Many spore forming bacteria are the causative agents of severe disease, such as Bacillus anthracis and anthrax. In these cases, the spore often acts as the infectious agent. Spores boast extreme resistance to chemical and UV damage among other bactericidal conditions. This is problematic due to the difficulty and economic costs of decontaminating exposure sites. The present work focuses on identifying and characterizing proteins active within spore germination, with a focus towards understanding the triggering of the major stages of germination. Understanding how each stage is initiated could allow for development of methods that induce these processes to efficiently germinate spores, thus facilitating cheap and effective decontamination. Sequencing of a spore transposon insertion library after exposure to germinants led to the identification of 42 genes with previously uncharacterized roles in spore germination. Fourteen of the genes, encoding proteins associated with the inner spore membrane, were further characterized. Mutants lacking these genes portrayed phenotypes consistent with failure of a GerA receptor-mediated germination response, and these genes affect the earliest stages of germination. Chemical cross-linking was used to characterize protein interactions important for stage II of spore germination. Site-directed in vivo crosslinking indicated that YpeB may exist as a multimer within the dormant spore. Further investigation of individual protein domains using bacterial two-hybrid analysis suggested that both N- and C-terminal domains of YpeB contribute to the formation of a multimer. In addition, the uncharacterized YpeB N-terminal domain was demonstrated to have strong self-association and may mediate self-association within the dormant spore. Additional genes that contribute to efficient initiation of spore germination in a GerA-dependent manner were identified via TnSeq. Chemical cross-linking of dormant spores was implemented to characterize protein interactions leading to stabilization and activation of an important enzyme that contributes to cortex degradation in stage II of germination. The presented studies employed a variety of techniques to provide additional insight into both stages of spore germination with a goal of furthering understanding of specific events that contribute to a loss of spore dormancy. / Doctor of Philosophy / Few bacterial species can undergo a specialized division process leading to the generation of a bacterial endospore. Endospores are dormant cells that boast resistance to a variety of environmental conditions that would otherwise cause bacterial cell death. These resistance traits make endospores immune to traditional bactericidal methods, making decontamination a nontrivial task. Further complicating the matter, spores are often the infectious particle of the associated disease, including hospital acquired diarrhea, infant botulism, anthrax, and many others. Presented work focuses on furthering understanding the process by which a dormant spore returns to a typical growing bacteria cell. Comprehension of major steps in this process may lead to novel methods for spore cleanup in which mechanisms within the spore are subverted to force a return to a typical bacterial cell state.

Food poisoning is mainly caused by pathogenic microorganisms and is now a severe problem worldwide. Therefore, rapid and sensitive methods are required to detect foodborne pathogens. A locally isolated bacterium, Bacillus licheniformis B38 was selected for this study. The spores of B. licheniformis B38 were induced by Schaeffer’s sporulation medium containing KCl, MgSO4.7H2O, Ca(NO3)4, MnCl2 and FeSO4. Schaeffer-Fulton endospore staining was used to differentiate spores and vegetative cells, where spores were stained green and vegetative cells were stained red. In order to separate the spores from the cells, a two-phase system was used to obtain pure spore suspension for following experiments. Spore coat proteins were extracted by SDS-8 M urea sample buffer and visualized by two different types of coomassie brilliant blue staining solutions. One of the staining solutions was more suitable for gel elution by diffusion. An ~10 kDa spore coat protein was selected for protein purification. Based on the given results, the protein purification by liquid chromatography was less convincing than using gel elution by diffusion technique. The two hypothetical protein sequences, P06552 and P45693, from the ~10 kDa spore coat protein were identified. In the preliminary study of B. licheniformis B38 spores detection by surface plasmon resonance, several binding parameters were studied. Dot blot was done to verify the reaction between the Bacillus spores polyclonal antibody against the B. licheniformis B38 spore coat protein. The most promising result was the binding of 0.1 mg/mL polyclonal antibody (analyte) to the 0.2 mg/mL spore coat protein at pH 2 (ligand) which showed 5.74 RU. The differences between a dot blot and a SPR detection techniques are described.

Si les flores contaminantes représentent la plupart du temps, dans les conserves, un risque industriel aujourd'hui maitrisé, la flore d'altération, de par sa résistance importante à la température, continue à constituer une cause de pertes économiques majeures. Pourtant cette dernière restait cependant peu caractérisée. En s'appuyant sur les travaux réalisés ces dernières années au sein de l'unité de microbiologie EMaiRIT'S du CTCPA (unité d'Expertise dans la Maitrise du Risque Industriel en Thermorésistants Sporulés du Centre Technique de la Conservation des Produits Agricoles), les principaux objectifs de cette thèse ont été (i) d'identifier et caractériser, en vue de sa maitrise ultérieure, la flore d'altération sporulante (ii) d'identifier l'origine de ces flores dans les conserveries et enfin (iii) de déterminer des moyens de maitrise.Pour cela, un état des lieux des bactéries sporulées d'altération des conserves a été effectué avec la collaboration de 122 conserveries sur plus de 10 ans en France. Cette caractérisation des espèces altérantes a permis l'élaboration d'un outil de biologie moléculaire (SporeTraQTM) afin d'identifier rapidement ces germes ou de pouvoir les détecter au sein d'une population complexe. En parallèle, l'amélioration de la connaissance de la thermorésistance de ces espèces, principale caractéristique de la flore sporulante, a été menée. A ce paramètre, il a été associé une relation avec la chimio résistance des spores. Identifiée, nous avons cherché à localiser cette flore d'altération au sein des usines à l'aide de plusieurs campagnes de prélèvements sur différents légumes. Au final, la flore spécifique du procédé de fabrication des conserves a été identifiée, caractérisée et localisée en vue d'améliorer la maitrise du risque microbien soit par une maitrise des contaminations et/ou un nettoyage plus performant (localisation au niveau d'étapes unitaires, efficacité de molécules sporicides) soir par un barème optimisé (en relation avec la thermorésistance). De plus, ce travail a été conduit au sein d'une approche bénéfice/risque représentant le futur de l'évolution des procédés agro-alimentaires associant amélioration de la qualité nutritionnelle et maintien de la maitrise sanitaire. Cette thèse s'appuie sur 5 publications de rang A. / Microbial contaminants of safety concern represent most of time, in canned food, an industrial risk which is well mastered. However, the spoilage flora, due to its high heat resistance, is responsible for major economic losses. Nevertheless, these bacteria remained poorly characterized. Based on the works realized during last 10 years within the EMaiRIT'S unit of microbiology of the CTCPA (expertise unit of the French Technical Center of the Preservation of Food, focused on Management of Industrial Risk liked to Heat Resistant Spores), the main objective of this thesis were: i) to identify and to characterize, with the aim of its later control, the spoilage spore forming bacteria florae ii) to identify the origin of these florae in canning factories and finally iii) to determine ways of control.For that purpose, a current inventory of spore forming bacteria in spoiled canned food was made with the cooperation of 122 canning factories over more than 10 years in France. This characterization of the spoilage species allowed the elaboration of a molecular biology tool (SporeTraQTM) for quick identification of these germs or their detection within a complex population. In parallel, the improvement of the knowledge about the heat resistance of these species, main characteristic of the spores, was led. In addition, the chemical resistance of spores was investigated. When identified, we tried to localize these spores on canning factories lines, with several sampling plans, on various vegetables. At the end, the specific spore forming bacteria related to the industrial canning process was identified, characterized and localized, allowing to improve the microbial risk control either by a more efficient cleaning, and through optimized process schedules. Furthermore, this work was driven within a benefic / risk approach representing the future of the food-processing evolution with improvement of the nutritional quality and the preservation of the sanitary control.This thesis leans on 5 publications of rank A.

No description available.

Chez Schizosaccharomyces pombe, le cycle méiotique est le mode de division cellulaire spécialisé qui permet la formation d’ascospores résistantes à différents stress lorsque les conditions environnementales ne sont pas propices à la multiplication cellulaire. Lors de mes travaux de thèse, mes objectifs consistaient à caractériser le rôle et le mécanisme d’action de la protéine Cuf2 lors du cycle méiotique chez S. pombe. Mes résultats ont montré que le gène cuf2[indice supérieur +] était exprimé exclusivement lors des divisions méiotiques et que la protéine se co-localisait de manière constitutive avec le matériel génétique. De plus, mes résultats ont dévoilé que Cuf2 participait à l’activation et à la répression de plusieurs gènes méiotiques selon un mécanisme de nature transcriptionnelle en s’associant spécifiquement avec leur région promotrice. Par la suite, mes résultats ont mis en évidence que Cuf2 interagissait physiquement avec Mei4, un facteur de transcription méiose-spécifique, au noyau des cellules méiotiques. Notamment, mes résultats ont montré que la présence de Mei4 et de son motif de liaison à l’ADN dénommé FLEX étaient nécessaires afin que Cuf2 puisse s’associer au promoteur de son gène cible fzr1[indice supérieur +] afin d’en activer l’expression. L’ensemble de mes résultats indiquent que Cuf2 et Mei4 interagissent aux promoteurs de certains gènes lors des divisions méiotiques afin d’en co-activer l’expression. D’ailleurs, mes résultats ont également montré que la fonction de Cuf2 était importante à la formation d’ascospores et à leur viabilité ; en absence de Cuf2, la majorité des ascospores présentent diverses aberrations et plus de la moitié d’entre elles sont non-viables. Globalement, mes résultats démontrent que Cuf2 est un régulateur critique de l’expression génique lors du cycle méiotique et que cette fonction est essentielle à la sporulation chez S. pombe.

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.

<p><em>Clostridium difficile</em> is a pathogen for both humans and animals and is often associated with antibiotic-associated diarrhea. Recently, several human cases of <em>C. difficile</em>-infection with increased mortality and morbidity have been reported. In studies performed in different countries <em>C. difficile</em> has been found in meat. Therefore the question whether <em>C. difficile</em> can be a zoonotic agent has been raised. The aim of this study was to optimize a method for isolation of <em>C. difficile</em> from faeces. When <em>C. difficile</em> is isolated from animals that do not have diarrhea the sample must be cultivated in an enrichment broth. Parameters influencing the enrichment were tested such as enrichment before and after spore selection, enrichment time, alcohol and heat chock for spore selection and if the samples had to be centrifuged or not before cultivation on agar plates. Enrichment in broth before spore selection was better than after. Heat and alcohol chock showed similar results, therefore you can chose which method you want. Cultivation from the pellet after centrifugation of the sample was better than cultivating directly from the inoculated broth. When the sample had low concentration of bacteria long enrichment time, 7 days or more, was best. The next step will be isolation of <em>C. difficile</em> from food-producing animals and humans and the strains will then be compared to se if the same strain is found in humans and in animals, to se if <em>C. difficile</em>-infection can be a zoonoz.</p>

Clostridium difficile is a pathogen for both humans and animals and is often associated with antibiotic-associated diarrhea. Recently, several human cases of C. difficile-infection with increased mortality and morbidity have been reported. In studies performed in different countries C. difficile has been found in meat. Therefore the question whether C. difficile can be a zoonotic agent has been raised. The aim of this study was to optimize a method for isolation of C. difficile from faeces. When C. difficile is isolated from animals that do not have diarrhea the sample must be cultivated in an enrichment broth. Parameters influencing the enrichment were tested such as enrichment before and after spore selection, enrichment time, alcohol and heat chock for spore selection and if the samples had to be centrifuged or not before cultivation on agar plates. Enrichment in broth before spore selection was better than after. Heat and alcohol chock showed similar results, therefore you can chose which method you want. Cultivation from the pellet after centrifugation of the sample was better than cultivating directly from the inoculated broth. When the sample had low concentration of bacteria long enrichment time, 7 days or more, was best. The next step will be isolation of C. difficile from food-producing animals and humans and the strains will then be compared to se if the same strain is found in humans and in animals, to se if C. difficile-infection can be a zoonoz.

Spore morphology of Polypodiaceae and Grammitidaceae from Taiwan were studied with light microscope and scanning electron microscope. Totally 18 genera and 62 species were observed. Polypodiaceae spores are ellipsoidal and monolete except Loxogramme grammitoides; with spore ornamentation tuberculate, verrucate, echinate, globule, rugate, undulate, foveolate and/or vermiculate. Grammitidaceae spores are global, trilete with spore ornamentation granulate, tuberculate and/or globules. The spore surface ornamentation can be used as taxonomic characters in Polypodiaceae and Grammitidaceae at familial, genera and species levels. Based on spore characters, Polypodiaceae and Grammitidaceae can be differentiated, Loxogrammeae and Drynariaeae are not separable from the rest of Polypodiaceae, and the current classification system of Lepisorieae and Microsoreae are not .internal consistent.

This work is devoted to formulation and development of a laser spectroscopic technique for rapid detection of biohazards, such as Bacillus anthracis spores. Coherent anti-Stokes Raman scattering (CARS) is used as an underlying process for active retrieval of species-specific characteristics of an analyte. Vibrational modes of constituent molecules are Raman-excited by a pair of ultrashort, femtosecond laser pulses, and then probed through inelastic scattering of a third, time-delayed laser field. We first employ the already known time-resolved CARS technique. We apply it to the spectroscopy of easy-to-handle methanol-water mixtures, and then continue building our expertise on solutions of dipicolinic acid (DPA) and its salts, which happen to be marker molecules for bacterial spores. Various acquisition schemes are evaluated, and the preference is given to multi-channel frequency-resolved detection, when the whole CARS spectrum is recorded as a function of the probe pulse delay. We demonstrate a simple detection algorithm that manages to differentiate DPA solution from common interferents. We investigate experimentally the advantages and disadvantages of near-resonant probing of the excited molecular coherence, and finally observe the indicative backscattered CARS signal from DPA and NaDPA powders. The possibility of selective Raman excitation via pulse shaping of the preparation pulses is also demonstrated. The analysis of time-resolved CARS experiments on powders and B. subtilis spores, a harmless surrogate for B. anthracis, facilitates the formulation of a new approach, where we take full advantage of the multi-channel frequency-resolved acquisition and spectrally discriminate the Raman-resonant CARS signal from the background due to other instantaneous four-wave mixing (FWM) processes. Using narrowband probing, we decrease the magnitude of the nonresonant FWM, which is further suppressed by the timing of the laser pulses. The devised technique, referred to as hybrid CARS, leads to a single-shot detection of as few as 104 bacterial spores, bringing CARS spectroscopy to the forefront of potential candidates for real-time biohazard detection. It also gives promise to many other applications of CARS, hindered so far by the presence of the overwhelming nonresonant FWM background, mentioned above.