Repeated Exposure of Aspergillus niger Spores to the Antifungal Bacterium Collimonas fungivorans Ter331 Selects for Delayed Spore Germination.

The bacterial strain Collimonas fungivorans Ter331 (CfTer331) inhibits mycelial growth and spore germination in Aspergillus niger N402 (AnN402). The mechanisms underlying this antagonistic bacterial-fungal interaction have been extensively studied, but knowledge on the long-term outcome of this interaction is currently lacking. Here, we used experimental evolution to explore the dynamics of fungal adaptation to recurrent exposure to CfTer331. Specifically, five single-spore isolates (SSIs) of AnN402 were evolved under three selection scenarios in liquid culture, i.e., (i) in the presence of CfTer331 for 80 growth cycles, (ii) in the absence of the bacterium for 80 cycles, and (iii) in the presence of CfTer331 for 40 cycles and then in its absence for 40 cycles. The evolved SSI lineages were then evaluated for phenotypic changes from the founder fungal strain, such as germinability with or without CfTer331. The analysis showed that recurrent exposure to CfTer331 selected for fungal lineages with reduced germinability and slower germination, even in the absence of CfTer331. In contrast, when AnN402 evolved in the absence of the bacteria, lineages with increased germinability and faster germination were favored. SSIs that were first evolved in the presence of CfTer331 and then in its absence showed intermediate phenotypes but overall were more similar to SSIs that evolved in the absence of CfTer331 for 80 cycles. This suggests that traits acquired from exposure to CfTer331 were reversible upon removal of the selection pressure. Overall, our study provides insights into the effects on fungi from the long-term coculture with bacteria. IMPORTANCE The use of antagonistic bacteria for managing fungal diseases is becoming increasingly popular, and thus there is a need to understand the implications of their long-term use against fungi. Most efforts have so far focused on characterizing the antifungal properties and mode of action of the bacterial antagonists, but the possible outcomes of the persisting interaction between antagonistic bacteria and fungi are not well understood. In this study, we used experimental evolution in order to explore the evolutionary aspects of an antagonistic bacterial-fungal interaction, using the antifungal bacterium Collimonas fungivorans and the fungus Aspergillus niger as a model system. We show that evolution in the presence or absence of the bacteria selects for fungal lineages with opposing and conditionally beneficial traits, such as slow and fast spore germination, respectively. Overall, our studies reveal that fungal responses to biotic factors related to antagonism could be to some extent predictable and reversible.

Targeted Delivery of Gene Silencing in Fungi Using Genetically Engineered Bacteria.

Exploiting RNA interference (RNAi) in disease control through non-transformative methods that overcome the hurdle of producing transgenic plants has attracted much attention over the last years. Here, we explored such a method and used non-pathogenic bacteria as a versatile system for delivering RNAi to fungi. Specifically, the RNaseIII-null mutant strain of Escherichia coli HT115(DE3) was transformed with two plasmid vectors that enabled the constitutive or IPTG-inducible production of double-stranded RNAs (dsRNAs) against genes involved in aflatoxins production in Aspergillus flavus (AflC) or virulence of Botrytis cinerea (BcSAS1). To facilitate the release of the dsRNAs, the bacterial cells were further genetically engineered to undergo a bacteriophage endolysin R-mediated autolysis, following a freeze-thaw cycle. Exposure under in vitro conditions of A. flavus or B. cinerea to living bacteria or their whole-cell autolysates induced silencing of AflC and BcSAS1 in a bacteria concentration-dependent manner, and instigated a reduction in aflatoxins production and mycelial growth, respectively. In planta applications of the living bacteria or their crude whole-cell autolysates produced similar results, thus creating a basis for translational research. These results demonstrate that bacteria can produce biologically active dsRNA against target genes in fungi and that bacteria-mediated RNAi can be used to control fungal pathogens.

Interruption of Aspergillus niger spore germination by the bacterially produced secondary metabolite collimomycin.

Collimonas fungivorans Ter331 (CfTer331) is a soil bacterium that produces collimomycin, a secondary metabolite that inhibits the vegetative growth of fungi. Here we show that CfTer331 can also interfere with fungal spore germination and that collimomycin biosynthesis is required for this activity. More specifically, in co-cultures of Aspergillus niger N402 (AnN402) co-nidiospores with CfTer331, the rate of transition from the isotropic to polarized stage of the germination process was reduced and the relatively few AnN402 conidiospores that completed the germination process were less likely to survive than those that were arrested in the isotropic phase. By contrast, a collimomycin-deficient mutant of CfTer331 had no effect on germination: in its presence, as in the absence or delayed presence of CfTer331, unhindered germination of conidiospores allowed rapid establishment of AnN402 mycelium and the subsequent acidification of the culture medium to the detriment of any bacteria present. However, when challenged early enough with CfTer331, the collimomycin-dependent arrest of the AnN402 germination process enabled CfTer331 to prevent AnN402 from forming mycelia and to gain dominance in the culture. We propose that the collimomycin-dependent arrest of spore germination represents an early intervention strategy used by CfTer331 to mitigate niche construction by fungi in nature.

Cloning of the Cytochrome b Gene From the Tomato Powdery Mildew Fungus Leveillula taurica Reveals High Levels of Allelic Variation and Heteroplasmy for the G143A Mutation.

Leveillula taurica is a major pathogen of tomato and several other crops that can cause substantial yield losses in favorable conditions for the fungus. Quinone outside inhibitor fungicides (QoIs) are routinely used for the control of the pathogen in tomato fields across California, but their recurrent use could lead to the emergence of resistance against these compounds. Here, we partially cloned the cytochrome b gene from L. taurica (Lt cytb) and searched within populations of the fungus collected from tomato fields across California for mutations that confer resistance to QoIs. A total of 21 single nucleotide polymorphisms (SNPs) were identified within a 704 bp fragment of the Lt cytb gene analyzed, of which five were non-synonymous substitutions. Among the most frequent SNPs encountered within field populations of the pathogen was the G143A substitution that confers high levels of resistance against QoIs in several fungi. The other four amino acid substitutions were novel mutations, whose effect on QoI resistance is currently unknown. Sequencing of the Lt cytb gene from individual single-cell conidia of the fungus further revealed that most SNPs, including the one leading to the G143A substitution, were present in a heteroplasmic state, indicating the co-existence of multiple mitotypes in single cells. Analysis of the field samples showed that the G143A substitution is predominantly heteroplasmic also within field populations of L. taurica in California, suggesting that QoI resistance in this fungus is likely to be quantitative rather than qualitative.

Factores asociados a la calidad de vida en mujeres con cáncer de mama. Medellín 2013 / Factors Associated with Quality of Life in Women with Breast Cancer. Medellin 2013 / Fatores associados a qualidade de vida em mulheres com cancro de mama. Medellín 2013

Resumen Introducción: el cáncer de mama es un problema de salud pública que genera diversos factores y sentimientos que afectan la calidad de vida. Objetivo: evaluar los factores asociados a la calidad de vida en pacientes con cáncer de mama. Métodos: estudio descriptivo de corte transversal que incluyó 82 pacientes. Se calculó la calidad de vida con WHQOL-BREF y los factores asociados se determinaron con un modelo de regresión logística binaria. Resultados: el 73.1 % calificó la calidad de vida mínimo como buena. El dominio físico fue el más afectado (65.1 ± 15.9), en tanto que el entorno fue el menos afectado (78.2 ± 14.2). Las variables que explican el deterioro de la calidad de vida fueron: riesgo edad <50 años, diagnóstico de cáncer avanzado al ingreso a la administradora de planes de beneficio (EAPB), mientras que los protectores fueron la satisfacción con el servicio y la terapia hormonal. Conclusión: es necesario enfatizar y tener mejor satisfacción con el servicio en mujeres menores de cincuenta años y que ingresaron con diagnóstico avanzado al programa.

Abstract Introduction: Breast cancer is a public health problem that generates various factors and feelings which affect the quality of life. Objective: to evaluate factors associated with quality of life in breast cancer patients. Methods: Descriptive cross-sectional study involving 82 patients. Quality of life was calculated with WHQOL-BREF and the associated factors were determined using a binary logistic regression model. Results: 73.1% rated quality of life at least as good. The physical sphere was the most affected (65.1 ± 15.9), while the environment was the least affected (78.2 ± 14.2). The variables that explain the deterioration of the quality of life were: age <50 years, diagnosis of advanced cancer upon admission to the benefit plan management (EAPB), while protective variables were satisfaction with the service and hormonal therapy. Conclusion: it becomes necessary to emphasize and to create a better satisfaction with the service in women under fifty and those who were admitted to the program with an advanced diagnosis.

Resumo Introdução: cancro de mama é um problema de saúde pública que gera diversos fatores e sentimentos que afetam a qualidade de vida. Objetivo: avaliar os fatores associados à qualidade de vida em pacientes com cancro de mama. Métodos: estudo descritivo de corte transversal que incluiu 82 pacientes. A qualidade de vida com WHQOL-BREF foi calculado e os fatores associados determinaram-se com um modelo de regressão logística binária. Resultados: o 73.1 % classificou a qualidade de vida como boa mínimo. O domínio físico foi o mais afetado (65.1 ± 15.9), enquanto o entorno foi o menos afetado (78.2 ± 14.2). As variáveis que explicam a deterioração da qualidade de vida foram: risco idade <50 anos, diagnóstico de câncer avançado ao ingresso à administradora de planos de benefício (EAPB), entanto os protetores foram satisfação com o serviço e a terapia hormonal. Conclusão: é preciso remarcar e ter melhor satisfação com o serviço em mulheres com menos de cinquenta anos e que ingressaram com diagnóstico avançado no programa.

Estudio piloto sobre la implementación de un programa lúdico en el contexto escolar para la estimulación del lenguaje / Pilot study of the implementation of an educational play-based program for language stimulation in the school setting

En el presente estudio se ha diseñado y aplicado un programa lúdico-educativo con el fin de estimular el lenguaje de niños de último curso de educación infantil con y sin dificultad específica. Este programa pretende responder a la necesidad de herramientas en el aula que, de forma atractiva y dinámica, actúen sobre las dificultades que pueden aparecer durante el desarrollo del lenguaje oral de los niños. Para ello, se empleó un diseño pre-post con una muestra de 25 alumnos de tercer curso de educación infantil. Las evaluaciones se realizaron a través de un registro de observación del lenguaje y de la Prueba de lenguaje oral de Navarra revisada. Las actividades del programa lúdico están basadas en el juego dirigido y se centran en aspectos como la atención, el reconocimiento visual, la lateralidad, el ritmo y la fluidez del habla, la discriminación auditiva, etc. Su duración ha sido de un mes, con 2 sesiones semanales de 45 min. Los resultados reflejan diferencias estadísticamente significativas en la mayoría de las variables objeto de intervención. Si bien se trata de un estudio piloto, los resultados parecen apoyar la utilidad de intervenciones breves de tipo lúdico-educativo aplicadas a nivel grupal en el contexto escolar para la estimulación del lenguaje (AU)

In the present study, an educational play-based program was designed and implemented to stimulate the language of children with or without specific difficulties in the last year of primary education. This program aimed to respond to the need for dynamic tools in the classroom to correct difficulties that may arise during the development of children's oral language. To do this, a pre-post design was used with a sample of 25 students in their third year of primary education. Evaluations were performed by the Revised Navarre oral language test and an observational procedure specifically designed for this study. The program's activities were based on directed play and focused on aspects such as attention, visual recognition, laterality, rhythm and fluency of speech, auditory discrimination, etc. The program lasted for 1 month with two weekly sessions of 45 min each.Statistically significant differences were found in most of the variables. Although this was a pilot study, the results seem to support the usefulness of brief interventions for language stimulation applied at the group level in schools (AU)

Cervicometría menor o igual a 25 mm para identificar parto a los siete días en pacientes con amenaza de parto prematuro. Estudio de cohorte retrospectivo en un hospital de cuarto nivel de complejidad en Medellín, Colombia, 2009-2012 / Cervical length measurement of 25 mm or less than for delivery identification within seven days in patients under threat of premature birth. Retrospective cohort study in a level IV hospital in Medellín, Colombia, 2009-2012

Objetivo: identificar si en mujeres con diagnóstico de amenaza de parto pretérmino y cervicometría menor o igual a 25 mm existe un punto de corte que permita mejorar la predicción del parto en los 7 días posteriores a la prueba.Materiales y métodos: estudio de cohorte retrospectivo en pacientes con embarazo único y edad gestacional entre 24 y 36+6 semanas con diagnóstico de amenaza de parto prematuro y cervicometría de 25 mm o menos al ingreso. Se excluyeron pacientes en trabajo de parto activo, con ruptura prematura de membranas, con historia de cerclaje o de tratamientos previos con progesterona. Se categorizó la medición en rangos de 0 a 5, 6 a 10, 11 a 15, 16 a 20 y 21 a 25 mm y se comparó la predicción de parto con la presencia o no de parto a los 7 días posprueba. Se estimó el cociente de probabilidades por categoría y el área bajo la curva.Resultados: la mediana del periodo de latencia al parto fue de 3 semanas (rango 1 a 5). Los cocientes de probabilidad para tener un parto antes de 7 días de realizada la medición fueron, para los diferentes punto de corte, de 3,3; 1,4; 1,3; 0,7 y 0,2, respectivamente. El área bajo la curva ROC para parto a los 7 días fue de 0,711.Conclusión: en mujeres con cérvix menor de 25 mm y APP no se encontró un punto de corte que permita diferenciar el mayor o menor riesgo de tener parto prematuro.

Objective: To identify a cut-off point in women under threat of preterm birth with cervical length of 25 mm or less in order to improve delivery prediction within 7 days of the test.Materials and methods: Retrospective cohort study in women with a single pregnancy between 24 and 36+6 weeks of gestation, diagnosed with threat of premature delivery and cervical length of 25 mm or less on admission. Patients in active labour or with premature membrane rupture, a history of cerclage or prior treatment with progesterone were excluded. Measurements were divided into range categories: 0 to 5, 6 to 10, 11 to 15, 16 to 20 and 21 to 25 mm, and birth prediction was compared with the presence or absence of birth within 7 days of the test. The likelihood ratio for each category and the area under the curve were estimated.Results: The median latency period for delivery was 3 weeks (range 1-5). For the different cut-off points, the probability quotients for going into labour before seven days after the measurement were 3.3, 1.4, 1,3, 0.7 and 0.2, respectively. The area under the curve (AUC) for delivery at seven days was 0.711.Conclusion: In women with cervical length under 25 mm and with a threat of preterm delivery, no cut-off point was found to help identify a greater or lesser risk of premature delivery.

Cultivable bacteria populations associated with leaves of banana and plantain plants and their antagonistic activity against Mycosphaerella fijiensis.

Mycosphaerella fijiensis is the etiological agent of Black Sigatoka, a fungal disease that affects production of banana and plantain crops in tropical regions. The sizes of cultivable epiphytic and endophytic bacterial populations, aerobic endospore forming bacteria (AEFB), and antagonist bacteria against M. fijiensis isolated from three Musa spp. cultivars from Urabá (Colombia) were studied, in order to find a suitable screening strategy to isolate antagonistic bacteria. Most of the variability found in the epiphytic and endophytic bacterial community sizes among fruit trees was explained by the cultivar differences. We found population sizes ranging from 1.25 × 10(3) to 9.64 × 10(5) CFU/g of fresh leaf and found that 44 % of total cultivable bacteria belong to the AEFB group. We isolated 648 AEFB from three different cultivars and assessed their antagonistic activity against M. fijiensis using the cell-free supernatant obtained from bacterial liquid cultures in three different in vitro assays. Five percent of those bacteria showed higher percent inhibition than the positive control Bacillus subtilis UA321 has (percent inhibition = 84 ± 5) in the screening phase. Therefore, they were selected as antagonistic bacteria against the pathogen. The strains with the highest percentage of antagonism were found in older leaves for the three cultivars, given support to recommend this group of leaves for future samplings. Some of these isolated bacteria affected the mycelium and ascospores morphology of the fungus. They also presented in vitro characteristics related to a successful colonization of the phylloplane such as indolic compounds, surfactant production, and biofilm formation, which makes them possible, potential candidates as biological control agents.