Développement et évaluation de méthodes d'isolation des spores de champignons contenues dans l'eau des rivières

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Real time detectionof airborne fungal spores and investigations into their dynamics in indoor air

Concern regarding the health effects of indoor air quality has grown in recent years, due to the increased prevalence of many diseases, as well as the fact that many people now spend most of their time indoors. While numerous studies have reported on the dynamics of aerosols indoors, the dynamics of bioaerosols in indoor environments are still poorly understood and very few studies have focused on fungal spore dynamics in indoor environments. Consequently, this work investigated the dynamics of fungal spores in indoor air, including fungal spore release and deposition, as well as investigating the mechanisms involved in the fungal spore fragmentation process. In relation to the investigation of fungal spore dynamics, it was found that the deposition rates of the bioaerosols (fungal propagules) were in the same range as the deposition rates of nonbiological particles and that they were a function of their aerodynamic diameters. It was also found that fungal particle deposition rates increased with increasing ventilation rates. These results (which are reported for the first time) are important for developing an understanding of the dynamics of fungal spores in the air. In relation to the process of fungal spore fragmentation, important information was generated concerning the airborne dynamics of the spores, as well as the part/s of the fungi which undergo fragmentation. The results obtained from these investigations into the dynamics of fungal propagules in indoor air significantly advance knowledge about the fate of fungal propagules in indoor air, as well as their deposition in the respiratory tract. The need to develop an advanced, real-time method for monitoring bioaerosols has become increasingly important in recent years, particularly as a result of the increased threat from biological weapons and bioterrorism. However, to date, the Ultraviolet Aerodynamic Particle Sizer (UVAPS, Model 3312, TSI, St Paul, MN) is the only commercially available instrument capable of monitoring and measuring viable airborne micro-organisms in real-time. Therefore (for the first time), this work also investigated the ability of the UVAPS to measure and characterise fungal spores in indoor air. The UVAPS was found to be sufficiently sensitive for detecting and measuring fungal propagules. Based on fungal spore size distributions, together with fluorescent percentages and intensities, it was also found to be capable of discriminating between two fungal spore species, under controlled laboratory conditions. In the field, however, it would not be possible to use the UVAPS to differentiate between different fungal spore species because the different micro-organisms present in the air may not only vary in age, but may have also been subjected to different environmental conditions. In addition, while the real-time UVAPS was found to be a good tool for the investigation of fungal particles under controlled conditions, it was not found to be selective for bioaerosols only (as per design specifications). In conclusion, the UVAPS is not recommended for use in the direct measurement of airborne viable bioaerosols in the field, including fungal particles, and further investigations into the nature of the micro-organisms, the UVAPS itself and/or its use in conjunction with other conventional biosamplers, are necessary in order to obtain more realistic results. Overall, the results obtained from this work on airborne fungal particle dynamics will contribute towards improving the detection capabilities of the UVAPS, so that it is capable of selectively monitoring and measuring bioaerosols, for which it was originally designed. This work will assist in finding and/or improving other technologies capable of the real-time monitoring of bioaerosols. The knowledge obtained from this work will also be of benefit in various other bioaerosol applications, such as understanding the transport of bioaerosols indoors.

Stunted cotton (Gossypium hirsutum L.) fully recovers biomass and yield of seed cotton after delayed root inoculation with spores of an arbuscular mycorrhizal fungus (Glomus mosseae)

In the northern grain and cotton region of Australia, poor crop growth after long periods of fallow, called 'long-fallow' disorder, is caused by a decline of natural arbuscular-mycorrhizal fungi (AMF). When cotton was grown in large pots containing 22 kg of Vertisol from a field recently harvested from cotton in Central Queensland, plants in pasteurised soil were extremely stunted compared with plants in unpasteurised soil. We tested the hypothesis that this extreme stunting was caused by the absence of AMF and examined whether such stunted plants could recover from subsequent treatment with AMF spores and/or P fertiliser. At 42 days after sowing, the healthy cotton growing in unpasteurised soil had 48% of its root-length colonised with AMF, whereas the stunted cotton had none. After inoculation with AMF spores (6 spores/g soil of Glomus mosseae) and/or application of P fertiliser (50 mg P/kg soil) at 45 days after sowing, the stunted plants commenced to improve about 25 days after treatment, and continued until their total dry matter and seed cotton production equalled that of plants growing in unpasteurised soil with natural AMF. In contrast, non-mycorrhizal cotton grown without P fertiliser remained stunted throughout and produced no bolls and only 1% of the biomass of mycorrhizal cotton. Even with the addition of P fertiliser, non-mycorrhizal cotton produced only 64% of the biomass and 58% of the seed cotton (lint + seed) of mycorrhizal cotton plants. These results show that cotton is highly dependent on AMF for P nutrition and growth in Vertisol (even with high rates of P fertiliser), but can recover from complete lack of AMF and consequent stunting during at least the first 45 days of growth when treated with AMF spores and/or P fertiliser. This corroborates field observations in the northern region that cotton may recover from long-fallow disorder caused by low initial levels of AMF propagules in the soil as the AMF colonisation of its roots increases during the growing season.

Bacillus cereus spores and cereulide in food-borne illness

B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≤ pH 1) environment. The spores are highly hydrophobic and therefore make them tend to stick to all kinds of surfaces, steel, plastics and live cells. In favorable conditions the spores of B. cereus may germinate into vegetative cells capable of producing food poisoning toxins. The toxins can be heat-labile protein formed after ingestion of the contaminated food, inside the gastrointestinal tract (diarrhoeal toxins), or heat stable peptides formed in the food (emesis causing toxin, cereulide). Cereulide cannot be inactivated in foods by cooking or any other procedure applicable on food. Cereulide in consumed food causes serious illness in human, even fatalities. In this thesis, B. cereus strains originating from different kinds of foods and environments and 8 different countries were inspected for their capability of forming cereulide. Of the 1041 isolates from soil, animal feed, water, air, used bedding, grass, dung and equipment only 1.2 % were capable of producing cereulide, whereas of the 144 isolates originating from foods 24 % were cereulide producers. Cereulide was detected by two methods: by its toxicity towards mammalian cells (sperm assay) and by its peculiar chemical structure using liquid-chromatograph-mass spectrometry equipment. B. cereus is known as one of the most frequent bacteria occurring in food. Most foods contain more than one kind of B. cereus. When randomly selected 100 isolates of B. cereus from commercial infant foods (dry formulas) were tested, 11% of these produced cereulide. Considering a frequent content of 103 to 104 cfu (colony forming units) of B. cereus per gram of infant food formula (dry), it appears likely that most servings (200 ml, 30 g of the powder reconstituted with water) may contain cereulide producers. When a reconstituted infant formula was inoculated with >105 cfu of cereulide producing B. cereus per ml and left at room temperature, cereulide accumulated to food poisoning levels (> 0.1 mg of cereulide per serving) within 24 hours. Paradoxically, the amount of cereulide (per g of food) increased 10 to 50 fold when the food was diluted 4 - 15 fold with water. The amount of the produced cereulide strongly depended on the composition of the formula: most toxin was formed in formulas with cereals mixed with milk, and least toxin in formulas based on milk only. In spite of the aggressive cleaning practices executed by the modern dairy industry, certain genotypes of B. cereus appear to colonise the silos tanks. In this thesis four strategies to explain their survival of their spores in dairy silos were identified. First, high survival (log 15 min kill ≤ 1.5) in the hot alkaline (pH >13) wash liquid, used at the dairies for cleaning-in-place. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterized by slow germination in rich medium and well preserved viability when exposed to heating at 90 ºC. Fourth, spores capable of germinating at 8 ºC and possessing the psychrotolerance gene, cspA. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus. In this thesis it was shown that cereulide producing B. cereus was capable of inhibiting the growth of cereulide non-producing B. cereus occurring in the same food. This phenomenon, called antagonism, has long been known to exist between B. cereus and other microbial species, e.g. various species of Bacillus, gram-negative bacteria and plant pathogenic fungi. In this thesis intra-species antagonism of B. cereus was shown for the first time. This brother-killing did not depend on the cereulide molecule, also some of the cereulide non-producers were potent antagonists. Interestingly, the antagonistic clades were most frequently found in isolates from food implicated with human illness. The antagonistic property was therefore proposed in this thesis as a novel virulence factor that increases the human morbidity of the species B. cereus, in particular of the cereulide producers.

Inactivation of Aspergillus flavus spores by curcumin-mediated photosensitization

Minimizing fungal infection is essential to the control of mycotoxin contamination of foods and feeds but many potential control methods are not without their own safety concerns for the consumers. Photodynamic inactivation is a novel light-based approach which offers a promising alternative to conventional methods for the control of mycotoxigenic fungi. This study describes the use of curcumin to inactivate spores of Aspergillus flavus, one of the major aflatoxin producing fungi in foods and feeds. Curcumin is a natural polyphenolic compound from the spice turmeric (Curcuma longa). In this study the plant has shown to be an effective photosensitiser when combined with visible light (420 nm). The experiment was conducted in in vitro and in vivo where A. flavus spores were treated with different photosensitiser concentration and light dose both in buffer solution and on maize kernels. Comparison of fungal load from treated and untreated samples was determined, and reductions of fungal spore counts of up to 3 log CFU ml−1 in suspension and 2 log CFU g−1 in maize kernels were obtained using optimal dye concentrations and light dose combinations. The results in this study indicate that curcumin-mediated photosensitization is a potentially effective method to decontaminate A. flavus spores in foods and feeds.

Probing biomolecular interaction forces using an anharmonic acoustic technique for selective detection of bacterial spores.

Receptor-based detection of pathogens often suffers from non-specific interactions, and as most detection techniques cannot distinguish between affinities of interactions, false positive responses remain a plaguing reality. Here, we report an anharmonic acoustic based method of detection that addresses the inherent weakness of current ligand dependant assays. Spores of Bacillus subtilis (Bacillus anthracis simulant) were immobilized on a thickness-shear mode AT-cut quartz crystal functionalized with anti-spore antibody and the sensor was driven by a pure sinusoidal oscillation at increasing amplitude. Biomolecular interaction forces between the coupled spores and the accelerating surface caused a nonlinear modulation of the acoustic response of the crystal. In particular, the deviation in the third harmonic of the transduced electrical response versus oscillation amplitude of the sensor (signal) was found to be significant. Signals from the specifically-bound spores were clearly distinguishable in shape from those of the physisorbed streptavidin-coated polystyrene microbeads. The analytical model presented here enables estimation of the biomolecular interaction forces from the measured response. Thus, probing biomolecular interaction forces using the described technique can quantitatively detect pathogens and distinguish specific from non-specific interactions, with potential applicability to rapid point-of-care detection. This also serves as a potential tool for rapid force-spectroscopy, affinity-based biomolecular screening and mapping of molecular interaction networks.

Probing biomolecular interaction forces using an anharmonic acoustic technique for selective detection of bacterial spores

Receptor-based detection of pathogens often suffers from non-specific interactions, and as most detection techniques cannot distinguish between affinities of interactions, false positive responses remain a plaguing reality. Here, we report an anharmonic acoustic based method of detection that addresses the inherent weakness of current ligand dependant assays. Spores of Bacillus subtilis (Bacillus anthracis simulant) were immobilized on a thickness-shear mode AT-cut quartz crystal functionalized with anti-spore antibody and the sensor was driven by a pure sinusoidal oscillation at increasing amplitude. Biomolecular interaction forces between the coupled spores and the accelerating surface caused a nonlinear modulation of the acoustic response of the crystal. In particular, the deviation in the third harmonic of the transduced electrical response versus oscillation amplitude of the sensor (signal) was found to be significant. Signals from the specifically-bound spores were clearly distinguishable in shape from those of the physisorbed streptavidin-coated polystyrene microbeads. The analytical model presented here enables estimation of the biomolecular interaction forces from the measured response. Thus, probing biomolecular interaction forces using the described technique can quantitatively detect pathogens and distinguish specific from non-specific interactions, with potential applicability to rapid point-of-care detection. This also serves as a potential tool for rapid force-spectroscopy, affinity-based biomolecular screening and mapping of molecular interaction networks. © 2011 Elsevier B.V.

Seeding nets with neutral spores of the red alga Porphyra umbilicalis (L.) Kutzing for use in integrated multi-trophic aquaculture (IMTA)

Nets in traditional Porphyra mariculture are seeded with conchospores derived from the conchocelis phase, and spend a nursery period in culture tanks or calm coastal waters until they reach several centimeters in length. Some species of Porphyra can regenerate the foliose phase directly through asexual reproduction, which suggests that the time, infrastructure, and costs associated with conchocelis culture might be avoided by seeding nets with asexual spores. Here, we present work from a short-term mariculture study using nets seeded with asexual spores (neutral spores) of a native Maine species of Porphyra. Porphyra umbilicalis (L.) Kutzing was selected for this proof of concept research because of its reproductive biology, abundance across seasons in Maine, and evidence of its promise as a mariculture crop. We studied the maturation, release, and germination of the neutral spores to develop an appropriate seeding protocol for nets, followed by development of a nursery raceway to provide an easily manipulated environment for the seeded nets. Neutral spores were produced throughout the year on the central Maine coast,however, there was a temporal variability in the number and survival of released neutral spores, depending upon thallus position in the intertidal zone. Small thalli were strictly vegetative, but most thalli reproduced by neutral spores- sexual reproduction was absent. Neutral spores germinated quickly at 10 and 15 'C, but germination was delayed at 5 degrees C. Unlike some algal zygotes and spores, neutral spores of R umbilicalis required light to germinate; however, irradiances of 25 and 100 mu mol photons M-2 S-1 were equally sufficient for germination. Rafts of seeded nets were deployed in Cobscook Bay, Maine, at two distances from salmon aquaculture pens and at a control site on a nearby, fallow aquaculture site (no salmon). There was no difference in nitrogen content of harvested thalli; however, both the density and the surface area of harvested thalli were different among the sites. The possible causes of these differences are discussed in the context of potential use of P umbilicalis in IMTA. (C) 2007 Elsevier B.V. All rights reserved.