Les spores fongiques sont à l'origine d'infections nosocomiales affectant le pronostic vital de patients immunodéprimés, et peuvent se transmettre par l'air. C'est pourquoi nous nous sommes intéressé à la déposition et au réenvol de spores d'"Aspergillus", responsables de pathologies gravissimes comme l'aspergillose pulmonaire invasive. Nous avons lors de nos expérimentations utilisé deux méthodes d'aérosolisation : le nébuliseur Collison standard, nécessitant la mise en solution des spores, ainsi qu'un prototype permettant de souffler directement sur les cultures fongiques Ceci nous a permis de mesurer la vitesse de sédimentation des spores, et d'évaluer l'efficacité et la rémanence de traitements fongicides en utilisant un protocole original mettant en œuvre des conditions réalistes.Un dispositif expérimental a été mis au point afin de soumettre des spores déposées sur une surface à un flux d'air tangentiel, et de filmer leur réenvol ( http://tinyurl.com/bla9ynz ), et un critère prédictif théorique de détachement a été exhibé. Des simulations numériques de l'écoulement autour de sphères idéales ont complété cette étude en nous donnant accès à des paramètres critiques inaccessibles expérimentalement.Nous avons finalement appliqué les résultats de nos investigations à la problématique des infections nosocomiales aérotransmises, et découvert que les ventilateurs de refroidissement d'appareils électroniques sont un réservoir de pathogènes et une source de contamination croisée potentielle. Des expériences en milieu contrôlé associées à une campagne de prélèvements en milieu hospitalier ont mis à jour ce nouveau et important risque de contamination.

An algorithmic method for the recognition of fungal spore cells in microscopic images, as well as its development and its origin, are described and demonstrated. The process is designed for a machine vision project which automatically identifies fungal spores within field samples for epidemiological simulation models. The method consists of a three-pass system that successfully recognizes spores in any position and which is tolerant of occlusion. / The algorithm, as implemented, demonstrated an accuracy of $ pm$5.3% on low quality images which is less than the assumed error of humans performing the same task. The processing speed also compared favorably with the performance of humans. / The method developed presents a framework of description that, through the first two passes, highlights certain distinctive aspects within an image. Those highlighted aspects are then recognized by the third pass. The system is loosely based on biological vision, is extremely versatile and could be adapted for the recognition of virtually any object in a digitized image.

Fluorescence techniques are being considered for the detection and identification of bacterial spores. This thesis sets out to empirically characterize the detailed autofluorescence spectroscopic properties of spores and their target molecules. The multiwavelength fluorescence studies from a unique endogenous biomarker, dipicolinic acid (DPA) and its calcium salt (CaDPA) in bacterial spores are found to be useful for fluorescence characterization of spores. A systematic determination of the fluorescence profile of the major chemical components of Bacillus spores and the effect of UV irradiation on them has been performed in dry samples, wet paste and in aqueous solution. The thesis applies reliable tools for accurately describing complex nature of spectral profile from bacterial spores, and for interpreting and identifying their spectral properties. We show that multiwavelength fluorescence technique combined with Principal Component Analysis (PCA) clearly indicates identifiable grouping among dry and wet Bacillus spore species. Differences are also observed between dried, wet and redried spores, indicating the stark effect of hydration on fluorescence fingerprints. The study revealed that changes in fluorescence of spores due to hydration/drying were reversible and supports a recent model of a dynamic and dormant spore structure. The spectra were analysed with PCA, revealing several spectroscopically characteristic features enabling spore species separation. The identified spectral features could be attributed to specific spore chemical components by comparing the spore sample signals with spectra obtained from the target molecules. PCA indicated underlying spectral patterns strongly related to species and the derived components were correlated with the chemical composition of the spore samples. More importantly, we examined and compared the fluorescence of normal spores with a mutant of the same strain whose spores lack DPA. We discovered that the dramatic fluorescence enhancement of Bacillus spores can be caused by UV irradiation in the spectral region of this unique biomarker without any pre treatment. Differences between spectra of spores, spore strains and other biological samples are very marked and are due to the dominance of the dipicolinate features in the spore spectra. This could lead to a cheap, more sensitive, faster and reagentless bacterial spore detector.

The full exploitation of bacterial whole-cell biosensing systems in field applications requires the survival of bacterial cells and long term-preservation of their sensing ability during transportation and on-site storage of such analytical systems. Specifically, there is a need for rapid, simple and inexpensive biosensing systems for monitoring human health and the environment in remote areas which often suffer from harsh atmospheric conditions and inadequate commercial distribution and storage facilities. Our laboratory has previously reported the successful use of bacterial spores as vehicles for the long-term preservation and storage of whole-cell biosensing systems at room temperature. In the present research, we have accomplished a year-long study to investigate the effect of extreme climatic conditions on the stability of spores-based whole-cell biosensing systems. The spores were stored in laboratory conditions that simulated those found in real harsh environments and germination ability and analytical performance of the spore-based sensing systems upon storage in such conditions was monitored. Our results proved that the intrinsic resistance of spores to harsh environmental conditions helped maintain the integrity of the sensor bacteria. The revived active cells actually retained their analytical performance during the course of the twelve-month storage study.

Spores of thermophilic bacilli are a common concern during the manufacture of milk powder. Spores are believed to occur in high numbers in milk powder due to their ability to survive pasteurisation, attach to stainless steel surfaces, germinate, grow as biofilms and subsequently enter the product stream and thereby contaminate the final product. In this study, thirty one thermophilic bacilli isolates were obtained from a New Zealand milk powder production line and identified as either Anoxybacillus flavithermus or Geobacillus spp. using random amplified polymorphic DNA (RAPD) and species-specific PCR. Sporulation media and a polyethylene glycol two-phase separation system were modified to produce high yields of spores free from debris. The spores of four Geobacillus spp. isolates (CGT-8, D4, E7 and E11) were characterised in terms of structure (electron microscopy), surface charge (zeta potential), hydrophobicity (contact angle and microbial adhesion to hexadecane) and attenuated total reflectance infrared spectroscopy (ATR-IR). Spores from three of the four isolates possessed an exosporium while the fourth did not. However the integrity of the exosporium varied over time. The spores were negatively charged (-10 to -20 mV) at neutral pH and high ionic strength (0.1 M KC1). Both hydrophobicity assays revealed that the spores of the four isolates were relatively hydrophilic while ATR-IR revealed the spores' surfaces consisted of protein and polysaccharides. The influence of these spore characteristics on adhesion to a variety of substrata under high flow rates was examined using the extended Derjaguin, Landau, Verwey and Overbeek (XDLVO) theory. Spores generally attached in higher numbers to hydrophobic surfaces compared to hydrophilic surfaces, however this observation was more prevalent for isolate D4. This result indicated that a single mechanism could not describe the adhesion of spores from different strains. A series of glass surfaces with modified characteristics were produced in order to test the antifouling properties on the adhesion of D4 spores. Spores suspended in a high ionic strength medium (0.1 M KC1) attached in greater numbers (1 Log₁₀ CFU cm⁻�) to positively charged and hydrophobic surfaces compared with negatively charged and hydrophilic surfaces. A clean in place (CIP) procedure, reduced spore numbers on hydrophobic and hydrophilic surfaces by 1.5 and by 2.0 Log₁₀ CFU cm⁻�, respectively. When spores were suspended in milk, there was little difference in the number of spores attaching to the different surfaces (ie. 3.5 to 3.8 Log₁₀ CFU cm⁻�), and spore removal from surfaces via a CIP regime was unchanged (1.5 to 2.0 Log₁₀ CFU cm⁻� reduction) compared with spores that attached in simple 1:1 electrolyte media. The effects of a caustic wash on spore surface characteristics and adhesion was determined. There was a significant reduction in spore viability (2 Log₁₀ CFU mL⁻�) after a 30 min caustic wash at 65 �C in the current study, however surviving spores displayed a greater propensity to attach to stainless steel. Surface characterisation results revealed an increase in hydrophobicity and a greater negative charge on the spores' surface after treatment with NaOH. Surviving spores could potentially recontaminate sections of the plant which are cleaned with this recycled caustic wash solution, thereby seeding surfaces with spores at the beginning of the next processing run. In conclusion, while surfaces that reduce spore adhesion and enhance removal can be produced, exposure to complex solutions such as milk can reduce the anti-fouling effectiveness of such surfaces to spore adhesion.

Spores of thermophilic bacilli are a common concern during the manufacture of milk powder. Spores are believed to occur in high numbers in milk powder due to their ability to survive pasteurisation, attach to stainless steel surfaces, germinate, grow as biofilms and subsequently enter the product stream and thereby contaminate the final product. In this study, thirty one thermophilic bacilli isolates were obtained from a New Zealand milk powder production line and identified as either Anoxybacillus flavithermus or Geobacillus spp. using random amplified polymorphic DNA (RAPD) and species-specific PCR. Sporulation media and a polyethylene glycol two-phase separation system were modified to produce high yields of spores free from debris. The spores of four Geobacillus spp. isolates (CGT-8, D4, E7 and E11) were characterised in terms of structure (electron microscopy), surface charge (zeta potential), hydrophobicity (contact angle and microbial adhesion to hexadecane) and attenuated total reflectance infrared spectroscopy (ATR-IR). Spores from three of the four isolates possessed an exosporium while the fourth did not. However the integrity of the exosporium varied over time. The spores were negatively charged (-10 to -20 mV) at neutral pH and high ionic strength (0.1 M KC1). Both hydrophobicity assays revealed that the spores of the four isolates were relatively hydrophilic while ATR-IR revealed the spores' surfaces consisted of protein and polysaccharides. The influence of these spore characteristics on adhesion to a variety of substrata under high flow rates was examined using the extended Derjaguin, Landau, Verwey and Overbeek (XDLVO) theory. Spores generally attached in higher numbers to hydrophobic surfaces compared to hydrophilic surfaces, however this observation was more prevalent for isolate D4. This result indicated that a single mechanism could not describe the adhesion of spores from different strains. A series of glass surfaces with modified characteristics were produced in order to test the antifouling properties on the adhesion of D4 spores. Spores suspended in a high ionic strength medium (0.1 M KC1) attached in greater numbers (1 Log₁₀ CFU cm⁻�) to positively charged and hydrophobic surfaces compared with negatively charged and hydrophilic surfaces. A clean in place (CIP) procedure, reduced spore numbers on hydrophobic and hydrophilic surfaces by 1.5 and by 2.0 Log₁₀ CFU cm⁻�, respectively. When spores were suspended in milk, there was little difference in the number of spores attaching to the different surfaces (ie. 3.5 to 3.8 Log₁₀ CFU cm⁻�), and spore removal from surfaces via a CIP regime was unchanged (1.5 to 2.0 Log₁₀ CFU cm⁻� reduction) compared with spores that attached in simple 1:1 electrolyte media. The effects of a caustic wash on spore surface characteristics and adhesion was determined. There was a significant reduction in spore viability (2 Log₁₀ CFU mL⁻�) after a 30 min caustic wash at 65 �C in the current study, however surviving spores displayed a greater propensity to attach to stainless steel. Surface characterisation results revealed an increase in hydrophobicity and a greater negative charge on the spores' surface after treatment with NaOH. Surviving spores could potentially recontaminate sections of the plant which are cleaned with this recycled caustic wash solution, thereby seeding surfaces with spores at the beginning of the next processing run. In conclusion, while surfaces that reduce spore adhesion and enhance removal can be produced, exposure to complex solutions such as milk can reduce the anti-fouling effectiveness of such surfaces to spore adhesion.

[Truncated abstract] Palynology has been used in a number of criminal cases where pollen and spores (palynomorphs) on clothing has featured as evidence. Pollen and spores are microscopic, generally morphologically unique to a plant genus and often species, resistant to decay, produced in large amounts and are components of soil. These unique features of pollen and spores make palynology a highly valuable forensic tool. Clothing is an excellent collector of pollen and spores as they become trapped in the fabric weave when clothing is brushed against flowering plants, comes into contact with dust, soil or air-borne pollen. Most forensic palynologists have found that palynomorphs from a crime scene may remain on clothing after washing or several days wear. No empirical research has been conducted on the retention of palynomorphs on clothing under differing conditions. Research of this kind is required to provide support for the future presentation and validation of palynological evidence in court. This project examined the relative retention of palynomorphs on clothing that had been worn during a simulated assault in a sheltered garden on the grounds of St George's College, Western Australia. Three replicate control soil samples each were collected from the actual assault scene and the whole garden to provide a baseline palynological profile for comparison to the experimental (Evidentiary) clothing samples. Forty pollen samples from the predominant species of plants in the garden and surrounds were collected, processed and databased as a reference for palynomorph identification. Standard T-shirts and jeans were chosen as the research clothing. During the simulated assault the knees of the jeans and the backs of the T-shirts came into abrasive contact with the soil of the garden for approximately one minute. The clothing then underwent three 'conditions' to simulate 'real life' situations. Three clothing sets were immediately collected after the assault (E1), three sets were worn for a period of three days after the assault (E2) and three sets were washed after the assault (E3). ... The Background clothing samples did not have a profile similar to the research garden but the profiles collected from each set reflected the areas to which they were worn. The number of palynomorphs per gram of garden soil ranged from thousands to tens-of-thousands of palynomorphs. The total number of palynomorphs collected by the E1 samples ranged from 100,000 to millions per clothing item. The E2 samples retained 1000's to tens-of-thousands of palynomorphs and the E3 samples retained 100's to 1000's of palynomorphs. The background clothing samples collected 1000's to tens-of-thousands of palynomorphs. These results confirm that jeans and T-shirts worn during an assault then worn for a period of days, or washed, will still contain pollen and spores characteristic of the assault area. This highlights the importance of investigating police enquiring where and for how long clothing of interest has been worn before and after an event, or if the clothing has been washed since the event. The results of this study will provide forensic palynologists with supportive data for future casework involving clothing.

Fluorescence techniques are being considered for the detection and identification of bacterial spores. This thesis sets out to empirically characterize the detailed autofluorescence spectroscopic properties of spores and their target molecules. The multiwavelength fluorescence studies from a unique endogenous biomarker, dipicolinic acid (DPA) and its calcium salt (CaDPA) in bacterial spores are found to be useful for fluorescence characterization of spores. A systematic determination of the fluorescence profile of the major chemical components of Bacillus spores and the effect of UV irradiation on them has been performed in dry samples, wet paste and in aqueous solution. The thesis applies reliable tools for accurately describing complex nature of spectral profile from bacterial spores, and for interpreting and identifying their spectral properties. We show that multiwavelength fluorescence technique combined with Principal Component Analysis (PCA) clearly indicates identifiable grouping among dry and wet Bacillus spore species. Differences are also observed between dried, wet and redried spores, indicating the stark effect of hydration on fluorescence fingerprints. The study revealed that changes in fluorescence of spores due to hydration/drying were reversible and supports a recent model of a dynamic and dormant spore structure. The spectra were analysed with PCA, revealing several spectroscopically characteristic features enabling spore species separation. The identified spectral features could be attributed to specific spore chemical components by comparing the spore sample signals with spectra obtained from the target molecules. PCA indicated underlying spectral patterns strongly related to species and the derived components were correlated with the chemical composition of the spore samples. More importantly, we examined and compared the fluorescence of normal spores with a mutant of the same strain whose spores lack DPA. We discovered that the dramatic fluorescence enhancement of Bacillus spores can be caused by UV irradiation in the spectral region of this unique biomarker without any pre treatment. Differences between spectra of spores, spore strains and other biological samples are very marked and are due to the dominance of the dipicolinate features in the spore spectra. This could lead to a cheap, more sensitive, faster and reagentless bacterial spore detector.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 3 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 5 uppsatser.