Putative ancient microorganisms from amber nuggets

Evolutionary microbiology studies based on the isolation of ancient DNA and/or microbial samples are scarce due to the difficulty of finding well preserved biological specimens. However, amber is a fossil resin with natural preserving properties for microbial cells and DNA. The visualization by transmission electron microscopy of different microorganismlike specimens found in amber nuggets from both the Miocene and the Cretaceous periods was accompanied by studies of ancient DNA obtained from the nuggets. After the design of specific primers based on the present sequences of both genes in Saccharomyces cerevisiae, the ancestral AGP2 sequence from the Miocene, as well as the 18S rRNA from the Cretaceous, were amplified. [Int Microbiol 2007; 10(2):117-122

Isolation and taxonomic study of a new canthaxanthin-containing bacterium, Gordonia jacobaea MV-1 sp. nov

This article describes the isolation and taxonomic study of a coryneform isolate of a new Gordonia species (G. jacobaea), strain MV-1, which accumulates several carotenoids, including the ketocarotenoid trans-canthaxanthin. Identification of this new isolate by morphobiochemical methods did not allow unambiguous taxon assignment, but sequencing of the 16S rRNA gene clearly pointed to the genus Gordonia, Gordonia sputi being the closest fit. Differences in certain transversions/ transitions in otherwise very well-conserved sequences of the described Gordonia species supported the proposal of this new taxon. The fact that both the best growth and best pigmentation were obtained with glucose, an inexpensive carbon source and at an industrially suitable temperature, suggests that this new bacterial strain may have good potential for the industrial production of canthaxanthin

Influence of culture conditions of Gordonia jacobaea MV-26 on canthaxanthin production

Commercial interest in the use of natural pigments isolated from microorganisms has increased in recent years; hence, molecules belonging to the polyisoprenoid group (i.e. β-carotene, astaxanthin, and canthaxanthin) have been the focus of much attention. The bacterium Gordonia jacobaea readily synthesizes and accumulates large amounts of canthaxanthin (β-β´-carotene-4,4´-dione), which is widely used in the food and cosmetics industries. In the present work, the effects of different low-cost raw materials on fermentation and canthaxanthin accumulation by a hyperpigmented strain of G. jacobaea were studied. Canthaxanthin production and peak levels of accumulation varied according to the different media used. [Int Microbiol 2005; 8(1):55-58

A New and Efficient Enrichment Method for Metagenomic Sequencing of Monkeypox Virus

[Abstract] Background The methodology described in previous literature for Monkeypox virus (MPXV) sequencing shows low efficiency when using metagenomic approaches. The aim of the present study was to evaluate a new fine-tuned method for extraction and enrichment of genomic MPXV DNA using clinical samples and to compare it to a non-enrichment metagenomic approach. Results A new procedure that allows sample enrichment in MPXV DNA, avoiding wasting the sequencing capacity in human DNA, was designed. This procedure consisted of host DNA depletion using a saponin/NaCl combination treatment and DNase, together with high g-force centrifugations. After typical quality control, samples using the enrichment method contained around 96% of reads not classified as human DNA, while the non-enrichment protocol showed around 5-10%. When reads not belonging to Orthopoxvirus were removed, enriched samples kept about 50% of the original read counts, while non-enriched ones kept only 2-7%. Conclusions Results showed a very significant improvement in sequencing efficiency, increasing the number of reads belonging to MPXV, the depth of coverage and the trustworthiness of the consensus sequences. This, in turn, allows for more samples to be included in a single cartridge, reducing costs and time to diagnosis, which can be very important factors when dealing with a contagious disease.This work was supported by a grant from the SERGAS-Galician Healthcare Service (Program “Innova Saúde”) to GB, by CIBERINFEC and also by Instituto de Salud Carlos III (ISCIII) through the projects PI20/00413 to MP and PI21/00704 to G

Automation Proposal for the Intermediate Steps in the 16S FFPE Samples Analysis Pipeline

Cursos e Congresos, C-155[Abstract] In the day-to-day work of bioinformatics, the use of integrated software packages, which encompass a wide range of tools, enables the development of pipelines for omics data analysis. Within the various existing pipelines, we focus on the analysis of the 16S rRNA gene as it allows for the study of diversity and taxonomy of prokaryotic microorganisms such as Bacteria and Archaea. However, these pipelines often involve a sequence of multiple tools that require intermediate steps before further processing can proceed, as in the case between Cutadapt and DADA2. In fact, in a typical pipeline, the values for DADA2 input arguments ’trunc-len-f’ and ’trunc-len-r’ are extracted from the output of Cutadapt. The best approach for selecting optimal values (aka the trimming positions) is graphically visualizing Cutadapt output and manually selecting the most accurate trimming position length. Therefore, we propose the automation of this specific intermediate step between Cutadapt and DADA2 tools, by selecting values displayed in the graphs that meet the filtering criteria. This automation has been incorporated into a custom pipeline for the analysis of the microbiome in 16S paired-end samples from colorectal cancer patients, and could potentially serve as a standardization approach in these processesThe authors of this paper extend their sincere appreciation to the collaborative efforts and contributions of the meiGAbiome Group, aswell as the entire team of medical and anatomopathologists. Finally, we are deeply grateful to the patients whose selfless donations have made this and numerous other studies possibl

Quorum Sensing as a Target for Controlling Surface Associated Motility and Biofilm Formation in Acinetobacter Baumannii ATCC® 17978TM

[Abstract] The important nosocomial pathogen Acinetobacter baumannii presents a quorum sensing (QS) system (abaI/abaR) mediated by acyl-homoserine-lactones (AHLs) and several quorum quenching (QQ) enzymes. However, the roles of this complex network in the control of the expression of important virulence-related phenotypes such as surface-associated motility and biofilm formation is not clear. Therefore, the effect of the mutation of the AHL synthase AbaI, and the exogenous addition of the QQ enzyme Aii20J on surface-associated motility and biofilm formation by A. baumannii ATCC® 17978TM was studied in detail. The effect of the enzyme on biofilm formation by several multidrug-resistant A. baumannii clinical isolates differing in their motility pattern was also tested. We provide evidence that a functional QS system is required for surface-associated motility and robust biofilm formation in A. baumannii ATCC® 17978TM. Important differences were found with the well-studied strain A. nosocomialis M2 regarding the relevance of the QS system depending on environmental conditions The in vitro biofilm-formation capacity of A. baumannii clinical strains was highly variable and was not related to the antibiotic resistance or surface-associated motility profiles. A high variability was also found in the sensitivity of the clinical strains to the action of the QQ enzyme, revealing important differences in virulence regulation between A. baumannii isolates and confirming that studies restricted to a single strain are not representative for the development of novel antimicrobial strategies. Extracellular DNA emerges as a key component of the extracellular matrix in A. baumannii biofilms since the combined action of the QQ enzyme Aii20J and DNase reduced biofilm formation in all tested strains. Results demonstrate that QQ strategies in combination with other enzymatic treatments such as DNase could represent an alternative approach for the prevention of A. baumannii colonization and survival on surfaces and the prevention and treatment of infections caused by this pathogen.Xunta de Galicia; ED481A-2015/311Xunta de Galicia; IN606B-2019/010Biotechnology and Biological Sciences Research Council (United Kingdom); BB/R012415/1Xunta de Galicia; ED481A-2019/19

Pneumonia infection in mice reveals the involvement of the feoA gene in the pathogenesis of Acinetobacter baumannii

[Abstract] Acinetobacter baumannii has emerged in the last decade as an important nosocomial pathogen. To identify genes involved in the course of a pneumonia infection, gene expression profiles were obtained from A. baumannii ATCC 17978 grown in mouse infected lungs and in culture medium. Gene expression analysis allowed us to determine a gene, the A1S_0242 gene (feoA), over-expressed during the pneumonia infection. In the present work, we evaluate the role of this gene, involved in iron uptake. The inactivation of the A1S_0242 gene resulted in an increase susceptibility to oxidative stress and a decrease in biofilm formation, in adherence to A549 cells and in fitness. In addition, infection of G. mellonella and pneumonia in mice showed that the virulence of the Δ0242 mutant was significantly attenuated. Data presented in this work indicated that the A1S_0242 gene from A. baumannii ATCC 17978 strain plays a role in fitness, adhesion, biofilm formation, growth, and, definitively, in virulence. Taken together, these observations show the implication of the feoA gene plays in the pathogenesis of A. baumannii and highlight its value as a potential therapeutic target.This work has been funded by Projects PI15/00860 to GB, CP13/00226 to AB, PI11/01034 to MP and P14/00059 and PI17/01482 to MP and AB, all integrated in the National Plan for Scientific Research, Development and Technological Innovation 2013–2016 and funded by the ISCIII – General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) “A way of making Europe”. The study was also funded by the project IN607A 2016/22 (Consellería de Cultura, Educación e Ordenación Universitaria) to G.B. Also supported by Planes Nacionales de I+D+i 2008–2011 / 2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015/0014 and REIPI RD16/0016/006) co-financed by European Development Regional Fund “A way to achieve Europe” and operative program Intelligent Growth 2014–2020. J. A. Vallejo was financially supported by the Sara Borrell Programme (ISCIII, Spain CD13/00373), J.C. Vázquez-Ucha was financially supported by the Miguel Servet Programme (ISCIII, Spain CP13/00226) and M. Martínez-Guitián was financially supported by the grant Clara Roy (Spanish Society of Clinical Microbiology and Infectious Diseases). We thank M. I. Voskuil (Dept. of Immunology and Microbiology, University of Colorado Medical School, CO, USA) for providing pMo130.Xunta de Galicia. Consellería de Cultura, Educación e Ordenación Universitaria; IN607A 2016/2

A rapid and simple method for constructing stable mutants of Acinetobacter baumannii

<p>Abstract</p> <p>Background</p> <p><it>Acinetobacter baumannii </it>is a multidrug-resistant bacterium responsible for nosocomial infections in hospitals worldwide. Study of mutant phenotypes is fundamental for understanding gene function. The methodologies developed to inactivate <it>A. baumannii </it>genes are complicated and time-consuming; sometimes result in unstable mutants, and do not enable construction of double (or more) gene knockout mutant strains of <it>A. baumannii</it>.</p> <p>Results</p> <p>We describe here a rapid and simple method of obtaining <it>A. baumannii </it>mutants by gene replacement via double crossover recombination, by use of a PCR product that carries an antibiotic resistance cassette flanked by regions homologous to the target locus. To demonstrate the reproducibility of the approach, we produced mutants of three different chromosomal genes (<it>omp33</it>, <it>oxyR</it>, and <it>soxR</it>) by this method. In addition, we disrupted one of these genes (<it>omp33</it>) by integration of a plasmid into the chromosome by single crossover recombination, the most widely used method of obtaining <it>A. baumannii </it>mutants. Comparison of the different techniques revealed absolute stability when the gene was replaced by a double recombination event, whereas up to 40% of the population reverted to wild-type when the plasmid was disrupting the target gene after 10 passages in broth without selective pressure. Moreover, we demonstrate that the combination of both gene disruption and gene replacement techniques is an easy and useful procedure for obtaining double gene knockout mutants in <it>A. baumannii</it>.</p> <p>Conclusions</p> <p>This study provides a rapid and simple method of obtaining stable mutants of <it>A. baumannii </it>free of foreign plasmidic DNA, which does not require cloning steps, and enables construction of multiple gene knockout mutants.</p

Draft Genome Sequences of Two Epidemic OXA-48-Producing Klebsiella pneumoniae Clinical Strains Isolated during a Large Outbreak in Spain

[Abstract] We report here the draft genome sequences of Klebsiella pneumoniae strains Kp1803 and Kp3380 isolated during a large outbreak at A Coruña Hospital in Spain. The final genome assemblies for Kp1803 and Kp3380 comprise approximately 6.6 and 6.1 Mb, respectively, and both strains have G+C contents of 57.2%.This work was supported by grants from the Spanish Society of Infectious Diseases and Clinical Microbiology to A.P. and by projects PI11/01034 and P14/00059 to M.P., integrated in the National Plan for Scientific Research, Development and Technological Innovation 2013–2016 and funded by the Instituto de Salud Carlos III (ISCII), General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) “A Way of Making Europe.” We also thank the Spanish Network for Research in Infectious Diseases (REIPI RD12/0015/0014 to G.B.), cofinanced by the European Development Regional Fund (EDRF) and by the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad

Characterization of a limestone in a batch fluidized bed reactor for sulfur retention under oxy-fuel operating conditions

12 FiguresCO2 and SO2 are some of the main polluting gases emitted into atmosphere in combustion processes using fossil fuel for energy production. The former is one of the major contributors to build-up the greenhouse effect implicated in global climate change and the latter produces acid rain. Oxy-fuel combustion is a technology, which consists in burning the fuel with a mix of pure O2 and recirculated CO2. With this technology the CO2 concentration in the flue gas may be enriched up to 95%, becoming possible an easy CO2 recovery. In addition, oxy-fuel combustion in fluidized beds allows in situ desulfurization of combustion gases by supplying calcium based sorbent.In this work, the effect of the principal operation variables affecting the sulfation reaction rate in fluidized bed reactors (temperature, CO2 partial pressure, SO2 concentration and particle size) under typical oxy-fuel combustion conditions have been analyzed in a batch fluidized bed reactor using a limestone as sorbent. It has been observed that sulfur retention can be carried out by direct sulfation of the CaCO3 or by sulfation of the CaO (indirect sulfation) formed by CaCO3 calcination. Direct sulfation and indirect sulfation operating conditions depended on the temperature and CO2 partial pressure. The rate of direct sulfation rose with temperature and the rate of indirect sulfation for long reaction times decreased with temperature. An increase in the CO2 partial pressure had a negative influence on the sulfation conversion reached by the limestone due to a higher temperature was needed to work in conditions of indirect sulfation. Thus, it is expected that the optimum temperature for sulfur retention in oxy-fuel combustion in fluidized bed reactors be about 925-950°C. Sulfation reaction rate rose with decreasing sorbent particle size and increasing SO2 concentration.This research has been supported by Spanish Ministry of Science and Innovation (MICINN, Project: CTQ2008-05399/PPQ) and by FEDER. M. de las Obras-Loscertales thanks MICINN for the F.P.I. fellowship.Peer Reviewe