A comprehensive metatranscriptome analysis pipeline and its validation using human small intestine microbiota datasets

Background Next generation sequencing (NGS) technologies can be applied in complex microbial ecosystems for metatranscriptome analysis by employing direct cDNA sequencing, which is known as RNA sequencing (RNA-seq). RNA-seq generates large datasets of great complexity, the comprehensive interpretation of which requires a reliable bioinformatic pipeline. In this study, we focus on the development of such a metatranscriptome pipeline, which we validate using Illumina RNA-seq datasets derived from the small intestine microbiota of two individuals with an ileostomy. Results The metatranscriptome pipeline developed here enabled effective removal of rRNA derived sequences, followed by confident assignment of the predicted function and taxonomic origin of the mRNA reads. Phylogenetic analysis of the small intestine metatranscriptome datasets revealed a strong similarity with the community composition profiles obtained from 16S rDNA and rRNA pyrosequencing, indicating considerable congruency between community composition (rDNA), and the taxonomic distribution of overall (rRNA) and specific (mRNA) activity among its microbial members. Reproducibility of the metatranscriptome sequencing approach was established by independent duplicate experiments. In addition, comparison of metatranscriptome analysis employing single- or paired-end sequencing methods indicated that the latter approach does not provide improved functional or phylogenetic insights. Metatranscriptome functional-mapping allowed the analysis of global, and genus specific activity of the microbiota, and illustrated the potential of these approaches to unravel syntrophic interactions in microbial ecosystems. Conclusions A reliable pipeline for metatransciptome data analysis was developed and evaluated using RNA-seq datasets obtained for the human small intestine microbiota. The set-up of the pipeline is very generic and can be applied for (bacterial) metatranscriptome analysis in any chosen niche

Effect of nickel and cobalt on methanogenic enrichment cultures and role of biogenic sulfide in metal toxicity attenuation

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2017.01341/full#supplementary-materialMetals play an important role in microbial metabolism by acting as cofactors for many enzymes. Supplementation of biological processes with metals may result in improved performance, but high metal concentrations are often toxic to microorganisms. In this work, methanogenic enrichment cultures growing on H2/CO2 or acetate were supplemented with trace concentrations of nickel (Ni) and cobalt (Co), but no significant increase in methane production was observed in most of the tested conditions. However, high concentrations of these metals were detrimental to methanogenic activity of the cultures. Cumulative methane production (after 6 days of incubation) from H2/CO2 was 40% lower in the presence of 8 mM of Ni or 30 mM of Co, compared to controls without metal supplementation. When acetate was used as substrate, cumulative methane production was also reduced: by 18% with 8 mM of Ni and by 53% with 30 mM of Co (after 6 days of incubation). Metal precipitation with sulphide was further tested as a possible method to alleviate metal toxicity. Anaerobic sludge was incubated with Co (30 mM) and Ni (8 mM) in the presence of sulphate or sulphide. The addition of sulphide helped to mitigate the toxic effect of the metals. Methane production from H2/CO2 was negatively affected in the presence of sulphate, possibly due to competition of hydrogenotrophic methanogens by sulphate-reducing bacteria. However, in the enrichment cultures growing on acetate, biogenically produced sulphide had a positive effect and more methane was produced in these incubations than in similar assays without sulphate addition. The outcome of competition between methanogens and sulphate-reducing bacteria is a determinant factor for the success of using biogenic sulphide as detoxification method.The research was financially supported by the People Program (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007-2013 under REA agreement 289193. Research of AS and DS is supported by a ERC grant (project 323009) of the European Union Seventh Framework Program FP7 and a Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO).info:eu-repo/semantics/publishedVersio

Carbono y nitrógeno recalcitrante en un suelo con dos sistemas de labranza: efectos de largo plazo

Los cambios de las formas más activas de la materia orgánica (MO) a formas intermedias o pasivas favorece el secuestro de carbono (C) en los suelos. Los objetivos del trabajo fueron i) evaluar los cambios de largo plazo en las fracciones recalcitrantes de C y nitrógeno (N) y ii) calcular el índice de recalcitrancia (IR) de estas fracciones en un suelo ubicado en un sitio subhúmedo del sudoeste bonaerense. La experiencia se realizó sobre un ensayo de larga duración en un suelo Argiudol Típico con dos sistemas de labranza contrastantes: labranza convencional (LC) y siembra directa (SD). Se tomaron muestras de suelo en los años 2003, 2009 y 2015 en las profundidades de 0-5 y 0-20 cm. Se determinó C orgánico total (COT), N total (Nt), C y N orgánico recalcitrante (COR y NOR). Además se calculó el IR de C (IRC) y de N (IRN). Los niveles de COT y Nt fueron mayores en SD en ambas profundidades y para todos los años. El COR en la profundidad 0-5 cm mostró diferentes efectos tanto por la labranza como por los años En 0-20 cm, sólo mostró diferencias significativas (p<0,05) entre años, sin efecto por la labranza. Por otra parte, el NOR evidenció diferencias altamente significativas entre sistemas de labranza y años en 0-5 cm, y diferencias significativas entre años para 0-20 cm. El IRC en superficie mostró diferencias entre los sistemas de labranza. La mayor acumulación del C y N en SD no resultó en una mayor humificación de las fracciones lábiles a formas estables. El secuestro de C en superficie mostró variabilidad con respecto al tiempo, mientras que en 0-20 cm no demostró estar asociado a los sistemas de labranza pero sí al efecto del año.Publicado en las Actas de las III Jornadas Nacionales de Suelos de Ambientes Semiáridos

Potential nitrogen mineralization in soils with sunflower seed husk application

El objetivo de este estudio fue evaluar el potencial de mineralización de nitrógeno (N) de suelos contrastantes luego del aporte de diferentes dosis de cascaras de semillas de girasol (CG) (Helianthus annuus L.), residuo abundante del sudoeste bonaerense (SOB). Se realizaron incubaciones aeróbicas de largo plazo (37 semanas), utilizando tres suelos diferentes (0-20 cm) y distintas dosis de CG calculadas con base en su contenido de N orgánico: 100 (D1), 200 (D2) y 400 (D3) kg N ha-1. Los suelos contrastaron en el contenido de fracciones granulométricas (S1: arena 509 g kg-1; limo 320 g kg-1 y arcilla171 g kg-1; S2: arena 351 g kg-1; limo 448 g kg-1 y arcilla 201 g kg-1; S3: arena 827 g kg-1, limo 107 g kg-1 y arcilla 66 g kg-1). Las características químicas de CG: conductividad eléctrica= 1,6 dS m-1 ; pH= 5,6; materia orgánica (MO) = 958 g kg-1 ; N total= 7,8 g kg-1, relación C:N= 79. Los suelos demostraron diferencias significativas en el N potencialmente mineralizable (N0) con valores que fueron S1>S2>S3, al igual que en la tasa de mineralización (k) (S1 y S3= 0,0036 semana-1, S2=0,0056 semana-1). Se halló una interacción significativa en los aportes de CG y suelos, por lo que se analizó la incorporación en cada suelo. Cuando se aplicó CG, se observó una respuesta positiva sobre S3 con D1 y D2. Para S1 y S2, la aplicación de CG no aumentó el N0, aunque tampoco produjo un detrimento. La dinámica de la mineralización de N acumulada demostró efectos diferentes al aporte de CG con respecto al tipo de suelo. El aporte de CG al suelo incrementó el potencial de mineralización de N en el suelo arenoso-franco, sin disminuir el potencial en los suelos restantes, por lo que podría ser utilizada como enmienda orgánica bajo estas condiciones.The objective of this study was to evaluate the potential nitrogen (N) mineralization of contrasting soils after the addition of different sunflower seed hulls (CG) (Helianthus annuus L.) doses, an abundant residue of the southwest of Buenos Aires Province (SOB). Long-term aerobic incubations (37 weeks) were performed, using three different soils (0-20 cm) and various CG levels based on their organic N content: 100 (D1), 200 (D2) and 400 (D3) kg N ha-1. Soils differs in granulometric fractions (S1: sand 509 g kg-1; silt 320 g kg-1 and clay 171 g kg-1; S2: sand 351 g kg-1; silt 448 g kg-1 and clay 201 g kg-1; S3: sand 827 g kg-1, silt 107 g kg-1 and clay 66 g kg-1). e CG chemical characteristics: electrical conductivity= 1.6 dS m-1; pH= 5.6; organic matter (MO) = 958 g kg-1; total N= 7.8 g kg-1, C:N ratio = 79. Significant differences were found in potentially mineralizable N (N.) for three soils, whose values were S1>S2>S3; as well as in the mineralization rate (.) (S1 and S3= 0.0036 week-1 , S2=0.0056 week-1). Significant interaction was found with the CG contribution and soils, so the contribution was analyzed for each soil. When GC was applied, a positive response was observed on S3 with D1 and D2. For S1 and S2, the application of GC did not increase the potential for N mineralization, however, it had not a detrimental effect on the potential. Dynamics of accumulated N mineralization showed different effects of CG addition with respect to soil type. The CG addition to soil increased the potential N mineralization in the sandy-loam soil, without reducing the potential in the remaining soils, which is why it could be used as an organic amendment under these conditions.Fil: Martinez, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Agronomía; ArgentinaFil: Moisés, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Agronomía; ArgentinaFil: Duval, Matias Ezequiel. Universidad Nacional del Sur. Departamento de Agronomía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; ArgentinaFil: Garcia, Ramiro Javier. Universidad Nacional del Sur. Departamento de Agronomía; ArgentinaFil: Potemsky, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; ArgentinaFil: Galantini, Juan Alberto. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentin

Multi-employment in health in the province of Buenos Aires: cross-sectional study of professions involved in COVID-19 patient care

INTRODUCCIÓN: La pandemia de COVID-19 expuso el impacto del multiempleo en los servicios de salud. El objetivo de este estudio fue caracterizar el multiempleo en médicos y enfermeros que trabajaron en internación general y cuidados críticos durante la pandemia en la provincia de Buenos Aires. MÉTODOS: Se realizó un estudio exploratorio, descriptivo y transversal. Los datos se recolectaron en el primer trimestre de 2021 con una encuesta estructurada anónima autoadministrada mediante muestreo no probabilístico en cadena. RESULTADOS: El 96,3% de los médicos y el 68,1% de los enfermeros declararon estar multiempleados. La media de empleos fue 3,1 para médicos (entre 1 y 5) y 1,9 para enfermeros (entre 1 y 3). Los enfermeros declararon trabajos con mayor carga horaria, predominio del empleo público y contratación estable. Los médicos multiempleados manifestaron mayor diversidad en la forma de contratación. En ambas profesiones el multiempleo es motivado principalmente por razones de índole económica. DISCUSIÓN: El multiempleo es difícil de medir mediante los registros oficiales. La proporción de multiempleo autorreportado en este estudio supera las cifras comunicadas por las estadísticas e informes oficiales. Aun así, las diferencias halladas entre médicos y enfermeros respecto al número de empleos son coincidentes con estudios previos. El problema del multiempleo requiere ser abordado desde la desprecarización del empleo, con mejoras en las condiciones económicas y de trabajo.INTRODUCTION: The COVID-19 pandemic exposed the impact of multi-employment in health services. The objective of this study was to characterize multi-employment among physicians and nurses working in general hospitalization and critical care during the pandemic in the province of Buenos Aires. METHODS: An exploratory, descriptive, cross-sectional study was conducted. Data were collected in the first quarter of 2021 with a structured anonymous self-administered survey using non-probabilistic chain sampling. RESULTS: A total of 96.3% of physicians and 68.1% of nurses reported being multi-employed. The mean number of jobs was 3.1 for physicians (between 1 and 5) and 1.9 for nurses (between 1 and 3). Nurses reported having jobs with longer hours, mostly public and stable employment. Multi-employed physicians reported greater variety in the form of hiring. In both professions, multi-employment is mainly motivated by economic reasons. DISCUSSION: Multi-employment is difficult to measure through official records. The proportion of self-reported multiemployment in this study exceeds the figures shown by official statistics and reports. The differences found between physicians and nurses regarding the number of jobs are still consistent with previous studies. The problem of multi-employment needs to be tackled by making employment less precarious, with improvements in economic and working conditions.Fil: Chaz Sardi, María Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Sur; ArgentinaFil: Martinez, Cintia Karina. Universidad Nacional del Sur; ArgentinaFil: Mirofsky, Matías Alberto. Sociedad Argentina de Medicina; ArgentinaFil: Lopez, Francisco Javier. Federacion Medica de la Provincia de Buenos Aires; ArgentinaFil: Garzaniti, Ramiro. Provincia de Buenos Aires. Ministerio de Salud; ArgentinaFil: Gubilei, Eliana Soledad. Provincia de Buenos Aires. Ministerio de Salud; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Lhopital, Cintia. Provincia de Buenos Aires. Ministerio de Salud; ArgentinaFil: González, Verónica. Provincia de Buenos Aires. Ministerio de Salud; ArgentinaFil: Duré, María Isabel. Provincia de Buenos Aires. Ministerio de Salud; ArgentinaFil: Garcia Dieguez, Marcelo. Universidad Nacional del Sur; Argentin

Multi-employment in health in the province of Buenos Aires: cross-sectional study of professions involved in COVID-19 patient care

INTRODUCCIÓN: La pandemia de COVID-19 expuso el impacto del multiempleo en los servicios de salud. El objetivo de este estudio fue caracterizar el multiempleo en médicos y enfermeros que trabajaron en internación general y cuidados críticos durante la pandemia en la provincia de Buenos Aires. MÉTODOS: Se realizó un estudio exploratorio, descriptivo y transversal. Los datos se recolectaron en el primer trimestre de 2021 con una encuesta estructurada anónima autoadministrada mediante muestreo no probabilístico en cadena. RESULTADOS: El 96,3% de los médicos y el 68,1% de los enfermeros declararon estar multiempleados. La media de empleos fue 3,1 para médicos (entre 1 y 5) y 1,9 para enfermeros (entre 1 y 3). Los enfermeros declararon trabajos con mayor carga horaria, predominio del empleo público y contratación estable. Los médicos multiempleados manifestaron mayor diversidad en la forma de contratación. En ambas profesiones el multiempleo es motivado principalmente por razones de índole económica. DISCUSIÓN: El multiempleo es difícil de medir mediante los registros oficiales. La proporción de multiempleo autorreportado en este estudio supera las cifras comunicadas por las estadísticas e informes oficiales. Aun así, las diferencias halladas entre médicos y enfermeros respecto al número de empleos son coincidentes con estudios previos. El problema del multiempleo requiere ser abordado desde la desprecarización del empleo, con mejoras en las condiciones económicas y de trabajo.INTRODUCTION: The COVID-19 pandemic exposed the impact of multi-employment in health services. The objective of this study was to characterize multi-employment among physicians and nurses working in general hospitalization and critical care during the pandemic in the province of Buenos Aires. METHODS: An exploratory, descriptive, cross-sectional study was conducted. Data were collected in the first quarter of 2021 with a structured anonymous self-administered survey using non-probabilistic chain sampling. RESULTS: A total of 96.3% of physicians and 68.1% of nurses reported being multi-employed. The mean number of jobs was 3.1 for physicians (between 1 and 5) and 1.9 for nurses (between 1 and 3). Nurses reported having jobs with longer hours, mostly public and stable employment. Multi-employed physicians reported greater variety in the form of hiring. In both professions, multi-employment is mainly motivated by economic reasons. DISCUSSION: Multi-employment is difficult to measure through official records. The proportion of self-reported multiemployment in this study exceeds the figures shown by official statistics and reports. The differences found between physicians and nurses regarding the number of jobs are still consistent with previous studies. The problem of multi-employment needs to be tackled by making employment less precarious, with improvements in economic and working conditions.Fil: Chaz Sardi, María Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Sur; ArgentinaFil: Martinez, Cintia Karina. Universidad Nacional del Sur; ArgentinaFil: Mirofsky, Matías Alberto. Sociedad Argentina de Medicina; ArgentinaFil: Lopez, Francisco Javier. Federacion Medica de la Provincia de Buenos Aires; ArgentinaFil: Garzaniti, Ramiro. Provincia de Buenos Aires. Ministerio de Salud; ArgentinaFil: Gubilei, Eliana Soledad. Provincia de Buenos Aires. Ministerio de Salud; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Lhopital, Cintia. Provincia de Buenos Aires. Ministerio de Salud; ArgentinaFil: González, Verónica. Provincia de Buenos Aires. Ministerio de Salud; ArgentinaFil: Duré, María Isabel. Provincia de Buenos Aires. Ministerio de Salud; ArgentinaFil: Garcia Dieguez, Marcelo. Universidad Nacional del Sur; Argentin

Integrated In Vitro and In Silico Modeling Delineates the Molecular Effects of a Synbiotic Regimen on Colorectal-Cancer-Derived Cells

By modulating the human gut microbiome, prebiotics and probiotics (combinations of which are called synbiotics) may be used to treat diseases such as colorectal cancer (CRC). Methodological limitations have prevented determining the potential combina- torial mechanisms of action of such regimens. We expanded our HuMiX gut-on-a-chip model to co-culture CRC-derived epithelial cells with a model probiotic under a simulated prebiotic regimen, and we integrated the multi-omic results with in silico metabolic modeling. In contrast to individual prebi- otic or probiotic treatments, the synbiotic regimen caused downregulation of genes involved in procarci- nogenic pathways and drug resistance, and reduced levels of the oncometabolite lactate. Distinct ratios of organic and short-chain fatty acids were produced during the simulated regimens. Treatment of primary CRC-derived cells with a molecular cocktail reflecting the synbiotic regimen attenuated self-renewal ca- pacity. Our integrated approach demonstrates the potential of modeling for rationally formulating synbi- otics-based treatments in the future

Birth mode is associated with earliest strain-conferred gut microbiome functions and immunostimulatory potential

The effects of caesarean section delivery on mother-to-neonate transmission of microbiota are unclear. Here the authors show that caesarean section delivery can affect the transmission of specific microbial strains and the immunomodulatory potential of the microbiota

Mining microbial metatranscriptomes for expression of antibiotic resistance genes under natural conditions

Antibiotic resistance genes are found in a broad range of ecological niches associated with complex microbiota. Here we investigated if resistance genes are not only present, but also transcribed under natural conditions. Furthermore, we examined the potential for antibiotic production by assessing the expression of associated secondary metabolite biosynthesis gene clusters. Metatranscriptome datasets from intestinal microbiota of four human adults, one human infant, 15 mice and six pigs, of which only the latter have received antibiotics prior to the study, as well as from sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment, were investigated. We found that resistance genes are expressed in all studied ecological niches, albeit with niche-specific differences in relative expression levels and diversity of transcripts. For example, in mice and human infant microbiota predominantly tetracycline resistance genes were expressed while in human adult microbiota the spectrum of expressed genes was more diverse, and also included beta-lactam, aminoglycoside and macrolide resistance genes. Resistance gene expression could result from the presence of natural antibiotics in the environment, although we could not link it to expression of corresponding secondary metabolites biosynthesis clusters. Alternatively, resistance gene expression could be constitutive, or these genes serve alternative roles besides antibiotic resistance.Peer reviewe

Systematic characterization of human gut microbiome-secreted molecules by integrated multi-omics

The human gut microbiome produces a complex mixture of biomolecules that interact with human physiology and play essential roles in health and disease. Crosstalk between micro-organisms and host cells is enabled by different direct contacts, but also by the export of molecules through secretion systems and extracellular vesicles. The resulting molecular network, comprised of various biomolecular moieties, has so far eluded systematic study. Here we present a methodological framework, optimized for the extraction of the microbiome-derived, extracellular biomolecular complement, including nucleic acids, (poly)peptides, and metabolites, from flash-frozen stool samples of healthy human individuals. Our method allows simultaneous isolation of individual biomolecular fractions from the same original stool sample, followed by specialized omic analyses. The resulting multi-omics data enable coherent data integration for the systematic characterization of this molecular complex. Our results demonstrate the distinctiveness of the different extracellular biomolecular fractions, both in terms of their taxonomic and functional composition. This highlights the challenge of inferring the extracellular biomolecular complement of the gut microbiome based on single-omic data. The developed methodological framework provides the foundation for systematically investigating mechanistic links between microbiome-secreted molecules, including those that are typically vesicle-associated, and their impact on host physiology in health and disease