Bacillus anthracis, the etiological agent of anthrax, is a spore- forming, toxin- producing bacterium. Currently, treatment of B. anthracis infections requires a 60- day antibiotic regimen. However, better therapeutics are required. Therefore, this study looked at the effect of levofloxacin and linezolid on B. anthracis cell viability, toxin production and spore formation using in vitro static models and a pharmacodynamic model. It was hypothesized that the combination would be the most effective at preventing toxin and spore production resulting in greater bacterial killing. However, these studies suggest otherwise. Nevertheless, clinically, the combination therapy may be more effective in rapid killing of vegetative B. anthracis and may be able to reduce the duration of therapy (by reducing the likelihood of spore survival). Therefore, the clinical benefit of combined therapy on long-term recurrence cannot be determined from these in vitro models. Further investigation with combination therapy is warranted. / October 2015

1) Einleitung 2) Pathogenese und Klinik des Botulismus 3) Vorkommen von C. botulinum und des Botulismus 4) Beeinflussung von Spore, vegetativer Zelle und BoNT 5) Diagnostik, Therapie und Prophylaxe 6) Diskussion

Doctor of Philosophy(PhD), / Airborne fungi are ubiquitous in the environment and human exposure is inevitable. Such fungi differ greatly in their taxonomic, physical, ecological and pathogenic characteristics. Currently, 69 000 species have been taxonomically classified and more than 80 of these are recognised to be aeroallergen sources. Many strategies have evolved to sample, identify and interpret fungal exposure to these species, however no strategy serves all purposes as exposure is a complex and dynamic process confounded by spatial, temporal and geographic variations in airborne counts, in addition to the inadequacies of the immunodiagnostic techniques available. To date, the interpretation of personal exposure and sensitisation to fungal allergens has been restricted to a few select species and the contribution of other genera, airborne hyphae and fragmented conidia to allergic disease are all poorly understood. The aim of the thesis was to utilize the Halogen Immunoassay (HIA) to diagnose fungal allergic sensitisation, to investigate the distribution and factors influencing allergens of fungi in the air and to understand what is actually inhaled in exposure settings. The novelty of the HIA derives from its unique ability to provide allergen sources that are actively secreted by the collected fungal spores and hyphae, which are bound to protein binding membranes (PBM) and then immunoprobed. In Chapter 2, the HIA was compared to the commercial in vitro Pharmacia UniCap assay (CAP) and the in vivo skin prick test (SPT), using 30 sera from subjects SPT positive to Aspergillus fumigatus and/or Alternaria alternata and 30 who were SPT negative to these fungi but sensitised to non-fungal allergens. Sera were analysed by CAP and the HIA against A. alternata, A. fumigatus, Cladosporium herbarum and Epicoccum purpurascens and compared statistically. Between 3% and 7% of SPT negative sera were identified to have specific IgE towards A. fumigatus and A. iv alternata, respectively. For the SPT positive sera, significant associations were found between the HIA and CAP scores for all fungal species tested (P<0.0001). Correlations between the HIA and SPT however, were weakly correlated for A. alternata (rs = 0.44, P<0.05) but not for A. fumigatus. In Chapter 3, personal exposure to indoor fungal aerosols was examined using the HIA to identify the fungal components that people were allergic to. Personal air sampling pumps (PASs) collected airborne fungal propagules onto PBMs for 2.5 hours indoors (n=21). Collected fungi were incubated overnight in a humid chamber to promote the germination of conidia. The membranes were then immunostained with pooled human Alternaria species-positive sera. All air samples contained fungal hyphae that expressed soluble allergens and were significantly higher in concentration than counts of conidia of individual well-characterised allergenic genera. Approximately 25% of all hyphae expressed detectable allergen compared to non-stained hyphae (P<0.05) and the resultant localisation of immunostaining was heterogeneous among hyphae. Fungal conidia of ten genera that were previously uncharacterised as allergen sources accounted for 8% of the total conidia that demonstrated IgE binding. In Chapter 4, the number and identity of fungi inhaled by 34 adults in an outdoor community setting was measured over 2 hour periods by people wearing Intra-nasal air samplers (INASs) and compared to fungal counts made with a Burkard spore trap and filter air samplers worn on the lapel. Using INAS, the most prevalent fungi inhaled belonged to soil borne spores of Alternaria, Arthrinium, Bipolaris, Cladosporium, Curvularia, Epicoccum, Exserohilum, Fusarium, Pithomyces, Spegazzinia, Tetraploa and Xylariaceae species, in addition to hyphal fragments. These results showed that inhaled exposure in most people varied in a 2-fold range with 10-fold outliers. In addition, the INAS and personal air filters agreed more with each other than with Burkard spore trap counts. The analysis was further confounded by different sampling efficiencies, locations of devices and ability to visualise and count fungal propagules. In Chapter 5, a double immunostaining technique based on the HIA was developed and applied to the conidia, hyphae and fungal fragments of A. alternata, A. fumigatus and Penicillium chrysogenum to discriminate between sources of allergens, v using IgE and to identify the fungi, using a fungal-specific antibody. The localisation of immunostaining was heterogeneous between both conidia and the state of germination with greater concentrations of double immunostaining detected following germination for each fungal species (P<0.0001). Fragmented A. alternata hyphae and morphologically indiscernible fragments could be identified for the first time using this technique. In Chapter 6, the factors affecting the release of allergen from the spores of eleven different species were studied. For nine of eleven species, between 5.7% and 92% of spores released allergen before germination. Ungerminated spores of P. chrysogenum and Trichoderma viride did not release detectable allergen. After germination, all spores that germinated eluted allergen from their hyphae. Upon germination there was a significant increase in the percentage of spores eluting detectable allergen (P<0.0001) and the localisation of allergen along the hyphae varied between species. Increased elution of allergen post germination might be a common feature of many species of allergenic fungi following inhalation. Additionally, Chapter 6 explored the extent to which inhaled spores or hyphae germinate after deposition in the nasal cavity and thus cause exposure to allergens. Twenty subjects had their noses lavaged at three separate intervals, (1) at the beginning of the experiment, (2) after one hour indoors and (3) after one hour outdoors. The recovery of spores and hyphal fragments from the nasal cavity varied between individuals and was significantly greater after outdoor exposures. Germinated fungal spores were recovered often in high concentrations for Aspergillus-Penicillium species, however the proportion between ungerminated and germinated spores were much lower for other genera recovered. Conclusions: Our analysis of cultured and wild-type fungi presents a new paradigm of natural fungal exposure, which in addition to commonly recognized species, implicates airborne hyphae, fragmented conidia and the conidia of a much more diverse range of genera as airborne allergens. Exposure is heterogeneous between individuals in the same geographic locality and the spectrum of fungal genera inhaled differs with the method of analysis. Many of the spores inhaled are likely to be allergenic, however upon germination there is an increased elution of allergen and this might be a common vi feature of many fungal species following inhalation. This project also provides novel techniques to diagnose fungal allergy by immunostaining wild-type fungi to which a patient is exposed with the patient’s own serum. Such an immunoassay combines environmental with serological monitoring on a patient specific basis and potentially avoids many problems associated with extract variability, based on the performance of current diagnostic techniques for fungal allergy.

Bacillus subtilis (ATCC 19659) were damaged by exposure to various concentrations of glutaraldehyde, as shown by decreased germination rates. The damage caused was repaired or otherwise obviated by the presence of sodium lactate in the holding medium. When two different salts of lactic acid were compared for ability to overcome the effect of glutaraldehyde, it was found that calcium salt of lactate was more effective than the sodium salt. The damage repair system involved l-alanine, lactate and either the sodium or calcium ions. The study involved in determining the difference in efficiency of spore repair was due to an organic or an amino acii snowed that the presence of two carboxylic functional groups did not effectively alter the reactivity.

An array of Bacillus licheniformis strains were isolated from a commercial powdered milk process. Bacteriophages exhibiting activity against B. licheniformis were isolated from cattle manure and effluent samples destined for a lagoon at a dairy farm. After sequencing, 8 of the 10 phages were found to be novel and genetically differentiated. Transmission electron scanning microscopy (TSEM) was performed. All bacteriophages were of the family Herelleviridae with contractile tail sheaths ranging from 80µm to 150µm and, surprisingly, survived a common fluid milk processing treatment used to inactivate vegetative cells. The survival of the phage after high temperature short time pasteurization of 73℃ for 20 s shows that the use of bacteriophages in milk to control B. licheniformis could be applied as a potential quality control, retarding the germination of spores and reduction of final spore counts in products with long run times such as dairy powders.

No description available.

Bacillus anthracis is a spore-forming bacterium that is included on the list of select agents compiled by the Centers for Disease Control. When a B. anthracis spore germinates, a protective layer of peptidoglycan known as the cortex must be depolymerized by germination-specific lytic enzymes (GSLEs) before the bacterium can become a metabolically active vegetative cell. By exploiting cortex lytic enzymes it may be possible to control germination. This could be beneficial in elucidating ways to enhance current decontamination methods. In this work we created in-frame deletion mutants to study not only the role of one GSLE, SleL, but by creating multi-deletion mutants, we were able to analyze how the protein cooperates with other lytic enzymes to efficiently hydrolyze the cortical PG. We determined that SleL plays an auxiliary role in complete peptidoglycan hydrolysis, secondary to cortex lytic enzymes CwlJ1, CwlJ2, and SleB. The loss of sleL results in a delay in the loss of optical density during germination. However, spores are capable of completing germination as long as CwlJ1 or SleB remains active. HPLC analysis of muropeptides collected from B. anthracis sleL strains indicates that SleL is an N-acetylglucosamidase that acts on cortical PG to produce small muropeptides which are quickly released from the germinating spore. By analyzing the in vitro and in vivo activities of SleL we confirmed the enzymatic activity of the protein, characterized its substrates, and studied the roles of its putative LysM domains in substrate binding and spore-protein association. We were able to show that purified SleL is capable of depolymerizing partially digested spore PG resulting in the production of N-acetylglucosaminidase products that are readily released as small muropeptides. In vitro, loss of the LysM domain(s) decreases hydrolysis effectiveness. The reduction in hydrolysis is likely due to LysM domains being involved in substrate recognition and PG binding. When the SleL derivatives are expressed in vivo those proteins lacking one or both LysM domains do not associate with the spore, suggesting that LysM is involved in directing protein localization. / Ph. D.

The Bacillus anthracis spore is highly resistant to environmental stresses, but cannot cause anthrax until it successfully germinates. An essential step of germination, degradation of the cortex peptidoglycan layer, is carried out by germination-specific lytic enzymes (GSLEs). While the GSLEs of several other Bacillus species have been investigated, they have not been characterized in the pathogen B. anthracis. In this work three GSLEs, CwlJ1, CwlJ2, and SleB are identified in B. anthracis and are investigated in order to better understand their functions. Genetic manipulation of cwlJ1, cwlJ2, and sleB was fundamental to this work. First, reporter gene fusions revealed that all three are expressed during spore formation and that CwlJ1 is likely the most abundant GSLE in the spore. Second, gene deletions eliminating each GSLE enabled the observation of mutant phenotypes during spore cortex degradation. CwlJ1 and SleB were identified as the most critical GSLEs for successful germination. High-performance liquid chromatography and mass spectroscopy revealed that SleB is required for lytic transglycosylase activity, but CwlJ1's mode of action was unclear. Multiple mutations of all of the GSLEs revealed that CwlJ2 is the least active GSLE, but that it participates in germination in response to Ca-Dipicolinic acid; a role it shares with the more dominant CwlJ1. Purification of the CwlJ1 and SleB proteins permitted in vitro assays of enzymatic activity as measured by changes in substrate optical density, solubility, and product formation. While CwlJ1 was recalcitrant to these methods, it was observed to cause cortex hydrolysis independently. SleB was more amenable and it was discovered to contain a peptidoglycan-binding domain that is primarily responsible for substrate binding, and a lytic transglycosylase domain that facilitates cortex-specific hydrolysis by recognizing muramic-δ-lactam. Future research will include determining the structure of SleB through x-ray crystallography and the identification of CwlJ1 activity by refining the protein purification method. The results of this and future research into CwlJ1, CwlJ2, and SleB may lead to a means to initiate spore germination prior to host infection. This will greatly ease spore decontamination measures, lower risk of infection, and discourage the use of B. anthracis spores as a biological weapon. / Ph. D.

The global food supply is being stressed by climate change, a growing population, and harmful diseases. One risk to vital cereal crops such as wheat and barley is Fusarium head blight (FHB), caused by the fungal plant pathogen Fusarium graminearum. Ascospores of the fungus are released from perithecia on the residues of corn and small grains and can be transported long distances (>500 m) through the atmosphere. The overall objective of this work was to assess the influence of meteorological conditions and perithecial maturity on ascospore release. The research focuses on F. graminearum because of its damaging impact to staple crops and the global ubiquity of FHB. The first specific objective was to apply state-of-the-science techniques to identify causal meteorological variables of ascospore release. We analyzed field measurements of airborne ascospores against meteorological conditions at Virginia Tech's Kentland Farm, Blacksburg, Virginia, USA and used convergent cross mapping and multivariate state space reconstruction to identify significant causal agents within this complicated natural and dynamic system. We identified relative humidity, solar radiation, wind speed, and air temperature as predictors of ascospore release. Our second research objective was to understand the impact of varying meteorological conditions on ascospore release under controlled environmental conditions. We assessed ascospore release in a chamber with controlled temperature (15°C and 25°C) and relative humidity (60%, 75%, and 95%). Ascospores released from ascospore-producing structures (perithecia) were captured on microscope slides placed inside of 3D-printed ascospore discharge devices. Results showed the sensitivity of ascospore release to relative humidity and temperature, with cool temperature and high relative humidity resulting in greater quantities of ascospores released. Our third research objective was to determine the relationship between the maturity, the number of ascospores, and the hardness of perithecia. A mechanical compression testing instrument was used to investigate the hardness of perithecia at various stages of maturity, producing a mean perithecium compression constant quantifying the uniaxial compression force required to rupture a perithecium. Results indicated that old perithecia contain the greatest amount of ascospores and exhibit increased resiliency, requiring greater forces to rupture, compared to young perithecia. This research has illustrated the complexities of F. graminearum ascospore release by describing the impact of several meteorological conditions and perithecial maturity on the timing and quantity of released ascospores. Collectively, our results may inform wheat growers on the nature and timing of ascospore release, which could help inform FHB management decisions in the future. / Ph. D.

La détection et l’identification de micro-organismes pathogènes est un réel enjeu de santé public tant au niveau alimentaire, que clinique ou d’intérêt national tel qu’illustré en biodéfense. Dans tous ces domaines, il est important d‘avoir des méthodes d’identification et de détection qui soient à la fois rapide, sensible et robuste. Cette thèse a pour objectif de contribuer au développement d’une approche rapide d’identification de micro-organismes sans a priori par spectrométrie de masse en tandem. Cette approche innovante, appelée phylopeptidomique, repose sur l’alliance de la peptidomique, i.e. analyse à large échelle des peptides provenant de la digestion enzymatique d’un échantillon biologique, et de la phylogénie des organismes cellulaires. Après extraction des protéines présentes dans l’échantillon à ausculter, des peptides sont générés et analysés par spectrométrie de masse en tandem. La déconvolution des signaux MS/MS à l’aide du logiciel « µOrg.ID » développé en propre au laboratoire permet d’identifier et quantifier les organismes présents dans l’échantillon en fonction des organismes indexés dans les bases de données. L’étude du protéome de Bacillus atrophaeus, agent simulant de l’anthrax, sous forme sporulée et végétative a permis d’illustrer une méthode d’identification de biomarqueurs protéiques permettant de déterminer le ratio entre les deux formes dans un échantillon. La limite de détection de la phylopeptidomique dans des échantillons purs et des échantillons en mélange équimolaire a été établie sur des bactéries modèles d’intérêts médical et environnemental. La limite de détection de spores de Bacillus atrophaeus en présence de 14 matrices interférentes (alimentaires, environnementales et autres) a permis de mettre en évidence les avantages et limitations de l’approche. Enfin, un mélange artificiel standardisé de 24 organismes a été développé afin d’évaluer les outils de bio-informatique en métaprotéomique. / The detection and identification of pathogenic microorganisms is a real public health issue for the food industry and the clinics or national interest as illustrated in the biodefense field. Thus, it is important to have identification and detection methods that are fast, sensitive and robust. This PhD thesis aims at contributing to the development of a rapid approach to identify microorganisms without any a priori by tandem mass spectrometry. This innovative approach, called phylopeptidomics, is based on the combination of peptidomics, i.e. large scale analysis of peptides derived from the enzymatic digestion of a biological sample, and the phylogeny of cellular organisms. After extraction of the proteins from the sample of interest, peptides are generated and analyzed by tandem mass spectrometry. The deconvolution of MS/MS signals using the "μOrg.ID" software developed in the laboratory enables the identification and quantification of organisms present in the sample according to the organisms indexed in generalist databases. The study of the proteome of Bacillus atrophaeus, a simulant agent of anthrax, in sporulated and vegetative form, has provided an illustration of a new method of identification of protein biomarkers, which allows determining the ratio between both forms. The limit of detection of phylopeptidomics in pure samples and equimolar mixtures was established with model bacteria of medical and environmental interests. The limit of detection of B. atrophaeus spores in the presence of 14 interfering matrices (food, environmental and others) has highlighted the advantages and limitations of the approach. Finally, a standardized artificial mixture of 24 organisms was developed in order to evaluate bioinformatics tools in metaproteomics.