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Journal ArticleDOI

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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Citations
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Journal ArticleDOI
TL;DR: Four Escherichia coli strains harboring the blaCTX–M–65 gene identified among 49 isolates from beef and pork collected at retail are characterized by whole-genome sequencing and showed that they are clonally related and belong to two sub-lineages.
Abstract: The emergence and dissemination of resistance to third and fourth-generation cephalosporins among Enterobacteriaceae from different sources impose a global public health threat. Here, we characterized by Whole Genome Sequencing four Escherichia coli strains harbouring the blaCTX-M-65 gene identified among 49 isolates from beef and pork collected at retail. The genomic content was determined using the Center for Genomic Epidemiology web tools. Additionally, the prediction and reconstruction of plasmids were conducted, the genetic platform of the blaCTX-M-65 gene was investigated, and the phylogenetic analysis was carried out using 17 other genomes with the same sequence type and harbouring the blaCTX-M-65 gene. All strains harboured blaCTX-M-65, blaOXA1 and blaTEM1B, and one also carried the blaSHV-12 gene. Other resistance genes, namely qnrS2, aac(6')-Ib-c, dfrA14, sul2, tetA and mphA were present in all genomes; the mcr-1.1 gene was identified in the colistin-resistant strains. They belonging to sequence type 2179, phylogenetic group B1, serotype O9:H9, and carried plasmids IncI, IncFIC(FII), and IncFIB. All strains share an identical genetic environment having the IS903 and ISEcp1flanking the blaCTX-M-65 gene. It seems likely that the blaCTX-M-65 gene is located in the chromosome in all isolates based on deep in silico analysis. Our findings showed that the strains are clonally related and belonging to two sub-lineages. This study reports the emergence of CTX-M-65 producing E. coli in Portugal in food products of animal origin. The chromosomal location of the blaCTX-M-65 gene may ensure a stable spread of resistance in the absence of selective pressure.

14 citations


Cites background from "CARD 2020: antibiotic resistome sur..."

  • ..., 1990) and The Comprehensive Antibiotic Resistance Database (CARD) (Alcock et al., 2019)....

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  • ...The sequencing of fragments was performed in an automatic sequencer ABI3100 (Applied Biosystems), and the identification of resistance genes was determined using the Basic Local Alignment Search Tool (BLAST) from the NCBI website (Altschul et al., 1990) and The Comprehensive Antibiotic Resistance Database (CARD) (Alcock et al., 2019)....

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Journal ArticleDOI
02 Feb 2021-Mbio
TL;DR: In this paper, an assembly-independent and spike-in facilitated metagenomic quantification approach was used to screen and quantify over 2,000 genes simultaneously, while delivering absolute gene concentrations comparable to those for quantitative PCR (qPCR) DNA.
Abstract: We demonstrate that an assembly-independent and spike-in facilitated metagenomic quantification approach can be used to screen and quantify over 2,000 genes simultaneously, while delivering absolute gene concentrations comparable to those for quantitative PCR (qPCR) DNA extracted from dairy manure slurry, digestate, and compost was spiked with genomic DNA from a marine bacterium and sequenced using the Illumina HiSeq4000 We compared gene copy concentrations, in gene copies per mass of sample, of five antimicrobial resistance genes (ARGs) generated with (i) our quantitative metagenomic approach, (ii) targeted qPCR, and (iii) a hybrid quantification approach involving metagenomics and qPCR-based 16S rRNA gene quantification Although qPCR achieved lower quantification limits, the metagenomic method avoided biases caused by primer specificity inherent to qPCR-based methods and was able to detect orders of magnitude more genes than is possible with qPCR assays We used the approach to simultaneously quantify ARGs in the Comprehensive Antimicrobial Resistance Database (CARD) We observed that the total abundance of tetracycline resistance genes was consistent across different stages of manure treatment on three farms, but different samples were dominated by different tetracycline resistance gene familiesIMPORTANCE qPCR and metagenomics are central molecular techniques that have offered insights into biological processes for decades, from monitoring spatial and temporal gene dynamics to tracking ARGs or pathogens Still needed is a tool that can quantify thousands of relevant genes in a sample as gene copies per sample mass or volume We compare a quantitative metagenomic approach with traditional qPCR approaches in the quantification of ARG targets in dairy manure samples By leveraging the benefits of nontargeted community genomics, we demonstrate high-throughput absolute gene quantification of all known ARG sequences in environmental samples

14 citations

Journal ArticleDOI
06 Jan 2021
TL;DR: In this article, the authors characterized the genomic features of two clinical Enterobacter cloacae complex (ECC) isolates, co-producing VIM and MCR enzymes, in Italy.
Abstract: Background: the co-production of carbapenemases and mcr-genes represents a worrisome event in the treatment of Enterobacteriaceae infections. The aim of the study was to characterize the genomic features of two clinical Enterobacter cloacae complex (ECC) isolates, co-producing VIM and MCR enzymes, in Italy. Methods: species identification and antibiotic susceptibility profiling were performed using MALDI-TOF and broth microdilution methods, respectively. Transferability of the blaVIM- and mcr- type genes was verified through conjugation experiment. Extracted DNA was sequenced using long reads sequencing technology on the Sequel I platform (PacBio). Results: the first isolate showed clinical resistance against ertapenem yet was colistin susceptible (EUCAST 2020 breakpoints). The mcr-9.2 gene was harbored on a conjugative IncHI2 plasmid, while the blaVIM-1 determinant was harbored on a conjugative IncN plasmid. The second isolate, resistant to both carbapenems and colistin, harbored: mcr-9 gene and its two component regulatory genes for increased expression on the chromosome, mcr-4.3 on non-conjugative (yet co-transferable) ColE plasmid, and blaVIM-1 on a non-conjugative IncA plasmid. Conclusions: to our knowledge, this is the first report of co-production of VIM and MCR in ECC isolates in Italy.

14 citations

Journal ArticleDOI
TL;DR: In this article, the authors examine the current state of Oxford Nanopore Technologies (ONT) products and their interaction with third-party software/databases to assess their capabilities for bacterial pathogen identification and antimicrobial resistance (AMR) prediction.
Abstract: Extended turnaround times and large economic costs hinder the usage of currently applied screening methods for bacterial pathogen identification (ID) and antimicrobial susceptibility testing. This review provides an overview of current detection methods and their usage in a clinical setting. Issues of timeliness and cost could soon be circumvented, however, with the emergence of detection methods involving single molecule sequencing technology. In the context of bringing diagnostics closer to the point of care, we examine the current state of Oxford Nanopore Technologies (ONT) products and their interaction with third-party software/databases to assess their capabilities for ID and antimicrobial resistance (AMR) prediction. We outline and discuss a potential diagnostic workflow, enumerating (1) rapid sample prep kits, (2) ONT hardware/software and (3) third-party software and databases to improve the cost, accuracy and turnaround times for ID and AMR. Multiple studies across a range of infection types support that the speed and accuracy of ONT sequencing is now such that established ID and AMR prediction tools can be used on its outputs, and so it can be harnessed for near real time, close to the point-of-care diagnostics in common clinical circumstances.

14 citations

Journal ArticleDOI
27 Dec 2020
TL;DR: In this paper, the complete genome of P. acidilactici HN9 was reported and compared with other bacterial strains belonging to the genus Pediococcus, including type II-A CRISPR/CRISPR-associated (Cas).
Abstract: Pediococcus acidilactici HN9 is a beneficial lactic acid bacterium isolated from Nhang, a traditional Thai-style fermented beef. In this study, the molecular properties of P. acidilactici HN9 were characterized to provide insights into its potential probiotic activity. Specifically, this work sought to report the complete genome of P. acidilactici HN9 and perform a comparative genome analysis with other bacterial strains belonging to the genus Pediococcus. Genomic features of HN9 were compared with those of all other bacterial Pediococcus strains to examine the adaptation, evolutionary relationships, and diversity within this genus. Additionally, several bioinformatic approaches were used to investigate phylogenetic relationships, genome stability, virulence factors, bacteriocin production, and antimicrobial resistance genes of the HN9 strain, as well as to ensure its safety as a potential starter culture in food applications. A 2,034,522 bp circular chromosome and two circular plasmids, designated pHN9-1 (42,239-bp) and pHN9-2 (30,711-bp), were detected, and used for pan-genome analysis, as well as for identification of bacteriocin-encoding genes in 129 strains belonging to all Pediococcus species. Two CRISPR regions were identified in P. acidilactici HN9, including type II-A CRISPR/CRISPR-associated (Cas). This study provides an in-depth analysis on P. acidilactici HN9, facilitating a better understanding of its adaptability to different environments and its mechanism to maintain genome stability over time.

14 citations

References
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Journal ArticleDOI
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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Journal ArticleDOI
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

Journal ArticleDOI
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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Journal ArticleDOI
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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Journal ArticleDOI
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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