Mucorales species cause debilitating, life-threatening sinopulmonary diseases in immunocompromised patients and penetrating wounds in trauma victims. Common antifungal agents against mucormycosis have significant toxicity and are often ineffective. To evaluate treatments against mucormycosis, sporangiospores are typically used for in vitro assays and in pre-clinical animal models of pulmonary infections. However, in clinical cases of wound mucormycosis caused by traumatic inoculation, hyphal elements found in soil are likely the form of the inoculated organism. In this study, Galleria mellonella larvae were infected with either sporangiospores or hyphae of Rhizopus arrhizus and Lichtheimia corymbifera. Hyphal infections resulted in greater and more rapid larval lethality than sporangiospores, with an approximate 10–16-fold decrease in LD₅₀ of hyphae for R. arrhizus (p = 0.03) and L. corymbifera (p = 0.001). Liposomal amphotericin B, 10 mg/kg, was ineffective against hyphal infection, while the same dosage was effective against infections produced by sporangiospores. Furthermore, in vitro, antifungal susceptibility studies show that minimum inhibitory concentrations of several antifungal agents against hyphae were higher when compared to those of sporangiospores. These findings support using hyphal elements of Mucorales species for virulence testing and antifungal drug screening in vitro and in G. mellonella for studies of wound mucormycosis.

Wound-invasive fungal diseases (WIFDs), especially mucormycosis, have emerged as life-threatening infections during recent military combat operations. Many combat-relevant fungal pathogens are refractory to current antifungal therapy. Therefore, animal models of WIFDs are urgently needed to investigate new therapeutic solutions. Our study establishes combat-relevant murine models of wound mucormycosis using Rhizopus arrhizus and Lichtheimia corymbifera, two Mucorales species that cause wound mucormycosis worldwide. These models recapitulate the characteristics of combat-related wounds from explosions, including blast overpressure exposure, full-thickness skin injury, fascial damage, and muscle crush. The independent inoculation of both pathogens caused sustained infections and enlarged wounds. Histopathological analysis confirmed the presence of necrosis and fungal hyphae in the wound bed and adjacent muscle tissue. Semi-quantification of fungal burden by colony-forming units corroborated the infection. Treatment with liposomal amphotericin B, 30 mg/kg, effectively controlled R. arrhizus growth and significantly reduced residual fungal burden in infected wounds (p < 0.001). This study establishes the first combat-relevant murine model of wound mucormycosis, paving the way for developing and evaluating novel antifungal therapies against combat-associated WIFDs.

Bloodstream infections are a significant threat to soldiers wounded in combat and contribute to preventable deaths. Novel and combination therapies that can be delivered on the battlefield or in lower roles of care are urgently needed to address the threat of bloodstream infection among military personnel. In this manuscript, we tested the antibacterial capability of silver ions (Ag⁺), with long-appreciated antibacterial properties, against ESKAPEE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species, and Escherichia coli) pathogens. We used the GENESYS (RAIN LLC) device to deliver Ag⁺ to Gram-positive and Gram-negative ESKAPEE organisms grown in broth, human blood, and serum. Following the Ag⁺ treatment, we quantified the antibacterial effects by quantifying colony-forming units. We found that Ag⁺ was bactericidal against 5 Gram-negative organisms, K pneumoniae, A baumannii, P aeruginosa, E cloacae, and E coli, and bacteriostatic against 2 Gram-positive organisms, E faecium and S aureus. The whole blood and serum inhibited the bactericidal activity of Ag⁺ against a common agent of bloodstream infection, P aeruginosa. Finally, when Ag⁺ was added in conjunction with antibiotic in the presence of whole blood, there was no significant effect of Ag⁺ over antibiotic alone. Our results confirmed that Ag⁺ has broad-spectrum antibacterial properties. However, the therapeutic value of Ag⁺ may not extend to the treatment of bloodstream infections because of the inhibition of Ag⁺ activity in blood and serum.

Introduction: Extensive trauma, commonly seen in wounded military Service Members, often leads to a severe sterile inflammation termed systemic inflammatory response syndrome (SIRS), which can progress to multiple organ dysfunction syndrome (MODS) and death. MODS is a serious threat to wounded Service Members, historically causing 10% of all deaths in trauma admissions at a forward deployed combat hospital. The importance of this problem will be exacerbated in large-scale combat operations, in which evacuation will be delayed and care of complex injuries at lower echelons of care may be prolonged. The main goal of this study was to optimize an existing mouse model of lethal SIRS/MODS as a therapeutic screening platform for the evaluation of immunomodulatory drugs. Materials and Methods: Male C57BL/6 mice were euthanized, and the bones and muscles were collected and blended into a paste termed tissue–bone matrix (TBX). The TBX at 12.5%–20% relative to body weight of each recipient mouse was implanted into subcutaneous pouches created on the dorsum of anesthetized animals. Mice were observed for clinical scores for up to 48 hours postimplantation and euthanized at the preset point of moribundity. To test effects of anesthetics on TBX-induced mortality, animals received isoflurane or ketamine/xylazine (K/X). In a separate set of studies, mice received TBX followed by intraperitoneal injection with 20 mg/kg or 40 mg/kg Eritoran or a placebo carrier. All Eritoran studies were performed in a blinded fashion. Results: We observed that K/X anesthesia significantly increased the lethality of the implanted TBX in comparison to inhaled anesthetics. Although all the mice anesthetized with isoflurane and implanted with 12.5% TBX survived for 24 hours, 60% of mice anesthetized with K/X were moribund by 24 hours postimplantation. To mimic more closely the timing of lethal SIRS/MODS following polytrauma in human patients, we extended observation to 48 hours. We performed TBX dose–response studies and found that as low as 15%, 17.5%, and 20% TBX caused moribundity/mortality in 50%, 80%, and 100% mice, respectively, over a 48-hour time period. With 17.5% TBX, we tested if moribundity/mortality could be rescued by anti-inflammatory drug Eritoran, a toll-like receptor 4 antagonist. Neither 20 mg/kg nor 40 mg/kg doses of Eritoran were found to be effective in this model. Conclusions: We optimized a TBX mouse model of SIRS/MODS for the purpose of evaluating novel therapeutic interventions to prevent trauma-related pathophysiologies in wounded Service Members. Negative effects of K/X on lethality of TBX should be further evaluated, particularly in the light of widespread use of ketamine in treatment of pain. By mimicking muscle crush, bone fracture, and necrosis, the TBX model has pleiotropic effects on physiology and immunology that make it uniquely valuable as a screening tool for the evaluation of novel therapeutics against trauma-induced SIRS/MODS.