Rapid vaccination using an acetalated dextran microparticulate subunit vaccine confers protection against triplicate challenge by bacillus anthracis.

PURPOSE: A rapid immune response is required to prevent death from Anthrax, caused by Bacillus anthracis. METHOD: We formulated a vaccine carrier comprised of acetalated dextran microparticles encapsulating recombinant protective antigen (rPA) and resiquimod (a toll-like receptor 7/8 agonist). RESULTS: We were able to protect against triplicate lethal challenge by vaccinating twice (Days 0, 7) and then aggressively challenging on Days 14, 21, 28. A significantly higher level of antibodies was generated by day 14 with the encapsulated group compared to the conventional rPA and alum group. Antibodies produced by the co-encapsulated group were only weakly-neutralizing in toxin neutralization; however, survival was not dependent on toxin neutralization, as all vaccine formulations survived all challenges except control groups. Post-mortem culture swabs taken from the hearts of vaccinated groups that did not produce significant neutralizing titers failed to grow B. anthracis. CONCLUSIONS: Results indicate that protective antibodies are not required for rapid protection; indeed, cytokine results indicate that T cell protection may play a role in protection from anthrax. We report the first instance of use of a particulate carrier to generate a rapid protective immunity against anthrax.

Bacillus anthracis spore interactions with mammalian cells: relationship between germination state and the outcome of in vitro.

BACKGROUND: During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium. RESULTS: Using an in vitro model of infection, we evaluated the influence of the germination state of B. anthracis spores, as controlled by defined culture conditions, on the outcome of infection. Spores prepared from B. anthracis Sterne 7702 germinated in a variety of common cell culture media supplemented with fetal bovine serum (FBS) while, in the absence of FBS, germination was strictly dependent on medium composition. RAW264.7 macrophage-like cells internalized spores to the same extent in either germinating or non-germinating media. However, significantly more viable, intracellular B. anthracis were recovered from cells infected under non-germinating conditions compared to germinating conditions. At the same time, RAW264.7 cells demonstrated a significant loss in viability when infected under non-germinating conditions. CONCLUSIONS: These results suggest that the outcome of host cell infection is sensitive to the germination state of spores at the time of uptake. Moreover, this study demonstrates the efficacy of studying B. anthracis spore infection of host cells within a defined, non-germinating, in vitro environment.

An Observational Study of Sepsis in Takeo Province Cambodia: An in-depth examination of pathogens causing severe infections.

The world's most consequential pathogens occur in regions with the fewest diagnostic resources, leaving the true burden of these diseases largely under-represented. During a prospective observational study of sepsis in Takeo Province Cambodia, we enrolled 200 patients over an 18-month period. By coupling traditional diagnostic methods such as culture, serology, and PCR to Next Generation Sequencing (NGS) and advanced statistical analyses, we successfully identified a pathogenic cause in 46.5% of our cohort. In all, we detected 25 infectious agents in 93 patients, including severe threat pathogens such as Burkholderia pseudomallei and viral pathogens such as Dengue virus. Approximately half of our cohort remained undiagnosed; however, an independent panel of clinical adjudicators determined that 81% of those patients had infectious causes of their hospitalization, further underscoring the difficulty of diagnosing severe infections in resource-limited settings. We garnered greater insight as to the clinical features of severe infection in Cambodia through analysis of a robust set of clinical data.

Inhibition of Bacillus anthracis spore outgrowth by nisin.

The lantibiotic nisin has previously been reported to inhibit the outgrowth of spores from several Bacillus species. However, the mode of action of nisin responsible for outgrowth inhibition is poorly understood. By using B. anthracis Sterne 7702 as a model, nisin acted against spores with a 50% inhibitory concentration (IC(50)) and an IC(90) of 0.57 microM and 0.90 microM, respectively. Viable B. anthracis organisms were not recoverable from cultures containing concentrations of nisin greater than the IC(90). These studies demonstrated that spores lose heat resistance and become hydrated in the presence of nisin, thereby ruling out a possible mechanism of inhibition in which nisin acts to block germination initiation. Rather, germination initiation is requisite for the action of nisin. This study also revealed that nisin rapidly and irreversibly inhibits growth by preventing the establishment of oxidative metabolism and the membrane potential in germinating spores. On the other hand, nisin had no detectable effects on the typical changes associated with the dissolution of the outer spore structures (e.g., the spore coats, cortex, and exosporium). Thus, the action of nisin results in the uncoupling of two critical sequences of events necessary for the outgrowth of spores: the establishment of metabolism and the shedding of the external spore structures.

High-throughput, single-cell analysis of macrophage interactions with fluorescently labeled Bacillus anthracis spores.

The engulfment of Bacillus anthracis spores by macrophages is an important step in the pathogenesis of inhalational anthrax. However, from a quantitative standpoint, the magnitude to which macrophages interact with and engulf spores remains poorly understood, in part due to inherent limitations associated with commonly used assays. To analyze phagocytosis of spores by RAW264.7 macrophage-like cells in a high-throughput, nonsubjective manner, we labeled B. anthracis Sterne 7702 spores prior to infection with an Alexa Fluor 488 amine-reactive dye in a manner that did not alter their germination, growth kinetics, and heat resistance. Using flow cytometry, large numbers of cells exposed to labeled spores were screened to concurrently discriminate infected from uninfected cells and surface-associated from internalized spores. These experiments revealed that spore uptake was not uniform, but instead, highly heterogeneous and characterized by subpopulations of infected and uninfected cells, as well as considerable variation in the number of spores associated with individual cells. Flow cytometry analysis of infections demonstrated that spore uptake was independent of the presence or absence of fetal bovine serum, a germinant that, while routinely used in vitro, complicates the interpretation of the outcome of infections. Two commonly used macrophage cell lines, RAW264.7 and J774A.1 cells, were compared, revealing significant disparity between these two models in the rates of phagocytosis of labeled spores. These studies provide the experimental framework for investigating mechanisms of spore phagocytosis, as well as quantitatively evaluating strategies for interfering with macrophage binding and uptake of spores.

Melioidosis in lower provincial Cambodia: A case series from a prospective study of sepsis in Takeo Province.

Melioidosis is a severe infectious disease caused by the gram-negative soil bacterium Burkholderia pseudomallei. Melioidosis is well known to be a major cause of morbidity and mortality in Southeast Asia, particularly in Thailand. However, melioidosis remains underreported in surrounding areas such as Cambodia. We report a case series of melioidosis in seven patients from Takeo Province, Cambodia. The patients, aged 24-65 years, were enrolled from May 2014 to May 2015 during a one year prospective study of sepsis at Takeo Provincial Hospital. They presented with fever, rigors, dyspnea, fatigue, diaphoresis, productive cough, and skin abscesses. Six of the seven patients were also hyponatremic. B. pseudomallei was cultured from the blood of six patients and the sputum of one patient. In this manuscript, we provide a detailed description of the clinical presentation, case management and laboratory confirmation of B. pseudomallei, as well as discuss the difficulties of identifying and treating melioidosis in low resource settings.

Multiphoton laser scanning microscopy as a tool for Xenopus oocyte research.

Multiphoton laser scanning microscopy (MPLSM) has become an increasingly invaluable tool in fluorescent optical imaging. There are several distinct advantages to implementing MPLSM as a Xenopus oocyte research tool. MPLSM increases signal-to-noise ratio and therefore increases image quality because there is no out-of-focus fluorescence as would be created in conventional or confocal microscopy. All the light that is generated can be collected and used to generate an image because point detection of descanned fluorescence is not required. This is particularly useful when imaging deep into tissue sections, as is necessary for Xenopus oocytes, which are notoriously large (approximately 1-mm diameter). Because multiphoton lasers use pulsed energy in the infrared wavelengths, the energy can also travel further into tissues with much less light scattering. Because there is no out-of-focus excitation, phototoxicity, photodamage, and photobleaching are significantly reduced, which is particularly important for long-term experiments that require the same region to be scanned repeatedly. Finally, multiple fluorophores can be simultaneously excited because of the broader absorption spectra of multiphoton dyes. In this chapter, we describe the advantages and disadvantages of using MPLSM to image Xenopus oocytes as compared to conventional and confocal microscopy. The practical application of imaging oocytes is demonstrated with specific examples.

Identification and functional analysis of Salmonella enterica serovar Typhimurium PmrA-regulated genes.

The PmrA-PmrB two-component regulatory system of Salmonella enterica serovar Typhimurium is activated in vivo and plays an important role in resistance to cationic antimicrobial peptides. Resistance is partly mediated by modifications to the lipopolysaccharide. To identify new PmrA-regulated genes, microarray analysis was undertaken comparing cDNA derived from PmrA-constitutive and PmrA-null strains. A combination of RT-PCR and transcriptional analysis confirmed the inclusion of six new loci in the PmrA-PmrB regulon: STM1253, STM1269, STM4118, STM0459, STM3968 and STM4568. These loci did not affect the ability to grow in high iron conditions, the ability to modify lipid A with aminoarabinose, or virulence. STM4118, a putative phosphoethanolamine phosphotransferase, had a minor effect on polymyxin resistance, whereas the remaining genes had no role in polymyxin resistance. Although several of the identified loci lacked the consensus PmrA binding site, PmrA was demonstrated to bind the promoter of a PmrA-activated gene lacking the consensus site. A more complete definition of the PmrA-PmrB regulon will provide a better understanding of its role in host and non-host environments.

Salmonella enterica serovar typhimurium resistance to bile: identification and characterization of the tolQRA cluster.

Salmonella enterica serovar Typhimurium is resistant to the action of bile salts, and resistance to bile is enhanced in strains in which the PhoP-PhoQ (PhoPQ) two-component regulatory system has been activated. To identify genes necessary for bile resistance, MudJ transposon mutagenesis was performed on a strain containing a phoP mutation that results in constitutive expression of PhoP-activated genes. After screening >10,000 mutants for the loss of growth on Luria-Bertani broth-bile plates, 14 bile-sensitive mutants were identified. Of these 14 mutants, 3 were found to retain the bile sensitivity phenotype upon P22 transduction, to possess wild-type growth characteristics, and to contain a smooth lipopolysaccharide. Southern hybridization experiments showed that all three strains contained unique insertions. DNA sequencing of the transposon-chromosomal-DNA fusion junctions of these strains showed all to be linked to the putative Salmonella orf1-tolQRA operon, with insertions in tolQ, orf1, and a gene upstream of the orf1-tolQRA operon not previously associated with Tol function (orfX). Through the use of transcriptional fusions, none of the putative tol (or tol-associated) genes were shown to be regulated by PhoPQ, bile, or the RcsC-RcsB two-component system; however, all of the genes (orfX, orf1, tolQRA) are predicted to be cotranscribed. This is the first identification of Salmonella serovar Typhimurium Tol homologs and the first demonstration of their role in bile resistance in this organism. In addition, the observed regulation, operon arrangement, and phenotypes associated with these tol genes demonstrate significant differences from their Escherichia coli homologs.