Dominance of phage particles carrying antibiotic resistance genes in the viromes of retail food sources

The growth of antibiotic resistance has stimulated interest in understanding the mechanisms by which antibiotic resistance genes (ARG) are mobilized. Among them, studies analyzing the presence of ARGs in the viral fraction of environmental, food and human samples, and reporting bacteriophages as vehicles of ARG transmission, have been the focus of increasing research. However, it has been argued that in these studies the abundance of phages carrying ARGs has been overestimated due to experimental contamination with non-packaged bacterial DNA or other elements such as outer membrane vesicles (OMVs). This study aims to shed light on the extent to which phages, OMVs or contaminating non-packaged DNA contribute as carriers of ARGs in the viromes. The viral fractions of three types of food (chicken, fish, and mussels) were selected as sources of ARG-carrying phage particles, whose ability to infect and propagate in an Escherichia coli host was confirmed after isolation. The ARG-containing fraction was further purified by CsCl density gradient centrifugation and, after removal of DNA outside the capsids, ARGs inside the particles were confirmed. The purified fraction was stained with SYBR Gold, which allowed the visualization of phage capsids attached to and infecting E. coli cells. Phages with Myoviridae and Siphoviridae morphology were observed by electron microscopy. The proteins in the purified fraction belonged predominantly to phages (71.8% in fish, 52.9% in mussels, 78.7% in chicken sample 1, and 64.1% in chicken sample 2), mainly corresponding to tail, capsid, and other structural proteins, whereas membrane proteins, expected to be abundant if OMVs were present, accounted for only 3.8–21.4% of the protein content. The predominance of phage particles in the viromes supports the reliability of the protocols used in this study and in recent findings on the abundance of ARG-carrying phage particles.This work was supported by the Spanish Ministerio de Ciencia e Innovación (PID2020-113355GB-I00), the Agencia Estatal de Investigación (AEI) and the European regional fund (ERF). The study was partially supported by the Generalitat de Catalunya (2017SGR170). PB-P has a grant from the Spanish Ministry of Economy, Industry and Competitiveness (BES-2017-081296), SM-C has a grant from Colciencias (Republic of Colombia) and LR-R is lecturer of the Serra-Hunter program, Generalitat de Catalunya. MDR-B has a Margarita Salas fellowship from the Spanish Ministerio de Universidades

Data-driven research into the inaccuracy of traditional models of thermal comfort in offices

The accurate prediction of thermal sensation among office workers, at design and post-occupancy stages, is crucial for controlling indoor temperature efficiently and correcting deficiencies in workspaces, ensuring healthy and productive working conditions. Traditional analytical comfort models are still the best tool for this purpose given their potential for interpretation. However, their reliability is undermined by their poor accuracy. Based on 304 data series of point-in-time measurements of quantitative and qualitative comfort-related parameters collected in an experimental campaign in three office buildings, one air-conditioned and two in free evolution, in San Luis Potosí (Mexico), this work aims to identify the major error-causing factors of steady and adaptive comfort models. The divergences between predicted and reported thermal sensation were set as a dependant variable of two multiple regressions, one for each model. Eighteen independent demographic, environmental, contextual and subjective variables were considered. No multicollinearity problems were identified. Our findings show that contextual factors and humidity perception were relevant in the adaptive model error. Clothing insulation highly impacted the accuracy of both models while age and body mass were not statistically significant for either of them. Metabolic rate was the factor with the greatest influence in the error of the steady model. Although not covered, other influential factors played a key role in models’ accuracy and further research is needed to integrate these in a new generation of more accurate and flexible analytical models

Ancient saltern metagenomics: tracking changes in microbes and their viruses from the underground to the surface

Microbial communities in hypersaline underground waters derive from ancient organisms trapped within the evaporitic salt crystals and are part of the poorly known subterranean biosphere. Here, we characterized the viral and prokaryotic assemblages present in the hypersaline springs that dissolve Triassic-Keuper evaporite rocks and feed the Anana Salt Valley (Araba/Alava, Basque Country, Spain). Four underground water samples (around 23% total salinity) with different levels of exposure to the open air were analysed by means of microscopy and metagenomics. Cells and viruses in the spring water had lower concentrations than what are normally found in hypersaline environments and seemed to be mostly inactive. Upon exposure to the open air, there was an increase in activity of both cells and viruses as well as a selection of phylotypes. The underground water was inhabited by a rich community harbouring a diverse set of genes coding for retinal binding proteins. A total of 35 viral contigs from 15 to 104 kb, representing partial or total viral genomes, were assembled and their evolutionary changes through the spring system were followed by SNP analysis and metagenomic island tracking. Overall, both the viral and the prokaryotic assemblages changed quickly upon exposure to the open air conditions.We would like to thank Anana Salt Valley Foundation, and Andoni Erkiaga Agirre, its director at the time of sampling, for their kind help. Thanks to Leire Arana, Edorta Loma and Kika Colom for their help with sampling and to Eduardo Gonzalez-Pastor for telling us about the Anana Salt Valley. We thank Heather Maughan for the professional English editing and the critical reading of the manuscript and Esther Rubio-Portillo for her help with statistical analyses. This work was funded by the Spanish Ministry of Science, Innovation and Universities grant MICROMATES (PGC2018-096956-B-C41 and C44, to J.A./F.S. and R.R.-M.), which was also supported with European Regional Development Fund (FEDER) funds, and by the Generalitat Valenciana grant PROMETEO/2017/129. Documen

Predominance of deterministic microbial community dynamics in salterns exposed to different light intensities

While the dynamics of microbial community assembly driven by environmental perturbations have been extensively studied, our understanding is far from complete, particularly for light‐induced perturbations. Extremely halophilic communities thriving in coastal solar salterns are mainly influenced by two environmental factors—salt concentrations and high sunlight irradiation. By experimentally manipulating light intensity through the application of shading, we showed that light acts as a deterministic factor that ultimately drives the establishment of recurrent microbial communities under near‐saturation salt concentrations. In particular, the stable and highly change‐resistant communities that established under high‐light intensities were dominated (>90% of metagenomic reads) by Haloquadratum spp. and Salinibacter spp. On the other hand, under 37‐fold lower light intensity, different, less stable and change‐resistant communities were established, mainly dominated by yet unclassified haloarchaea and relatively diverse photosynthetic microorganisms. These communities harboured, in general, much lower carotenoid pigment content than their high‐irradiation counterparts. Both assemblage types appeared to be highly resilient, re‐establishing when favourable conditions returned after perturbation (i.e. high‐irradiation for the former communities and low‐irradiation for the latter ones). Overall, our results revealed that stochastic processes were of limited significance to explain these patterns.This study was funded by the Spanish Ministry of Economy projects CGL2012-39627-C03-03 CLG2015_66686-C3-1-P and PGC2018-096956-B-C41 (to R.R.M.), CGL2015_66686-C3-3-P (to J.A.) and CGL2015_66686-C3-2-P (to J.E.G.P.), which were also supported with European Regional Development Fund (FEDER) funds. R.A. was funded by the Max Planck Society. KTK’s research was supported, in part, by the U.S. National Science Foundation (Award No. 1831582). T.V.P. received a pre-doctoral fellowship (No. BES-2013-064420) from the Spanish Government Ministry for Finance and Competition. R.R.M. acknowledges the financial support of the sabbatical stay at Georgia Tech supported by the Grant PRX18/00048 of the Ministry of Sciences, Innovation and Universities

Environmental dissolved DNA harbours meaningful biological information on microbial community structure

Extracellular DNA (eDNA) comprises all the DNA molecules outside cells. This component of microbial ecosystems may serve as a source of nutrients and genetic information. Hypersaline environments harbour one of the highest concentrations of eDNA reported for natural systems, which has been attributed to the physicochemical preservative effect of salts and to high viral abundance. Here, we compared centrifugation and filtration protocols for the extraction of dissolved DNA (dDNA, as opposed to eDNA that also includes DNA from free viral particles) from a solar saltern crystallizer pond (CR30) water sample. The crystallizer dDNA fraction has been characterized, for the first time, and compared with cellular and viral metagenomes from the same location. High‐speed centrifugation affected CR30 dDNA concentration and composition due to cell lysis, highlighting that protocol optimization should be the first step in dDNA studies. Crystallizer dDNA, which accounted for lower concentrations than those previously reported for hypersaline anoxic sediments, had a mixed viral and cellular origin, was enriched in archaeal DNA and had a distinctive taxonomic composition compared to that from the cellular assemblage of the same sample. Bioinformatic analyses indicated that nanohaloarchaeal viruses could be a cause for these differences.This research was supported by the Spanish Ministry of Science, Innovation and Universities grant MICROMATES (PGC2018-096956-B-C44), which was also supported with European Regional Development Fund (FEDER) funds, and by the Generalitat Valenciana grant PROMETEO/2017/129. B.A.-R. is a ACIF (Generalitat Valenciana) fellow

Ancient saltern metagenomics: tracking changes in microbes and their viruses from the underground to the surface

Microbial communities in hypersaline underground waters derive from ancient organisms trapped within the evaporitic salt crystals and are part of the poorly known subterranean biosphere. Here, we characterized the viral and prokaryotic assemblages present in the hypersaline springs that dissolve Triassic-Keuper evaporite rocks and feed the Añana Salt Valley (Araba/Alava, Basque Country, Spain). Four underground water samples (around 23% total salinity) with different levels of exposure to the open air were analyzed by means of microscopy and metagenomics. Cells and viruses in the spring water had lower concentrations than what are normally found in hypersaline environments and seemed to be mostly inactive. Upon exposure to the open air, there was an increase in activity of both cells and viruses as well as a selection of phylotypes. The underground water was inhabited by a rich community harboring a diverse set of genes coding for retinal binding proteins. A total of 35 viral contigs from 15 to 104 Kb, representing partial or total viral genomes, were assembled and their evolutionary changes through the spring system were followed by SNP analysis and metagenomic island tracking. Overall, both the viral and the prokaryotic assemblages changed quickly upon exposure to the open air conditions.This work was funded by the Spanish Ministry of Science, Innovation and Universities grant MICROMATES (PGC2018-096956-B-C41 and C44, to J.A./F.S. and R.R.M), which was also supported with European Regional Development Fund (FEDER) funds, and by the Generalitat Valenciana grant PROMETEO/2017/129

Prokaryotic and viral community structure in the singular chaotropic salt lake Salar de Uyuni

Salar de Uyuni (SdU) is the largest hypersaline salt flat and the highest lithium reservoir on Earth. In addition to extreme temperatures and high UV irradiance, SdU has high concentrations of chaotropic salts which can be important factors in controlling microbial diversity. Here, for the first time we characterize the viral diversity of this hypersaline environment during the two seasons, as well as the physicochemical characteristics and the prokaryotic communities of the analysed samples. Most of the selected samples showed a peculiar physicochemical composition and prokaryotic diversity, mostly different from each other even for samples from locations in close proximity or the same season. In contrast to most hypersaline systems Bacteria frequently outnumbered Archaea. Furthermore, an outstanding percentage of members of Salinibacter sp., likely a species different from the cosmopolitan Salinibacter ruber, was obtained in most of the samples. Viral communities displayed the morphologies normally found in hypersaline environments. Two seasonal samples were chosen for a detailed metagenomic analysis of the viral assemblage. Both viral communities shared common sequences but were dominated by sample‐specific viruses, mirroring the differences also observed in physicochemical and prokaryotic community composition. These metaviromes were distinct from those detected in other hypersaline systems analysed to date.This research was supported by the MINECO projects CLG2015_66686-C3-3 (to JA) and CGL2015-66242-R (to RA), which were also supported with European Regional Development Fund (FEDER) funds

Recovering microbial genomes from metagenomes in hypersaline environments: The Good, the Bad and the Ugly

Current metagenomic tools allow the recovery of microbial genomes directly from the environment. This can be accomplished by binning metagenomic contigs according to their coverage and tetranucleotide frequency, followed by an estimation of the bin quality. The public availability of bioinformatics tools, together with the decreasing cost of next generation sequencing, are democratizing this powerful approach that is spreading from specialized research groups to the general public. Using metagenomes from hypersaline environments, as well as mock metagenomes composed of Archaea and Bacteria frequently found in these systems, we have analyzed the advantages and difficulties of the binning process in these extreme environments to tackle microbial population diversity. These extreme systems harbor relatively low species diversity but high intraspecific diversity, which can compromise metagenome assembly and therefore the whole binning process. The main goal is to compare the output of the binning process with what is previously known from the analyzed samples, based on years of study using different approaches. Several scenarios have been analyzed in detail: (i) a good quality bin from a species highly abundant in the environment; (ii) an intermediate quality bin with incongruences that can be solved by further analyses and manual curation, and (iii) a low-quality bin to investigate the failure to recover a very abundant microbial genome as well as some possible solutions. The latter can be considered the “great metagenomics anomaly” and is mainly due to assembly problems derived from the microdiversity of naturally co-existing populations in nature.This research was supported by the Spanish Ministry of Economy project CLG2015 66686-C3-3 (to JA), which also included European Regional Development Fund (FEDER) funds

Prokaryotic and viral community structure in the singular chaotropic salt lake Salar de Uyuni

Salar de Uyuni (SdU) is the largest hypersaline salt flat and the highest lithium reservoir on Earth. In addition to extreme temperatures and high UV irradiance, SdU has high concentrations of chaotropic salts which can be important factors in controlling microbial diversity. Here, for the first time we characterize the viral diversity of this hypersaline environment during the two seasons, as well as the physicochemical characteristics and the prokaryotic communities of the analysed samples. Most of the selected samples showed a peculiar physicochemical composition and prokaryotic diversity, mostly different from each other even for samples from locations in close proximity or the same season. In contrast to most hypersaline systems Bacteria frequently outnumbered Archaea. Furthermore, an outstanding percentage of members of Salinibacter sp., likely a species different from the cosmopolitan Salinibacter ruber, was obtained in most of the samples. Viral communities displayed the morphologies normally found in hypersaline environments. Two seasonal samples were chosen for a detailed metagenomic analysis of the viral assemblage. Both viral communities shared common sequences but were dominated by sample‐specific viruses, mirroring the differences also observed in physicochemical and prokaryotic community composition. These metaviromes were distinct from those detected in other hypersaline systems analysed to date

Metagenómica de una salina ancestral: monitorización de los cambios de las comunidades microbianas desde el subsuelo hasta la superficie

Resumen del trabajo presentado al XXVIII Congreso Nacional de Microbiología, celebrado virtualmente del 28 de junio al 2 de julio de 2021.Ministerio de ciencia e innovación Gobierno de España (MICROMATES PGC2018-096956- B-C44), incluyendo fondos (FEDER), y Generalitat Valenciana PROMETEO /2017/2019.Peer reviewe