Aspectos moleculares del desarrollo de biofilms, la colonización de la planta y la emisión de compuestos orgánicos volátiles en bacterias rizosféricas. Molecular aspects of biofilm development, plant colonization and microbial volatile organic emission in rhizospheric bacteria.

Las bacterias que habitan la rizosfera y que poseen la capacidad de provocar un efecto positivo sobre las plantas son denominadas en su conjunto como Rizobacterias Promotoras del Crecimiento Vegetal (PGPR). Estas bacterias han desarrollado diferentes estrategias para adaptarse a diversas condiciones ambientales. La capacidad para responder a variaciones en la disponibilidad nutricional permite la persistencia de la bacteria en el suelo y mejora sus posibilidades para colonizar la planta hospedadora. En la naturaleza, a menudo las bacterias se encuentran en estructuras de comunidades de microorganismos interconectados denominados biofilms, con un estilo de vida diferente al de la vida en forma planctónica. La formación del biofilm podría representar una estrategia de supervivencia de la rizobacteria a condiciones adversas del suelo. Por Microscopía Confocal de Barrido Láser (CLSM), hemos observado que Rhizobium leguminosarum desarrolla un biofilm característico sobre una superficie abiótica. Hemos identificado algunos de los factores genéticos que influyen en su formación. El presente proyecto propone avanzar en el conocimiento de los factores ambientales y genéticos que influyen sobre la capacidad de las rizobacterias para formar biofilms y su impacto en la interacción con las plantas. A través de enfoques genéticos (mutacionales y de expresión génica) y análisis por CLSM nos proponemos acercarnos a un modelo de los factores de superficie, extracelulares y regulatorios propios de la bacteria que influyen en las propiedades de adhesión y la formación de biofilms. Por último, se intentará correlacionar la emisión de compuestos orgánicos volátiles por las bacterias rizosféricas con ciertos aspectos de la promoción del crecimiento de las plantas.

Novel role of a family of major facilitator transporters in biofilm development and virulence of Candida albicans.

The QDR (quinidine drug resistance) family of genes encodes transporters belonging to the MFS (major facilitator superfamily) of proteins. We show that QDR transporters, which are localized to the plasma membrane, do not play a role in drug transport. Hence, null mutants of QDR1, QDR2 and QDR3 display no alterations in susceptibility to azoles, polyenes, echinocandins, polyamines or quinolines, or to cell wall inhibitors and many other stresses. However, the deletion of QDR genes, individually or collectively, led to defects in biofilm architecture and thickness. Interestingly, QDR-lacking strains also displayed attenuated virulence, but the strongest effect was observed with qdr2∆, qdr3∆ and in qdr1/2/3∆ strains. Notably, the attenuated virulence and biofilm defects could be reversed upon reintegration of QDR genes. Transcripts profiling confirmed differential expression of many biofilm and virulence-related genes in the deletion strains as compared with wild-type Candida albicans cells. Furthermore, lipidomic analysis of QDR-deletion mutants suggests massive remodelling of lipids, which may affect cell signalling, leading to the defect in biofilm development and attenuation of virulence. In summary, the results of the present study show that QDR paralogues encoding MFS antiporters do not display conserved functional linkage as drug transporters and perform functions that significantly affect the virulence of C. albicans.

Data quality in biofilm high-throughput routine analysis: intralaboratory protocol adaptation and experiment reproducibility

Biofilm research is growing more diverse and dependent on high-throughput technologies and the large-scale production of results aggravates data substantiation. In particular, it is often the case that experimental protocols are adapted to meet the needs of a particular laboratory and no statistical validation of the modified method is provided. This paper discusses the impact of intra-laboratory adaptation and non-rigorous documentation of experimental protocols on biofilm data interchange and validation. The case study is a non-standard, but widely used, workflow for Pseudomonas aeruginosa biofilm development, considering three analysis assays: the crystal violet (CV) assay for biomass quantification, the XTT assay for respiratory activity assessment, and the colony forming units (CFU) assay for determination of cell viability. The ruggedness of the protocol was assessed by introducing small changes in the biofilm growth conditions, which simulate minor protocol adaptations and non-rigorous protocol documentation. Results show that even minor variations in the biofilm growth conditions may affect the results considerably, and that the biofilm analysis assays lack repeatability. Intra-laboratory validation of non-standard protocols is found critical to ensure data quality and enable the comparison of results within and among laboratories.

Comparing different methods to measure biofilm thickness: the techniques are: optical coherence tomography, confocal laser scanning microscopy and low load compression test

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Impairment of the Bacterial Biofilm Stability by Triclosan

The accumulation of the widely-used antibacterial and antifungal compound triclosan (TCS) in freshwaters raises concerns about the impact of this harmful chemical on the biofilms that are the dominant life style of microorganisms in aquatic systems. However, investigations to-date rarely go beyond effects at the cellular, physiological or morphological level. The present paper focuses on bacterial biofilms addressing the possible chemical impairment of their functionality, while also examining their substratum stabilization potential as one example of an important ecosystem service. The development of a bacterial assemblage of natural composition – isolated from sediments of the Eden Estuary (Scotland, UK) – on non-cohesive glass beads (,63 mm) and exposed to a range of triclosan concentrations (control, 2 – 100 mg L21) was monitored over time by Magnetic Particle Induction (MagPI). In parallel, bacterial cell numbers, division rate, community composition (DGGE) and EPS (extracellular polymeric substances: carbohydrates and proteins) secretion were determined. While the triclosan exposure did not prevent bacterial settlement, biofilm development was increasingly inhibited by increasing TCS levels. The surface binding capacity (MagPI) of the assemblages was positively correlated to the microbial secreted EPS matrix. The EPS concentrations and composition (quantity and quality) were closely linked to bacterial growth, which was affected by enhanced TCS exposure. Furthermore, TCS induced significant changes in bacterial community composition as well as a significant decrease in bacterial diversity. The impairment of the stabilization potential of bacterial biofilm under even low, environmentally relevant TCS levels is of concern since the resistance of sediments to erosive forces has large implications for the dynamics of sediments and associated pollutant dispersal. In addition, the surface adhesive capacity of the biofilm acts as a sensitive measure of ecosystem effects

Streptococcus mutans Biofilm Adhesion on Composite Resin Surfaces After Different Finishing and Polishing Techniques

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ability of Salmonella spp. to Produce Biofilm Is Dependent on Temperature and Surface Material

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Susceptibility of Candida albicans and Candida glabrata biofilms to silver nanoparticles in intermediate and mature development phases

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The importance of pneumococcal capsule in inate immunity and biofilm formation

Chapter 1 gives an overview about Streptococcus pneumoniae, its role as a human pathogen and its virulence factors. Additionally, biofilm development and its relevance in clinics are introduced, and the innate immune response to pneumococcus as well as bacterial-viral interactions in the upper respiratory tract are also discussed. Chapter 2 emphasizes the three main topics of this thesis: the role of capsule and pneumolysin in the immune response in the respiratory tract, biofilm formation of S. pneumoniae serotypes and commensal streptococci in vitro, and host innate immune responses to RSV and S. pneumoniae during in vitro co-infections. Aims and hypotheses are provided here. Chapter 3 is divided into two parts: First, the release of the pro-inflammatory cytokines CXCL8 and IL-6 from the human pharyngeal epithelial cell line Detroit 562 and from human bronchial epithelial cells (iHBEC) is described in response to S. pneumoniae. Capsule was shown to suppress the release of both cytokines in both cell lines tested, but release was much less from iHBEC cells. During intranasal colonization of mice, suppression of CXCL8 release by the capsule was also observed in vivo, but the effect was only measured in the absence of pneumolysin. Long term, stable nasopharyngeal carriage in a mouse model resulted in the dissemination of nonencapsulated pneumococci into the lungs, whereas encapsulated strains remained in the nasopharynx. The S. pneumoniae capsule thus plays a role in modulation of the pro-inflammatory immune response in the respiratory tract. Second, results on immunological cells and immune regulation in a long term, stable nasopharyngeal carriage mouse model are presented. Mice were infected with encapsulated or nonencapsulated pneumococcal strains, and after 1, 3, 8 and 15 days, were sacrificed to evaluate the numbers of CD45+ cells, neutrophils, macrophages, FoxP3+ regulatory T-cells and CD3+ T-cells in the nasal mucosa as well as the amount of secreted IL-10 in the nasopharynx. Nasopharyngeal colonization which is effectively silent resulted in the stimulation of FoxP3+ regulatory T-cells and IL-10 release associated with immune homeostasis, whereas lung infiltration was required to increase the number of neutrophils and macrophages resulting in a stronger innate immune response in the nasal mucosa. Chapter 4 contains results of mono- and co-stimulation using RSV and pneumococci or pneumococcal virulence factors on the human bronchial epithelial cell line BEAS-2B. An increase in CXCL8 and IL-6 levels was measured for mixed stimulations of RSV and pneumococcus when encapsulated bacteria were used. Increasing pneumolysin concentrations resulted in enhanced CXCL8 levels. Priming of bronchial epithelial cells with RSV opens the door for more severe pneumococcal infections. Chapter 5 is composed of two parts: The first part describes initial biofilm formation of serotypes 6B and 7F in a static model in vitro. Biofilms of both serotypes contained SCVs, but only serotype 6B increased in SCV formation between 16 and 65h of incubation. SCV stability was tested by passaging clones in complex medium, where SCV production is not associated with advantages in growth. Serotype 6B lost the SCV phenotype indicating a fast adaptation to a changing nutritional environment. Limitations of our in vitro model are discussed. The second part is about initial biofilm formation of mixed culture growth of S. pneumoniae with commensal streptococci. Competition dominates this process. S. oralis and pneumococcus compete for nutrients, whereas mixed species growth of S. mitis or S. pseudopneumoniae with S. pneumoniae is mainly influenced by other factors. In Chapter 6 the findings of chapters 3, 4 and 5 are discussed and an outlook for further studies is provided. Chapters 7, 8, 9, 10 and 11 contain the references, the acknowledgements, the curriculum vitae, the appendix and the declaration of originality.

Characterization of cell-to-cell signaling-deficient Pseudomonas aeruginosa strains colonizing intubated patients.

Cell-to-cell signaling involving N-acyl-homoserine lactone compounds termed autoinducers (AIs) is instrumental to virulence factor production and biofilm development by Pseudomonas aeruginosa. In order to determine the importance of cell-to-cell signaling during the colonization of mechanically ventilated patients, we collected 442 P. aeruginosa pulmonary isolates from 13 patients. Phenotypic characterization showed that 81% of these isolates produced the AI-dependent virulence factors elastase, protease, and rhamnolipids. We identified nine genotypically distinct P. aeruginosa strains. Six of these strains produced AIs [N-butanoyl-homoserine lactone or N-(3-oxo-dodecanoyl)-homoserine lactone] and extracellular virulence factors (elastase, total exoprotease, rhamnolipid, hydrogen cyanide, or pyocyanin) in vitro. Three of the nine strains were defective in the production of both AIs and extracellular virulence factors. Two of these strains had mutational defects in both the lasR and rhlR genes, which encode the N-acyl-homoserine lactone-dependent transcriptional regulators LasR and RhlR, respectively. The third of these AI-deficient strains was only mutated in the lasR gene. Our observations suggest that most, but not all, strains colonizing intubated patients are able to produce virulence factors and that mutations affecting the cell-to-cell signaling circuit are preferentially located in the transcriptional regulator genes.

Two GacA-dependent small RNAs modulate the quorum-sensing response in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the GacS/GacA two-component system positively controls the quorum-sensing machinery and the expression of extracellular products via two small regulatory RNAs, RsmY and RsmZ. An rsmY rsmZ double mutant and a gacA mutant were similarly impaired in the synthesis of the quorum-sensing signal N-butanoyl-homoserine lactone, the disulfide bond-forming enzyme DsbA, and the exoproducts hydrogen cyanide, pyocyanin, elastase, chitinase (ChiC), and chitin-binding protein (CbpD). Both mutants showed increased swarming ability, azurin release, and early biofilm development.

The small RNA PhrS stimulates synthesis of the Pseudomonas aeruginosa quinolone signal.

Quorum sensing, a cell-to-cell communication system based on small signal molecules, is employed by the human pathogen Pseudomonas aeruginosa to regulate virulence and biofilm development. Moreover, regulation by small trans-encoded RNAs has become a focal issue in studies of virulence gene expression of bacterial pathogens. In this study, we have identified the small RNA PhrS as an activator of PqsR synthesis, one of the key quorum-sensing regulators in P. aeruginosa. Genetic studies revealed a novel mode of regulation by a sRNA, whereby PhrS uses a base-pairing mechanism to activate a short upstream open reading frame to which the pqsR gene is translationally coupled. Expression of phrS requires the oxygen-responsive regulator ANR. Thus, PhrS is the first bacterial sRNA that provides a regulatory link between oxygen availability and quorum sensing, which may impact on oxygen-limited growth in P. aeruginosa biofilms.

Étude de la virulence et de la résistance aux antibiotiques des Staphylococcus aureus résistants à la méthicilline chez le porc à l'abattoir au Québec

Depuis quelques années et dans plusieurs pays, un nouveau type de Staphylococcus aureus résistant à la méthicilline (SARM), le séquence type (ST) 398, a été fréquemment retrouvé chez les porcs et chez les fermiers en contact avec ces porcs. Au Canada, très peu d’informations sont disponibles concernant le SARM d’origine porcine. Une première étude dans notre laboratoire a permis de récolter 107 isolats de SARM provenant de deux abattoirs porcins du Québec. Le présent travail vise à caractériser les gènes de virulence et de résistance aux antibiotiques de ces SARM, d’étudier leur formation de biofilm en relation avec la spécificité du groupe agr et de vérifier la localisation plasmidique et la transférabilité de ces gènes à des souches de SARM d’origine humaine. Plusieurs souches ont démontré différents patrons phénotypiques de résistance aux antibiotiques. Vingt-quatre souches représentatives de ces isolats ont été soumises à une caractérisation plus approfondie par une étude génotypique en utilisant une biopuce à ADN et un grand nombre de gènes de virulence a été détecté codant pour des entérotoxines staphylococcales, des leucocidines, des hémolysines, des auréolysines, des facteurs d’immunoévasion, des superantigènes, des facteurs d’adhésion et des facteurs impliqués dans la formation de biofilm. Des gènes de résistance envers les aminoglycosides, les macrolides, les lincosamides, les tétracyclines et les biocides ont été également détectés par biopuce et leur localisation plasmidique a par la suite été déterminée. La transférabilité de ces gènes de souches porcines à des souches de SARM d’origine humaine a été démontrée par conjugaison bactérienne; ainsi le transfert horizontal de certains gènes de résistance aux antibiotiques et de virulence a été observé. Ces travaux de recherche apportent une meilleure connaissance de la résistance aux antibiotiques et de la virulence des SARM d’origine porcine et de leur potentiel de contribution à l’émergence de certaines résistances et facteurs de virulence chez le SARM d’origine humaine.

An engineering model of the human colon

The development and performance of a three-stage tubular model of the large human intestine is outlined. Each stage comprises a membrane fermenter where flow of an aqueous polyethylene glycol solution on the outside of the tubular membrane is used to control the removal of water and metabolites (principally short chain fatty acids) from, and thus the pH of, the flowing contents on the fermenter side. The three stage system gave a fair representation of conditions in the human gut. Numbers of the main bacterial groups were consistently higher than in an existing three-chemostat gut model system, suggesting the advantages of the new design in providing an environment for bacterial growth to represent the actual colonic microflora. Concentrations of short chain fatty acids and Ph levels throughout the system were similar to those associated with corresponding sections of the human colon. The model was able to achieve considerable water transfer across the membrane, although the values were not as high as those in the colon. The model thus goes some way towards a realistic simulation of the colon, although it makes no pretence to simulate the pulsating nature of the real flow. The flow conditions in each section are characterized by low Reynolds numbers: mixing due to Taylor dispersion is significant, and the implications of Taylor mixing and biofilm development for the stability, that is the ability to operate without washout, of the system are briefly analysed and discussed. It is concluded that both phenomena are important for stabilizing the model and the human colon.

Efetividade de vernizes fotopolimerizáveis experimentais na adesão e formação de biofilme multiespécies e sua associação com a escovação na remoção e recolonização do biofilme de uma resina acrílica para base de prótese

Pós-graduação em Reabilitação Oral - FOAR