Dissertations / Theses on the topic 'Fungal-bacterial interactions'

Systemic acquired resistance (SAR) is a form of inducible defense response triggered upon localized infection that confers broad-spectrum disease resistance against secondary infections. Several factors are known to regulate SAR and these include phenolic phytohormone salicylic acid (SA), phosphorylated sugar glycerol-3-phosphate (G3P), and dicarboxylic acid azelaic acid (AzA). This study evaluated a role for free radicals nitric oxide (NO) and reactive oxygen species (ROS) in SAR. Normal accumulation of both NO and ROS was required for normal SAR and mutations preventing NO/ROS accumulation and/or biosynthesis compromised SAR. A role for NO and ROS was further established using pharmacological approaches. Notably, both NO and ROS conferred SAR in a concentration dependent manner. This was further established using genetic mutants that accumulated high levels of NO. NO/ROS acted upstream of G3P and in parallel to SA. Collectively, these results suggest that NO and ROS are essential components of the SAR pathway.

The Candida genus is full of fungi that are subtle parts of the human microbiome, but they can cause complications if they overgrow within the body—specifically the mouth and throat, the genitalia, and the entire body through infection of the bloodstream. Candida species are a rising problem for many across the world, and this can be seen in the recent threat of Candida auris hospitalizing patients and being regularly resistant to anti-fungal medications. Beyond C. auris, Candida albicans is the most common Candida species that humans must combat because it causes the most infections in humans—mostly vaginal yeast infections. C. albicans does have natural competitors that can either inhibit its growth or kill it in general, and the competition that we took advantage of was with the Alcaligenes species. Alcaligenes faecalis and Alcaligenes viscolactis have been shown to at least inhibit C. albicans growth and maybe even kill the fungus. This rate of infection from C. albicans places it at the forefront of Candida research, and we attempted to further this research by utilizing both A. faecalis and A. viscolactis to create a co-infection model for Caenorhabditis elegans—a simple nematode lifeform. It is known that A. faecalis and A. viscolactis do not commonly adversely affect humans, so little research has been done concerning their clinical effects. We were looking to find a possible answer to C. albicans infections beyond antifungal drugs because we know that antibiotic resistance is on the rise. We performed liquid assays to test the survivability of C. elegans nematodes in various bacterial/fungal circumstances. We subjected batches of C. elegans to E. coli OP50 as a control, A. faecalis, A. viscolactis, C. albicans, A. faecalis and C. albicans, and A. viscolactis and C. albicans. This procedure was followed in order to determine the viability of using the Alcaligenes species to either help the C. elegans survive the infection or prevent them from getting infected at all. After following through with the project, we found that there was a noticeable increase in the survivability of C. elegans when subjected to both one of the Alcaligenes species and C. albicans as opposed to the C. albicans alone. The data, although early, shows the possibility of Alcaligenes species being used to combat C. albicans infections in lifeforms.

Bacteria and fungi have acquired the ability to interact and survive in many hostile environments both found in nature, as well as, the human body. Candida albicans, an opportunistic fungal pathogen, causes a variety of infections in immunocompromised or immunosuppressed individuals, but also asymptomatically colonizes 80% of the population within the intestinal tract, oral cavity, as well as, the female genitourinary system. A unique capability of C. albicans is the ability to change its morphology from benign circular yeast form, to oval pseudohyphal form, to cylindrical tissue-penetrating hyphal form. Our laboratory has previously identified a bacterium, Alcaligenes faecalis, which displays inhibitory characteristics towards C. albicans. A review of the literature shows that some bacteria have the ability to inhibit C. albicans, but only when in the hyphal form. We therefore wanted to explore if the morphological state of C. albicans dictated the degree of inhibition A. faecalis is able to exude, or simply “does morphology matter?” To determine this effect, a series of both solid and liquid media experiments were performed using a wild-type (able to convert between morphologies) strain of C. albicans, a mutant strain of C. albicans locked into the yeast morphological state, and a mutant strain of C. ablicans locked into the hyphal morphological state. For solid media experiments, the different strains of C. albicans were made into a lawn on agar plates, A. faecalis was spotted onto the lawns, and, after 24 hours, observed for signs of inhibition. For liquid media experiments, C. albicans strains were inoculated alone or co-cultured with A. faecalis for 24 hours and plated to enumerate colony forming units. Our results indicate that: (1) the morphological state of C. albicans is not a determining factor, which is a unique finding compared to other published reports; (2) Both A. faecalis and the closely related A. viscolactis both inhibit C. albicans showing that this is a shared ability among the Alcaligenes genus. (3) that the ability to inhibit C. albicans is thru some form of contact dependent mechanism, as the cell free supernatant of A. faecalis has no inhibitory action. Currently, the exact mechanism for this interaction is unknown, but could be one of the secretion systems bacteria use for interactions with other microbes. As there are very limited treatments for fungal infections and severe side-effects associated with current antifungals, exploiting these mechanisms are medically relevant to human health as they could potentially lead to novel treatments for problematic human fungal pathogens.

Dans les écosystèmes forestiers, la décomposition du bois est un processus majeur, notamment impliqué dans le cycle du carbone et des nutriments. Les champignons basidiomycètes saprotrophes, incluant les pourritures blanches, sont les principaux agents de cette décomposition dans les forêts tempérées. Bien que peu étudiées, des communautés bactériennes sont également présentes dans le bois en décomposition et cohabitent avec ces communautés fongiques. L'impact des interactions bactéries-champignons sur le fonctionnement d'une niche écologique a été décrit dans de nombreux environnements. Cependant, leur rôle dans le processus de décomposition du bois n'a été que très peu investigué. A partir d'expériences en microcosme et en utilisant une approche non cultivable, il a été démontré que la présence du champignon Phanerochaete chrysosporium influençait significativement la structure et la diversité des communautés bactériennes associées au processus de décomposition du hêtre (Fagus sylvatica). Par une approche cultivable, cet effet mycosphère a été confirmé, se traduisant par une augmentation de la densité des communautés bactériennes en présence du champignon ainsi que par une modification de la diversité fonctionnelle de ces communautés. Enfin, une approche polyphasique a été développée, combinant l'analyse des propriétés physico-chimiques du bois et des activités enzymatiques extracellulaires. Les résultats de cette expérience ont révélé que l'association de P. chrysosporium avec une communauté bactérienne issue de la mycosphère de ce dernier aboutissait à une dégradation plus importante du matériau bois par rapport à la dégradation par le champignon seul, démontrant pour la première fois des interactions bactéries-champignons synergiques dans le bois en décomposition
Wood decomposition is an important process in forest ecosystems in terms of their carbon and nutrient cycles. In temperate forests, saprotrophic basidiomycetes such as white-rot fungi are the main wood decomposers. While they have been less studied, bacterial communities also colonise decaying wood and coexist with these fungal communities. Although the impact of bacterial-fungal interactions on niche functioning has been highlighted in a wide range of environments, little is known about their role in wood decay. Based on microcosm experiments and using a culture-independent approach, we showed that the presence of the white-rot fungus Phanerochaete chrysosporium significantly modified the structure and diversity of the bacterial communities associated with the degradation of beech wood (Fagus sylvatica). Using a culture-dependent approach, it was confirmed that in the presence of the fungus the mycosphere effect resulted in increased bacterial abundance and modified the functional diversity of the fungal-associated bacterial communities. Lastly, a polyphasic approach simultaneously analysing wood physicochemical properties and extracellular enzyme activities was developed. This approach revealed that P. chrysosporium associated with a bacterial community isolated from its mycosphere was more efficient in degrading wood compared to the fungus on its own, highlighting for the first time synergistic bacterial-fungal interactions in decaying wood

Les macroalgues hébergent de nombreux micro-organismes, collectivement désignés sous le terme de microbiote algal, qui ont un rôle essentiel dans le développement et l’état de santé de leur hôte. Dans ce travail, nous avons exploré le microbiote fongique et bactérien d’algues brunes, ainsi que l’impact des interactions bactérie-champignon sur la médiation chimique et, en particulier, sur le quorum sensing bactérien. Par des approches de metabarcoding ciblant l’ADNr 16S et l’ITS2, nous avons montré que les communautés fongiques et bactériennes associées à la macroalgue brune Saccharina latissima étaient très riches, principalement composées de quelques OTUs dominants, et d’une grande abondance d’OTUs « rares ». De manière intéressante, les communautés fongiques comme bactériennes différaient de celles de l’eau de mer environnante et paraissaient spécifiques des tissus algaux. Cependant, de grandes variations intra et interindividuelles de composition ont été observées au sein des échantillons de tissus d’algue. Ainsi, ce qui définit la spécificité des communautés microbiennes reste à préciser. En parallèle, nous avons exploré la médiation chimique au sein de l’endomicrobiote de quatre algues brunes : Saccharina latissima, Laminaria digitata, Pelvetia canaliculata et Ascophylum nodosum, et révélé que de nombreux endophytes fongiques et bactériens isolés synthétisaient des métabolites interférant avec les systèmes de quorum sensing bactériens, en les induisant ou les inhibant. De plus, les bioessais basés sur les biosenseurs, couplés à une approche métabolomique, effectués sur les co-cultures, ont mis en évidence en quoi les interactions bactéries-champignons au sein de l’endomicrobiote d’algues brunes pouvaient affecter la production de médiateurs chimiques, et notamment de molécules interférant avec le quorum sensing bactérien. Ensemble, ces résultats suggèrent que le quorum sensing pourrait jouer un rôle clé dans le réseau complexe d’interactions au sein du microbiote algal, et ainsi dans l’équilibre hôte-microbiote
Macroalgae host various microorganisms, collectively referred as the algal microbiota, which play an essential role in the development and health status of their host. In this work, we explored the bacterial and fungal microbiota of brown algae, as well as the impact of bacterial fungal interactions on the chemical mediation and, in particular, on the bacterial quorum sensing. Using 16S rDNA-based and ITS2-based metabarcoding approaches we showed that the fungal and bacterial communities associated with the brown macroalgae Saccharina latissima were very rich, mainly composed of few dominant OTUs, and a large abundance of “rare” OTUs. Interestingly, both fungal and bacterial communities differed from the ones of the surrounding seawater and appeared specific to the algal tissues. However, high intra and interindividual variations of composition were observed among the algal tissue samples. Thus what define the specificity of the microbial communities remains to be clarified. In parallel, we explored the chemical mediation within the cultivable endomicrobiota of four brown algae: Saccharina latissima, Laminaria digitata, Pelvetia canaliculata and Ascophylum nodosum, and pointed out that many of the isolated bacterial and fungal endophytes could synthetize metabolites interfering with bacterial quorum sensing systems, either inducing or inhibiting them. Additionally, biosensor-based bioassays coupled with metabolomics approaches performed on co-culture experiments, highlighted how bacterial-fungal interactions within the endomicrobiota of brown algae could affect the production of chemical mediators, including those interfering with bacterial quorum sensing. Altogether, the results suggest that the quorum sensing could play a key role in the complex network of interactions within the algal microbiota, and thus in the host-microbiota equilibrium

Minor Groove Binders are a group of compound that bind to the minor grooves of DNA. Some of these compounds are able to bind to specific sequences on DNA, causing toxicity to cells, and this has generated an interest in their use as antimicrobial or anticancer agents. They are generally characterised by their recognisable crescent shape. The focus of this research is the effect of two minor groove binders AIK 19/56/2 and AIK 20/25/1 on the survival of various bacterial cell types as well as mammalian and fungal cells. This has been examined using growth curves, killing curves, fluorescent microscopy, HPLC spectra and topoisomerase I inhibition have been used. These experiments have given an insight into the mechanism of action of these compounds.

This dissertation comprises a series of studies designed to explore the associations between plants and the endophytic fungi they harbor in their above-ground tissues. By viewing endophyte diversity in ecologically and economically important hosts through the lenses of phylogenetic biology, microbiology, and biotechnology, this body of work links plant ecology with newly discovered symbiotic units comprised of endophytic fungi and the bacteria that inhabit them.This work begins with a large-scale survey of endophytic fungi from native and non-native Cupressaceae in Arizona and North Carolina. After isolating over 400 strains of endophytes, I inferred the evolutionary relationships among these fungi using both Bayesian and parsimony analyses. In addition to showing that native and introduced plants contained different endophytes, I found that the endophytes themselves harbor additional microbial symbionts, recovering members of the beta- and gamma-proteobacterial orders Burkholderiales, Xanthomonadales, and Enterobacteriales and numerous novel, previously uncultured bacteria. This work finds that phylogenetically diverse bacterial endosymbionts occur within living hyphae of multiple major lineages of ascomycetous endophytes.A focus on 29 fungal/bacterial associations revealed that bacterial and fungal phylogenies are incongruent with each other and did not reflect the phylogenetic relationships of host plants. Instead, both endophyte and bacterial assemblages were strongly structured by geography, consistent with local horizontal transmission. Endophytes could be cured of their bacterial endosymbionts using antibiotics, providing a tractable experimental system for comparisons of growth and metabolite production under varying conditions. Studies of seven focal fungal/bacterial pairs showed that bacteria could significantly alter growth of fungi at different nutrient and temperature levels in vitro, and that different members of the same bacterial lineages interact with different fungi in different ways.Focusing on one isolate, I then describe for the first time the production of indole-3-acetic acid (IAA) by a non-pathogenic, foliar endophytic fungus (Pestalotiopsis neglecta), suggesting a potential benefit to the host plant harboring this fungus. I show that this fungus is inhabited by an endohyphal bacterium (Luteibacter sp.) and demonstrate that mycelium containing this bacterium produces significantly more IAA in vitro than the fungus alone. I predict that the general biochemical pathway used by the fungal-endohyphal complex is L-tryptophan-dependent and measure effects of IAA production in vivo, focusing on root and shoot growth in tomato seedlings.

Le calvados est une eau-de-vie obtenue par distillation du cidre. Le cidre de distillation résulte d’une fermentation naturelle spontanée non maîtrisée, qui permet toutes les interactions possibles entre les différentes communautés microbiennes. En fin de fermentation alcoolique, le glycérol produit peut être dégradé et converti en 3-hydroxypropionaldehyde (3-HPA) par Lactobacillus collinoides via l’enzyme glycérol déshydratase codée par l’opéron pdu. Lors de la distillation, la déshydratation du 3-HPA en acroléine entraîne des désordres organoleptiques connus sous le nom de piqûre acroléique en développant un goût d’amertume dans les calvados. Ces travaux de thèse ont eu pour premier objectif d’apporter une réelle nouveauté dans l’étude de la diversité de dix cidres de distillation normands lors de la fermentation par une approche de métagénomique et d’établir des liens entre la composition microbienne et la qualité aromatique. Cette approche a permis de mettre en évidence plus de 40 genres bactériens et fongiques et des différences significatives ont été constatées au sein de ces populations entre les cidres des différents producteurs au cours du processus de fermentation, suggérant que les pratiques d’élaboration mènent à une typicité du cidre et donc à celle du calvados. L’étude de la composition des communautés microbiennes en lien avec la composition aromatique des cidres de distillation et celle des micro- distillats a été effectuée. Trois groupes de dynamiques et compositions microbiennes, directement liées aux producteurs, émergent conduisant à des compositions aromatiques différentes. L’analyse comparative de huit génomes de souches de Lactobacillus collinoides isolées à partir de cidres de distillation portant sur les métabolismes associés à la fermentation et sur la capacité des souches à métaboliser le glycérol a été effectuée. Deux types d’opéron pdu se profilent et cette variabilité génétique observée entre les souches de L. collinoides suggère des capacités différentes de dégradation du glycérol. Un plan expérimental (D-optimal) faisant varier 6 facteurs (glucose, fructose, azote, éthanol, acide malique, pH) a été élaboré pour étudier le métabolisme du glycérol chez ces souches. Trois profils génétiques et métaboliques ont pu être mis en évidence. Les souches de L. collinoides possédant l’opéron de type I sont toujours capables de dégrader le glycérol, contrairement à celles porteuses de l’opéron de type II. Ces travaux ouvrent des perspectives intéressantes quant à la présence et la physiologie de L. collinoides lors de l’élaboration du cidre et son implication dans la piqûre acroléique
Calvados is a Normandy brandy made by the distillation of cider. Ciders destined for distillation resultfrom spontaneous fermentation without any human intervention, leading to various interactions between thedifferent microbial communities. At the end of alcoholic fermentation, glycerol produced can be degraded andconverted to 3-hydroxypropionaldehyde (3-HPA) by Lactobacillus collinoides via the glycerol dehydrataseenzyme encoded by the pdu operon. During distillation, dehydration of 3-HPA to acrolein leads to organolepticdisorders, developing a bitterness taste in calvados known as “piqûre acroléique”. The first objective of this thesiswas to unveil the diversity of ten ciders for distillation during fermentation using a high throughput sequencing(16S, ITS1) approach and to establish links between microbial composition and aromatic quality. This approachallowed us to identify more than 40 bacterial and fungal and significant differences were found among fungal andbacterial populations between producers during the fermentation process, suggesting that the practices lead to thetypicity of cider, and therefore to the typicity of calvados. The study of the microbial composition was correlatedwith the aromatic composition of ciders for distillation during the fermentation process and of the micro-distillates.Three groups of microbial dynamics and compositions, directly related to the producers, were observed leading todifferent aromatic compositions. A comparative analysis of eight genomes of Lactobacillus collinoides strainsisolated from the ciders according to the different metabolisms associated with fermentation as well as on theability of the strains to metabolize glycerol was performed. Two types of operon pdu were highlighted and thisgenetic variability between L. collinoides strains suggests different abilities for glycerol degradation. Anexperimental design (D-optimal) by varying 6 factors (glucose, fructose, nitrogen, ethanol, malic acid, pH) wasdeveloped to study the metabolism of glycerol in these strains. 3 genetic and metabolic profiles were identified. L.collinoides strains with operon type I are always able to degrade glycerol, contrarily to those harbouring the operonof type II. This work opens up interesting perspectives regarding the presence and physiology of L. collinoidesduring the development of cider and its involvement in the “piqûre acroléique”

Le dépérissement du pommier est une maladie tellurique causée par plusieurs espèces Pythiacée. Il est responsable de graves dommages et de pertes d'arbres dans de nombreux vergers en Tunisie. Comme la lutte chimique contre cette maladie pose des problèmes éco toxicologiques et les moyens prophylactiques ont des efficacités limitées, d’autres moyens de lutte sont activement recherchés. L’objectif de cette thèse était d’identifier un nouvel agent de lutte biologique contre les oomycètes responsables de cette maladie à partir d’une collection d’isolats fongiques et bactériens Tunisiens comme alternative au contrôle chimique. Les isolats fongiques étudiés appartenaient aux genres Trichoderma, Aspergillus et Penicillium spp., les isolats bactériens appartenaient au genre Bacillus spp. clade subtilis. Les isolats fongiques ont fortement inhibé la croissance in vitro des Pythiacées (> 40%) par rapport aux isolats bactériens ; en particulier, les filtrats de culture des isolats A. westerdijkiae A7 et T. simmonsii A2. L'évaluation de l’activité préventive et curative contre les Pythiacées sur des porte-greffes du pommier de ces deux isolats fongiques et de la souche Bacillus B2 a montré que T. simmonsii A2 était le plus efficace lorsqu'il était appliqué à titre préventif. De même, la combinaison de la souche Bacillus B2 et de l’isolat A. westerdijkiae A7 a induit une bonne protection contre les Pythiacées en préventif. La combinaison des isolats T. simmonsii A2 et A. westerdijkiae A7 a entraîné une meilleure protection en traitement curatif, alors que la combinaison des trois isolats ensemble réduisait fortement l’activité protectrice. Notre étude révèle le potentiel des isolats Tunisiens, seuls ou en combinaison, en tant qu'agents de lutte biologique contre le dépérissement du pommier ainsi qu'un effet bénéfique supplémentaire sur la croissance végétale observé au niveau des racines et de la longueur des tiges. Ainsi, nous avons sélectionnés les meilleurs candidats agissant par antibiose pour l'identification des principaux agents actifs responsables de l'activité anti-oomycète. L'isolat A. westerdijkiae A7 a été retenu avec 100% d'inhibition de la croissance mycélienne des isolats de Pythiacées testés. Plusieurs étapes de purification successives du filtrat de culture ont permis d’identifier l'acide pénicillique (acide 3-méhoxy-5-méthyl-4-oxo-2,5-hexadiénoïque) en tant que principale molécule responsable de l’inhibition la croissance mycélienne des Pythiacées testées. Étant donné que l’acide pénicillique possède des propriétés toxiques pour la santé humaine et animale, l’isolat A. westerdijkiae A7 ne pourra pas être utilisé en tant que BCA puisqu’il produit cette mycotoxine malgré nos résultats prometteurs in planta. L’ensemble de ces travaux montre le potentiel anti-oomycète des micro-organismes in vitro qui peut différer de l’activité protectrice contre les Pythiacées in planta. Ils révèlent également la nécessité de caractériser l’ingrédient actif pour les études de toxicité nécessaire au développement d’un produit de bio-contrôle
Apple dieback is a telluric disease caused by several Pythiaceae species. It is responsible for serious damage and loss of trees in many orchards in Tunisia. As the chemical control of this disease poses eco-toxicological problems and the prophylactic means have limited effectiveness, other means of fight are actively sought. The objective of this thesis was to identify a new biological control agent against oomycetes responsible for this disease from a collection of Tunisian fungal and bacterial isolates as an alternative to chemical control. The fungal isolates studied belonged to the genera Trichoderma, Aspergillus and Penicillium spp., The bacterial isolates to the genus Bacillus spp. clade subtilis. Fungal isolates strongly inhibited the growth of Pythiaceae in vitro (> 40%) compared to bacterial isolates; in particular, the culture filtrates of isolates A. westerdijkiae A7 and T. simmonsii A2. The evaluation of the preventive and curative activity against Pythiaceae on apple rootstocks of these two fungal isolates and of the Bacillus B2 strain showed that T. simmonsii A2 was the most effective when applied preventively. Likewise, the combination of the Bacillus B2 strain and the A. westerdijkiae A7 isolate induced good protection against Pythiaceae as a preventive measure. The combination of the T. simmonsii A2 and A. westerdijkiae A7 isolates resulted in better protection in curative therapy, while the combination of the three isolates together greatly reduced the protective activity. Our study reveals the potential of Tunisian isolates, alone or in combination, as biological control agents against apple dieback as well as an additional beneficial effect on plant growth observed at the level of the roots and the length of the stems. Thus, we have selected the best candidates acting by antibiosis for the identification of the main active agents responsible for anti-oomycete activity. The A. westerdijkiae A7 isolate was retained with 100% inhibition of mycelial growth of the Pythiaceae isolates tested. Several successive purification steps of the culture filtrate made it possible to identify penicillic acid (3-mehoxy-5-methyl-4-oxo-2,5-hexadienoic acid) as the main molecule responsible for inhibiting growth mycelia of the Pythiaceae tested. Since penicillic acid has toxic properties for human and animal health, isolate A. westerdijkiae A7 cannot be used as BCA since it produces this mycotoxin despite our promising results in planta. All of this work shows the anti-oomycete potential of microorganisms in vitro, which may differ from the protective activity against Pythiaceae in planta. They also reveal the need to characterize the active molecule for the toxicity studies necessary for the development of a biocontrol product

Tese de doutoramento em Engenharia Química e Biológica
Water is indispensable to life and a safe and accessible supply must be available to all. The presence of microorganisms is a threat to this commitment. Biofilms are the main reservoir of microorganisms inside water distribution systems and they are extremely ecologically diverse. Biofilm formation in drinking water has mainly been studied regarding bacteria, however, filamentous fungi and bacteria can coexist inside these systems forming inter-kingdom biofilms. The ecology of a biofilm is a complex function of different factors, including the presence of microbial metabolites excreted by its inhabitants. The present work aimed to study the ability of filamentous fungi to interact and influence bacteria whilst forming inter-kingdom biofilms. For this purpose, different methods to analyse biofilms were applied, including total biomass, metabolic activity, bacterial colony forming units and epifluorescence microscopy. The study on the effect of quorum sensing molecules and fungal secondary metabolites on inter-kingdom biofilm formation and development was also done. In addition, an RT-qPCR method to quantify a gene involved in the patulin biosynthetic pathway was performed and the expression levels were compared with the patulin production in inter-kingdom biofilms. The results revealed fungal stage development is important in the first 24 h of biofilm formation. Inter-kingdom biofilm formation is microorganism dependent and inter-kingdom biofilms may provide an advantage to the opportunistic bacterium Acinetobacter calcoaceticus to replicate and proliferate. Methylobacterium oryzae biofilm formation and development was more susceptible to the presence of exogenous molecules than A. calcoaceticus biofilms. The effect of M. oryzae on Penicillium expansum biofilms is dependent on the time of interaction. More mature P. expansum biofilms appear to be more resistant to the inhibitory effect that M. oryzae causes towards idh gene expression and patulin production. M. oryzae affects patulin production by acting at the transcriptional level of the idh gene. In conclusion, a possible protection role of filamentous fungi towards opportunistic bacteria inside complex biofilms is reported. Moreover, quorum sensing and quenching molecules play a vital role in inter-kingdom communication and/or microbial assembly processes that influence biofilm formation between filamentous fungi and bacteria.
A água é indispensável à vida e um fornecimento seguro deve estar disponível para todos. A presença de microrganismos é uma ameaça a este compromisso. Os biofilmes são a principal fonte de microrganismos em sistemas de distribuição de água e são ecologicamente muito ricos. A formação de biofilmes em sistemas de água de consumo tem sido estudada principalmente em relação às bactérias, contudo, nestes sistemas, fungos filamentosos e bactérias interagem formando biofilmes mistos. A ecologia de um biofilme é extremamente complexa e é suscetível a diferentes fatores como a presença de metabolitos microbianos excretados pelos seus habitantes. O trabalho aqui apresentado teve como objetivo o estudo da capacidade de fungos filamentosos interagirem e influenciarem bactérias enquanto formam biofilmes mistos. Com este propósito, diferentes métodos de análise de biofilmes foram usados, incluindo biomassa total, atividade metabólica, unidades formadoras de colónias bacterianas, e microscopia de epifluorescência. Foi também realizado o estudo do efeito de moléculas de quórum sensing e metabolitos secundários produzidos por fungos na formação e desenvolvimento de biofilmes mistos. Adicionalmente, foi usado um método de RT-qPCR para quantificar um gene relacionado com a via biossintética da patulina e os valores de expressão relativa foram comparados com a produção de patulina em biofilmes mistos. Os resultados revelaram que o estado de desenvolvimento do fungo filamentoso aquando da inoculação é importante nas primeiras 24 h de formação de biofilme. A formação de biofilmes mistos é dependente dos microrganismos presentes e pode conferir uma vantagem a bactérias oportunistas como a Acinetobacter calcoaceticus para se replicar e proliferar. A formação e desenvolvimento de biofilmes da Methylobacterium oryzae demonstrou ser mais suscetível à presença de moléculas exógenas do que os biofilmes da A. calcoaceticus. O efeito da M. oryzae em biofilmes do Penicillium expansum é dependente do momento de interação. Biofilmes fúngicos mais maduros parecem ser mais resistentes ao efeito inibitório que a M. oryzae causa na expressão do gene idh e na produção de patulina. Esta bactéria afeta a produção de patulina atuando ao nível transcricional do gene idh. Em conclusão, é aqui reportado um possível efeito protetor que o fungo filamentoso confere a bactérias oportunistas em biofilmes mistos. Além disso, a presença de moléculas de quórum sensing e de metabolitos secundários desempenham um papel fundamental por parte dos diferentes microrganismos em biofilmes mistos, que resultam na regulação de processos que levam à sua formação e estabelecimento.
I would also like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for having financially supported the work presented in this thesis through the FCT Doctoral Program in Applied and Environmental Microbiology (DP_AEM) under grant no. PD/BD/128033/2016. Furthermore, this work was also supported by FCT under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte