Evaluation of viable cells in Pseudomonas aeruginosa biofilms by colony count and live/dead staining

Pseudomonas aeruginosa is a human pathogen capable to form robust biofilms. P. aeruginosa biofilms represent a serious problem because of the adverse effects on human health and industry, from sanitary and economic points of view. Typical strategies to break down biofilms have been long used, such as the use of disinfectants or antibiotics, but also, according to their high resistance to standard antimicrobial approaches, alternative strategies employing photocatalysis or control of biofilm formation by modifying surfaces, have been proposed. Colony forming units (cfu) counting and live/dead staining, two classic techniques used for biofilm quantification, are detailed in this work. Both methods assess cell viability, a key factor to analyze the microbial susceptibility to given treatment, then, they represent a good approach for evaluation of an antibiofilm strategy.Fil: Pezzoni, Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; Argentina. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; ArgentinaFil: Pizarro, Ramón Augusto. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; ArgentinaFil: Costa, Cristina Susana. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; Argentin

Role of quorum sensing in UVA-induced biofilm formation in Pseudomonas aeruginosa

Pseudomonas aeruginosa, a versatile bacterium present in terrestrial and aquatic environments and a relevant opportunistic human pathogen, is largely known for the production of robust biofilms. The unique properties of these structures complicate biofilm eradication, because they make the biofilms very resistant to diverse antibacterial agents. Biofilm development and establishment is a complex process regulated by multiple regulatory genetic systems, among them is quorum sensing (QS), a mechanism employed by bacteria to regulate gene transcription in response to population density. In addition, environmental factors such as UVA radiation (400–315 nm) have been linked to biofilm formation. In this work, we further investigate the mechanism underlying the induction of biofilm formation by UVA, analysing the role of QS in this phenomenon. We demonstrate that UVA induces key genes of the Las and Rhl QS systems at the transcriptional level. We also report that pelA and pslA genes, which are essential for biofilm formation and whose transcription depends in part on QS, are significantly induced under UVA exposure. Finally, the results demonstrate that in a relA strain (impaired for ppGpp production), the UVA treatment does not induce biofilm formation or QS genes, suggesting that the increase of biofilm formation due to exposure to UVA in P. aeruginosa could rely on a ppGpp-dependent QS induction.Fil: Pezzoni, Magdalena. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; ArgentinaFil: Pizarro, Ramón Augusto. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); ArgentinaFil: Costa, Cristina Susana. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); Argentin

Detection of Catalase Activity by Polyacrylamide Gel Electrophoresis (PAGE) in Cell Extracts from Pseudomonas aeruginosa

Bacteria in nature and as pathogens commonly face oxidative stress which causes damage to proteins, lipids and DNA. This damage is produced by the action of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), singlet oxygen, superoxide anion and hydroxyl radical. ROS are generated by antimicrobials, environmental factors (e.g., ultraviolet radiation, osmotic stress), aerobic respiration, and host phagocytes during infective processes. Pseudomonas aeruginosa, a versatile bacterium, is a prevalent opportunistic human pathogen which possesses several defense strategies against ROS. Among them, two catalases (KatA and KatB) have been well characterized by their role on the defense against multiple types of stress. In this protocol, KatA and KatB activities are detected by polyacrylamide gel electrophoresis (PAGE). It is also suggested that the detection of KatB is elusive.Fil: Pezzoni, Magdalena. Comisión Nacional de Energía Atómica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pizarro, R. A.. Comisión Nacional de Energía Atómica; ArgentinaFil: Costa, C. S.. Comisión Nacional de Energía Atómica; Argentin

Presencia de espinas en la molleja de pichones de cotorra Argentina (Myiopsitta m. monachus)

Fil: Aramburú, Rosana Mariel. División Zoología Vertebrados. Facultad de Ciencias Naturales y Museo. Universidad Nacional de La Plata; ArgentinaFil: Pezzoni, Magdalena. División Zoología Vertebrados. Facultad de Ciencias Naturales y Museo. Universidad Nacional de La Plata; ArgentinaFil: Calvo, Sebastián. División Zoología Vertebrados. Facultad de Ciencias Naturales y Museo. Universidad Nacional de La Plata; ArgentinaFil: Arambarri, Angélica Margarita. División Instituto Spegazzini. Facultad de Ciencias Naturales y Museo. Universidad Nacional de La Plata; Argentin

Role of the quorum sensing mechanism in the response of pseudomonas aeruginosa to lethal and sublethal UVA irradiation

The role of quorum sensing (QS) in the response of Pseudomonas aeruginosa to UVA radiation was investigated in the PAO1 strain and derivatives defective in the synthesis of the QS signals 3OC12-HSL (lasI strain), C4-HSL (rhlI strain) or both (lasI rhlI strain). Cell viability measurements demonstrated that the double mutant was significantly more sensitive to UVA than single mutants, which in turn showed reduced cell survival with regard to the PAO1 strain. Irradiation under nitrogen atmosphere and chemiluminescence measurements indicated the oxidative nature of the UVA-induced damage. The activity of the antioxidant enzymes catalase and superoxide dismutase was assayed in these strains before and after irradiation, and a strong correlation between catalase levels and UVA sensitivity was observed. When a sublethal UVA dose was applied to PAO1, a growth delay was observed and this mechanism depended on the rhl system. Moreover, low doses of UVA irradiation triplicated the level of C4-HSL in log PAO1 cells. It is concluded that QS is fundamental in the defense against the toxic effects of UVA in P. aeruginosa. The induction of the QS system by UVA independently of cell density could function as an adaptative strategy to withstand this hostile environmental condition.Fil: Costa, Cristina Susana. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); ArgentinaFil: Pezzoni, Magdalena. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández, Rubén O.. Comisión Nacional de Energía Atómica; ArgentinaFil: Pizarro, Ramón Augusto. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); Argentin

Response to lethal UVA radiation in the Antarctic bacterium Pseudomonas extremaustralis: polyhydroxybutyrate and cold adaptation as protective factors

Pseudomonas extremaustralis is an Antarctic bacterium with high stress resistance, able to grow under cold conditions. It is capable to produce polyhydroxyalkanoates (PHAs) mainly as polyhydroxybutyrate (PHB) and, to a lesser extent, medium-chain length polyhydroxyalkanoates (mclPHAs). In this work, we analyzed the role of PHAs and cold adaptation in the survival of P. extremaustralis after lethal UVA exposure. P. extremaustralis presented higher radiation resistance under polymer accumulation conditions. This result was also observed in the derivative mutant strain PHA−, deficient for mclPHAs production. On the contrary, the PHB− derivative mutant, deficient for PHB production, showed high sensitivity to UVA exposure. Complementation of the PHB− strain restored the wild-type resistance level, indicating that the UVA-sensitive phenotype is due to the lack of PHB. All strains exhibited high sensitivity to radiation when cultured under PHAs non-accumulation conditions. A slight decrease in PHB content was observed after UVA exposure in association with increased survival. The scattering of UVA radiation by intracellular PHAs granules could also result in bacterial cell protection. In addition, cold conditions improved UVA tolerance, probably depending on PHB mobilization. Results showed that PHB accumulation is crucial in the resistance to UVA in P. extremaustralis. Mechanisms involved probably entail depolymerization and light scattering acting as a screen, both conferring protection against oxidative stress.Fil: Tribelli, Paula Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Pezzoni, Magdalena. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Brito, María Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Montesinos, Victor Nahuel. Comisión Nacional de Energía Atómica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Costa, Cristina Susana. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; ArgentinaFil: López, Nancy Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin

Transforming an inert nanopolymer into broad-spectrum bactericidal by superstructure tuning

Poloxamer block copolymers (also known as Pluronic®) are particularly useful for drug delivery and self-assembly techniques. These nanopolymers are generally considered to be biologically inert and they were used to generate only bacteria repellent surfaces but keeps bacteria alive and as a latent threat. However, the inherent capabilities of these nanopolymers to kill bacteria have been largely overlooked. Here, we report that Pluronic shaped as superstructures (self-organized array of micelles) in fact possess a broad-spectrum bactericidal activity (capability of killing bacteria) similar to that shown for some antibiotics. This further represents the first report that shows that appropriate control of superstructured mesophase architecture is a key parameter for bactericidal efficacy. Based on this finding, we have developed a highly bactericidal coating (>99.9 % kill) against all tested Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Salmonella typhimurium LT2, Escherichia coli K12 and Pseudomonas aeruginosa PAO1) bacteria which moreover allows the adhesion and proliferation of mammalian cells. The inexpensiveness and ease of production make these versatile nanopolymer structures a powerful tool for the development of a new generation of highly effective antimicrobial coatings.Fil: Scilletta, Natalia Antonela. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia - Nodo Constituyentes | Comision Nacional de Energia Atomica. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia - Nodo Constituyentes.; ArgentinaFil: Pezzoni, Magdalena. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Desimone, Martín Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; ArgentinaFil: Soler Illia, Galo Juan de Avila Arturo. Universidad Nacional de San Martin. Instituto de Nanosistemas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Catalano, Paolo Nicolás. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia - Nodo Constituyentes | Comision Nacional de Energia Atomica. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia - Nodo Constituyentes.; ArgentinaFil: Bellino, Gabriel Martín. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia - Nodo Constituyentes | Comision Nacional de Energia Atomica. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia. Unidad Ejecutora Instituto de Nanociencia y Nanotecnologia - Nodo Constituyentes.; Argentin

Effect of temperature on microcystin-LR removal and lysis activity on <i>Microcystis aeruginosa</i> (cyanobacteria) by an indigenous bacterium belonging to the genus <i>Achromobacter</i>

Microcystis is a frequent cyanobacterium bloom-forming with cosmopolitan distribution which can produce a hepatotoxin group called microcystins (MCs). These MCs are resistant to the traditional processes employed in the water treatment plants and they are often detected after conventional treatments. Because of this, the bio-removal studies have obtained a great interest in the last decades. In this work, a bacterial strain namely LG1 with the ability to remove microcystin-LR (MC-LR) under laboratory conditions was isolated from Rio de la Plata River and it was identified as Achromobacter spp. This ubiquitous bacterium was able to remove 79.5% MC-LR in 7 days with average removal time of 3.33 ± 0.08, 3.06 ± 0.05, and 2.77 ± 0.05 days at 28, 32, and 36 ± 1 °C, being higher at high temperature (36 °C) with an activation energy = 16.79 ± 1.99 kJ mol⁻¹. LG1 grew better at higher temperature (from 28 to 36 ± 1 °C) increasing the specific growth rate (μ) and reducing 2-fold the lag phase duration (LPD) without significant differences (p > 0.05) between maximum population density (MPD). In addition, LG1 showed a lysis activity on two M. aeruginosa native strains in 7 days measured as chlorophyll a (Chl-a) concentration. The lysis activity increased around 2-fold when increasing the temperature from 28 to 36 ± 1 °C. This is the first report of an indigenous bacterium belonging to the genusAchromobacter spp. isolated from the Rio de la Plata River with the capacity to remove MC-LR and lysis activity on M. aeruginosa.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Criotecnología de Alimento

Exposure to low doses of UVA increases biofilm formation in Pseudomonas aeruginosa

The establishment of bacterial biofilms on abiotic surfaces is a complex process regulated by multiple genetic regulators and environmental factors which are able to modulate the passage of planktonic cells to a sessile state. Solar ultraviolet-A radiation (UVA, 315-400) is one of the main environmental stress factors that bacteria must face at the Earth´s surface. The deleterious effects of UVA are mainly due to oxidative damage. This paper reports that exposure to low UVA doses promotes biofilm formation in three prototypical strains of Pseudomonas aeruginosa, a relevant opportunistic human pathogen. It demonstrates that exposure of planktonic cells to sublethal doses of UVA can increase cell surface hydrophobicity and swimming motility, two parameters known to favor cell adhesion. These results suggest that UVA radiation acts, at least in part, by promoting the first stages of biofilm development.Fil: Pezzoni, Magdalena. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pizarro, Ramón Augusto. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); ArgentinaFil: Costa, Cristina Susana. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); Argentin

UVA as environmental signal for alginate production in Pseudomonas aeruginosa: role of this polysaccharide in the protection of planktonic cells and biofilms against lethal UVA doses

Pseudomonas aeruginosa is an extremely versatile microorganism that survives in a wide variety of niches. It is capable to respond rapidly to changes in the environment by producing secondary metabolites and virulence factors, including alginate. Alginate is an extracellular polysaccharide that protects the bacteria from antibiotics and oxidative agents, and enhances cell adhesion to solid surfaces in the process of biofilm formation. In the present study, we analyzed the role of alginate in the response of P. aeruginosa to lethal doses of ultraviolet-A (UVA) radiation, the major fraction of solar UV radiation reaching the Earth’s surface. We also studied the role of alginate in the context of the adaptive responses generated when P. aeruginosa is exposed to sublethal doses of UVA radiation. The survival studies demonstrated that alginate has a key role in the resistance of P. aeruginosa to the oxidative stress generated by lethal UVA doses, both in planktonic cells and in static biofilms. In addition, the presence of alginate proved to be essential in the occurrence of adaptive responses such as induction of biofilm formation and cross-protection against hydrogen peroxide and sodium hypochlorite, both generated by exposure to low UVA doses. Finally, we demonstrated that the increase of biofilm formation is accompanied by an increase in alginate concentration in the biofilm matrix, possibly through the ppGpp-dependent induction of genes related to alginate regulation (algR and algU) and biosynthesis (algD operon). Given the importance of alginate in biofilm formation and its protective roles, better understanding of the mechanisms associated to its functions and synthesis is relevant, given the normal exposure of P. aeruginosa to UVA radiation and other types of oxidative stresses.Fil: Pezzoni, Magdalena. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lemos, Martin. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; ArgentinaFil: Pizarro, Ramón Augusto. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; ArgentinaFil: Costa, Cristina Susana. Comisión Nacional de Energía Atómica. Gerencia de Área de Aplicaciones de la Tecnología Nuclear. Departamento de Radiobiología; Argentin