a good biocompatibility activity in vivo, setting the stage for developing new strategies for the treatment of chronic wound infections.

Human breast milk isolated lactic acid bacteria: antimicrobial and immunomodulatory activity on the Galleria mellonella burn wound model.

Managing burn injuries is a challenge in healthcare. Due to the alarming increase in antibiotic resistance, new prophylactic and therapeutic strategies are being sought. This study aimed to evaluate the potential of live Lactic Acid Bacteria for managing burn infections, using Galleria mellonella larvae as an alternative preclinical animal model and comparing the outcomes with a common antibiotic. The antimicrobial activity of LAB isolated from human breast milk was assessed in vitro against Pseudomonas aeruginosa ATCC 27853. Additionally, the immunomodulatory effects of LAB were evaluated in vivo using the G. mellonella burn wound infection model. In vitro results demonstrated the antimicrobial activity of Lactic Acid Bacteria against P. aeruginosa. In vivo results show that their prophylactic treatment improves, statistically significant, larval survival and modulates the expression of immunity-related genes, Gallerimycin and Relish/NF-κB, strain-dependently. These findings lay the foundation and suggest a promising alternative for burn wound prevention and management, reducing the risk of antibiotic resistance, enhancing immune modulation, and validating the potential G. mellonella as a skin burn wound model.

Antibacterial Efficacy of a Chitosan-Based Hydrogel Modified With Epsilon-Poly-l-Lysine Against Pseudomonas aeruginosa in a Murine-Infected Burn Wound Model.

Because antibiotic resistance is increasing worldwide and the leading cause of death in burn patients is an infection, an urgent need exists for nonantibiotic approaches to eliminate multidrug-resistant bacteria from burns to prevent their systemic dissemination and sepsis. We previously demonstrated the significant antibiofilm activity of a chitosan (CS) hydrogel containing the antimicrobial peptide epsilon-poly-l-lysine (EPL) against multidrug-resistant Pseudomonas aeruginosa using ex vivo porcine skin. In this study, we evaluated the in vivo antibacterial efficacy of a CS/EPL hydrogel against P. aeruginosa in a murine burn wound infection model. Full-thickness burns were created on the dorsum using a heated brass rod and were inoculated with bioluminescent, biofilm-forming P. aeruginosa (Xen41). Mice were treated with CS/EPL, CS, or no hydrogel applied topically 2 or 24 hours after inoculation to assess the ability to prevent or eradicate existing biofilms, respectively. Dressing changes occurred daily for 3 days, and in vivo bioluminescence imaging was performed to detect and quantitate bacterial growth. Blood samples were cultured to determine systemic infection. In vitro antibacterial activity and cytotoxicity against human primary dermal fibroblasts, keratinocytes, and mesenchymal stem cells were also assessed. CS/EPL treatment initiated at early or delayed time points showed a significant reduction in bioluminescence imaging signal compared to CS on days 2 and 3 of treatment. Mice administered CS/EPL had fewer bloodstream infections, lower weight loss, and greater activity than the untreated and CS groups. CS/EPL reduced bacterial burden by two orders of magnitude in vitro and exhibited low cytotoxicity against human cells. A topical hydrogel delivering the antimicrobial peptide EPL demonstrates in vivo efficacy to reduce but not eradicate established P. aeruginosa biofilms in infected burn wounds. This biocompatible hydrogel shows promise as an antimicrobial barrier dressing for the sustained protection of burn wounds from external bacterial contamination.

Injectable Antibacterial Hydrogel with Asiaticoside-Loaded Liposomes and Ultrafine Silver Nanosilver Particles Promotes Healing of Burn-Infected Wounds.

Post-injury infection and wound healing are recurrent daily life problems. Therefore, the necessity of developing a biomaterial with antibacterial and wound-healing properties is paramount. Based on the special porous structure of hydrogel, we modified recombinant collagen and quaternary ammonium chitosan and fused them with silver nanoparticles (Ag@mental-organic framework (MOF)) with antibacterial properties, and asiaticoside-loaded liposomes (Lip@AS) with anti-inflammatory/vascularization effects to form the rColMA/QCSG/LIP@AS/Ag@MOF (RQLAg) hydrogel. The prepared hydrogel possessed good sustainable release capabilities of Ag+ and AS and exhibited concentration-dependent swelling properties, pore size, and compressive strength. Cellular experiments showed that the hydrogel exhibited good cell compatibility and promoted cell migration, angiogenesis, and M1 macrophage polarization. Additionally, the hydrogels exhibited excellent antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in vitro. In vivo, Sprague Dawley (SD) rats burn-wound infection model showed that the RQLAg hydrogel could efficiently promote wound healing and had stronger healing promoting abilities than those of Aquacel Ag. In summary, the RQLAg hydrogel is expected to be an excellent material for accelerating open wound healing and preventing bacterial infections. This article is protected by copyright. All rights reserved.

Potential antibacterial activity and healing effect of topical administration of bone marrow and adipose mesenchymal stem cells encapsulated in collagen-fibrin hydrogel scaffold on full-thickness burn wound infection caused by Pseudomonas aeruginosa

Burns injuries are prone to hospital-acquired infections, and Pseudomonas aeruginosa is one of the most common causes of mortality and morbidity in patients with burn injuries. Thus, this study aimed to analyze the effects of topical treatment with bone marrow (BM-MSC) and adipose mesenchymal stem cells (AD-MSC) encapsulated in collagen and fibrin scaffolds in a Balb/c model of burn wound infection. Extraction of stem cells from adipose and bone marrow tissue of rats was performed and cells were characterized using standard methods. Then, collagen, fibrin and collagen-fibrin scaffolds were constructed and the extracted cells were encapsulated in all three scaffolds. Then, 3rd degree burn was induced in mice and 1.5 × 108 (CFU/ml) of P. aeruginosa was introduced to the burn wound. Subsequently, after 24 h of inducing wound infection, encapsulated MSCs were introduced as dressings to burn wound infection and microbial load as well as rate of wound infection healing was measured. The results of this study showed that the use of BM-MSC and AD-MSC encapsulated in collagen-fibrin scaffold reduced the bacteria load down to 54 and 21 CFU/gr, respectively (P < 0.05). Moreover, BM-MSC and AD-MSC encapsulated in collagen-fibrin showed 80% and 75% wound healing, respectively (P < 0.05). Also, we found no significant between cell origin and healing. Encapsulation of MSCs into collagen-fibrin scaffolds could be effective not only against P. aeruginosa infection, but also healing and regeneration of burn wound.

Pseudomonas aeruginosa PAO1 outer membrane vesicles-diphtheria toxoid conjugate as a vaccine candidate in a murine burn model

Pseudomonas aeruginosa is an opportunistic pathogen considered a common cause of nosocomial infection with high morbidity and mortality in burn patients. Immunoprophylaxis techniques may lower the mortality rate of patients with burn wounds infected by P. aeruginosa; consequently, this may be an efficient strategy to manage infections caused by this bacterium. Several pathogenic Gram-negative bacteria like P. aeruginosa release outer membrane vesicles (OMVs), and structurally OMV consists of several antigenic components capable of generating a wide range of immune responses. Here, we evaluated the immunogenicity and efficacy of P. aeruginosa PA-OMVs (PA-OMVs) conjugated with the diphtheria toxoid (DT) formulated with alum adjuvant (PA-OMVs-DT + adj) in a mice model of burn wound infection. ELISA results showed that in the group of mice immunized with PA-OMVs-DT + adj conjugated, there was a significant increase in specific antibodies titer compared to non-conjugated PA-OMVs or control groups. In addition, the vaccination of mice with PA-OMVs-DT + adj conjugated generated greater protective effectiveness, as seen by lower bacterial loads, and eightfold decreased inflammatory cell infiltration with less tissue damage in the mice burn model compared to the control group. The opsonophagocytic killing results confirmed that humoral immune response might be critical for PA-OMVs mediated protection. These findings suggest that PA-OMV-DT conjugated might be used as a new vaccine against P. aeruginosa in burn wound infection.

Mussel-inspired hydrogel with injectable self-healing and antibacterial properties promotes wound healing in burn wound infection

Burn wound infections cause serious problems for public health. More than 180,000 patients die from burns every year worldwide. In addition, the difficulty of healing wounds and wound infections caused by burns affects the mental health of patients. Therefore, it is very important to develop a wound dressing that can promote wound repair and exhibits good antibacterial effects. Here, we used oxidized konjac glucomannan (OKGM), γ-poly(glutamic acid) modified with dopamine and L-cysteine (γ-PGA-DA-Cys) and ε-polylysine (ε-PL) to produce an OKGM/γ-PGA-DA-Cys/ε-PL (OKPP) hydrogel. This hydrogel was produced by thiol-aldehyde addition and Schiff-base reactions and has the ability to be injected and self-heal. The results showed that the hydrogel exhibits good antibacterial effects on Pseudomonas aeruginosa and Staphylococcus aureus and has antioxidant effects in vitro. Moreover, the hydrogel also exhibits good adhesion. In a burn wound infection model, the hydrogel promoted wound healing and reduced the production of inflammation. These results proved that the hydrogel has clinical potential as a wound dressing for burn wound infection.

Photo-Crosslinked Antimicrobial Hydrogel Exhibiting Wound Healing Ability and Curing Infections In Vivo.

With the increasing focus on healthcare research in the current times, therapeutic and biomaterial interventions for healing of wounds and mitigation of wound-associated infections have seen expedited progress. Conventional approaches consist of release-active gels, which demonstrate leaching of antimicrobials, such as antibiotics, metal ions, etc. However, these systems suffer from the disadvantages of burst release, reservoir exhaustion, and associated toxicity. In this report, intrinsically antimicrobial hydrogel (HyDex) is developed by one-pot UV crosslinking of methacrylated dextran, polyethylene glycol diacrylate, and cationic lipophilic methacrylate with varied hydrophobic chain, which displays broad-spectrum antimicrobial activity, hemostatic ability, and rapid wound closure efficacy. The optimized hydrogel exhibits potent antimicrobial efficacy against multidrug-resistant Gram-positive and Gram-negative bacteria as well as against pathogenic fungus Candida albicans. The HyDex hydrogel shows rapid arrest of bleeding in mice liver puncture model. The hydrogel kills carbapenem-resistant Acinetobacter baumannii in a murine model of burn wound infection with >99% reduction in bacterial burden. Furthermore, this hydrogel displays significant reduction in inflammatory responses, with accelerated wound healing in rat deep wound model. Collectively, these results imply the excellent promise held by lead hydrogel to be developed for tackling deep tissue wounds, notorious infections, and resulting inflammatory responses.

Light-activated molecular machines are fast-acting broad-spectrum antibacterials that target the membrane.

The increasing occurrence of antibiotic-resistant bacteria and the dwindling antibiotic research and development pipeline have created a pressing global health crisis. Here, we report the discovery of a distinctive antibacterial therapy that uses visible (405 nanometers) light-activated synthetic molecular machines (MMs) to kill Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, in minutes, vastly outpacing conventional antibiotics. MMs also rapidly eliminate persister cells and established bacterial biofilms. The antibacterial mode of action of MMs involves physical disruption of the membrane. In addition, by permeabilizing the membrane, MMs at sublethal doses potentiate the action of conventional antibiotics. Repeated exposure to antibacterial MMs is not accompanied by resistance development. Finally, therapeutic doses of MMs mitigate mortality associated with bacterial infection in an in vivo model of burn wound infection. Visible light–activated MMs represent an unconventional antibacterial mode of action by mechanical disruption at the molecular scale, not existent in nature and to which resistance development is unlikely.

PaP1, a Broad-Spectrum Lysin-Derived Cationic Peptide to Treat Polymicrobial Skin Infections.

Most skin infections, including those complicating burns, are polymicrobial involving multiple causative bacteria. Add to this the fact that many of these organisms may be antibiotic-resistant, and a simple skin lesion or burn could soon become life-threatening. Membrane-acting cationic peptides from Gram-negative bacteriophage lysins can potentially aid in addressing the urgent need for alternative therapeutics. Such peptides natively constitute an amphipathic region within the structural composition of these lysins and function to permit outer membrane permeabilization in Gram-negative bacteria when added externally. This consequently allows the lysin to access and degrade the peptidoglycan substrate, resulting in rapid hypotonic lysis and bacterial death. When separated from the lysin, some of these cationic peptides kill sensitive bacteria more effectively than the native molecule via both outer and cytoplasmic membrane disruption. In this study, we evaluated the antibacterial properties of a modified cationic peptide from the broad-acting lysin PlyPa01. The peptide, termed PaP1, exhibited potent in vitro bactericidal activity toward numerous high priority Gram-positive and Gram-negative pathogens, including all the antibiotic-resistant ESKAPE pathogens. Both planktonic and biofilm-state bacteria were sensitive to the peptide, and results from time-kill assays revealed PaP1 kills bacteria on contact. The peptide was bactericidal over a wide temperature and pH range and could withstand autoclaving without loss of activity. However, high salt concentrations and complex matrices were found to be largely inhibitory, limiting its use to topical applications. Importantly, unlike other membrane-acting antimicrobials, PaP1 lacked cytotoxicity toward human cells. Results from a murine burn wound infection model using methicillin-resistant Staphylococcus aureus or multidrug-resistant Pseudomonas aeruginosa validated the in vivo antibacterial efficacy of PaP1. In these studies, the peptide enhanced the potency of topical antibiotics used clinically for treating chronic wound infections. Despite the necessity for additional preclinical drug development, the collective data from our study support PaP1 as a potential broad-spectrum monotherapy or adjunctive therapy for the topical treatment of polymicrobial infections and provide a foundation for engineering future lysin-derived peptides with improved antibacterial properties.

Polymyxin B-Triggered Assembly of Peptide Hydrogels for Localized and Sustained Release of Combined Antimicrobial Therapy.

Repurposing old antibiotics into more effective and safer formulations is an emergent approach to tackle the growing threat of antimicrobial resistance. Herein, a peptide hydrogel is reported for the localized and sustained release of polymyxin B (PMB), a decade-old antibiotic with increasing clinical utility for treating multidrug-resistant Gram-negative bacterial infections. The hydrogel is assembled by additing PMB solution into a rationally designed peptide amphiphile (PA) solution and its mechanical properties can be adjusted through the addition of counterions, envisioning its application in diverse infection scenarios. Sustained release of PMB from the hydrogel over a 5-day period and prolonged antimicrobial activities against Gram-negative bacteria are observed. The localized release of active PMB from the hydrogel is shown to be effective in vivo for treating Pseudomonas aeruginosa infection in the Galleria mellonella burn wound infection model, dramatically reducing the mortality from 93% to 13%. Complementary antimicrobial activity against Gram-positive Staphylococcus aureus and enhanced antimicrobial effect against the Gram-negative Acinetobacter baumannii are observed when an additional antibiotic fusidic acid is incorporated into the hydrogen network. These results demonstrate the potential of the PMB-triggered PA hydrogel as a versatile platform for the localized and sustained delivery of combined antimicrobial therapies.

AB569, a Novel, Topical Bactericidal Gel Formulation, Kills Pseudomonas aeruginosa and Promotes Wound Healing in a Murine Model of Burn Wound Infection.

ABSTRACTCutaneous thermal injuries from burns/explosives are a major cause of morbidity and mortality and represent a monumental burden on our current health care system. Injury severity is predominantly due to potentially lethal sepsis caused by multidrug-resistant (MDR) bacteria such as Pseudomonas aeruginosa (MDR-PA). Thus, there is a critical need to develop novel and effective antimicrobials for the (i) prevention, (ii) treatment, and (iii) healing of such wounds that are complicated by MDR-P. aeruginosa and other bacterial infections. AB569 is a novel bactericidal tandem consisting of acidified NaNO2 (A-NO2–) and Na2-EDTA. Here, we first show that AB569 acts synergistically to kill all human burn wound strains of P. aeruginosa in vitro. This was found to be due, in part, to the generation of A-NO2–-mediated nitric oxide (NO) formation coupled with the metal chelating properties of Na2-EDTA. Using a murine scald burn wound model of P. aeruginosa infection, an AB569-Solosite gel formulation eradicated all bacteria. Futher, we also demonstrate enhanced AB569-mediated wound healing by not only accelerating wound contraction, but also by reducing levels of the proinflammatory cytokines interleukin-6 (IL-6) and IL-1β while increasing the levels of anti-inflammatory cytokine, IL-10, and granulocyte-colony-stimulating factor (G-CSF). We also observed better epidermal restoration in AB569-treated wounds. Taken together, we conclude that this study provides solid foundational evidence that AB569 can be used topically to treat highly problematic dermal insults, including wound, burn, blast, and likely, diabetic infections in civilian and military populations, and help relieve the economical burden that MDR organisms have on the global health care system.

Targeting the gut to prevent sepsis from a cutaneous burn.

Severe burn injury induces gut barrier dysfunction and subsequently a profound systemic inflammatory response. In the present study, we examined the role of the small intestinal brush border enzyme, intestinal alkaline phosphatase (IAP), in preserving gut barrier function and preventing systemic inflammation after burn wound infection in mice. Mice were subjected to a 30% total body surface area dorsal burn with or without intradermal injection of Pseudomonas aeruginosa. Mice were gavaged with 2000 units of IAP or vehicle at 3 and 12 hours after the insult. We found that both endogenously produced and exogenously supplemented IAP significantly reduced gut barrier damage, decreased bacterial translocation to the systemic organs, attenuated systemic inflammation, and improved survival in this burn wound infection model. IAP attenuated liver inflammation and reduced the proinflammatory characteristics of portal serum. Furthermore, we found that intestinal luminal contents of burn wound–infected mice negatively impacted the intestinal epithelial integrity compared with luminal contents of control mice and that IAP supplementation preserved monolayer integrity. These results indicate that oral IAP therapy may represent an approach to preserving gut barrier function, blocking proinflammatory triggers from entering the portal system, preventing gut-induced systemic inflammation, and improving survival after severe burn injuries.

119 A Nitric Oxide Releasing Bio-active Wound Dressing for Burn Wound Infection Treatment

Abstract Introduction The future of multi-domain battlefield operation requires wound dressings to prevent infection at the point of injury. Majority of antimicrobial agents only target wound infection while other healing events are left to their natural fates. Nitric oxide (NO) acts against both gram-positive and -negative bacteria and has the potential to positively affect wound healing. In this study we have developed a novel wound dressing integrated with a NO donor - S-nitroso- glutathione (GSNO) in a hybrid formulation of alginate and poly(vinyl alcohol) (PVA) to prevent/treat burn wound infection. Methods The NO releasing wound dressing was fabricated using PVA, alginate, and glycerol, crosslinked with CaCl2 incorporating GSNO. Thereafter, release kinetics were measured up to 4 days. The antibacterial efficacy was determined against both P. aeruginosa and S. aureus. Then the biocompatibility of the NO wound dressing was assessed using in vitro fibroblast proliferation and wound healing assay. Finally, the efficacy of the wound dressing was assessed in vivo using a 3-cm diameter porcine burn wound infection model. Results The Alginate-PVA-GSNO dressing showed a desired physiological level NO flux of 4.42 × 10-10 mol cm-2 min-1 for 72 hours. Alginate−PVA−GSNO dressings showed ~3 log reduction in S. aureus and ~2 log reduction in P. aeruginosa CFU/mg when compared to control. The NO-releasing dressing improved fibroblast proliferation and migration resulting in complete closure of the wound within 48 h in vitro. The safety and efficacy of NO-releasing dressing were successfully established in the both P. aeruginosa and S. aureus infected porcine burn wounds. Histological assessments are carried out to determine the effect of NO-releasing dressing on overall healing process. Conclusions This study shows Alginate−PVA-GSNO wound dressing provides antimicrobial and wound healing properties in vitro. Preliminary in vivo wound healing studies established the safety and efficacy profile of NO-releasing dressing to treat burn wounds. Applicability of Research to Practice An easy to apply, field deployable and effective antimicrobial wound dressing is still a major requisite for combat burn wounds. NO delivering alginate-PVA based wound dressing may be an ideal candidate to inhibit infection as well as promote the wound healing process.

Comparative Analysis of the Host Response in a Rat Model of Deep-Partial and Full-Thickness Burn Wounds With Pseudomonas aeruginosa Infection.

Burn wound injury affects soldiers and civilians alike, often resulting in a dynamic, but un-orchestrated, host response that can lead to infection, scarring, and potentially death. To mitigate these factors, it is important to have a clinically relevant model of burn wound infection that can be utilized for advancing burn wound treatments. Our previous reports have demonstrated the ability of Pseudomonas aeruginosa to generate a biofilm infection within a modified Walker-Mason rat burn model of deep-partial (DPT) and full-thickness (FT) burn wounds (10% total body surface area) in male Sprague-Dawley rats (350–450 g). Here, we further define this model with respect to the host response when challenged with P. aeruginosa infection between the two burn types. Following burn injury and immediate surface exposure to P. aeruginosa, inflammation at the local and systemic levels were monitored for an 11 days period. Compared to burn-only groups, infection with P. aeruginosa further promoted local inflammation in both DPT and FT burn wounds, which was evident by enhanced cellular influx (including neutrophils and monocytes), increased levels of several pro-inflammatory cytokines (IL-1β, IL-6, GRO/KC, andMIP-1α), and reduced IL-10. Systemically, only minor changes were seen in circulating white blood cells and cytokines; however, increases in high mobility group box-1 (HMGB-1) and hyaluronan, as well as decreases in fibronectin were noted particularly in FT burns. Compared to the burn-only group, P. aeruginosa infection resulted in sustained and/or higher levels of HMGB-1 and hyaluronan. Combined with our previous work that defined the burn depth and development of P. aeruginosa biofilms within the wound, this study further establishes this model by defining the host response to the burn and biofilm-infection. Furthermore, this characterization shows several similarities to what is clinically seen and establishes this model for future use in the development and testing of novel therapeutics for burn wound treatment at home and on the battlefield.

Lysine-Based Small Molecule Sensitizes Rifampicin and Tetracycline against Multidrug-Resistant Acinetobacter baumannii and Pseudomonas aeruginosa.

The priority pathogen list published by the World Health Organization (WHO) has categorized carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa as the top two critical pathogens, and hence, the development of novel antibacterial strategies to tackle such bacteria is highly necessary. Toward this aim, herein we report the efficacy of the combination of a lysine-based membrane-active small molecule, D-LANA-14 (d-lysine conjugated aliphatic norspermidine analogue bearing tetradecanoyl chain) and the obsolete/inactive antibiotics (such as tetracycline and rifampicin) to combat these superbugs. The combination of D-LANA-14 and the antibiotics tetracycline or rifampicin showed not only synergistic activity against growing planktonic cells of meropenem-resistant A. baumannii and P. aeruginosa clinical isolates but was also able to disrupt their established biofilms. More importantly, this synergistic effect was retained under the in vivo scenario, wherein the combination showed excellent efficacy in mice model of burn-wound infection with a drastic reduction of bacterial burden. A combined treatment of D-LANA-14 (40 mg/kg) and rifampicin (40 mg/kg) showed 4.9 log and 4.0 log reduction in A. baumannii and P. aeruginosa viability, respectively. On the contrary, individual treatment of D-LANA-14 decreased bacterial burden by 2.3 log (A. baumannii) and 1.3 log (P. aeruginosa) and rifampicin reduced about 3.0 log (A. baumannii) and 1.6 log (P. aeruginosa). Owing to the membrane-active nature imparted by D-LANA-14, bacteria could not develop resistance against the combined treatment, whereas a high-level of resistance development was observed against the last resort Gram-negative antibiotic, colistin. Taken together, the results therefore indicate a great potential of this novel combination to be developed as therapeutic regimen to combat infections caused by critical Gram-negative pathogens.

In vivo activity and low toxicity of the second-generation antimicrobial peptide DGL13K

Antimicrobial peptides have been evaluated as possible alternatives to traditional antibiotics. The translational potential of the antimicrobial peptide DGL13K was tested with focus on peptide toxicity and in vivo activity in two animal models. DGL13K was effective against Pseudomonas aeruginosa, Staphylococcus aureus and methicillin-resistant S. aureus with minimal bactericidal concentrations similar to the minimal inhibitory concentration. The peptide showed low toxicity to human red blood cells and HEK cells with median lethal dose around 1 mg/ml. The median lethal dose in greater wax moth larvae (Galleria mellonella) was about 125mg/kg while the peptide caused no skin toxicity in a mouse model. A novel high-throughput luminescence assay was used to test peptide activity in infected G. mellonella, thus reducing vertebrate animal use. DGL13K killed P. aeruginosa in both the G. mellonella model and a mouse burn wound infection model, with bacterial viability 3-10-fold lower than in untreated controls. Future experiments will focus on optimizing peptide delivery, dose and frequency to further improve the antibacterial effect.

Fms-like tyrosine kinase-3 ligand increases resistance to burn wound infection through effects on plasmacytoid dendritic cells

BackgroundPatients experiencing large thermal injuries are susceptible to opportunistic infections that can delay recovery and lead to sepsis. Dendritic cells (DC) are important for the detection of pathogens and activation of the innate and acquired immune responses. DCs are significantly decreased in burn patients early after injury, and the development of sepsis is associated with persistent DC depletion. In a murine model of burn wound infection, the enhancement of DCs after injury by treatment with the DC growth factor Fms-like tyrosine kinase-3 ligand (FL) enhances neutrophil migration to infection, improves bacterial clearance, and increases survival in a DC-dependent manner. FL expands the production of both conventional DCs (cDC) and plasmacytoid DCs (pDC). It has been established that cDCs are required for some of the protective effects of FL after burn injury. This study was designed to determine the contribution of the pDC subset.MethodsMice were subjected to full-thickness scald burns under deep anesthesia and were provided analgesia. pDCs were depleted by injection of anti-plasmacytoid dendritic cell antigen-1 antibodies. Survival, bacterial clearance, and neutrophil responses in vivo and in vitro were measured.ResultsDepletion of preexisting pDCs, but not FL-expanded pDCs, abrogated the beneficial effects of FL on survival, bacterial clearance, and neutrophil migration in response to burn wound infection. This requisite role of pDCs for FL-mediated enhancement of neutrophil migratory capacity is not due to direct effects of pDCs on neutrophils. cDCs, but not pDCs, directly increased neutrophil migratory capacity after co-culture.ConclusionsThe protective effects of FL treatment after burn injury are mediated by both pDCs and cDCs. Pharmacological enhancement of both DC subtypes by FL is a potential therapeutic intervention to enhance immune responses to infection and improve outcome after burn injury.

Antibodies raised against divalent type b flagellin and pilin provide effective immunotherapy against Pseudomonas aeruginosa infection of mice with burn wounds

Burn wound infections caused by multidrug-resistant Pseudomonas aeruginosa strains are a serious challenge to therapy because of the complex pathogenesis and paucity of new effective antibiotics. Therefore, there is renewed interest in developing antibody-based therapeutic strategies. Immunotherapy strategies typically target selected virulence factors that are expressed by the majority of clinical strains of P. aeruginosa, particularly because virulence factors mediate infection. Here we used a murine model of burn wound infection to evaluate the efficacy of antibodies raised against the divalent type b flagellin and PilA (flagellin b + PilA), as acute virulence factors, to prevent and treat infection. Antibodies to flagellin b + PilA exhibited superior synergistic effects that improved opsono-phagocytosis and cell invasion compared with antibodies to each monovalent flagellin b or PilA. Further, when used for prophylaxis, the antibodies against flagellin b + PilA and combined therapeutic and prophylactic regimens markedly improved the survival of mice infected with disparate P. aeruginosa strains from 91.6% to 100% compared with treatment using imipenem. Therefore, antibodies against flagellin b + PilA interfere with the activities of their respective cognate individual target antigens and enhance coverage against clinical strains of P. aeruginosa that may not express one of these two virulence factors.