Abstract
The Comprehensive Antibiotic Resistance Database (CARD; https://card.mcmaster.ca) is a curated resource providing reference DNA and protein sequences, detection models and bioinformatics ...tools on the molecular basis of bacterial antimicrobial resistance (AMR). CARD focuses on providing high-quality reference data and molecular sequences within a controlled vocabulary, the Antibiotic Resistance Ontology (ARO), designed by the CARD biocuration team to integrate with software development efforts for resistome analysis and prediction, such as CARD’s Resistance Gene Identifier (RGI) software. Since 2017, CARD has expanded through extensive curation of reference sequences, revision of the ontological structure, curation of over 500 new AMR detection models, development of a new classification paradigm and expansion of analytical tools. Most notably, a new Resistomes & Variants module provides analysis and statistical summary of in silico predicted resistance variants from 82 pathogens and over 100 000 genomes. By adding these resistance variants to CARD, we are able to summarize predicted resistance using the information included in CARD, identify trends in AMR mobility and determine previously undescribed and novel resistance variants. Here, we describe updates and recent expansions to CARD and its biocuration process, including new resources for community biocuration of AMR molecular reference data.
The Comprehensive Antibiotic Resistance Database (CARD; card.mcmaster.ca) combines the Antibiotic Resistance Ontology (ARO) with curated AMR gene (ARG) sequences and resistance-conferring mutations ...to provide an informatics framework for annotation and interpretation of resistomes. As of version 3.2.4, CARD encompasses 6627 ontology terms, 5010 reference sequences, 1933 mutations, 3004 publications, and 5057 AMR detection models that can be used by the accompanying Resistance Gene Identifier (RGI) software to annotate genomic or metagenomic sequences. Focused curation enhancements since 2020 include expanded β-lactamase curation, incorporation of likelihood-based AMR mutations for Mycobacterium tuberculosis, addition of disinfectants and antiseptics plus their associated ARGs, and systematic curation of resistance-modifying agents. This expanded curation includes 180 new AMR gene families, 15 new drug classes, 1 new resistance mechanism, and two new ontological relationships: evolutionary_variant_of and is_small_molecule_inhibitor. In silico prediction of resistomes and prevalence statistics of ARGs has been expanded to 377 pathogens, 21,079 chromosomes, 2,662 genomic islands, 41,828 plasmids and 155,606 whole-genome shotgun assemblies, resulting in collation of 322,710 unique ARG allele sequences. New features include the CARD:Live collection of community submitted isolate resistome data and the introduction of standardized 15 character CARD Short Names for ARGs to support machine learning efforts.
Genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly important to monitor the transmission and adaptive evolution of the virus. The accessibility of ...high-throughput methods and polymerase chain reaction (PCR) has facilitated a growing ecosystem of protocols. Two differing protocols are tiling multiplex PCR and bait capture enrichment. Each method has advantages and disadvantages but a direct comparison with different viral RNA concentrations has not been performed to assess the performance of these approaches. Here we compare Liverpool amplification, ARTIC amplification, and bait capture using clinical diagnostics samples. All libraries were sequenced using an Illumina MiniSeq with data analyzed using a standardized bioinformatics workflow (SARS-CoV-2 Illumina GeNome Assembly Line; SIGNAL). One sample showed poor SARS-CoV-2 genome coverage and consensus, reflective of low viral RNA concentration. In contrast, the second sample had a higher viral RNA concentration, which yielded good genome coverage and consensus. ARTIC amplification showed the highest depth of coverage results for both samples, suggesting this protocol is effective for low concentrations. Liverpool amplification provided a more even read coverage of the SARS-CoV-2 genome, but at a lower depth of coverage. Bait capture enrichment of SARS-CoV-2 cDNA provided results on par with amplification. While only two clinical samples were examined in this comparative analysis, both the Liverpool and ARTIC amplification methods showed differing efficacy for high and low concentration samples. In addition, amplification-free bait capture enriched sequencing of cDNA is a viable method for generating a SARS-CoV-2 genome sequence and for identification of amplification artifacts.
Vaccination has transformed public health, most notably including the eradication of smallpox. Despite its profound historical importance, little is known of the origins and diversity of the viruses ...used in smallpox vaccination. Prior to the twentieth century, the method, source and origin of smallpox vaccinations remained unstandardised and opaque. We reconstruct and analyse viral vaccine genomes associated with smallpox vaccination from historical artefacts. Significantly, we recover viral molecules through non-destructive sampling of historical materials lacking signs of biological residues. We use the authenticated ancient genomes to reveal the evolutionary relationships of smallpox vaccination viruses within the poxviruses as a whole.
While increasing rates of antimicrobial resistance undermine our current arsenal of antibiotics, resistance-modifying agents (RMAs) hold promise to extend the lifetime of these important molecules. ...We here provide a standardized nomenclature for RMAs within the Comprehensive Antibiotic Resistance Database in aid of RMA discovery, data curation, and genome mining.
Early life stage mortality is one of the problems faced by Atlantic cod aquaculture. However, our understanding of immunity in early life stage fish is still incomplete, and the information available ...is restricted to a few species. In the present work we investigated the expression of immune-relevant transcripts in Atlantic cod during early development. The transcripts subjected to QPCR analysis in the present study were previously identified as putative anti-viral or anti-bacterial genes in Atlantic cod using suppression subtractive hybridization (SSH) libraries, QPCR, and/or microarrays. Of the 11 genes involved in this study, only atf3, cxc chemokine and gaduscidin-1 were not detected at the transcript level in all developmental stages investigated from unfertilized egg to early larval stage. Adam22, hamp, il8, irf1, irf7, lgp2, sacsin, and stat1 transcripts were detected in unfertilized egg and 7h post-fertilization (~2-cell stage) embryos, showing maternal contribution of these immune-relevant transcripts to the early embryonic transcriptome. The Atlantic cod genes included in this study presented diverse transcript expression profiles throughout embryonic and early larval development. For example, adam22 and sacsin transcripts rose abruptly during blastula/gastrula stage and were then expressed at relatively high levels through subsequent embryonic and early larval developmental stages. A peak in irf1 and irf7 transcript expression during early segmentation suggests that these interferon pathway genes play developmental stage-specific roles during cod embryogenesis. Stat1 had increasing transcript expression throughout blastula/gastrula, segmentation, and early larval developmental stages. Atf3, cxc chemokine, gaduscidin-1, and il8 transcripts rose approximately 2–3 fold during hatching, supporting the hypothesis that there is preparation at the immune-relevant transcript expression level to deal with environmental pathogens that may be encountered during early larval development. The specific roles that interferon pathway and other immune-relevant genes play in early life stage cod, and the potential impact of their dynamic transcript expression on immune competence of Atlantic cod embryos and larvae, remain unclear and warrant further study.
► Immune-relevant gene expression was studied in eggs and early development using QPCR. ► Diverse developmental expression profiles of immune-relevant genes were observed. ► Irf1, irf7, il8, and cxc chemokine transcripts were induced during segmentation. ► Atf3, gaduscidin-1, il8, and cxc chemokine transcripts were induced during hatching. ► Expression profiles of some genes suggest developmental stage-specific roles.
Enterococcus faecium
is a ubiquitous opportunistic pathogen that is exhibiting increasing levels of antimicrobial resistance (AMR). Many of the genes that confer resistance and pathogenic functions ...are localized on mobile genetic elements (MGEs), which facilitate their transfer between lineages. Here, features including resistance determinants, virulence factors and MGEs were profiled in a set of 1273
E. faecium
genomes from two disparate geographic locations (in the UK and Canada) from a range of agricultural, clinical and associated habitats. Neither lineages of
E. faecium
, type A and B, nor MGEs are constrained by geographic proximity, but our results show evidence of a strong association of many profiled genes and MGEs with habitat. Many features were associated with a group of clinical and municipal wastewater genomes that are likely forming a new human-associated ecotype within type A. The evolutionary dynamics of
E. faecium
make it a highly versatile emerging pathogen, and its ability to acquire, transmit and lose features presents a high risk for the emergence of new pathogenic variants and novel resistance combinations. This study provides a workflow for MGE-centric surveillance of AMR in
Enterococcus
that can be adapted to other pathogens.
The Comprehensive Antibiotic Resistance Database (CARD; http://arpcard.mcmaster.ca) is a manually curated resource containing high quality reference data on the molecular basis of antimicrobial ...resistance (AMR), with an emphasis on the genes, proteins and mutations involved in AMR. CARD is ontologically structured, model centric, and spans the breadth of AMR drug classes and resistance mechanisms, including intrinsic, mutation-driven and acquired resistance. It is built upon the Antibiotic Resistance Ontology (ARO), a custom built, interconnected and hierarchical controlled vocabulary allowing advanced data sharing and organization. Its design allows the development of novel genome analysis tools, such as the Resistance Gene Identifier (RGI) for resistome prediction from raw genome sequence. Recent improvements include extensive curation of additional reference sequences and mutations, development of a unique Model Ontology and accompanying AMR detection models to power sequence analysis, new visualization tools, and expansion of the RGI for detection of emergent AMR threats. CARD curation is updated monthly based on an interplay of manual literature curation, computational text mining, and genome analysis.
Identification of the nucleotide sequences encoding antibiotic resistance elements and determination of their association with antibiotic resistance are critical to improve surveillance and monitor ...trends in antibiotic resistance. Current methods to study antibiotic resistance in various environments rely on extensive deep sequencing or laborious culturing of fastidious organisms, both of which are heavily time-consuming operations. An accurate and sensitive method to identify both rare and common resistance elements in complex metagenomic samples is needed. Referencing the sequences in the Comprehensive Antibiotic Resistance Database, we designed a set of 37,826 probes to specifically target over 2,000 nucleotide sequences associated with antibiotic resistance in clinically relevant bacteria. Testing of this probe set on DNA libraries generated from multidrug-resistant bacteria to selectively capture resistance genes reproducibly produced higher numbers of reads on target at a greater length of coverage than shotgun sequencing. We also identified additional resistance gene sequences from human gut microbiome samples that sequencing alone was not able to detect. Our method to capture the resistome enables a sensitive means of gene detection in diverse environments where genes encoding antibiotic resistance represent less than 0.1% of the metagenome.
Abstract
Background
The Public Health Alliance for Genomic Epidemiology (PHA4GE) (https://pha4ge.org) is a global coalition that is actively working to establish consensus standards, document and ...share best practices, improve the availability of critical bioinformatics tools and resources, and advocate for greater openness, interoperability, accessibility, and reproducibility in public health microbial bioinformatics. In the face of the current pandemic, PHA4GE has identified a need for a fit-for-purpose, open-source SARS-CoV-2 contextual data standard.
Results
As such, we have developed a SARS-CoV-2 contextual data specification package based on harmonizable, publicly available community standards. The specification can be implemented via a collection template, as well as an array of protocols and tools to support both the harmonization and submission of sequence data and contextual information to public biorepositories.
Conclusions
Well-structured, rich contextual data add value, promote reuse, and enable aggregation and integration of disparate datasets. Adoption of the proposed standard and practices will better enable interoperability between datasets and systems, improve the consistency and utility of generated data, and ultimately facilitate novel insights and discoveries in SARS-CoV-2 and COVID-19. The package is now supported by the NCBI’s BioSample database.
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