The highly infectious and deadly pathogen, Francisella tularensis, is classified by the CDC as a Category A bioterrorism agent. Inhalation of a single bacterium results in an acute pneumonia with a 30-60% mortality rate without treatment. Due to the prevalence of antibiotic resistance, there is a strong need for new types of antibacterial drugs. Resazurin is commonly used to measure bacterial and eukaryotic cell viability through its reduction to the fluorescent product resorufin. When tested on various bacterial taxa at the recommended concentration of 44 µM, a potent bactericidal effect was observed against various Francisella and Neisseria species, including the human pathogens type A F. tularensis (Schu S4) and N. gonorrhoeae. As low as 4.4 µM resazurin was sufficient for a 10-fold reduction in F. tularensis growth. In broth culture, resazurin was reduced to resorufin by F. tularensis. However, resorufin also suppressed the growth of F. tularensis suggesting that the process of reducing resazurin was not responsible for the observed antimicrobial effect. Replication of F. tularensis in primary human macrophages and non-phagocytic cells was abolished following treatment with 44 μM resazurin indicating this compound could be an effective therapy for tularemia in vivo.

Medical devices, such as contact lenses, bring bacteria in direct contact with human cells. Consequences of these host-pathogen interactions include the alteration of mammalian cell surface architecture and induction of cellular death that renders tissues more susceptible to infection. Gram-negative bacteria known to induce cellular blebbing by mammalian cells, Pseudomonas and Vibrio species, do so through a type III secretion system-dependent mechanism. This study demonstrates that a subset of bacteria from the Enterobacteriaceae bacterial family induce cellular death and membrane blebs in a variety of cell types via a type V secretion-system dependent mechanism. Here, we report that ShlA-family cytolysins from Proteus mirabilis and Serratia marcescens were required to induce membrane blebbling and cell death. Blebbing and cellular death were blocked by an antioxidant and RIP-1 and MLKL inhibitors, implicating necroptosis in the observed phenotypes. Additional genetic studies determined that an IgaA family stress-response protein, GumB, was necessary to induce blebs. Data supported a model where GumB and shlBA are in a regulatory circuit through the Rcs stress response phosphorelay system required for bleb formation and pathogenesis in an invertebrate model of infection and proliferation in a phagocytic cell line. This study introduces GumB as a regulator of S. marcescens host-pathogen interactions and demonstrates a common type V secretion system-dependent mechanism by which bacteria elicit surface morphological changes on mammalian cells. This type V secretion-system mechanism likely contributes bacterial damage to the corneal epithelial layer, and enables access to deeper parts of the tissue that are more susceptible to infection. Bacteria must overcome host defenses to cause infection. This is especially true for corneal infections where bacteria must penetrate the epithelium in order to gain access to the stroma where bacteria can rapidly multiply, induce inflammation, and cause vision loss. Members of the Enterobacteriaceae commonly cause contact lens associated infections, but the mechanisms by which they damage corneal cells are largely unknown. Here we present evidence that Serratia marcescens and Proteus mirabilis are able to induce dramatic morphological changes in mammalian corneal cells that correlates with rapid cellular death. Secretion of ShlA-like cytolysins via type V secretion was responsible for this phenotype, and this effect was regulated by the conserved Rcs phosphorelay stress response system, including IgaA-family protein GumB. This study provides a model for stress-mediated regulation of cytolysins that induce epithelial damage and promote ocular infection.

The phenoxazine dye resazurin exhibits bactericidal activity against the Gram-negative pathogens Francisella tularensis and Neisseria gonorrhoeae. One resazurin derivative, resorufin pentyl ether, significantly reduces vaginal colonization by Neisseria gonorrhoeae in a mouse model of infection. The narrow spectrum of bacteria susceptible to resazurin and its derivatives suggests these compounds have a novel mode of action. To identify potential targets of resazurin and mechanisms of resistance, we isolated mutants of F. tularensis subsp. holarctica live vaccine strain (LVS) exhibiting reduced susceptibility to resazurin and performed whole genome sequencing. The genes pilD (FTL_0959) and dipA (FTL_1306) were mutated in half of the 46 resazurin-resistant (RZR) strains sequenced. Complementation of select RZR LVS isolates with wild-type dipA or pilD partially restored sensitivity to resazurin. To further characterize the role of dipA and pilD in resazurin susceptibility, a dipA deletion mutant, ΔdipA, and pilD disruption mutant, FTL_0959d, were generated. Both mutants were less sensitive to killing by resazurin compared to wild-type LVS with phenotypes similar to the spontaneous resazurin-resistant mutants. This study identified a novel role for two genes dipA and pilD in F. tularensis susceptibility to resazurin.

Distal radius fracture (DRF) is the most common fracture among pediatric patients, usually occurring after a fall onto an outstretched hand. The growing incidence of DRFs highlights the need to explore improved treatment methods to ensure proper healing and realignment. Current immobilization methods are inconsistent and have negative impacts on patient satisfaction. Drawbacks of fiberglass casts include thermal injuries, discomfort, and increased risk of skin infection. Although prefabricated splints have less downsides than fiberglass casts, skin irritation and odor are still a concern. 3D-printed orthoses present a viable alternative to traditional immobilization methods for DRFs, especially in the pediatric population. By scanning the fractured radius, the orthosis is tailored specifically to the patient's anatomy. 3D-printed orthoses are water resistant, breathable, and tend to weigh less. As a result, they offer improved hygiene, comfort, and patient satisfaction while reducing complications seen with more traditional methods of immobilization. Preliminary research suggests that 3D-printed orthoses are as effective as traditional immobilization methods in pediatric patients with DRFs. However, additional studies are needed to further assess the patient satisfaction, compliance, and durability of 3D-printed orthoses compared with traditional immobilization methods in the pediatric population.

Legg-Calvé-Perthes disease (LCPD) is a condition marked by temporary blood flow disruption to the proximal femur, commonly afflicting children aged 15 years and younger. The etiology of the disease is often idiopathic and involves the development of avascular necrosis of the femoral head, subsequently leading to bone weakening and deformity. Obesity exacerbates LCPD, correlating with delayed diagnosis, increased disease severity, and bilateral involvement. Leptin, growth hormone, and other inflammatory mediator alterations in obese individuals contribute to the pathogenesis. Treatment ranges from conservative measures to surgery, with particular challenges in obese patients. An improved understanding of the impact of obesity on LCPD progression is crucial for tailored management and optimal outcomes.

Neisseria gonorrhoeae has developed resistance to every antibiotic currently approved for the treatment of gonorrhea, prompting the development of new therapies. The phenoxazine dye resazurin exhibits robust antimicrobial activity against N. gonorrhoeae in vitro but fails to limit vaginal colonization by N. gonorrhoeae in a mouse model. The lack of in vivo efficacy may be due to oxygen limitation as in vitro susceptibility assays with resazurin are conducted under atmospheric oxygen while a microaerophilic environment is present in the vagina. Here, we utilized broth microdilution assays to determine the susceptibility of N. gonorrhoeae to resazurin under low and atmospheric oxygen conditions. The minimal inhibitory concentration of resazurin for multiple N. gonorrhoeae clinical isolates was significantly higher under low oxygen. This effect was specific to resazurin as N. gonorrhoeae was equally susceptible to other antibiotics under low and atmospheric oxygen conditions. The reduced susceptibility of N. gonorrhoeae to resazurin under low oxygen was largely attributed to reduced oxidative stress, as the addition of antioxidants under atmospheric oxygen mimicked the reduced susceptibility to resazurin observed under low oxygen. Together, these data suggest oxygen concentration is an important factor to consider when evaluating the efficacy of new antibiotics against N. gonorrhoeae in vitro.