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.
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.
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.
The growing threat of antimicrobial resistance (AMR) calls for new epidemiological surveillance methods, as well as a deeper understanding of how antimicrobial resistance genes (ARGs) have been ...transmitted around the world. The large pool of sequencing data available in public repositories provides an excellent resource for monitoring the temporal and spatial dissemination of AMR in different ecological settings. However, only a limited number of research groups globally have the computational resources to analyze such data. We retrieved 442 Tbp of sequencing reads from 214,095 metagenomic samples from the European Nucleotide Archive (ENA) and aligned them using a uniform approach against ARGs and 16S/18S rRNA genes. Here, we present the results of this extensive computational analysis and share the counts of reads aligned. Over 6.76∙10
8
read fragments were assigned to ARGs and 3.21∙10
9
to rRNA genes, where we observed distinct differences in both the abundance of ARGs and the link between microbiome and resistome compositions across various sampling types. This collection is another step towards establishing global surveillance of AMR and can serve as a resource for further research into the environmental spread and dynamic changes of ARGs.
Microbial communities have huge impacts on their ecosystems and local environments spanning from marine and soil communities to the mammalian gut. Bacteriophages (phages) are important drivers of ...population control and diversity in the community, but our understanding of complex microbial communities is halted by biased detection techniques. Metagenomics have provided a method of novel phage discovery independent of in vitro culturing techniques and have revealed a large proportion of understudied phages. Here, five jumbophage genomes, that were previously assembled in silico from pig faecal metagenomes, are detected and observed directly in their natural environment using a modified phageFISH approach, and combined with methods to decrease bias against large-sized phages (e.g., jumbophages). These phages are uncultured with unknown hosts. The specific phages were detected by PCR and fluorescent in situ hybridisation in their original faecal samples as well as across other faecal samples. Co-localisation of bacterial signals and phage signals allowed detection of the different stages of phage life cycle. All phages displayed examples of early infection, advanced infection, burst, and free phages. To our knowledge, this is the first detection of jumbophages in faeces, which were investigated independently of culture, host identification, and size, and based solely on the genome sequence. This approach opens up opportunities for characterisation of novel in silico phages in vivo from a broad range of gut microbiomes.
•An analysis of farm dust from 79 pig and poultry farms using metagenomics.•Farm dust resistomes are compared to animal fecal and farmers stool resistomes.•Farm dust bacterial microbiomes have an ...abundant and rich resistome.•Animal fecal resistomes are an important, but not the only, source of farm dust ARGs.•Antimicrobial usage in the animals is positively associated with dust resistome abundance.
Livestock farms are a reservoir of antimicrobial resistant bacteria from feces. Airborne dust-bound bacteria can spread across the barn and to the outdoor environment. Therefore, exposure to farm dust may be of concern for animals, farmers and neighboring residents. Although dust is a potential route of transmission, little is known about the resistome and bacterial microbiome of farm dust.
We describe the resistome and bacterial microbiome of pig and poultry farm dust and their relation with animal feces resistomes and bacterial microbiomes, and on-farm antimicrobial usage (AMU). In addition, the relation between dust and farmers’ stool resistomes was explored.
In the EFFORT-study, resistomes and bacterial microbiomes of indoor farm dust collected on Electrostatic Dust fall Collectors (EDCs), and animal feces of 35 conventional broiler and 44 farrow-to-finish pig farms from nine European countries were determined by shotgun metagenomic analysis. The analysis also included 79 stool samples from farmers working or living at 12 broiler and 19 pig farms and 46 human controls. Relative abundance of and variation in resistome and bacterial composition of farm dust was described and compared to animal feces and farmers’ stool.
The farm dust resistome contained a large variety of antimicrobial resistance genes (ARGs); more than the animal fecal resistome. For both poultry and pigs, composition of dust resistomes finds (partly) its origin in animal feces as dust resistomes correlated significantly with fecal resistomes. The dust bacterial microbiome also correlated significantly with the dust resistome composition. A positive association between AMU in animals on the farm and the total abundance of the dust resistome was found. Occupational exposure to pig farm dust or animal feces may contribute to farmers’ resistomes, however no major shifts in farmers resistome towards feces or dust resistomes were found in this study.
Poultry and pig farm dust resistomes are rich and abundant and associated with the fecal resistome of the animals and the dust bacterial microbiome.
Reliable methods for monitoring antimicrobial resistance (AMR) in livestock and other reservoirs are essential to understand the trends, transmission and importance of agricultural resistance. ...Quantification of AMR is mostly done using culture-based techniques, but metagenomic read mapping shows promise for quantitative resistance monitoring.
We evaluated the ability of: (i) MIC determination for Escherichia coli; (ii) cfu counting of E. coli; (iii) cfu counting of aerobic bacteria; and (iv) metagenomic shotgun sequencing to predict expected tetracycline resistance based on known antimicrobial consumption in 10 Danish integrated slaughter pig herds. In addition, we evaluated whether fresh or manure floor samples constitute suitable proxies for intestinal sampling, using cfu counting, qPCR and metagenomic shotgun sequencing.
Metagenomic read-mapping outperformed cultivation-based techniques in terms of predicting expected tetracycline resistance based on antimicrobial consumption. Our metagenomic approach had sufficient resolution to detect antimicrobial-induced changes to individual resistance gene abundances. Pen floor manure samples were found to represent rectal samples well when analysed using metagenomics, as they contain the same DNA with the exception of a few contaminating taxa that proliferate in the extraintestinal environment.
We present a workflow, from sampling to interpretation, showing how resistance monitoring can be carried out in swine herds using a metagenomic approach. We propose metagenomic sequencing should be part of routine livestock resistance monitoring programmes and potentially of integrated One Health monitoring in all reservoirs.
Abstract
The emergence of antimicrobial resistance (AMR) is one of the biggest health threats globally. In addition, the use of antimicrobial drugs in humans and livestock is considered an important ...driver of antimicrobial resistance. The commensal microbiota, and especially the intestinal microbiota, has been shown to have an important role in the emergence of AMR. Mobile genetic elements (MGEs) also play a central role in facilitating the acquisition and spread of AMR genes. We isolated
Escherichia coli
(n = 627) from fecal samples in respectively 25 poultry, 28 swine, and 15 veal calf herds from 6 European countries to investigate the phylogeny of
E. coli
at country, animal host and farm levels. Furthermore, we examine the evolution of AMR in
E. coli
genomes including an association with virulence genes, plasmids and MGEs. We compared the abundance metrics retrieved from metagenomic sequencing and whole genome sequenced of
E. coli
isolates from the same fecal samples and farms. The
E. coli
isolates in this study indicated no clonality or clustering based on country of origin and genetic markers; AMR, and MGEs. Nonetheless, mobile genetic elements play a role in the acquisition of AMR and virulence genes. Additionally, an abundance of AMR was agreeable between metagenomic and whole genome sequencing analysis for several AMR classes in poultry fecal samples suggesting that metagenomics could be used as an indicator for surveillance of AMR in
E. coli
isolates and vice versa.
Candidate Phyla Radiation (CPR) bacteria are small, likely episymbiotic organisms found across Earth's ecosystems. Despite their prevalence, the distribution of CPR lineages across habitats and the ...genomic signatures of transitions among these habitats remain unclear. Here, we expand the genome inventory for Absconditabacteria (SR1), Gracilibacteria, and Saccharibacteria (TM7), CPR bacteria known to occur in both animal-associated and environmental microbiomes, and investigate variation in gene content with habitat of origin. By overlaying phylogeny with habitat information, we show that bacteria from these three lineages have undergone multiple transitions from environmental habitats into animal microbiomes. Based on co-occurrence analyses of hundreds of metagenomes, we extend the prior suggestion that certain Saccharibacteria have broad bacterial host ranges and constrain possible host relationships for Absconditabacteria and Gracilibacteria. Full-proteome analyses show that animal-associated Saccharibacteria have smaller gene repertoires than their environmental counterparts and are enriched in numerous protein families, including those likely functioning in amino acid metabolism, phage defense, and detoxification of peroxide. In contrast, some freshwater Saccharibacteria encode a putative rhodopsin. For protein families exhibiting the clearest patterns of differential habitat distribution, we compared protein and species phylogenies to estimate the incidence of lateral gene transfer and genomic loss occurring over the species tree. These analyses suggest that habitat transitions were likely not accompanied by large transfer or loss events but rather were associated with continuous proteome remodeling. Thus, we speculate that CPR habitat transitions were driven largely by availability of suitable host taxa and were reinforced by acquisition and loss of some capacities.
Studying the genetic differences between related microorganisms from different environment types can indicate factors associated with their movement among habitats. This is particularly interesting for bacteria from the Candidate Phyla Radiation because their minimal metabolic capabilities require associations with microbial hosts. We found that shifts of Absconditabacteria, Gracilibacteria, and Saccharibacteria between environmental ecosystems and mammalian mouths/guts probably did not involve major episodes of gene gain and loss; rather, gradual genomic change likely followed habitat migration. The results inform our understanding of how little-known microorganisms establish in the human microbiota where they may ultimately impact health.
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.
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.
•Food chain workers are exposed to antimicrobial resistant (AMR) bacteria in livestock.•We describe faecal resistomes and microbiomes of farmers and slaughterhouse workers.•Faecal AMR gene loads in ...pork exposed workers exceed those in broiler farmers/controls.•Resistome and microbiome differences are shown between and within occupational groups.•On-farm working hours and working/living on a pig farm determinethe faecal resistome.•Contact with livestock may be a determinant for human AMR gene carriage.
By studying the entire human faecal resistome and associated microbiome, the diversity and abundance of faecal antimicrobial resistance genes (ARGs) can be comprehensively characterized. Prior culture-based studies have shown associations between occupational exposure to livestock and carriage of specific antimicrobial resistant bacteria. Using shotgun metagenomics, the present study investigated 194 faecal resistomes and bacteriomes from humans occupationally exposed to ARGs in livestock (i.e. pig and poultry farmers, employees and family members and pig slaughterhouse workers) and a control population (Lifelines cohort) in the Netherlands. In addition, we sought to identify determinants for the human resistome and bacteriome composition by applying a combination of multivariate (NMDS, PERMANOVA, SIMPER and DESeq2 analysis) and multivariable regression analysis techniques.
Pig slaughterhouse workers and pig farmers carried higher total ARG abundances in their stools compared to broiler farmers and control subjects. Tetracycline, β-lactam and macrolide resistance gene clusters dominated the resistome of all studied groups. No significant resistome alpha diversity differences were found among the four populations. However, the resistome beta diversity showed a separation of the mean resistome composition of pig and pork exposed workers from broiler farmers and controls, independent of their antimicrobial use. We demonstrated differences in resistome composition between slaughter line positions, pig versus poultry exposed workers, as well as differences between farmers and employees versus family members. In addition, we found a significant correlation between the bacteriome and resistome, and significant differences in the bacteriome composition between and within the studied subpopulations. Finally, an in-depth analysis of pig and poultry farms – of which also farm livestock resistomes were analysed – showed positive associations between the number of on-farm working hours and human faecal AMR loads.
We found that the total normalized faecal ARG carriage was larger in persons working in the Dutch pork production chain compared to poultry farmers and controls. Additionally, we showed significant differences in resistome and bacteriome composition of pig and pork exposed workers compared to a control group, as well as within-population (farms, slaughterhouse) compositional differences. The number of on-farm working hours and the farm type (pig or broiler) that persons live or work on are determinants for the human faecal resistome. Overall, our results may suggest direct or indirect livestock contact as a determinant for human ARG carriage. Future studies should further focus on the connection between the human and livestock resistome (i.e. transmission routes) to substantiate the evidence for livestock-associated resistome acquisition.
Bacteriophages (phages) dramatically shape microbial community composition, redistribute nutrients via host lysis and drive evolution through horizontal gene transfer. Despite their importance, much ...remains to be learned about phages in the human microbiome. We investigated the gut microbiomes of humans from Bangladesh and Tanzania, two African baboon social groups and Danish pigs; many of these microbiomes contain phages belonging to a clade with genomes >540 kilobases in length, the largest yet reported in the human microbiome and close to the maximum size ever reported for phages. We refer to these as Lak phages. CRISPR spacer targeting indicates that Lak phages infect bacteria of the genus Prevotella. We manually curated to completion 15 distinct Lak phage genomes recovered from metagenomes. The genomes display several interesting features, including use of an alternative genetic code, large intergenic regions that are highly expressed and up to 35 putative transfer RNAs, some of which contain enigmatic introns. Different individuals have distinct phage genotypes, and shifts in variant frequencies over consecutive sampling days reflect changes in the relative abundance of phage subpopulations. Recent homologous recombination has resulted in extensive genome admixture of nine baboon Lak phage populations. We infer that Lak phages are widespread in gut communities that contain the Prevotella species, and conclude that megaphages, with fascinating and underexplored biology, may be common but largely overlooked components of human and animal gut microbiomes.
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