Requirement of a Plasmid-Encoded Catalase for Survival of \u3cem\u3eRhizobium etli\u3c/em\u3e CFN42 in a Polyphenol-Rich Environment

Nitrogen-fixing bacteria collectively called rhizobia are adapted to live in polyphenol-rich environments. The mechanisms that allow these bacteria to overcome toxic concentrations of plant polyphenols have not been clearly elucidated. We used a crude extract of polyphenols released from the seed coat of the black bean to simulate a polyphenol-rich environment and analyze the response of the bean-nodulating strain Rhizobium etli CFN42. Our results showed that the viability of the wild type as well as that of derivative strains cured of plasmids p42a, p42b, p42c, and p42d or lacking 200 kb of plasmid p42e was not affected in this environment. In contrast, survival of the mutant lacking plasmid p42f was severely diminished. Complementation analysis revealed that the katG gene located on this plasmid, encoding the only catalase present in this bacterium, restored full resistance to testa polyphenols. Our results indicate that oxidation of polyphenols due to interaction with bacterial cells results in the production of a high quantity of H2O2, whose removal by the katG-encoded catalase plays a key role for cell survival in a polyphenol-rich environment

Housekeeping genes essential for pantothenate biosynthesis are plasmid-encoded in Rhizobium etli and Rhizobium leguminosarum

<p>Abstract</p> <p>Background</p> <p>A traditional concept in bacterial genetics states that housekeeping genes, those involved in basic metabolic functions needed for maintenance of the cell, are encoded in the chromosome, whereas genes required for dealing with challenging environmental conditions are located in plasmids. Exceptions to this rule have emerged from genomic sequence data of bacteria with multipartite genomes. The genome sequence of <it>R. etli </it>CFN42 predicts the presence of <it>panC </it>and <it>panB </it>genes clustered together on the 642 kb plasmid p42f and a second copy of <it>panB </it>on plasmid p42e. They encode putative pantothenate biosynthesis enzymes (pantoate-β-alanine ligase and 3-methyl-2-oxobutanoate hydroxymethyltransferase, respectively). Due to their ubiquitous distribution and relevance in the central metabolism of the cell, these genes are considered part of the core genome; thus, their occurrence in a plasmid is noteworthy. In this study we investigate the contribution of these genes to pantothenate biosynthesis, examine whether their presence in plasmids is a prevalent characteristic of the <it>Rhizobiales </it>with multipartite genomes, and assess the possibility that the <it>panCB </it>genes may have reached plasmids by horizontal gene transfer.</p> <p>Results</p> <p>Analysis of mutants confirmed that the <it>panC </it>and <it>panB </it>genes located on plasmid p42f are indispensable for the synthesis of pantothenate. A screening of the location of <it>panCB </it>genes among members of the <it>Rhizobiales </it>showed that only <it>R. etli </it>and <it>R. leguminosarum </it>strains carry <it>panCB </it>genes in plasmids. The <it>panCB </it>phylogeny attested a common origin for chromosomal and plasmid-borne <it>panCB </it>sequences, suggesting that the <it>R. etli </it>and <it>R. leguminosarum panCB </it>genes are orthologs rather than xenologs. The <it>panCB </it>genes could not totally restore the ability of a strain cured of plasmid p42f to grow in minimal medium.</p> <p>Conclusions</p> <p>This study shows experimental evidence that core <it>panCB </it>genes located in plasmids of <it>R. etli </it>and <it>R. leguminosarum </it>are indispensable for the synthesis of pantothenate. The unusual presence of <it>panCB </it>genes in plasmids of <it>Rhizobiales </it>may be due to an intragenomic transfer from chromosome to plasmid. Plasmid p42f encodes other functions required for growth in minimal medium. Our results support the hypothesis of cooperation among different replicons for basic cellular functions in multipartite rhizobia genomes.</p

Modelación de la cinética de reacción para la producción de polihidroxialcanoatos microbianos mediante Bacillus megaterium

Abstract. The production of poly(3-hydroxybutyrate) PHA by Bacillus. megaterium depends exclusively on the concentration of the carbon source (glucose), so it is proposed to use mathematical simulation models for the kinetics applied in the production of microbial polyhydroxyalkanoates. Bacteria can be isolated from Californian red worm humus, and a logistic model including an inhibition factor and a constant associated with cell maintenance is used for biomass growth. For product formation kinetics, the Leudeking-Piret model is proposed, where the product formation coefficient depends on cell growth and the constant associated with cell maintenance, both of which are determined by the fermentation pH, and correspond to associated and non-associated growth, respectively. The model for substrate consumption considers that cells metabolize substrate for growth, product synthesis and energy generation, as well as for internal pH control activities and exchange of cellular components. Kinetic equations are proposed to estimate experimental results of this case study based on the logistic, Leudeking-Piret and substrate consumption models, to determine the values of biomass and product yields, depending on the substrate used in the stoichiometry of PHA production. The next stage contemplates the application of UV-Vis spectrophotometry to estimate cell growth in Colony Forming Units (CFU) and its comparison with the McFarland scale to quantify equivalently the number of bacterial cells.La producción de poli-(3-hidroxibutirato) PHA por Bacillus. megaterium depende exclusivamente de la concentración de la fuente de carbono (glucosa), por lo que se propone utilizar modelos matemáticos de simulación para la cinética aplicada en la producción de polihidroxialcanoatos microbianos. Las bacterias pueden aislarse del humus de la lombriz roja californiana, y para el crecimiento de biomasa se utiliza un modelo logístico que incluye un factor de inhibición y una constante asociada al mantenimiento celular. En la cinética de formación de producto se propone el modelo de Leudeking-Piret, donde el coeficiente de formación del producto depende del crecimiento celular y la constante asociada al mantenimiento celular, ambas están determinadas por el pH de la fermentación, y corresponden al crecimiento asociado y no asociado respectivamente. El modelo para consumo de sustrato considera que las células lo metabolizan para crecimiento, síntesis de producto y generación de energía, así como para actividades de control de pH interno e intercambio de componentes celulares. Se plantea ecuaciones de cinética para estimar resultados experimentales de este caso de estudio basadas en los modelos logístico, de Leudeking-Piret y de consumo de sustrato, para determinar los valores de los rendimientos de biomasa y de producto, en función al sustrato utilizado en la estequiometria de la producción de PHA. La siguiente etapa contempla la aplicación de la espectrofotometría UV-Vis para estimar el crecimiento celular en Unidades Formadoras de Colonias (UFC) y su comparación con la escala de McFarland para cuantificar de forma equivalente el número de células bacterianas

Análisis del comportamiento del suelo, implementando cemento alternativo base pumicita en la estabilización de un material tipo base

The present work shows the behavior of a base layer in its stabilization by implementing a pumicite-based cement, with the objective of analyzing the modifications in the physical properties, through tests of consistency limits using alternative cements, in a base material, evaluating the behavior of compaction, with the use of an alternative cement, perform the treatment of the alternative material substitute for cement, by grinding and sifting, as well as obtaining the exact percentage of use of alternative cement when applying it to the stabilization of the base material. For this, several mixtures of pumicite-based cementing material and composite portland cement were made with different percentages of additions, which were 4%, 6%, and 8% respectively, and based on the tests carried out, choose the one that best way it behaves. In order to reduce the implementation of composite Portland cement, thus reducing the pollution that its manufacture brings with it. The mixture that led to a better soil-cement reaction was the addition of 6%, 50% composite portland cement, 50% pumicite-based cementing material, presenting better behavior in the base-type material, thus partially replacing the use of composite portland cement.El presente trabajo muestra el comportamiento de una capa base en su estabilización implementando un cemento base pumicita, con el objetivo de analizar las modificaciones en las propiedades físicas, a través de ensayos de límites de consistencia utilizando cementos alternativos, en un material de base, evaluar el comportamiento de la compactación, con la utilización de un cemento alternativo, realizar el tratamiento del material alternativo sustituto al cemento, mediante molienda y tamizado, así como obtener el porcentaje exacto de utilización de cemento alternativo al aplicarlo en la estabilización del material de base. Para esto, se realizaron varias mezclas de material cementante base pumicita y cemento portland compuesto con diferentes porcentajes de adiciones, las cuales fueron de 4%, 6%, y 8% respectivamente, y en base a las pruebas realizadas, elegir la que de mejor manera se comporte. Con la finalidad de reducir la implementación de cemento Portland compuesto, reduciendo así la contaminación que trae consigo su fabricación. Siendo la mezcla que conllevo a una mejor reacción suelo-cemento la de adición del 6%, 50% cemento portland compuesto, 50% material cementante base pumicita, presentando mejor comportamiento en el material tipo base, de esta manera se logra sustituir parcialmente el uso de cemento portland compuesto

Polarimetric imaging for the detection of synthetic models of SARS-CoV-2: A proof of concept

Objective: To conduct a proof-of-concept study of the detection of two synthetic models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using polarimetric imaging. Approach: Two SARS-CoV-2 models were prepared as engineered lentiviruses pseudotyped with the G protein of the vesicular stomatitis virus, and with the characteristic Spike protein of SARS-CoV-2. Samples were prepared in two biofluids (saline solution and artificial saliva), in four concentrations, and deposited as 5-µL droplets on a supporting plate. The angles of maximal degree of linear polarization (DLP) of light diffusely scattered from dry residues were determined using Mueller polarimetry from87 samples at 405 nm and 514 nm. A polarimetric camera was used for imaging several samples under 380–420 nm illumination at angles similar to those of maximal DLP. Per-pixel image analysis included quantification and combination of polarization feature descriptors in 475 samples. Main results: The angles (from sample surface) of maximal DLP were 3° for 405 nm and 6° for 514 nm. Similar viral particles that differed only in the characteristic spike protein of the SARS-CoV-2, their corresponding negative controls, fluids, and the sample holder were discerned at 10-degree and 15-degree configurations. Significance: Polarimetric imaging in the visible spectrum may help improve fast, non-contact detection and identification of viral particles, and/or other microbes such as tuberculosis, in multiple dry fluid samples simultaneously, particularly when combined with other imaging modalities. Further analysis including realistic concentrations of real SARS-CoV-2 viral particles in relevant human fluids is required. Polarimetric imaging under visible light may contribute to a fast, cost-effective screening of SARS-CoV-2 and other pathogens when combined with other imaging modalities.12 página

Hyperspectral image processing for the identification and quantification of lentiviral particles in fluid samples

Optical spectroscopic techniques have been commonly used to detect the presence of biofilm-forming pathogens (bacteria and fungi) in the agro-food industry. Recently, near-infrared (NIR) spectroscopy revealed that it is also possible to detect the presence of viruses in animal and vegetal tissues. Here we report a platform based on visible and NIR (VNIR) hyperspectral imaging for non-contact, reagent free detection and quantification of laboratory-engineered viral particles in fluid samples (liquid droplets and dry residue) using both partial least square-discriminant analysis and artificial feed-forward neural networks. The detection was successfully achieved in preparations of phosphate buffered solution and artificial saliva, with an equivalent pixel volume of 4 nL and lowest concentration of 800 TU.mu L-1. This method constitutes an innovative approach that could be potentially used at point of care for rapid mass screening of viral infectious diseases and monitoring of the SARS-CoV- 2 pandemic.This research was funded by grants number COV20-00080 and COV20-00173 of the 2020 Emergency Call for Research Projects about the SARS-CoV-2 virus and the COVID-19 disease of the Institute of Health 'Carlos III', Spanish Ministry of Science and Innovation, and by grant number EQC2019-006240-P of the 2019 Call for Acquisition of Scientific Equipment, FEDER Program, Spanish Ministry of Science and Innovation. This work has been supported by the European Commission through the JRC HUMAINT project. ABR was supported by grant number RTI2018-094465-J funded by the Spanish National Agency of Research. The authors would like to gratefully acknowledge the assistance of the members of the EOD-CBRN Group of the Spanish National Police, whose identities cannot be disclosed, and who are represented here by JMNG. Authors thank continuous support from their institutions

Predictive Power of the "Trigger Tool" for the detection of adverse events in general surgery: a multicenter observational validation study

Background In spite of the global implementation of standardized surgical safety checklists and evidence-based practices, general surgery remains associated with a high residual risk of preventable perioperative complications and adverse events. This study was designed to validate the hypothesis that a new “Trigger Tool” represents a sensitive predictor of adverse events in general surgery. Methods An observational multicenter validation study was performed among 31 hospitals in Spain. The previously described “Trigger Tool” based on 40 specific triggers was applied to validate the predictive power of predicting adverse events in the perioperative care of surgical patients. A prediction model was used by means of a binary logistic regression analysis. Results The prevalence of adverse events among a total of 1,132 surgical cases included in this study was 31.53%. The “Trigger Tool” had a sensitivity and specificity of 86.27% and 79.55% respectively for predicting these adverse events. A total of 12 selected triggers of overall 40 triggers were identified for optimizing the predictive power of the “Trigger Tool”. Conclusions The “Trigger Tool” has a high predictive capacity for predicting adverse events in surgical procedures. We recommend a revision of the original 40 triggers to 12 selected triggers to optimize the predictive power of this tool, which will have to be validated in future studies

Spatiotemporal Characteristics of the Largest HIV-1 CRF02_AG Outbreak in Spain: Evidence for Onward Transmissions

Background and Aim: The circulating recombinant form 02_AG (CRF02_AG) is the predominant clade among the human immunodeficiency virus type-1 (HIV-1) non-Bs with a prevalence of 5.97% (95% Confidence Interval-CI: 5.41–6.57%) across Spain. Our aim was to estimate the levels of regional clustering for CRF02_AG and the spatiotemporal characteristics of the largest CRF02_AG subepidemic in Spain.Methods: We studied 396 CRF02_AG sequences obtained from HIV-1 diagnosed patients during 2000–2014 from 10 autonomous communities of Spain. Phylogenetic analysis was performed on the 391 CRF02_AG sequences along with all globally sampled CRF02_AG sequences (N = 3,302) as references. Phylodynamic and phylogeographic analysis was performed to the largest CRF02_AG monophyletic cluster by a Bayesian method in BEAST v1.8.0 and by reconstructing ancestral states using the criterion of parsimony in Mesquite v3.4, respectively.Results: The HIV-1 CRF02_AG prevalence differed across Spanish autonomous communities we sampled from (p &lt; 0.001). Phylogenetic analysis revealed that 52.7% of the CRF02_AG sequences formed 56 monophyletic clusters, with a range of 2–79 sequences. The CRF02_AG regional dispersal differed across Spain (p = 0.003), as suggested by monophyletic clustering. For the largest monophyletic cluster (subepidemic) (N = 79), 49.4% of the clustered sequences originated from Madrid, while most sequences (51.9%) had been obtained from men having sex with men (MSM). Molecular clock analysis suggested that the origin (tMRCA) of the CRF02_AG subepidemic was in 2002 (median estimate; 95% Highest Posterior Density-HPD interval: 1999–2004). Additionally, we found significant clustering within the CRF02_AG subepidemic according to the ethnic origin.Conclusion: CRF02_AG has been introduced as a result of multiple introductions in Spain, following regional dispersal in several cases. We showed that CRF02_AG transmissions were mostly due to regional dispersal in Spain. The hot-spot for the largest CRF02_AG regional subepidemic in Spain was in Madrid associated with MSM transmission risk group. The existence of subepidemics suggest that several spillovers occurred from Madrid to other areas. CRF02_AG sequences from Hispanics were clustered in a separate subclade suggesting no linkage between the local and Hispanic subepidemics