T3SS and alginate biosynthesis of Pseudomonas aeruginosa impair healing of infected rabbit wounds.

Pseudomonas aeruginosa (a Gram-negative bacterium) is an opportunistic pathogen found in many infected wounds and is known to impair healing. To test the hypothesis that knocking out P. aeruginosa genes that are overexpressed during wound infection can cripple a pathogen's ability to impair healing, we assessed two pathways: the Type III secretion system (T3SS) and alginate biosynthesis. We generated single- and double-mutant strains of ExsA (T3SS activator), AlgD (GDP- mannose 6-dehydrogenase of alginate biosynthesis) and their complemented strains and evaluated their pathogenicity in a rabbit ear full-thickness excision-wound infection model. Wounds were inoculated with different strains (wild type, mutants, and complementary strains) at 106 CFU/wound on post-wounding day 3. After 24 h, 5 days and 9 days post-infection, wounds were harvested for measuring bacterial counts (viable and total) and wound healing (epithelial gap). On day 9 post-infection, the viable counts of the double mutant, (exsA/algD)‾ were 100-fold lower than the counts of the wild type (PAO1), single mutants, or the complement double-mutant, (exsA/algD)‾/+. Also, when compared to wounds infected with wild type or control strains, wounds infected with the double-knockout mutant was less inhibitory to wound healing (p < 0.05). Additionally, the double mutant showed greater susceptibility to macrophage phagocytosis in vitro than all other strains (p < 0.001). In conclusion, compared to single gene knockouts, double knockout of virulence genes in T3SS pathway and alginate biosynthesis pathway is more effective in reducing P. aeruginosa pathogenicity and its ability to impair wound healing. This study highlights the necessity of a dual-targeted anti-virulence strategy to improve healing outcomes of P. aeruginosa-infected wounds.

Temporal shifts in the mycobiome structure and network architecture associated with a rat (Rattus norvegicus) deep partial-thickness cutaneous burn.

With a diverse physiological interface to colonize, mammalian skin is the first line of defense against pathogen invasion and harbors a consortium of microbes integral in maintenance of epithelial barrier function and disease prevention. While the dynamic roles of skin bacterial residents are expansively studied, contributions of fungal constituents, the mycobiome, are largely overlooked. As a result, their influence during skin injury, such as disruption of skin integrity in burn injury and impairment of host immune defense system, is not clearly delineated. Burn patients experience a high risk of developing hard-to-treat fungal infections in comparison to other hospitalized patients. To discern the changes in the mycobiome profile and network assembly during cutaneous burn-injury, a rat scald burn model was used to survey the mycobiome in healthy (n = 30) (sham-burned) and burned (n = 24) skin over an 11-day period. The healthy skin demonstrated inter-animal heterogeneity over time, while the burned skin mycobiome transitioned toward a temporally stabile community with declining inter-animal variation starting at day 3 post-burn injury. Driven primarily by a significant increase in relative abundance of Candida, fungal species richness and abundance of the burned skin decreased, especially in days 7 and 11 post-burn. The network architecture of rat skin mycobiome displayed community reorganization toward increased network fragility and decreased stability compared to the healthy rat skin fungal network. This study provides the first account of the dynamic diversity observed in the rat skin mycobiome composition, structure, and network assembly associated with postcutaneous burn injury.

Live attenuated Francisella novicida vaccine protects against Francisella tularensis pulmonary challenge in rats and non-human primates.

Francisella tularensis causes the disease tularemia. Human pulmonary exposure to the most virulent form, F. tularensis subsp. tularensis (Ftt), leads to high morbidity and mortality, resulting in this bacterium being classified as a potential biothreat agent. However, a closely-related species, F. novicida, is avirulent in healthy humans. No tularemia vaccine is currently approved for human use. We demonstrate that a single dose vaccine of a live attenuated F. novicida strain (Fn iglD) protects against subsequent pulmonary challenge with Ftt using two different animal models, Fischer 344 rats and cynomolgus macaques (NHP). The Fn iglD vaccine showed protective efficacy in rats, as did a Ftt iglD vaccine, suggesting no disadvantage to utilizing the low human virulent Francisella species to induce protective immunity. Comparison of specific antibody profiles in vaccinated rat and NHP sera by proteome array identified a core set of immunodominant antigens in vaccinated animals. This is the first report of a defined live attenuated vaccine that demonstrates efficacy against pulmonary tularemia in a NHP, and indicates that the low human virulence F. novicida functions as an effective tularemia vaccine platform.

A Soft Skin Adhesive (SSA) Patch for Extended Release of Pirfenidone in Burn Wounds.

As much as half or more of deep partial-thickness burn wounds develop hypertrophic scarring and contracture. Once formed, treatments are only minimally effective. Pirfenidone (Pf), indicated for treatment of idiopathic pulmonary fibrosis, is an anti-inflammatory and anti-fibrotic small molecule that potentially can be repurposed as a preventative against scarring in burn wounds. We present a drug-in-matrix patch with a soft skin adhesive (SSA) wound-contacting layer for multi-day drug delivery of Pf into burn wounds at the point of injury. Our patch construction consists of an SSA adhesive layer (Liveo™ MG7-9850, Dupont, Wilmington, DE, USA) for wound fixation, an acrylic co-polymer drug matrix (DURO-TAK 87-2852, Henkel, Düsseldorf, Germany) as the drug (Pf) reservoir, and an outermost protective polyurethane backing. By employing a drug-in-matrix patch design, Pf can be loaded as high as 2 mg/cm2. Compared to the acrylic co-polymer adhesive patch preparations and commercial films, adding an SSA layer markedly reduces skin stripping observed under scanning electron microscopy (SEM). Moreover, the addition of varying SSA thicknesses did not interfere with the in vitro release kinetics or drug permeation in ex vivo porcine skin. The Pf patch can be easily applied onto and removed from deep partial-thickness burn wounds on Duroc pigs. Continuous multi-day dosing of Pf by the patches (>200 µg/cm2/day) reduced proinflammatory biomarkers in porcine burn wounds. Pf patches produced by the manual laboratory-scale process showed excellent stability, maintaining intact physical patch properties and in vitro biological activity for up to one year under long-term (25 °C at 60% RH) and 6 months under accelerated (40 °C at 75% RH) test conditions. To manufacture our wound safe-and-extended-release patch, we present scale-up processes using a machine-driven automated roll-to-roll pilot scale coater.

Comparative Transcriptome Analysis of Superficial and Deep Partial-Thickness Burn Wounds in Yorkshire vs Red Duroc Pigs.

Hypertrophic scars are a common negative outcome of deep partial-thickness (DPT) burn wounds resulting in increased dermal thickness, wound area contracture, and inflammation of the affected area. The red Duroc and Yorkshire porcine breeds are common large animal models for studying dermal wounds due to their structural similarities to human skin; however, the porcine transcriptomic profiles of dermal burn wounds and healing process are not well known. In response, a longitudinal transcriptomic comparative study was conducted comparing red Duroc and Yorkshire superficial and DPT burn wounds to their respective control uninjured tissue. Using next-generation RNA sequencing, total RNAs were isolated from burn wound tissue harvested on 0, 3, 7, 15, 30, and 60 days postburn, and mRNA-seq and gene expression read counts were generated. Significant differentially expressed genes relative to uninjured tissue were defined, and active biological processes were determined using gene set enrichment analyses. Additionally, collagen deposition, α-smooth muscle actin (SMA) protein concentration, epidermal and dermal thickness measurements, and wound area changes in response to burn injury were characterized. Overall, the red Duroc pigs, in response to both burn wound types, elicited a more robust and prolonged inflammatory immune response, fibroblast migration, and proliferation, as well as heightened levels of extracellular matrix modulation relative to respective burn types in the Yorkshire pigs. Collectively, the red Duroc DPT burn wounds produce a greater degree of hypertrophic scar-like response compared with Yorkshire DPT burn wounds. These findings will facilitate future porcine burn studies down-selecting treatment targets and determining the effects of novel therapeutic strategies.

Identification of Metagenomics Structure and Function Associated With Temporal Changes in Rat (Rattus norvegicus) Skin Microbiome During Health and Cutaneous Burn.

The cutaneous skin microbiome is host to a vast ensemble of resident microbes that provide essential capabilities including protection of skin barrier integrity and modulation of the host immune response. Cutaneous burn-injury promotes alteration of cutaneous and systemic immune response that can affect both commensal and pathogenic microbes. A cross-sectional study of a limited number of burn patients revealed a difference in the bacteriome of burned versus control participants. Temporal changes of the skin microbiome during health and cutaneous burn-injury remains largely unknown. Furthermore, how this microbial shift relates to community function in the collective metagenome remain elusive. Due to cost considerations and reduced healing time, rodents are frequently used in burn research, despite inherent physiological differences between rodents and human skin. Using a rat burn model, a longitudinal study was conducted to characterize the rat skin bacterial residents and associated community functions in states of health (n = 30) (sham-burned) and when compromised by burn-injury (n = 24). To address the knowledge gap, traumatic thermal injury and disruption of cutaneous surface is associated with genus-level changes in the microbiota, reduced bacterial richness, and altered representation of bacterial genes and associated predicted functions across different skin microbial communities. These findings demonstrate that, upon burn-injury, there is a shift in diversity of the skin's organismal assemblages, yielding a core microbiome that is distinct at the genome and functional level. Moreover, deviations from the core community correlate with temporal changes post-injury and community transition from the state of cutaneous health to disease (burn-injury).

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis.

Pulmonary infection with the bacterium Francisella tularensis can lead to the serious and potentially fatal disease, tularemia, in humans. Due to the current lack of an approved tularemia vaccine for humans, research is focused on vaccine development utilizing appropriate animal models. The Fischer 344 rat has emerged as a model that reflects human susceptibility to F. tularensis infection, and thus is an attractive model for tularemia vaccine development. Intratracheal inoculation of the Fischer 344 rat with F. tularensis mimics pulmonary exposure in humans. The successful delivery into the rat trachea is critical for pulmonary delivery. A laryngoscope with illumination is used to properly intubate the tracheae of anesthetized rats; the correct placement within the trachea is determined by a simple device to detect breathing. Following intubation, the F. tularensis culture is delivered in a measured dose via syringe. This technique standardizes pulmonary delivery of F. tularensis within the rat trachea to evaluate vaccine efficacy.

Lipidation of the FPI protein IglE contributes to Francisella tularensis ssp. novicida intramacrophage replication and virulence.

Francisella tularensis is a Gram-negative bacterium responsible for the human disease tularemia. The Francisella pathogenicity island (FPI) encodes a secretion system related to type VI secretion systems (T6SS) which allows F. tularensis to escape the phagosome and replicate within the cytosol of infected macrophages and ultimately cause disease. A lipoprotein is typically found encoded within T6SS gene clusters and is believed to anchor portions of the secretion apparatus to the outer membrane. We show that the FPI protein IglE is a lipoprotein that incorporates (3)H-palmitate and localizes to the outer membrane. A C22G IglE mutant failed to be lipidated and failed to localize to the outer membrane, consistent with C22 being the site of lipidation. Francisella tularensis ssp. novicida expressing IglE C22G is defective for replication in macrophages and unable to cause disease in mice. Bacterial two-hybrid analysis demonstrated that IglE interacts with the C-terminal portion of the FPI inner membrane protein PdpB, and PhoA fusion analysis indicated the PdpB C-terminus is located within the periplasm. We predict this interaction facilitates channel formation to allow secretion through this system.