CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database
TL;DR: A new Resistomes & Variants module provides analysis and statistical summary of in silico predicted resistance variants from 82 pathogens and over 100 000 genomes, able to summarize predicted resistance using the information included in CARD, identify trends in AMR mobility and determine previously undescribed and novel resistance variants.
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.
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TL;DR: In this article, the authors investigated mechanisms of cross-resistance to chlorine and peracetic acid (PAA) disinfectants by antibiotic-resistant bacteria and found that resistant E. fergusonii showed an increase in gene expression of New Delhi metallo-β-lactamase (blaNDM-1) gene to chlorine, but there was no increase in expression to PAA.
6 citations
TL;DR: In this article, a genomic framework and genotyping scheme for Shigella sonnei is presented to efficiently identify genotype and resistance determinants from whole genome sequencing (WGS) data.
Abstract: Shigella sonnei is the most common agent of shigellosis in high-income countries, and causes a significant disease burden in low- and middle-income countries. Antimicrobial resistance is increasingly common in all settings. Whole genome sequencing (WGS) is increasingly utilised for S. sonnei outbreak investigation and surveillance, but comparison of data between studies and labs is challenging. Here, we present a genomic framework and genotyping scheme for S. sonnei to efficiently identify genotype and resistance determinants from WGS data. The scheme is implemented in the software package Mykrobe and tested on thousands of genomes. Applying this approach to analyse >4,000 S. sonnei isolates sequenced in public health labs in three countries identified several common genotypes associated with increased rates of ciprofloxacin resistance and azithromycin resistance, confirming intercontinental spread of highly-resistant S. sonnei clones and demonstrating the genomic framework can facilitate monitoring the spread of resistant clones, including those that have recently emerged, at local and global scales.
6 citations
TL;DR: In this article, the authors describe the genomic characteristics of methicillin-resistant Staphylococcus epidermidis (MRSE) isolated from clinical sources, to comprehend the genetic basis of antibiotic resistance, virulence, and potential pathogenicity.
Abstract: Staphylococcus epidermidis has become an important nosocomial pathogen. Multidrug resistance makes S. epidermidis infections difficult to treat. The study aims to describe the genomic characteristics of methicillin-resistant S. epidermidis (MRSE) isolated from clinical sources, to comprehend the genetic basis of antibiotic resistance, virulence, and potential pathogenicity. Sixteen MRSE underwent whole-genome sequencing, and bioinformatics analyses were carried out to ascertain their resistome, virulome, mobilome, clonality, and phylogenomic relationships. In all, 75% of isolates displayed multidrug resistance and were associated with the carriage of multiple resistance genes including mecA, blaZ, tet(K), erm(A), erm(B), erm(C), dfrG, aac(6')-aph(2''), and cat(pC221) conferring resistance to β-lactams, tetracyclines, macrolide-lincosamide-streptogramin B, aminoglycosides, and phenicols, which were located on both plasmids and chromosomes. Their virulence profiles were evidenced by the presence of genes involved in adherence/biofilm formation (icaA, icaB, icaC, atl, ebh, and ebp), immune evasion (adsA, capC, and manA), and antiphagocytosis (rmlC, cdsA, and A). The community-acquired SCCmec type IV was the most common SCCmec type. The CoNS belonged to seven multilocus sequence types (MLSTs) and carried a diversity of mobile genetic elements such as phages, insertion sequences, and plasmids. The bacterial anti-phage defense systems clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) immunity phage system and restriction-modification system (R-M system) and the arginine catabolic mobile element (ACME) involved in immune evasion and transport of virulence genes were also found. The insertion sequence, IS256, linked with virulence, was found in 56.3% of isolates. Generally, the isolates clustered according to STs, with some similarity but also considerable variability within isolates. Whole-genome sequencing and bioinformatics analysis provide insights into the likely pathogenicity and antibiotic resistance of S. epidermidis, necessitating surveillance of this emerging pathogen.
6 citations
TL;DR: The utility of mobile DNA sequencing technology supplemented with reference-based assembly in sequence typing and elucidating the basis of AMR is demonstrated, demonstrating the utility of real-time sequencing in the outbreak investigations, diagnosis and management of infections, especially in resource-limited settings.
Abstract: Background Africa has one of the highest incidences of gonorrhoea, but not much information is available on the relatedness with strains from other geographical locations. Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major public health threat, with the bacteria gaining resistance to most of the available antibiotics, compromising treatment across the world. Whole-genome sequencing is an efficient way of predicting AMR determinants and their spread in the human population. Previous studies on Kenyan gonococcal samples have focused on plasmid-mediated drug resistance and fluoroquinolone resistance using Illumina sequencing. Recent advances in next-generation sequencing technologies like Oxford Nanopore Technology (ONT) have helped in the generation of longer reads of DNA in a shorter duration with lower cost. However, long-reads are error-prone. The increasing accuracy ofbase-calling algorithms, high throughput, error-correction strategies, and ease of using the mobile sequencer in remote areas is leading to the adoption of the MinION sequencer (ONT), for routine microbial genome sequencing. Methods To investigate whether MinION-only sequencing is sufficient for diagnosis, genome sequencing and downstream analysis like inferring phylogenetic relationships and detection of AMR in resource-limited settings, we sequenced the genomes of fourteen clinical isolates suspected to be N. gonorrhoeae from Nairobi, Kenya. The isolates were tested using standard bacteriological methods for identification, interpreted using analytical profile index and antibiotic susceptibility tests had indicated ciprofloxacin and gentamycin resistance. Using whole-genome sequencing, the isolates were confirmed to be cases of N. gonorrhoeae (n=12), Additionally, we identified reads from N. meningitidis (n=2) and both of N. gonorrhoeae and Moraxella osloensis (n=3) in the sample (co-infections) respectively, which have been implicated in sexually transmitted infections in the recent years. The near-complete N. gonorrhoeae genomes (n=10) were analysed further for mutations/factors causing AMR using an in-house database of mutations curated from the literature. We attempted to understand the basis of drug resistance using homology modelling of AMR proteins, using known structures from other bacteria. Results We observe that Ciprofloxacin resistance is associated with multiple mutations in both gyrA and parC. We identified mutations conferring tetracycline (rpsJ) and Sulfonamide (folA) resistance in all the isolates and plasmids encoding beta-lactamase and tet(M) were identified in almost all of the strains. Phylogenetic analysis clustered the nine isolates into clades containing previously sequenced genomes from Kenya and countries across the world. Conclusion Here, we demonstrate the utility of mobile DNA sequencing technology supplemented with reference-based assembly in sequence typing and elucidating the basis of AMR. Bioinformatics profiling to predict AMR can be used along with routine AMR susceptibility tests in clinics. The workflow followed in the study, including AMR mutation dataset creation and the genome identification, assembly and analysis, can be used for the genome assembly and analysis of any clinical isolate. Further studies are required to determine the utility of real-time sequencing in the outbreak investigations, diagnosis and management of infections, especially in resource-limited settings. Data availability The raw reads generated for this study have been deposited in BioProject Accession: PRJNA660404 (https://www.ncbi.nlm.nih.gov). The biosample details are available under the ids SAMN15960547, SAMN15960548, SAMN15960549, SAMN15960550, SAMN15960551, SAMN15960552, SAMN15960553, SAMN15960554, SAMN15960555. The genomes and annotation files are available under the bioproject.
6 citations
15 Jun 2021
TL;DR: In this article, the genomes of sixty-three E. coli strains from the gut of patients with Crohn's disease and healthy subjects were sequenced and eighteen E coli-like metagenome-assembled genomes (MAGs) were reconstructed from the shotgun-metagenome sequencing data of fecal samples.
Abstract: Crohn’s disease (CD) is characterized by a chronic, progressive inflammation across the gastrointestinal tract with a series of exacerbations and remissions. A significant factor in the CD pathogenesis is an imbalance in gut microbiota composition, particularly the prevalence of Escherichia coli. In the present study, the genomes of sixty-three E. coli strains from the gut of patients with CD and healthy subjects were sequenced. In addition, eighteen E. coli-like metagenome-assembled genomes (MAGs) were reconstructed from the shotgun-metagenome sequencing data of fecal samples. The comparative analysis revealed the similarity of E. coli genomes regardless of the origin of the strain. The strains exhibited similar genetic patterns of virulence, antibiotic resistance, and bacteriocin-producing systems. The study showed antagonistic activity of E. coli strains and the metabolic features needed for their successful competition in the human gut environment. These observations suggest complex bacterial interactions within the gut which may affect the host and cause intestinal damage.
6 citations
References
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TL;DR: A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score.
88,255 citations
"CARD 2020: antibiotic resistome sur..." refers background in this paper
...The latter is described by CARD’s Model Ontology (MO, Supplementary Figure S1), which includes reference nucleotide and protein sequences, as well as additional search parameters including mutations conferring AMR (if applicable) and curated BLAST(P/N) (34,35) bit score cut-offs....
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TL;DR: Burrows-Wheeler Alignment tool (BWA) is implemented, a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps.
Abstract: Motivation: The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals.
Results: We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is ~10–20× faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package.
Availability: http://maq.sourceforge.net
Contact: [email protected]
43,862 citations
TL;DR: Bowtie 2 combines the strengths of the full-text minute index with the flexibility and speed of hardware-accelerated dynamic programming algorithms to achieve a combination of high speed, sensitivity and accuracy.
Abstract: As the rate of sequencing increases, greater throughput is demanded from read aligners. The full-text minute index is often used to make alignment very fast and memory-efficient, but the approach is ill-suited to finding longer, gapped alignments. Bowtie 2 combines the strengths of the full-text minute index with the flexibility and speed of hardware-accelerated dynamic programming algorithms to achieve a combination of high speed, sensitivity and accuracy.
37,898 citations
"CARD 2020: antibiotic resistome sur..." refers methods in this paper
...Metagenomics analysis (i.e. RGI bwt) uses Bowtie2 (40) or BWA (41) mapping of sequencing reads to CARD’s PHM reference sequences only, while annotation of genomes or assembly contigs predicts resistome using four of CARD’s AMR detection models: PHM, PVM, RVM and POM (note: RGI currently only scans for nonsynonymous substitutions; not frameshifts, deletions or insertions)....
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...RGI bwt) uses Bowtie2 (40) or BWA (41) mapping of sequencing reads to CARD’s PHM reference sequences only, while annotation of genomes or assembly contigs predicts resistome using four of CARD’s AMR detection models: PHM, PVM, RVM and POM (note: RGI currently only scans for nonsynonymous substitutions; not frameshifts, deletions or insertions)....
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TL;DR: The goals of the PDB are described, the systems in place for data deposition and access, how to obtain further information and plans for the future development of the resource are described.
Abstract: The Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource.
34,239 citations
"CARD 2020: antibiotic resistome sur..." refers methods in this paper
...In 2017, we described the CARD*Shark text-mining algorithm (26) for computer-assisted literature triage, which we have expanded based on the new ARO Drug Class classification tags....
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TL;DR: The new BLAST command-line applications, compared to the current BLAST tools, demonstrate substantial speed improvements for long queries as well as chromosome length database sequences.
Abstract: Sequence similarity searching is a very important bioinformatics task. While Basic Local Alignment Search Tool (BLAST) outperforms exact methods through its use of heuristics, the speed of the current BLAST software is suboptimal for very long queries or database sequences. There are also some shortcomings in the user-interface of the current command-line applications. We describe features and improvements of rewritten BLAST software and introduce new command-line applications. Long query sequences are broken into chunks for processing, in some cases leading to dramatically shorter run times. For long database sequences, it is possible to retrieve only the relevant parts of the sequence, reducing CPU time and memory usage for searches of short queries against databases of contigs or chromosomes. The program can now retrieve masking information for database sequences from the BLAST databases. A new modular software library can now access subject sequence data from arbitrary data sources. We introduce several new features, including strategy files that allow a user to save and reuse their favorite set of options. The strategy files can be uploaded to and downloaded from the NCBI BLAST web site. The new BLAST command-line applications, compared to the current BLAST tools, demonstrate substantial speed improvements for long queries as well as chromosome length database sequences. We have also improved the user interface of the command-line applications.
13,223 citations
"CARD 2020: antibiotic resistome sur..." refers background or methods in this paper
...The website also includes a built-in BLAST instance for comparing sequences to CARD reference sequences and a web instance of RGI for resistome prediction with data visualization tools (https:// card.mcmaster.ca/analyze)....
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...The RVM is functionally similar to the PVM, except it works for rRNA mutations and therefore uses a nucleotide reference sequence and a BLASTN bit score cut-off....
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...Briefly, RGI algorithmically predicts AMR genes and mutations from submitted genomes using a combination of open reading frame prediction with Prodigal (38), sequence alignment with BLAST (35) or DIAMOND (39), and curated resistance mutations included with the AMR detection model....
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...In the same time period, the CARD website hosted ∼45 000 BLAST analyses, ∼220 000 RGI analyses, ∼64 000 data file downloads, and ∼10,000 RGI software downloads....
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...We had determined that the asymptotic nature of the BLAST expectation value (E) gave it very low discriminatory power between different -lactamase gene families (nearly 13 of CARD’s content), but that the linear nature of the BLAST bit score (S′) allowed this level of discrimination....
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