Transcriptional profiling of the clpX mutant in Bacillus anthracis reveals regulatory connection with the lrgAB operon.

ClpX functions as either an independent chaperone or a component of the ClpXP protease, a conserved intracellular protease that acts as a global regulator in the bacterial cell by degrading regulatory proteins, stress response proteins and rate-limiting enzymes. Previously, we found that loss of clpX in Bacillus anthracis Sterne leads to increased susceptibility to antimicrobial agents that target the cell envelope. The aim of this study was to identify genes within the regulatory network of clpX that contribute to antimicrobial resistance. Using microarray analysis, we found 119 genes that are highly differentially expressed in the ∆clpX mutant, with the majority involved in metabolic, transport or regulatory functions. Several of these differentially expressed genes, including glpF, sigM, mrsA, lrgA and lrgB, are associated with cell wall-active antibiotics in other bacterial species. We focused on lrgA and lrgB, which form the lrgAB operon and are downregulated in ∆clpX, because loss of lrgAB increases autolytic activity and penicillin susceptibility in Staphylococcus aureus. While we observed no changes in autolytic activity in either ∆clpX or ∆lrgAB B. anthracis Sterne, we find that both mutants have increased susceptibility to the antimicrobial peptide LL-37 and daptomycin. However, phenotypes between ∆clpX and ∆lrgAB are not identical as ∆clpX also displays increased susceptibility to penicillin and nisin but ∆lrgAB does not. Therefore, while decreased expression of lrgAB may be partially responsible for the increased antimicrobial susceptibility seen in the ∆clpX mutant, disruption of other pathways must also contribute to this phenotype.

Enhancing the fathead minnow fish embryo toxicity test: Optimizing embryo production and assessing the utility of additional test endpoints.

The fathead minnow fish embryo toxicity (FET) test has been identified as a potential alternative to toxicity test methods that utilize older fish. However, several challenges have been identified with the fathead minnow FET test, including: 1) difficulties in obtaining appropriately-staged embryos for FET test initiation, 2) a paucity of data comparing fathead minnow FET test performance to the fathead minnow larval growth and survival (LGS) test and 3) a lack of sublethal endpoints that could be used to estimate chronic toxicity and/or predict adverse effects. These challenges were addressed through three study objectives. The first objective was to optimize embryo production by assessing the effect of breeding group composition (number of males and females) on egg production. Results showed that groups containing one male and four females produced the largest clutches, enhancing the likelihood of procuring sufficient numbers of embryos for FET test initiation. The second study objective was to compare the performance of the FET test to that of the fathead minnow LGS test using three reference toxicants. The FET and LGS tests were similar in their ability to predict the acute toxicity of sodium chloride and ethanol, but the FET test was found to be more sensitive than the LGS test for sodium dodecyl sulfate. The last objective of the study was to evaluate the utility and practicality of several sublethal metrics (i.e., growth, developmental abnormalities and growth- and stress-related gene expression) as FET test endpoints. Developmental abnormalities, including pericardial edema and hatch success, were found to offer the most promise as additional FET test endpoints, given their responsiveness, potential for predicting adverse effects, ease of assessment and low cost of measurement.

A Novel Hemiarthroplasty Design for Treatment of Post-traumatic Elbow Ankylosis and Distal Humerus Nonunion: A Case Report.

CASE: A 26 year old man sustained a blast injury to the right elbow, resulting in chronic distal humerus nonunion and post-traumatic ankylosis. After debridement and flap coverage, a custom distal humerus hemiarthroplasty construct with extramedullary orthogonal plating was used. Satisfaction and functional outcomes were maintained through 6 years of follow-up. CONCLUSION: This case presents a unique 1-stage surgical solution which demonstrates the potential for uncemented extramedullary hemiarthroplasty fixation with simultaneous compression plate osteosynthesis across a nonunion site. The potential for hemiarthroplasty designs to be linked to orthogonal plates preserves the intramedullary canal for future conversion to total elbow arthroplasty if necessary.

Galleria mellonella as an Infection Model for Bacillus anthracis Sterne.

Understanding bacterial virulence provides insight into the molecular basis behind infection and could identify new drug targets. However, assessing potential virulence determinants relies on testing in an animal model. The mouse is a well-validated model but it is constrained by the ethical and logistical challenges of using vertebrate animals. Recently the larva of the greater wax moth Galleria mellonella has been explored as a possible infection model for a number of pathogens. In this study, we developed G. mellonella as an infection model for Bacillus anthracis Sterne. We first validated two different infection assays, a survival assay and a competition assay, using mutants containing disruptions in known B. anthracis virulence genes. We next tested the utility of G. mellonella to assess the virulence of transposon mutants with unknown mutations that had increased susceptibility to hydrogen peroxide in in vitro assays. One of these transposon mutants also displayed significantly decreased virulence in G. mellonella. Further investigation revealed that this mutant had a disruption in the petrobactin biosynthesis operon (asbABCDEF), which has been previously implicated in both virulence and defense against oxidative stress. We conclude that G. mellonella can detect attenuated virulence of B. anthracis Sterne in a manner consistent with that of mammalian infection models. Therefore, G. mellonella could serve as a useful alternative to vertebrate testing, especially for early assessments of potential virulence genes when use of a mammalian model may not be ethical or practical.