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Gut microbial induction of host immune maturation exemplifies host-microbe mutualism. We colonized germ-free (GF) mice with mouse microbiota (MMb) or human microbiota (HMb) to determine whether small intestinal immune maturation depends on a coevolved host-specific microbiota. Gut bacterial numbers and phylum abundance were similar in MMb and HMb mice, but bacterial species differed, especially the Firmicutes. HMb mouse intestines had low levels of CD4(+) and CD8(+) T cells, few proliferating T cells, few dendritic cells, and low antimicrobial peptide expression--all characteristics of GF mice. Rat microbiota also failed to fully expand intestinal T cell numbers in mice. Colonizing GF or HMb mice with mouse-segmented filamentous bacteria (SFB) partially restored T cell numbers, suggesting that SFB and other MMb organisms are required for full immune maturation in mice. Importantly, MMb conferred better protection against Salmonella infection than HMb. A host-specific microbiota appears to be critical for a healthy immune system.
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Inmunidad Innata , Intestinos/inmunología , Intestinos/microbiología , Metagenoma , Animales , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Proliferación Celular , Femenino , Vida Libre de Gérmenes , Humanos , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Infecciones por Salmonella/inmunología , Especificidad de la Especie , Organismos Libres de Patógenos Específicos , Simbiosis , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: The possibility of recovering metagenome-assembled genomes (MAGs) from sequence reads allows for further insights into microbial communities and their members, possibly even analyzing such sequences with tools designed for single-isolate genomes. As result quality depends on sequence quality, performance of tools for single-isolate genomes on MAGs should be tested beforehand. Bioinformatics can be leveraged to quickly create varied synthetic test sets with known composition for this purpose. RESULTS: We present MAGICIAN, a flexible, user-friendly pipeline for the simulation of MAGs. MAGICIAN combines a synthetic metagenome simulator with a metagenomic assembly and binning pipeline to simulate MAGs based on user-supplied input genomes, allowing users to test performance of tools on MAGs while having a ground truth to compare results to. Using MAGICIAN, we found that even very slight (1%) changes in depth of coverage can drastically affect whether a genome can be recovered. We also demonstrate the use of simulated MAGs by evaluating the suitability of such genomes obtained with MAGICIAN's current default pipeline for analysis with the antimicrobial resistance gene identification tool ResFinder. CONCLUSIONS: Using MAGICIAN, it is possible to simulate MAGs which, while generally high in quality, reflect issues encountered with real-world data, thus providing realistic best-case data. Evaluating the results of ResFinder analysis of these genomes revealed a risk for plausible-looking false positives, which underlines the need for pipeline validation so that researchers are aware of the potential issues when interpreting real-world data. Furthermore, the effects of fluctuations in depth of coverage on genome recovery in our simulated "random sequencing" warrant further investigation and indicate random subsampling of reads may affect discovery of more genomes.
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Metagenoma , Microbiota , Simulación por Computador , Microbiota/genética , Metagenómica/métodos , Biología ComputacionalRESUMEN
Wastewaters serve as important hot spots for antimicrobial resistance and monitoring can be used to analyse the abundance and diversity of antimicrobial resistance genes at the level of large bacterial and human populations. In this study, whole genome sequencing of beta-lactamase-producing Escherichia coli and metagenomic analysis of whole-community DNA were used to characterize the occurrence of antimicrobial resistance in hospital, municipal and river waters in the city of Brno (Czech Republic). Cefotaxime-resistant E. coli were mainly extended-spectrum beta-lactamase (ESBL) producers (95.6%, n = 158), of which the majority carried blaCTX-M (98.7%; n = 151) and were detected in all water samples except the outflow from hospital wastewater treatment plant. A wide phylogenetic diversity was observed among the sequenced E. coli (n = 78) based on the detection of 40 sequence types and single nucleotide polymorphisms (average number 34,666 ± 15,710) between strains. The metagenomic analysis revealed a high occurrence of bacterial genera with potentially pathogenic members, including Pseudomonas, Escherichia, Klebsiella, Aeromonas, Enterobacter and Arcobacter (relative abundance >50%) in untreated hospital and municipal wastewaters and predominance of environmental bacteria in treated and river waters. Genes encoding resistance to aminoglycosides, beta-lactams, quinolones and macrolides were frequently detected, however blaCTX-M was not found in this dataset which may be affected by insufficient sequencing depth of the samples. The study pointed out municipal treated wastewater as a possible source of multi-drug resistant E. coli and antimicrobial resistance genes for surface waters. Moreover, the combination of two different approaches provided a more holistic view on antimicrobial resistance in water environments. The culture-based approach facilitated insight into the dynamics of ESBL-producing E. coli and the metagenomics shows abundance and diversity of bacteria and antimicrobial resistance genes vary across water sites.
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Escherichia coli , Aguas Residuales , Antibacterianos/farmacología , República Checa , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Hospitales , Humanos , Metagenómica , Filogenia , beta-Lactamasas/genéticaRESUMEN
Intestinal mucositis is a common side effect of chemotherapy leading to diarrhea, abdominal pain and increased risk of infections. The intestinal microbiota has been recognized as a key regulator of mucosal immune responses. Therefore, we hypothesized that intestinal microbial changes would be associated with enterocyte loss and systemic inflammation during induction treatment for childhood acute lymphoblastic leukemia (ALL). We prospectively included 51 children newly-diagnosed with ALL treated in Denmark in 2015-2018. Plasma C-reactive protein (CRP), plasma citrulline (marker of functional enterocytes mass) measurements and fecal samplings were performed on treatment Days 1, 8, 15, 22 and 29. Moreover, intestinal mucositis was scored by a trained nurse/physician. Fecal samples in patients and 19 healthy siblings were analyzed by 16S rRNA gene sequencing (V3-V4 region). Bacterial alpha diversity was lower in patients compared to siblings. It decreased from Day 1 to Days 8-22 and increased on Day 29. Shannon alpha diversity index was correlated with CRP on Days 15-29 (rho = -0.33-0.49; p < 0.05) and with citrulline on Days 15 and 29 (although with p values <0.06, rho = 0.32-0.34). The abundance of unclassified Enterococcus species (spp.) was correlated with CRP on Days 22-29 (rho = 0.42-0.49; p < 0.009), while the abundance of unclassified Lachnospiraceae spp. was correlated with citrulline on days 8-15 (rho = 0.48-0.62, p < 0.001). Systemic inflammation, enterocyte loss and relative abundance of unclassified Enterococcus spp. reached a peak around Day 15. In conclusion, specific changes in the microbiota were associated with the severity of enterocyte loss and systemic inflammation during chemotherapy.
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Bacterias/clasificación , Microbioma Gastrointestinal/efectos de los fármacos , Quimioterapia de Inducción/efectos adversos , Mucositis/inducido químicamente , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Análisis de Secuencia de ADN/métodos , Adolescente , Bacterias/genética , Bacterias/aislamiento & purificación , Estudios de Casos y Controles , Niño , Preescolar , ADN Bacteriano/genética , ADN Ribosómico/genética , Heces/microbiología , Femenino , Humanos , Lactante , Masculino , Mucositis/microbiología , Filogenia , Leucemia-Linfoma Linfoblástico de Células Precursoras/microbiología , Estudios Prospectivos , ARN Ribosómico 16S/genética , HermanosRESUMEN
Segmented filamentous bacteria (SFB) are host-specific intestinal symbionts that comprise a distinct clade within the Clostridiaceae, designated Candidatus Arthromitus. SFB display a unique life cycle within the host, involving differentiation into multiple cell types. The latter include filaments that attach intimately to intestinal epithelial cells, and from which "holdfasts" and spores develop. SFB induce a multifaceted immune response, leading to host protection from intestinal pathogens. Cultivation resistance has hindered characterization of these enigmatic bacteria. In the present study, we isolated five SFB filaments from a mouse using a microfluidic device equipped with laser tweezers, generated genome sequences from each, and compared these sequences with each other, as well as to recently published SFB genome sequences. Based on the resulting analyses, SFB appear to be dependent on the host for a variety of essential nutrients. SFB have a relatively high abundance of predicted proteins devoted to cell cycle control and to envelope biogenesis, and have a group of SFB-specific autolysins and a dynamin-like protein. Among the five filament genomes, an average of 8.6% of predicted proteins were novel, including a family of secreted SFB-specific proteins. Four ADP-ribosyltransferase (ADPRT) sequence types, and a myosin-cross-reactive antigen (MCRA) protein were discovered; we hypothesize that they are involved in modulation of host responses. The presence of polymorphisms among mouse SFB genomes suggests the evolution of distinct SFB lineages. Overall, our results reveal several aspects of SFB adaptation to the mammalian intestinal tract.
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Proteínas Bacterianas/genética , Genoma Bacteriano , Bacterias Grampositivas Formadoras de Endosporas/fisiología , Intestinos/microbiología , Análisis de la Célula Individual/métodos , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/metabolismo , Adaptación Fisiológica , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/metabolismo , Diferenciación Celular/genética , ADN Ribosómico , Células Epiteliales/microbiología , Bacterias Grampositivas Formadoras de Endosporas/genética , Ratones , Técnicas Analíticas Microfluídicas , Datos de Secuencia Molecular , Filogenia , Polimorfismo Genético , Análisis de Secuencia de ADNRESUMEN
Confined spatial patterns of microbial distribution are prevalent in nature, such as in microbial mats, soil communities, and water stream biofilms. The symbiotic two-species consortium of Pseudomonas putida and Acinetobacter sp. strain C6, originally isolated from a creosote-polluted aquifer, has evolved a distinct spatial organization in the laboratory that is characterized by an increased fitness and productivity. In this consortium, P. putida is reliant on microcolonies formed by Acinetobacter sp. C6, to which it attaches. Here we describe the processes that lead to the microcolony pattern by Acinetobacter sp. C6. Ecological spatial pattern analyses revealed that the microcolonies were not entirely randomly distributed and instead were arranged in a uniform pattern. Detailed time-lapse confocal microscopy at the single-cell level demonstrated that the spatial pattern was the result of an intriguing self-organization: small multicellular clusters moved along the surface to fuse with one another to form microcolonies. This active distribution capability was dependent on environmental factors (carbon source and oxygen) and historical contingency (formation of phenotypic variants). The findings of this study are discussed in the context of species distribution patterns observed in macroecology, and we summarize observations about the processes involved in coadaptation between P. putida and Acinetobacter sp. C6. Our results contribute to an understanding of spatial species distribution patterns as they are observed in nature, as well as the ecology of engineered communities that have the potential for enhanced and sustainable bioprocessing capacity.
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Acinetobacter/fisiología , Biopelículas/crecimiento & desarrollo , Consorcios Microbianos , Pseudomonas putida/fisiología , Microscopía Confocal , Imagen de Lapso de TiempoRESUMEN
Metagenomics is increasingly used to describe microbial communities in biological specimens. Ideally, the steps involved in the processing of the biological specimens should not change the microbiome composition in a way that it could lead to false interpretations of inferred microbial community composition. Common steps in sample preparation include sample collection, storage, DNA isolation, library preparation, and DNA sequencing. Here, we assess the effect of three library preparation kits and two DNA sequencing platforms. Of the library preparation kits, one involved a PCR step (Nextera), and two were PCR free (NEXTflex and KAPA). We sequenced the libraries on Illumina HiSeq and NextSeq platforms. As example microbiomes, two pig fecal samples and two sewage samples of which aliquots were stored at different storage conditions (immediate processing and storage at -80°C) were assessed. All DNA isolations were performed in duplicate, totaling 80 samples, excluding controls. We found that both library preparation and sequencing platform had systematic effects on the inferred microbial community composition. The different sequencing platforms introduced more variation than library preparation and freezing the samples. The results highlight that all sample processing steps need to be considered when comparing studies. Standardization of sample processing is key to generating comparable data within a study, and comparisons of differently generated data, such as in a meta-analysis, should be performed cautiously. IMPORTANCE Previous research has reported effects of sample storage conditions and DNA isolation procedures on metagenomics-based microbiome composition; however, the effect of library preparation and DNA sequencing in metagenomics has not been thoroughly assessed. Here, we provide evidence that library preparation and sequencing platform introduce systematic biases in the metagenomic-based characterization of microbial communities. These findings suggest that library preparation and sequencing are important parameters to keep consistent when aiming to detect small changes in microbiome community structure. Overall, we recommend that all samples in a microbiome study are processed in the same way to limit unwanted variations that could lead to false conclusions. Furthermore, if we are to obtain a more holistic insight from microbiome data generated around the world, we will need to provide more detailed sample metadata, including information about the different sample processing procedures, together with the DNA sequencing data at the public repositories.
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Metagenómica , Microbiota , Animales , Bacterias/genética , Sesgo , ADN , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Metagenómica/métodos , Microbiota/genética , Análisis de Secuencia de ADN/métodos , PorcinosRESUMEN
Poly(dimethylsiloxane) (PDMS) is an attractive, versatile, and convenient material for use in biomedical devices that are in direct contact with the user. A crucial component in such a device is its surface in terms of antimicrobial properties preventing infection. Moreover, due to its inherent hydrophobicity, PDMS is rather prone to microbial colonization. Thus, developing an antimicrobial PDMS surface in a simple, large-scale, and applicable manner is an essential step in fully exploiting PDMS in the biomedical device industry. Current chemical modification methods for PDMS surfaces are limited; therefore, we present herein a new method for introducing an atom transfer radical polymerization (ATRP) initiator onto the PDMS surface via the base-catalyzed grafting of [(chloromethyl)phenylethyl]trimethoxysilane to the PDMS. The initiator surface was grafted with poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) brushes via a surface-initiated supplemental activator and reducing agent ATRP (SI-SARA-ATRP). The use of sodium sulfite as a novel reducing agent in SI-SARA-ATRP allowed for polymerization during complete exposure to air. Moreover, a fast and linear growth was observed for the polymer over time, leading to a 400 nm thick polymer layer in a 120 min reaction time. Furthermore, the grafted PDMAEMA was quaternized, using various alkylhalides, in order to study the effect on surface antimicrobial properties. It was shown that antimicrobial activity not only depended highly on the charge density but also on the amphiphilicity of the surface. The fast reaction rate, high oxygen tolerance, increased antimicrobial activity, and the overall robustness and simplicity of the presented method collectively move PDMS closer to its full-scale exploitation in biomedical devices.
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The impact of immune mediators on weight homeostasis remains underdefined. Interrogation of resistance to diet-induced obesity in mice lacking a negative regulator of Toll-like receptor signaling serendipitously uncovered a role for B cell activating factor (BAFF). Here we show that overexpression of BAFF in multiple mouse models associates with protection from weight gain, approximating a log-linear dose response relation to BAFF concentrations. Gene expression analysis of BAFF-stimulated subcutaneous white adipocytes unveils upregulation of lipid metabolism pathways, with BAFF inducing white adipose tissue (WAT) lipolysis. Brown adipose tissue (BAT) from BAFF-overexpressing mice exhibits increased Ucp1 expression and BAFF promotes brown adipocyte respiration and in vivo energy expenditure. A proliferation-inducing ligand (APRIL), a BAFF homolog, similarly modulates WAT and BAT lipid handling. Genetic deletion of both BAFF and APRIL augments diet-induced obesity. Lastly, BAFF/APRIL effects are conserved in human adipocytes and higher BAFF/APRIL levels correlate with greater BMI decrease after bariatric surgery. Together, the BAFF/APRIL axis is a multifaceted immune regulator of weight gain and adipose tissue function.
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Factor Activador de Células B/genética , Obesidad/genética , Transducción de Señal/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Aumento de Peso/genética , Adipocitos/citología , Adipocitos/metabolismo , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/citología , Tejido Adiposo Blanco/metabolismo , Animales , Factor Activador de Células B/metabolismo , Células Cultivadas , Dieta Alta en Grasa/efectos adversos , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Obesidad/etiología , Obesidad/metabolismo , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismoRESUMEN
Metagenomics-based high-throughput sequencing (HTS) enables comprehensive detection of all species comprised in a sample with a single assay and is becoming a standard method for outbreak investigation. However, unlike real-time PCR or serological assays, HTS datasets generated for pathogen detection do not easily provide yes/no answers. Rather, results of the taxonomic read assignment need to be assessed by trained personnel to gain information thereof. Proficiency tests are important instruments of validation, harmonization, and standardization. Within the European Union funded project COMPARE [COllaborative Management Platform for detection and Analyses of (Re-) emerging and foodborne outbreaks in Europe], we conducted a proficiency test to scrutinize the ability to assess diagnostic metagenomics data. An artificial dataset resembling shotgun sequencing of RNA from a sample of contaminated trout was provided to 12 participants with the request to provide a table with per-read taxonomic assignments at species level and a report with a summary and assessment of their findings, considering different categories like pathogen, background, or contaminations. Analysis of the read assignment tables showed that the software used reliably classified the reads taxonomically overall. However, usage of incomplete reference databases or inappropriate data pre-processing caused difficulties. From the combination of the participants' reports with their read assignments, we conclude that, although most species were detected, a number of important taxa were not or not correctly categorized. This implies that knowledge of and awareness for potentially dangerous species and contaminations need to be improved, hence, capacity building for the interpretation of diagnostic metagenomics datasets is necessary.
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BACKGROUND: Worldwide, the number of emerging and re-emerging infectious diseases is increasing, highlighting the importance of global disease pathogen surveillance. Traditional population-based methods may fail to capture important events, particularly in settings with limited access to health care, such as urban informal settlements. In such environments, a mixture of surface water runoff and human feces containing pathogenic microorganisms could be used as a surveillance surrogate. METHOD: We conducted a temporal metagenomic analysis of urban sewage from Kibera, an urban informal settlement in Nairobi, Kenya, to detect and quantify bacterial and associated antimicrobial resistance (AMR) determinants, viral and parasitic pathogens. Data were examined in conjunction with data from ongoing clinical infectious disease surveillance. RESULTS: A large variation of read abundances related to bacteria, viruses, and parasites of medical importance, as well as bacterial associated antimicrobial resistance genes over time were detected. Significant increased abundances were observed for a number of bacterial pathogens coinciding with higher abundances of AMR genes. Vibrio cholerae as well as rotavirus A, among other virus peaked in several weeks during the study period whereas Cryptosporidium spp. and Giardia spp, varied more over time. CONCLUSION: The metagenomic surveillance approach for monitoring circulating pathogens in sewage was able to detect putative pathogen and resistance loads in an urban informal settlement. Thus, valuable if generated in real time to serve as a comprehensive infectious disease agent surveillance system with the potential to guide disease prevention and treatment. The approach may lead to a paradigm shift in conducting real-time global genomics-based surveillance in settings with limited access to health care.
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Bacterias/genética , Enfermedades Transmisibles/genética , Metagenoma/genética , Microbiología del Agua , Animales , Bacterias/patogenicidad , Enfermedades Transmisibles/microbiología , Enfermedades Transmisibles/parasitología , Enfermedades Transmisibles/virología , Farmacorresistencia Bacteriana/genética , Heces/microbiología , Heces/parasitología , Heces/virología , Humanos , Kenia/epidemiología , Metagenómica/métodos , Parásitos/genética , Parásitos/patogenicidad , Aceptación de la Atención de Salud , Aguas del Alcantarillado/microbiología , Aguas del Alcantarillado/parasitología , Aguas del Alcantarillado/virología , Virus/genética , Virus/patogenicidad , Agua/análisisRESUMEN
Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use metagenomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.
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Bacterias/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Genes Bacterianos , Metagenoma , Aguas del Alcantarillado/microbiología , África , Asia , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Monitoreo Epidemiológico , Europa (Continente) , Humanos , Metagenómica/métodos , Consorcios Microbianos/efectos de los fármacos , Consorcios Microbianos/genética , América del Norte , Oceanía , Salud Poblacional , Factores Socioeconómicos , América del SurRESUMEN
When grown as a biofilm in laboratory flow chambers Pseudomonas aeruginosa can develop mushroom-shaped multicellular structures consisting of distinct subpopulations in the cap and stalk portions. We have previously presented evidence that formation of the cap portion of the mushroom-shaped structures in P. aeruginosa biofilms occurs via bacterial migration and depends on type IV pili (Mol Microbiol 50: 61-68). In the present study we examine additional factors involved in the formation of this multicellular substructure. While pilA mutants, lacking type IV pili, are deficient in mushroom cap formation, pilH and chpA mutants, which are inactivated in the type IV pili-linked chemosensory system, showed only minor defects in cap formation. On the contrary, fliM mutants, which are non-flagellated, and cheY mutants, which are inactivated in the flagellum-linked chemotaxis system, were largely deficient in cap formation. Experiments involving DNase treatment of developing biofilms provided evidence that extracellular DNA plays a role in cap formation. Moreover, mutants that are deficient in quorum sensing-controlled DNA release formed microcolonies upon which wild-type bacteria could not form caps. These results constitute evidence that type IV pili, flagellum-mediated motility and quorum sensing-controlled DNA release are involved in the formation of mature multicellular structures in P. aeruginosa biofilms.
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Biopelículas/crecimiento & desarrollo , ADN Bacteriano/metabolismo , Fimbrias Bacterianas/genética , Flagelos/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Percepción de Quorum , Adhesión Bacteriana , Quimiotaxis , ADN Bacteriano/genética , Matriz Extracelular/genética , Matriz Extracelular/microbiología , Proteínas Fimbrias/genética , Genes Bacterianos , Prueba de Complementación Genética , Genotipo , Microscopía Confocal , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMEN
Explorations of complex microbiomes using genomics greatly enhance our understanding about their diversity, biogeography, and function. The isolation of DNA from microbiome specimens is a key prerequisite for such examinations, but challenges remain in obtaining sufficient DNA quantities required for certain sequencing approaches, achieving accurate genomic inference of microbiome composition, and facilitating comparability of findings across specimen types and sequencing projects. These aspects are particularly relevant for the genomics-based global surveillance of infectious agents and antimicrobial resistance from different reservoirs. Here, we compare in a stepwise approach a total of eight commercially available DNA extraction kits and 16 procedures based on these for three specimen types (human feces, pig feces, and hospital sewage). We assess DNA extraction using spike-in controls and different types of beads for bead beating, facilitating cell lysis. We evaluate DNA concentration, purity, and stability and microbial community composition using 16S rRNA gene sequencing and for selected samples using shotgun metagenomic sequencing. Our results suggest that inferred community composition was dependent on inherent specimen properties as well as DNA extraction method. We further show that bead beating or enzymatic treatment can increase the extraction of DNA from Gram-positive bacteria. Final DNA quantities could be increased by isolating DNA from a larger volume of cell lysate than that in standard protocols. Based on this insight, we designed an improved DNA isolation procedure optimized for microbiome genomics that can be used for the three examined specimen types and potentially also for other biological specimens. A standard operating procedure is available from https://dx.doi.org/10.6084/m9.figshare.3475406. IMPORTANCE Sequencing-based analyses of microbiomes may lead to a breakthrough in our understanding of the microbial worlds associated with humans, animals, and the environment. Such insight could further the development of innovative ecosystem management approaches for the protection of our natural resources and the design of more effective and sustainable solutions to prevent and control infectious diseases. Genome sequence information is an organism (pathogen)-independent language that can be used across sectors, space, and time. Harmonized standards, protocols, and workflows for sample processing and analysis can facilitate the generation of such actionable information. In this study, we assessed several procedures for the isolation of DNA for next-generation sequencing. Our study highlights several important aspects to consider in the design and conduct of sequence-based analysis of microbiomes. We provide a standard operating procedure for the isolation of DNA from a range of biological specimens particularly relevant in clinical diagnostics and epidemiology.
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The indigenous microbiota of the nasal cavity plays important roles in human health and disease. Patterns of spatial variation in microbiota composition may help explain Staphylococcus aureus colonization and reveal interspecies and species-host interactions. To assess the biogeography of the nasal microbiota, we sampled healthy subjects, representing both S. aureus carriers and noncarriers at three nasal sites (anterior naris, middle meatus, and sphenoethmoidal recess). Phylogenetic compositional and sparse linear discriminant analyses revealed communities that differed according to site epithelium type and S. aureus culture-based carriage status. Corynebacterium accolens and C. pseudodiphtheriticum were identified as the most important microbial community determinants of S. aureus carriage, and competitive interactions were only evident at sites with ciliated pseudostratified columnar epithelium. In vitro cocultivation experiments provided supporting evidence of interactions among these species. These results highlight spatial variation in nasal microbial communities and differences in community composition between S. aureus carriers and noncarriers.
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Antibiosis , Biota , Portador Sano/microbiología , Mucosa Nasal/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/crecimiento & desarrollo , Corynebacterium/crecimiento & desarrollo , HumanosRESUMEN
We review the recent advances in the understanding of the Pseudomonas aeruginosa biofilm lifestyle from studies using in vitro laboratory setups such as flow chambers and microtiter trays. Recent work sheds light on the role of nutrients, motility, and quorum sensing in structure formation in P. aeruginosa biofilms. The second messenger, c-di-GMP, is established as an important regulator of the synthesis of polysaccharide and protein components of the biofilm matrix. Extracellular DNA is shown to be an essential component of the biofilm matrix. It has become apparent that biofilm formation involves interactions between different subpopulations. The molecular mechanisms underlying the tolerance of biofilm bacteria to antimicrobial agents are beginning to be unraveled, and new knowledge has been obtained regarding the environmental cues and regulatory mechanisms involved in biofilm dispersal.