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.

Tularemia is a potentially fatal disease caused by the Category A bioterrorism agent Francisella tularensis. Aminoglycosides, fluoroquinolones, and tetracyclines can be used to treat tularemia; however, there is a high incidence of relapse and treatment failures when using these drugs. Furthermore, there is no tularemia vaccine licensed for use in the United States. Therefore, new antibiotics that target F. tularensis are being investigated. A novel family of resazurin-based antibiotics called resazomycins exhibit antimicrobial activity against F. tularensis and other Gram-negative pathogens including Neisseria gonorrhoeae. The mode of action of resazomycins has yet to be determined. To elucidate potential targets of resazurin (Rz), we screened for spontaneous Rz-resistant (Rzr) F. tularensis LVS mutants. Through the screen, 93% of all Rzr mutants sequenced contained mutations within the coding regions of FTL_0421, FTL_0895, and FTL_1504. In addition, 100% of all Rzr mutants sequenced contained a mutation approximately 50bp upstream of the gene FTL_0445. To understand the effect the mutation has on transcription of FTL_0445, RNA was isolated from wild-type LVS and an Rzr mutant (Rzr1). Quantitative reverse transcription PCR revealed FTL_0445 was upregulated in Rzr1 compared to wild-type LVS. To determine the role of FTL_0445 in Rz resistance, a FTL_0445 null deletion mutant is being generated in Rzr1 using standard molecular genetic techniques. Upon completion, the Rz sensitivity of the deletion mutant will be assessed using agar dilution assays. Understanding the role of FTL_0445 in Rz susceptibility would facilitate further development of these compounds as potential treatments for tularemia and gonorrhea.

The CDC classifies Francisella tularensis as a Category A bioterrorism agent. Due to the growing global threat of antibiotic resistant bacteria, novel therapeutics against F. tularensis must be developed. Resazomycins are resazurin (Rz)-based compounds that exhibit antimicrobial activity against F. tularensis and other gram-negative bacteria. The action of resazomycins is not well understood, but potential targets of the antibiotic were identified in a high throughput screen for Rz-resistant isolates. The dipA (FTL_1306) gene was identified as mutated in half of the 48 Rz-resistant (RZR) strains sequenced. To further investigate the role of dipA in Rz susceptibility, we introduced a wild-type copy of dipA into select RZR isolates (RZR1, 5, 43, and 46) that contain dipA mutations. The dipA gene was amplified by PCR from wild-type F. tularensis and cloned into the F. tularensis shuttle vector pABST to generate a construct (pABST-dipA) in which dipA will be constitutively expressed under control of the groEL promoter. The pABST-dipA plasmid was mobilized into each of the selected RZR isolates by electroporation. Western blotting indicated that expression of wild-type dipA was restored in RZR strains with the introduction of the pABST-dipA construct. The MIC of resazurin for the resulting RZR dipA-complemented strains was equivalent to that wild-type F. tularensis and significantly different from resistant mutants. Further investigation is needed to fully elucidate the contribution of dipA to the bactericidal action of resazomycins.

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.

In 1979, Gilula described three arcs that may aid in diagnosing disruption of the carpal bones in radiographic analysis. The first arc travels along the proximal boundaries of the proximal row of carpals. This study seeks to assess the first arc via geometric morphometric analysis to assess variance and sexual dimorphism. The study also assesses the potential for computer‐aided identification of carpal disruption, specifically, scapholunate ligament disruption. A total of 196 PA hand radiographs of dominant hands were assessed (153 females, 43 males). Discriminant function analysis revealed no significant difference in the contour of the first carpal arc between sexes (Mahalanobis distance = 22.4; T‐square = 16844.3; p = 0.8634). A principle component analysis was performed with the inclusion of an additional three radiographs demonstrating scapholunate ligament disruptions. All three scapholunate ligament disruptions placed outside of the 95% confidence ellipse of shape variance. Therefore, the dataset generated in this study may serve as a promising means of computer‐aided identification of derangement of the carpal bones in both females and males.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Extension of the index finger and thumb are typically independent movements that are not tethered by muscular attachment. This report demonstrates a case of an extensor pollicis et indicis communis found during the dissection of an 84 year‐old female body donor. The extensor pollicis et indicis communis is a rare muscular variation that originates in the posterior compartment of the forearm, traverses the fourth extensor compartment of the extensor retinaculum, and then bifurcates in the dorsum of the hand to insert at both the thumb and index finger. The action of this muscle is to extend the index and thumb simultaneously in addition to performing thumb reposition. Further, this variation is unique in that it would antagonize and potentially impede the act of pinching as well as opposition of the thumb— both functions that are well‐developed in humans. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .