A case of intoxication due to a highly cytotoxic Bacillus cereus strain isolated from cooked chicken

Outbreaks of Bacillus cereus infection/intoxication are not commonly reported because symptoms are often mild, and the disease is self-limiting. However, hypervirulent strains increase health risks. We report a case, which occurred in Argentina, of severe food The studied strain was highly cytotoxic, showed high ability to detach Caco-2 cells and was positive for the hblA, hblB, and hblC genes of the hbl complex, bceT, entS and ces. As it is considered that B. cereus emetic cluster evolved from a panmictic population of diarrhoeal strains, B. cereus MVL2011 could constitute an intermediate strain between diarrhoeal and emetic strains.Prepint aceptado el 14 de agosto de 2014 para su publicación

In vitro interaction between Bacillus megaterium strains and Caco-2 cells

To further our understanding of the virulence potential of Bacillus megaterium strains, cell association and invasion assays were conducted in vitro by infecting human enterocytes (Caco-2 cells) with 53 strains of this bacterium isolated from honey. Two series of experiments were performed: (i) necrosis and cell detachment assays with the supernatants of bacterial culture fi ltrates from 16-h cultures and (ii) adhesion/invasion assays in which cultured enterocytes incubated with bacteria from 3-h cultures were resuspended in Dulbecco’s modifi ed Eagle’s medium and chloramphenicol. The detachment of Caco-2 cells was evaluated by staining the cells with crystal violet. Necrosis was assessed by fl uorescence microscopy of cells labeled with propidium iodide. Association (adhesion plus invasion) was determined by plate counts and invasion in an aminoglycoside protection assay. The results showed that spent culture supernatants detached and necrotized Caco-2 cells in a strain-dependent manner. Seven out of 53 B. megaterium fi ltered culture supernatants caused complete cell detachment. Suspensions of these same bacterial strains adhered and invaded enterocytes in 2-h infection experiments. To our knowledge, this is the fi rst report on the interaction between B. megaterium and intestinal epithelial Caco-2 cells.Fil: López, Ana Claudia. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro en Investigación de Fitopatología; Argentina;Fil: Minnaard, Jessica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Centro de Investigaciones en Criotecnología de Alimentos (i); Argentina;Fil: Perez, Pablo Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Centro de Investigaciones en Criotecnología de Alimentos (i); Argentina; Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Microbiología General; Argentina;Fil: Alippi, Adriana Mónica. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro en Investigación de Fitopatología; Argentina

ALIMENTOS PROBIÓTICOS: INTERACCIÓN CON ANTICOAGULANTES ORALES

Los alimentos probióticos han adquirido relevancia debido a la gran demanda de los consumidores de alimentos saludables. En ese mismo contexto, existe una prevalencia de enfermedades crónicas para las cuales no hay cura pero sí tratamiento, y por ende hay una gran cantidad de pacientes polimedicados por diversas enfermedades crónicas (hipertensión e hipercolesterolemia son algunos ejemplos). Esto lleva a que tanto el consumidor como la industria demanden del conocimiento de la acción específica que cada microorganismo probiótico o sustancia bioactiva tiene sobre el individuo y cómo afectan los factores intrínsecos y extrínsecos del individuo al producto, así como las interacciones de los alimentos funcionales con los medicamentos. Esto es particularmente importante en medicamentos de estrecho margen terapéutico como algunos anticoagulantes, cuya dosificación es afectada por interacciones con los alimentos u otros medicamentos, variaciones genéticas del individuo y podría ser afectada por factores que alteren su solubilidad, ya que pertenecen a la clase II del Sistema de Clasificación Biofarmacéutica (SCB). Este sistema clasifica a las drogas orales según su solubilidad y permeabilidad; las drogas de la clase II son de baja solubilidad y alta permeabilidad y se conoce que las sales biliares aumentan su solubilidad.   Este plan de trabajo propone seleccionar cepas potencialmente probióticas con actividad enzimática sobre sales biliares, para luego estudiar in vitro en condiciones de simulación intestinal, el efecto que tiene el metabolismo de los ácidos biliares mediado por estas cepas sobre la solubilidad de formulaciones orales de anticoagulantes pertenecientes a la clase II del SCB (baja solubilidad y alta permeabilidad). Además, propone estudiar, en presencia de las bacterias probióticas y las drogas, la permeabilidad de monocapas intestinales, la modulación de los transportadores tipo ABC implicados y la respuesta celular (expresión y producción de citoquinas, expresión de citocromos, vías de señalización, distribución de moléculas de uniones estrechas). Finalmente, en base a los resultados obtenidos in vitro se seleccionará una combinación cepa-droga para evaluar in vivo la actividad anticoagulante y la respuesta del hospedador. Los resultados obtenidos en el plan propuesto contribuirán a la comprensión de la interacción de alimentos a base de probióticos con anticoagulantes orales, para ampliar la base del conocimiento que contribuya a establecer la posología de este tipo de medicamentos. &nbsp

Disassembly of F-actin cytoskeleton after interaction of Bacillus cereus with fully differentiated human intestinal Caco-2 cells

In the present study, the role of direct procaryote-eucaryote interactions in the virulence of Bacillus cereus was investigated. As a model of human enterocytes, differentiated Caco-2 cells were used. Infection of fully differentiated Caco-2 cells with B. cereus in the exponential phase of growth, in order to minimize the concentration of spores or sporulating microorganisms, shows that a strain-dependent cytopathic effect develops. Interestingly, addition of 3-h-old cultures of some strains resulted in complete detachment of the cultured cells after a 3-h infection whereas no such effect was found after a 3-h infection with 16-h-old cultures. Infection of enterocyte-like cells with B. cereus leads to disruption of the F-actin network and necrosis. Even though the effect of secreted factors cannot be ruled out, direct eucaryote-procaryote interaction seems to be necessary. In addition, we observed that some B. cereus strains were able to be internalized in Caco-2 cells. Our findings add a new insight into the mechanisms of virulence of B. cereus in the context of intestinal infection.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Criotecnología de Alimento

Biomodification of acenocoumarol by bifidobacteria

The increased interest of consumers in probiotic foods requires a deeper knowledge on the possible interactions with drugs, because their pharmacological properties could be modified. In this context, these studies are relevant for drugs such as acenocoumarol, whose dosage must be controlled due to, among other factors, food-drug interactions. Acenocoumarol is an oral anticoagulant with a narrow therapeutic range. The aim of the present research is to evaluate, in vitro, the effect of bifidobacteria on acenocoumarol. The drug was incubated with Bifidobacterium bifidum CIDCA 5310 or Bifidobacterium adolescentis CIDCA 5317 in MRS broth at 37°C for 24 h in anaerobic conditions. The effect of incubation with sterilized spent culture supernatants (SSCS) was also evaluated. Analysis by RP-HPLC showed that both bifidobacterial strains reduced the area of the acenocoumarol peak and two new peaks were evidenced. In addition, a decrease in the intensity of the bands at 1650, 1390 and 1110/cm was observed in the FTIR spectroscopic determinations. Moreover, a new band appeared at 1720/cm. No effect on the drug was observed when incubation was performed with SSCS. The present study showed a significant change in the concentration of the anticoagulant after incubation with bifidobacteria and results are compatible with biomodification of the drug due to enzymatic activity of bifidobacteria.Fil: Fragomeno, Melisa. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Assad, Sabrina Eliana. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Mobili, Pablo. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Peruzzo, Pablo Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Minnaard, Jessica. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Perez, Pablo Fernando. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentin

In vitro interaction between <i>Bacillus megaterium</i> strains and Caco-2 cells

To further our understanding of the virulence potential of Bacillus megaterium strains, cell association and invasion assays were conducted in vitro by infecting human enterocytes (Caco-2 cells) with 53 strains of this bacterium isolated from honey. Two series of experiments were performed: (i) necrosis and cell detachment assays with the supernatants of bacterial culture fi ltrates from 16-h cultures and (ii) adhesion/invasion assays in which cultured enterocytes incubated with bacteria from 3-h cultures were resuspended in Dulbecco’s modifi ed Eagle’s medium and chloramphenicol. The detachment of Caco-2 cells was evaluated by staining the cells with crystal violet. Necrosis was assessed by fl uorescence microscopy of cells labeled with propidium iodide. Association (adhesion plus invasion) was determined by plate counts and invasion in an aminoglycoside protection assay. The results showed that spent culture supernatants detached and necrotized Caco-2 cells in a strain-dependent manner. Seven out of 53 B. megaterium fi ltered culture supernatants caused complete cell detachment. Suspensions of these same bacterial strains adhered and invaded enterocytes in 2-h infection experiments. To our knowledge, this is the fi rst report on the interaction between B. megaterium and intestinal epithelial Caco-2 cells.Facultad de Ciencias Agrarias y Forestale

A case of intoxication due to a highly cytotoxic <i>Bacillus cereus</i> strain isolated from cooked chicken

Outbreaks of Bacillus cereus infection/intoxication are not commonly reported because symptoms are often mild, and the disease is self-limiting. However, hypervirulent strains increase health risks. We report a case, which occurred in Argentina, of severe food The studied strain was highly cytotoxic, showed high ability to detach Caco-2 cells and was positive for the hblA, hblB, and hblC genes of the hbl complex, bceT, entS and ces. As it is considered that B. cereus emetic cluster evolved from a panmictic population of diarrhoeal strains, B. cereus MVL2011 could constitute an intermediate strain between diarrhoeal and emetic strains.Facultad de Ciencias Agrarias y Forestale

Surface properties of bifidobacterial strains of human origin

The adherence of Bifidobacterium strains isolated from infant feces and commercial fermented dairy products to enterocyte-like cells was correlated with the autoagglutination and hemagglutination properties of these organisms. These results allowed us to define two groups: (i) cell-adherent bacteria showing hemagglutination and autoagglutination and (ii) non-cell-adherent, nonhemagglutinating, nonautoagglutinating bacteria. Glass adherence was shown to be nonspecific and was discarded as a criterion for selection of adherent cells. Hydrophobicity appeared to be necessary for adhesion to enterocyte-like cells and autoagglutination. Adhesive strains were highly hydrophobic, and the degree of adherence was slightly dependent on the surface potential. Cells autoagglutinated more when the electrostatic negative charges on the cell surface were shielded by a decrease in the pH from 7 to 2. However, in some strains negative charges at the cell surface were adjuvant to adhesion, thus suggesting that specific chemical interactions occurred. The present results provide a method for preliminary selection of bacteria potentially adherent to epithelial cells by means of autoagglutination.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Criotecnología de Alimento

Disassembly of F-actin cytoskeleton after interaction of Bacillus cereus with fully differentiated human intestinal Caco-2 cells

In the present study, the role of direct procaryote-eucaryote interactions in the virulence of Bacillus cereus was investigated. As a model of human enterocytes, differentiated Caco-2 cells were used. Infection of fully differentiated Caco-2 cells with B. cereus in the exponential phase of growth, in order to minimize the concentration of spores or sporulating microorganisms, shows that a strain-dependent cytopathic effect develops. Interestingly, addition of 3-h-old cultures of some strains resulted in complete detachment of the cultured cells after a 3-h infection whereas no such effect was found after a 3-h infection with 16-h-old cultures. Infection of enterocyte-like cells with B. cereus leads to disruption of the F-actin network and necrosis. Even though the effect of secreted factors cannot be ruled out, direct eucaryote-procaryote interaction seems to be necessary. In addition, we observed that some B. cereus strains were able to be internalized in Caco-2 cells. Our findings add a new insight into the mechanisms of virulence of B. cereus in the context of intestinal infection.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Criotecnología de Alimento