Efecto inhibidor de extractos de nalca sobre la formación de biopelículas de Helicobacter pylori J99 y de Escherichia coli aisladas de catéteres urinarios y/o infecciones urinarias

56 p.La infección crónica por Helicobacter pylori, un problema importante en salud pública en Chile, frecuentemente se asocia a patologías gastroduodenal inflamatorias crónicas y a cáncer gástrico. Actualmente los regímenes de erradicación más utilizados incluyen una combinación de un inhibidor de la bomba de protones y dos agentes antibacterianos. Como otras bacterias, H. pylori forma biopelículas in vivo e in vitro. Por otra parte, Escherichia coli, un microorganismo frecuente como agente etiológico de infección urinaria, forma biopelículas en diversas superficies, entre ellas los catéteres urinarios, considerados como una de las vías de entrada y ascenso de esta bacteria hacia el tracto urinario y de posterior infección. El objetivo de esta Tesis fue investigar la capacidad del extracto de nalca y de curcumina, un pigmento natural, para inhibir la formación de biopelículas por estos microorganismos mencionados, y la posibilidad de que la acción de este último compuesto sea potenciada por el extracto de nalca sobre la biopelícula de E. coli. Se indujo la formación de biopelícula por ambas bacterias sobre superficie de poliestireno y se ensayó la potencia inhibitoria de este proceso por extracto de nalca y por curcumina,ensayadas a concentraciones sub-inhibitorias por un método colorimétrico, basado en la retención de cristal violeta por la biopelícula, previa eliminación de las células bacterianas no adheridas. Los resultados obtenidos permiten concluir que los extractos de nalca y curcumina inhiben la formación de biopelícula formada por H. pylori J99, y en menor proporción por las cepas de E. coli ATCC 25922 y E. coli S1. Además, estos compuestos ensayados en asociación no mostraron evidencia de potenciación de su acción inhibitoria cuando se ensayaron sobre las cepas de E Coli./ABSTRACT: Chronic infection with Helicobacter pylori, an important problem of public health in Chile, is frequently associated with chronic inflamatory gastroduodenal pathologies as well as with gastric cancer. At present, those mostly used regimes for the eradication of the microorganism include a proton pump inhibitor plus two antibacterial agents. H. pylori, as well as other bacteria, grows in vivo and in vitro forming biofilms. On the other hand, Escherichia coli, a frequent microorganism producing urinary tract infection, also grows forming biofilms on different surfaces such as those of urinary catheters. Therefore, catheters is considered to be one the most important mean of bacterial entrance into the urinary tact and thus of producing the infection process. The objective of this Thesis was to investigate the inhibitory activity of extracts of nalca and curcumin, a natural pigment, on biofilm formation by these microorganisms. Also, the possibility of a synergic effect between curcumin and components of the nalca extract upon biofilm formation was investigated. Bacterial biofilm formation was induced upon polystyrene surfaces and the level of inhibition of biofilm formation by extracts of nalca and of curcumin was evaluated. The assays were performed using subinhibitory concentrations the extracts and measured by a colorimetric method based on the staining of the bacterial biofilm with crystal violet in the absence of non adhered bacterial cells previously removed by washing the surfaces with saline. The results of the experiments with H. pylori J99 indicated that extracts of nalca and curcumin inhibit, with different levels, the formation of biofilm. Strains of E.coli ATCC 25922 and E.coli S1 produced a similar effect but at a lower proportion. In addition, the compounds under study, when assayed in combinations upon the strains E.coli, did not exhibit potentiation effect of the inhibitory activity upon biofilm formation

Draft genome sequence of Weissella viridescens UCO-SMC3, isolated from the slime of helix aspersa Müller snails

This report describes the draft genome sequence of Weissella viridescens UCO-SMC3, isolated from Helix aspersa Müller slime. The reads were generated by a whole-genome sequencing (WGS) strategy on an Illumina MiSeq sequencer and were assembled into contigs with a total estimated size of 1,612,814 bp. A total of 2,455 genes were predicted, including 2,301 protein-coding sequences. The draft genome sequence of W. viridescens UCO-SMC3 will be useful for further studies of specific genetic features and for understanding the mechanisms of its beneficial properties in the skin.Fil: Garcia Cancino, Apolinaria. Universidad de Concepción; ChileFil: Albarracín, Leonardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán; Argentina. Tohoku University; JapónFil: Espinoza Monje, Marcela. Universidad de Concepción; ChileFil: Campos Martin, Jorge. Universidad de Concepción; ChileFil: Garcia Castillo,Valeria. Tohoku University; Japón. Universidad de Concepción; ChileFil: Nakano, Yukha. Tohoku University; JapónFil: Ikeda Ohtsubo, Wakako. Tohoku University; JapónFil: Gutierrez, Cristian. Universidad de Concepción; ChileFil: Morita, Hidetoshi. Okayama University; JapónFil: Kitazawa, Haruki. Tohoku University; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japó

Evaluation of the immunomodulatory activities of the probiotic strain lactobacillus fermentum UCO-979C

Lactobacillus fermentum UCO-979C, a strain isolated from a human stomach, was previously characterized by its potential probiotic properties. The UCO-979C strain displayed the ability to beneficially regulate the innate immune response triggered by Helicobacter pylori infection in human gastric epithelial cells. In this work, we conducted further in vitro studies in intestinal epithelial cells (IECs) and in vivo experiments in mice in order to characterize the potential immunomodulatory effects of L. fermentum UCO-979C on the intestinal mucosa. Results demonstrated that the UCO-979C strain is capable to differentially modulate the immune response of IECs triggered by Toll-like receptor 4 (TLR4) activation through the modulation of TLR negative regulators' expression. In addition, we demonstrated for the first time that L. fermentum UCO-979C is able to exert its immunomodulatory effect in the intestinal mucosa in vivo. The feeding of mice with L. fermentum UCO-979C significantly increased the production of intestinal IFN-γ, stimulated intestinal and peritoneal macrophages and increased the number of Peyer's patches CD4+ T cells. In addition, L. fermentum UCO-979C augmented intestinal IL-6, reduced the number of immature B220+CD24high B cells from Peyer's patches, enhanced the number of mature B B220+CD24low cells, and significantly increased intestinal IgA content. The results of this work revealed that L. fermentum UCO-979C has several characteristics making it an excellent candidate for the development of immunobiotic functional foods aimed to differentially regulate immune responses against gastric and intestinal pathogens.Fil: Garcia Castillo, Valeria. Tohoku University; Japón. Universidad de Concepción; Chile. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Komatsu, Ryoya. Tohoku University; JapónFil: Clua, Maria Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Indo, Yuhki. Tohoku University; JapónFil: Takagi, Michihiro. Tohoku University; JapónFil: Salva, Maria Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Islam, M. Aminul. Bangladesh Agricultural University; Bangladesh. Tohoku University; JapónFil: Alvarez, Gladis Susana. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Takahashi, Hideki. Tohoku University; JapónFil: Garcia Cancino, Apolinaria. Universidad de Concepción; ChileFil: Kitazawa, Haruki. Tohoku University; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japó

The Exopolysaccharide of Lactobacillus fermentum UCO-979C Is Partially Involved in Its Immunomodulatory Effect and Its Ability to Improve the Resistance against Helicobacter pylori Infection

Lactobacillus fermentum UCO-979C (Lf979C) beneficially modulates the cytokine response of gastric epithelial cells and macrophages after Helicobacter pylori infection in vitro. Nevertheless, no in vivo studies were performed with this strain to confirm its beneficial immunomodulatory effects. This work evaluated whether Lf979C improves protection against H. pylori infection in mice by modulating the innate immune response. In addition, we evaluated whether its exopolysaccharide (EPS) was involved in its beneficial effects. Lf979C significantly reduced TNF-α, IL-8, and MCP-1 and augmented IFN-γ and IL-10 in the gastric mucosa of H. pylori-infected mice. The differential cytokine profile induced by Lf979C in H. pylori-infected mice correlated with an improved reduction in the pathogen gastric colonization and protection against inflammatory damage. The purified EPS of Lf979C reduced IL-8 and enhanced IL-10 levels in the gastric mucosa of infected mice, while no effect was observed for IFN-γ. This work demonstrates for the first time the in vivo ability of Lf979C to increase resistance against H. pylori infection by modulating the gastric innate immune response. In addition, we advanced knowledge of the mechanisms involved in the beneficial effects of Lf979C by demonstrating that its EPS is partially responsible for its immunomodulatory effect.Fil: Garcia Castillo, Valeria. Universidad de Concepción; Chile. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Marcial, Guillermo Emilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Albarracín, Leonardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología; ArgentinaFil: Tomokiyo, Mikado. Tohoku University; JapónFil: Clua, Maria Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Takahashi, Hideki. Tohoku University; JapónFil: Kitazawa, Haruki. Tohoku University; JapónFil: García Cancino, Apolinaria. Universidad de Concepción; ChileFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japó

Efficient Selection of New Immunobiotic Strains With Antiviral Effects in Local and Distal Mucosal Sites by Using Porcine Intestinal Epitheliocytes

Previously, we evaluated the effect of the immunobiotic strain Lactobacillus rhamnosus CRL1505 on the transcriptomic response of porcine intestinal epithelial (PIE) cells triggered by the challenge with the Toll-like receptor 3 (TLR-3) agonist poly(I:C) and successfully identified a group of genes that can be used as prospective biomarkers for the screening of new antiviral immunobiotics. In this work, several strains of lactobacilli were evaluated according to their ability to modulate the expression of IFNα, IFNβ, RIG1, TLR3, OAS1, RNASEL, MX2, A20, CXCL5, CCL4, IL-15, SELL, SELE, EPCAM, PTGS2, PTEGES, and PTGER4 in PIE cells after the stimulation with poly(I:C). Comparative analysis of transcripts variations revealed that one of the studied bacteria, Lactobacillus plantarum MPL16, clustered together with the CRL1505 strain, indicating a similar immunomodulatory potential. Two sets of in vivo experiments in Balb/c mice were performed to evaluate L. plantarum MPL16 immunomodulatory activities. Orally administered MPL16 prior intraperitoneal injection of poly(I:C) significantly reduced the levels of the proinflammatory mediators tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and IL-15 in the intestinal mucosa. In addition, orally administered L. plantarum MPL16 prior nasal stimulation with poly(I:C) or respiratory syncytial virus infection significantly decreased the levels of the biochemical markers of lung tissue damage. In addition, reduced levels of the proinflammatory mediators TNF-α, IL-6, and IL-8 were found in MPL16-treated mice. Improved levels of IFN-β and IFN-γ in the respiratory mucosa were observed in mice treated with L. plantarum MPL16 when compared to control mice. The immunological changes induced by L. plantarum MPL16 were not different from those previously reported for the CRL1505 strain in in vitro and in vivo studies. The results of this work confirm that new immunobiotic strains with the ability of stimulating both local and distal antiviral immune responses can be efficiently selected by evaluating the expression of biomarkers in PIE cells.Fil: Albarracín, Leonardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Garcia-Castillo, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad de Concepción; ChileFil: Masumizu, Yuki. Tohoku University; JapónFil: Indo, Yuhki. Tohoku University; JapónFil: Islam, Md Aminul. Tohoku University; JapónFil: Suda, Yoshihito. Miyagi University; JapónFil: García Cancino, Apolinaria. Universidad de Concepción; ChileFil: Aso, Hisashi. Tohoku University; JapónFil: Takahashi, Hideki. Tohoku University; JapónFil: Kitazawa, Haruki. Tohoku University; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japó

DETECCIÓN Y CARACTERIZACION PARCIAL DE BACTERIOCINAS PRODUCIDAS POR LACTOBACILLUS SPP CON ACCION ANTI HELICOBACTER PYLORI

El objetivo del estudio fue determinar si las cepas de Lactobacillus spp. son capaces de inhibir el crecimiento de cepas de Helicobacter pylori procedentes de biopsias gástricas de pacientes. La metodología utilizada fue aislamiento, cultivo e identificación de Helicobacter pylori, Lactobacillus spp y la inhibición del crecimiento de helicobacter pylori mediada por cepas de lactobacillus spp., se muestreó un total de 200 pacientes de los cuales 135 fueron HP+ (Helicobacter pylori positivos). No se encontró diferencias significativas entre factores como la edad, sexo y procedencia geográfica con la infección por esta bacteria. Se logró aislar un total de 47 cepas de Lactobacillus spp de un total de 200 pacientes de los cuales 31 cepas pertenecían a pacientes que presentaban también a Helicobacter pylori. Sin embargo no se encontró relación a nivel estadístico por la prueba del Chi-cuadrado entre ambos grupos. Lactobacillus spp. mostró acción inhibitoria frente a Helicobacter pilori en un 89.4% y el espectro de inhibición microbiana producida por Lactobacillus spp. osciló entre 1.0 y 2.5 cm

Immunomodulatory properties of microcapsules containing Lactobacillus rhamnosus UCO-25A in planktonic o biofilm forms: impact on intestinal and systemic immunity

Previously, we performed a detailed characterization of the probiotic properties of Lactobacillus rhamnosus UCO-25A originally isolated from human stomach. We demonstrated that the UCO-25 strain has high hydrophobicity properties and is able to form biofilms on glass and cell surfaces. Moreover, we demonstrated that L. rhamnosus UCO-25A is a strain with immunomodulatory activity. Using a murine model, we observed that the oral administration of UCO-25A strain is able to increase the activity of peritoneal macrophages and intestinal antigen presenting cells as well as the levels of intestinal IgA (Ilabaca et al., 2018, submitted for publication). Then, our findings provided a scientific rationale for the use of L. rhamnosus UCO-25A in the development of new immunobiotic products. One of the major challenges in the production of new food or pharmaceutical products is to preserve the viability of immunobiotic strains in order they can exert their beneficial effects. Encapsulation is an interesting alternative to achieve this goal. Encapsulation can protect microorganisms from external environments and induce the release of them in a metabolically active state in the intestine.The aim of this work was to evaluate whether L. rhamnosus UCO-25A, administered in an encapsulated form, does preserve its immunomodulatory activities. Moreover, the immunomodulatory capacities of its planktonic o biofilm states were comparatively studied. Immunobiotic capsules were prepared with carob tree-gum and alginate through an extrusion process in the B-390/B395Pro Encapsulator. Capsules were then covered with low molecular weight chitosan (Type I). Female 5-week-old BALB/c mice were used for in vivo studies. L. rhamnosus UCO-25A encapsulated in planktonic (P25A) or biofilm (B25A) forms were administered at a concentration of 109 cells/mouse/day to different groups of mice for two consecutive days by gavage. Mice treated with PBS solution were used as controls. One day after the last treatment we evaluated: a) peritoneal macrophages phagocytic and microbicidal activities; b) IgA levels in intestinal fluid (IF); c) levels of IFN-γ and TNF-α in IF and serum, and d) immune cell populations in peritoneal cavity, Peyer´s patches and spleen by flow cytometry. Although P25A or B25A did not significantly change the number of peritoneal macrophages (F4/80+ cells) both treatments improved their phagocytic and microbicidal activity (% of phagocytosis: control=4.2±1.2, P25A=19.8±2.4, B25A=16.8±2.1) as well as their MHC-II expression. In addition, enhanced levels of IF and serum TNF-α and IFN-γ were detected, indicating a potent systemic stimulation as well (serum IFN-γ (pg/mL): control=65.2±4.3, P25A=187.3±4.7, B25A=145.6±3.9). It should be noted that P25A was more efficient to increase those parameters when compared to B25A. We also observed changes in antigen presenting cells of mice Peyer´s patches. Higher percentages of CD11b+CD86+ as well as CD11c+CD86+ cells were detected in mice treated with P25A or B25A when compared to controls. Both treatments were equally effective to modulate antigen presenting cells (% of CD86+ in CD11c+ cells: control=72.4±3.7, P25A=88.1±4.1, B25A=85.6±3.3). P25A or B25A were also able to stimulate intestinal humoral immunity as observed by the improved levels of IF IgA (IgA (ug/mL): control=2.1±0.2, P25A=3.1±0.1, B25A=2.7±0.1) and the percentages of B220+ and CD24+ cells in Peyer´s patches (% of B220+ cells: control=18.3±2.3, P25A=32.5±3.1, B25A=23.4±2.7). Once again, the P25A treatment was more efficient to increase those parameters when compared to B25A. No significant influence of P25A or B25A was observed on T cells as demonstrated by studies of CD4+ and CD8+ cells in Peyer´s patches and spleen.The results of this work show that the UCO-25A strain, encapsulated in a planktonic state, has optimal immunomodulatory properties demonstrated by its ability to stimulate immune cells in both intestinal and systemic compartments. These findings open the possibility to develop prototype immunobiotic products using encapsulated planktonic L. rhamnosus UCO-25A intended for use in humans.Fil: Ilabaca, Alejandra. Universidad de Concepción; ChileFil: Clua, Maria Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Salva, Maria Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Garcia, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad de Concepción; Chile. Tohoku University; JapónFil: Albarracín, Leonardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Kitazawa, Haruki. Tohoku University; JapónFil: Alvarez, Gladis Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; ArgentinaFil: Garcia Cancino, Apolinaria. Universidad de Concepción; ChileFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónXII Congress of the Latin American Association of Immunology; XXIII Congress of the Mexican Society of ImmunologyCancúnMéxicoLatin American Association of ImmunologyMexican Society of Immunolog

Characterization of the immunomodulatory and anti-Helicobacter pylori properties of the human gastric isolate Lactobacillus rhamnosus UCO-25A

The ability to form biofilms and the potential immunomodulatory properties of the human gastric isolate Lactobacillus rhamnosus UCO-25A were characterized in vitro. It was demonstrated that L. rhamnosus UCO-25A is able to form biofilms on abiotic and cell surfaces, and to modulate the inflammatory response triggered by Helicobacter pylori infection in gastric epithelial cells and THP-1 macrophages. L. rhamnosus UCO-25A exhibited a substantial antiinflammatory effect in both cell lines and improved IL-10 levels produced by challenged macrophages. Additionally, UCO-25A protected AGS cells against H. pylori infection with a higher pathogen inhibition when a biofilm was formed. Given the importance of inflammation in H. pylori-mediated diseases, the differential modulation of the inflammatory response in the gastric mucosa by an autochthonous strain is an attractive alternative for improving H. pylori eradication and reducing the severity of the diseases that arise from the resulting chronic inflammation.Fil: Garcia Castillo, Valeria. Universidad de Concepción; Chile. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Marín Vega, Ana María. Tohoku University; JapónFil: Ilabaca, Alejandra. Universidad de Concepción; ChileFil: Albarracín, Leonardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología; ArgentinaFil: Marcial, Guillermo Emilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Kitazawa, Haruki. Tohoku University; JapónFil: Garcia Cancino, Apolinaria. Tohoku University; JapónFil: Villena, Julio Cesar. Tohoku University; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentin