Plant surfaces as vehicles of Bacillus cereus responsible of human food poisoning

• Introduction A major concern in food safety is the contamination of fresh and stored food with spoiling bacteria that provoke human poisoning. Bacillus cereus is a common food-borne pathogen responsible of important poisoning outbreaks and severe bacteraemia and septicaemia. Poisoning caused by B. cereus is classified in two main categories: emetic and diarrheic. The emetic poisoning is correlated to the production of cereulide. This toxin is very heat stable, and it can be produced in the food contaminated by B. cereus cells. Diarrheic poisoning is provoked by the enterotoxin hemolysin BL, the non-hemolytic enterotoxin and the cytotoxin K. • Objective To study the interaction of B. cereus with plants as a bacteria reservoir, and in ready-to-eat fruits and vegetables. • Materials & Methods A collection of strains implicated in food-borne outbreaks were tested in vitro for a battery of phenotypes related to bacterial multicellular behaviour and thus interaction with host. 1. Solid or liquid media were used to study biofilm formation, motility or adhesion to surfaces. 2. Leaves, fruits and vegetables (melon leaf, cucumber leaf and fruit and endive) were used to study the persistence of B. cereus over time and their distribution and organization by electron microscopy. • Results All the strains behaved similarly in vitro, only some persisted on plant surfaces. Among them, the emetic strain AH187 was selected because bacterial cells persisted on a concentration of 104-105 CFU per gram of leaf, vegetable or fruit, with a sporulation rate of 40%. The electron microscopy images showed the organization of bacteria in well-developed biofilms with visible extracellular matrix. Finally, mass spectrometry analysis proved the presence of some isoforms of cereluide on the different surfaces. • Conclusion The fact that cells of B. cereus persist in leaf surface mainly as vegetative cells are indicative of their ability to adapt to the physico-chemical changeable phyllosphere, and thus to produce the emetic toxin cereulide. The presence of spores, and the formation of biofilms can be indicative of the versatile adhesive properties of this strain to diverse surfaces. Altogether are supportive of the importance of plant surfaces either as reservoir of bacterial cells or as vehicles for further contamination and food poisoning.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Nuevos datos sobre la floración, fructificación y germinación de fanerógamas marinas en Andalucía

Nuevos datos sobre la floración, fructificación y germinación de fanerógamas marinas en Andalucía. Se aportan todos los datos conocidos sobre las floraciones y fructificaciones de las distintas fanerógamas marinas presentes en las provincias de Almería y Granada. Se presentan datos sobre las floraciones de Posidonia oceanica (L.) Delile detectadas en la provincia de Almería en los últimos años, y un caso de floración en la provincia de Granada, así como las fructificaciones y frutos maduros de esta especie en tres años consecutivos (2001/02, 2002/03 y 2003/04), lo que nunca antes se había observado en las costas andaluzas. Se estudia la evolución del tamaño de los frutos a lo largo del desarrollo en P. oceanica, así como la presencia de los frutos en las playas, el número de frutos por infrutescencia, cuántos aparecen con dehiscencia, y el porcentaje de frutos parcialmente o totalmente depredados. Se detecta en Almería (y por primera vez en Andalucía) la germinación natural de semillas de P. oceanica y se estudia el desarrollo de las plántulas en acuario, se obtienen medidas precisas de los frutos y sus semillas, y se comparan con los de otros puntos del Mediterráneo. Se aportan también datos sobre las floraciones y fructificaciones de Cymodocea nodosa (Ucria) Ascher. en Almería; sobre la fructificación de Zostera marina L. en Almería, Granada y también en Málaga; sobre la floración de Zostera noltii Horn. en Almería; y sobre la floración y fructificación de Ruppia maritima L. var. maritima en lagunas litorales y salinas de Almería

Extracellular matrix components are required to protect bacillus subtilis from pseudomonas invasion and co-colonization of plants

Plants are colonized by a vast variety of microbes. Among them, bacteria are the most predominant and are able to adapt to environmental changes and interact with other microorganisms using a wide array of molecules, metabolic plasticity and secretion systems. One way bacteria have evolved to succeed in this competitive scenario is the formation of biofilms which provides protection to the cells, modulates the flux of signals and controls cellular differentiation. Thus, efforts are encouraged to really determine the functionality of the bacterial extracellular matrix. In this study, we have employed microbiological and microscopic techniques to study the interaction between Bacillus subtilis 3610 and Pseudomonas chlororaphis PCL1606. We demonstrate the important role of the extracellular matrix in protecting B. subtilis colonies from infiltration by Pseudomonas. Furthermore, time-lapse confocal laser scanning microscopy (CLSM) analyses of the bacterial interactions have permitted to complete the study of the bacterial behaviors and to measure bacterial expansion rates. Surprising, we find that the Pseudomonas type VI secretion system (T6SS) is required in the cell-to-cell contact with matrix-impaired B. subtilis cells, revealing a novel role for T6SS against Gram-positive bacteria. In response to P. chlororaphis infiltration, we find that B. subtilis activates sporulation and expresses motility-related genes. Confocal microscopy of the bacterial interactions using plant organs highlights the functional importance of these different bacterial strategies in their coexistence as stable bacterial communities. The findings further our understanding of the functional role played by biofilms in mediating bacterial social interactions.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Nuevos datos sobre la distribución de las fanerógamas marinas en las provincias de Almería y Granada (SE España)

Nuevos datos sobre la distribución de las fanerógamas marinas en las provincias de Almería y Granada (SE España). Se estudia la distribución de las distintas especies de fanerógamas marinas en el litoral de las provincias de Almería y Granada. Se detallan las áreas cubiertas por praderas de Posidonia oceanica (L.) Delile en la provincia de Almería y las localidades donde todavía se encuentran manchas de esta especie en la provincia de Granada. Se aportan datos sobre la distribución en la zona de Cymodocea nodosa (Ucria)Ascherson, así como nuevos y numerosos datos sobre la presencia en el área de estudio de Zostera marina L. y Zostera noltii Hornemann. Se relaciona la distribución de las distintas especies de fanerógamas con el frente Almería-Orán, como límite biogeográfico difícil de franquear en la distribución de la mayoría de ellas, y se revisa el estado de conservación de las praderas dentro de los espacios naturales protegidos que existen en la zona

Analysis of the molecular machinery implicated in multicellularity in bacillus cereus

Introduction: Bacillus cereus is a Gram-positive bacterium usually implicated in food poisoning outbreaks and human infections that sometimes result fatal. These events are closely related to the assembly of a biofilm that serves as a reservoir of cells, a nest for sporulation and protection from environmental stresses, host defenses or chemotherapy. Objectives: To perform a comprehensive comparative study of biofilm and planktonic cells to: i) delineate the molecular machinery implicated in the different steps of the biofilm life cycle, and ii) define new genes dedicated to biofilm formation. Materials & methods: Bacteria were grown under biofilm inducing conditions. Biofilm cells were separated from planktonic cells at different times and their whole mRNA was isolated, sequenced and analyzed. Results: Our results reveal a high number of genes associated to biofilm, many of them with unknown function, but highly conserved in others bacterial species. Besides, we found global changes in cell wall synthesis, metabolism and interspecies interaction molecules. ​ Conclusions: The interaction of B. cereus with other bacteria is conditioned by secondary metabolites, which are apparently overexpressed in biofilm. On the other hand, toxins are mainly expressed in planktonic cells, which are more oriented to interact with its hosts. These results reveal the defense and attacking positions of B. cereus in biofilm vs planktonic states.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

A short version of the amyloid-like protein TasA fibrillates and supports biofilm formation in Bacillus cereus

The formation of bacterial biofilms is a doable thanks to the assembly of an extracellular matrix that provides to the entire community with i) an outstanding architectonic structure and ii) protection to the cells from external aggressions. In Bacillus subtilis, a structural element dedicated to the formation of the extracellular matrix is the amyloid-like protein TasA. To form fibers, TasA needs the participation of the protein TapA. Indeed, a tapA mutant resembles phenotypically to a tasA mutant, which is wrinkle-less pellicles or colonies with no distinguishable morphological features. tasA is widely spread within the Bacillus genus, but tapA is absent in the heterogeneous group of Bacillus cereus which includes environmental and pathogenic members; some of them are responsible for important food intoxication outbreaks. Then, we asked whether TasA would still retain functionality in biofilm formation in B. cereus. Comparative genomic analysis showed a region in B. cereus containing two orthologous of tasA, tasA and calY, and the orthologous of sipW, that encodes a signal peptidase. Our mutagenic studies revealed that the entire region was relevant for biofilm formation, and electron microscopy proved the major propensity of TasA than CalY to form fibers in the cell surfaces. These findings also indicated that in B. cereus as opposed to B. subtilis, an accessory TapA protein is not necessary for the fibrillation of TasA. Indeed, the heterologous expression of this region of B. cereus restored the capacity of a B. subtilis tasA operon mutant or a single tasA mutant to form pellicles. These pellicles stained with the amyloid dye Congo Red and the cells were decorated with fibers, both findings suggestive of an amyloid-like nature of the B. cereus TasA. Intriguingly, in a B. subtilis tapA mutant, only the entire region of B. cereus fully rescued pellicle formation, fibrillation or Congo Red staining, to a lesser extent did sipw-tasA, and no restoration was observed with sipW-calY. These observations led us to speculate that TapA might cross seed the fibrillation of TasA or CalY in B. subtilis. In summary, TasA is relevant for biofilm formation in these two bacterial species, which appears to be governed by its polymerizing nature. The fact that we count with two bacterial species containing versions of TasA with subtle differences will be of great value in our studies of the mechanistic of polymerization of these bacterial amyloid-like fibers and their contribution to the assembly of the extracellular matrix.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Compounds produced by two robust Bacillus amyloliquefaciens biocontrol strains involved in antimicrobial activity and plant-growth promotion

Several members of the Bacillus genus are potential candidates to be used as biological control agents to combat pests or plant diseases. The bacterial attributes associated to Bacillus behaviour are mainly: the production of antimicrobial compounds, the plant-growth promotion capability and the induction of systemic resistance in plant host. In previous works, we have demonstrated this multifaceted biocontrol activity of B. amyloliquefaciens CECT8237 (UMAF6639) and CECT8238 (UMAF6614) strains, which contributes to plant protection against bacterial and fungal pathogens. In order to identify the bacterial features responsible for the outstanding biocontrol activity of these strains, their genomes were sequenced and analysed. Firstly, those features previously described for other Bacillus to be involved in the biocontrol activity were localized: i) Biosynthetic genes of secondary metabolites. Apart from the lipopeptides, formerly detected, we have demonstrated the production of other additional compounds that might participate in the antibiosis activity; ii) Biosynthetic genes of the volatile compounds 2,3-butanediol and acetoin, both involved in the induction of plant defence responses. Secondly, genetic singularities non-conserved within the Bacillus genus, which might contribute to the biocontrol ability of B. amyloliquefaciens CECT8237 and CECT8238 were identified in both genomes. Among them, we highlight two genomic regions hypothetically implicated in the production of non-characterized secondary metabolites. Ongoing studies are focused on elucidating the functionality of these uncharacterized regions, leading to a better understanding of the mechanisms of action involved in the robust biocontrol skills of these strains.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Elaboración del plan de mantenimiento para la línea de producción del ladrillo en la empresa ladrillera Arcillas LTDA

La elaboración del presente plan de mantenimiento preventivo para la empresa Ladrillera Arcillas LTDA. se desarrolla con el fin de que los operarios técnicos mecánicos puedan encontrar y prevenir fallas tempranas o inesperadas por medio de una lista de actividades datadas en un cronograma (Tablero de Control), de esta manera asegurar el correcto funcionamiento y cumplir objetivo por el cual han sido diseñadas las máquinas..

The expression profile of secondary metabolites in biofilms of B. amyloliquefaciens CECT 8237 biocontrol strain

The contribution of Bacillus amyloliquefaciens CECT 8237 (UMAF6639) strain to the plant protection against bacterial and fungal pathogens is mainly based on: i) the production of antimicrobial compounds, ii) the plant-growth promotion capability and iii) the induction of systemic resistance in plant host. In previous works, we demonstrated the relevant implication of the three families of lipopeptides in the biocontrol activity and biofilm formation on melon leaves. The analysis of the genome sequence revealed features previously identified in other Bacillus strains, such as genes related to biofilm formation, phytostimulation and induction of systemic resistance in the host plant, and novel genomic regions non-conserved within the Bacillus genus, and therefore with potential genes implicated in the biocontrol activity. Considering the relevance of biofilm formation and production of secondary metabolites in biocontrol, we analyzed the expression profile of several secondary metabolites produced by CECT 8237 in biofilm inducing conditions. To do so, we optimized an in situ detection method based on MALDI-TOF analysis of secondary metabolites within the bacterial colony and in supernatants and pellicles of B. amyloliquefaciens biofilms. We found a major accumulation of these secondary metabolites in the core and middle area of the colony and in the spent medium compared to pellicle. Further studies will help elucidating the real implication of these molecules in the bacterial ecology or in its mechanisms of defence, against competitors, and/or offense, against pathogens and its possible relation with the niche they occupy. This work was supported by grants from the Plan Nacional I+D+I (AGL-2012-31968) and Incentivos a Proyectos de Excelencia de la Junta de Andalucía (P10-AGR-57-97), both cofinanced by FEDER funds (EU). This work was also supported by grant from Koppert B.V. (8.06/60.4086) and European Research Council-Starting Grant BacBio ERC637971.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Estudio de la interacción de Bacillus cereus responsable de intoxicaciones eméticas en humanos con la superficie de hojas y frutos.

Bacillus cereus es una bacteria patógena de humanos que comúnmente se transmite por la ingesta de alimentos contaminados causando importantes brotes de intoxicación alimentaria. La persistencia y colonización de esta bacteria en frutos y vegetales supone un grave problema en la industria médica y alimentaria. En este trabajo, se ha estudiado el comportamiento de diferentes aislados procedentes de intoxicaciones alimentarias sobre la superficie de frutos y hojas de plantas como posibles vehículos de estos microorganismos a través de los cuales se producirían las toxiinfecciones alimentarias. Todos los aislados fueron inicialmente estudiados in vitro para una batería de fenotipos relacionados con el comportamiento multicelular bacteriano: Formación de biofilm, movilidad swarming/sliding o adhesión a superficie. De entre todos los aislados se seleccionaron aquellos con características distintivas (morfología de colonia, fuerte adhesión a las paredes de pocillos, o formación de película en la interfase aire-líquido) para estudios de interacción con plantas. En general no observamos una correlación entre el comportamiento in vitro y los resultados en planta, por lo que decidimos seleccionar una cepa emética por varios motivos: i) produce la toxina emética cereulide, y una enterotoxina no hemolítica, siendo por tanto de gran interés en seguridad alimentaria, ii) en las distintas plantas ensayadas se mantuvo a concentraciones de 105 UFC por gramo de hoja inoculada de los que un 40% apareció en la forma de esporas y iii) es una cepa manipulable genéticamente. Uno de los resultados más interesantes fue la aparición de un mutante espontáneo en la fracción más externa de la una colonia de la cepa emética crecida en medio de movilidad swarming. Este mutante daba lugar a una colonia con morfología totalmente diferente y mayor capacidad de movilidad. En los ensayos en planta, el mutante se comportó como el silvestre en cuanto a persistencia, formación de biofilm, o esporulación. La comparativa de los genomas sin embargo mostró la pérdida masiva de elementos transponibles, así como otros locus no caracterizados previamente. Con todos estos datos en nuestras manos nos encontramos posicionados para comprender que herramientas, conocidas o aún por conocer, utiliza esta cepa para interaccionar con la planta, y de qué forma coordina su persistencia con una eventual producción de toxinas que tienen como diana al hombre. Este trabajo está financiado por European Research Council-Starting Grant-2014 (8.06 UE/60.8003).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech