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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: The minimal inhibitory concentration values collected revealed intermediate resistance to aminoglycosides, whereas susceptibility was detected for different classes of β-lactams, quinolones, glycopeptide, macrolides, and sulfonamides in all strains.
Abstract: The existence of antibiotic-resistant bacteria in food products, particularly those carrying acquired resistance genes, has increased concerns about the transmission of these genes from beneficial microbes to human pathogens. In this study, we evaluated the antibiotic resistance-susceptibility patterns of 16 antibiotics in eight S. thermophilus strains, whose genome sequence is available, using phenotypic and genomic approaches. The minimal inhibitory concentration values collected revealed intermediate resistance to aminoglycosides, whereas susceptibility was detected for different classes of β-lactams, quinolones, glycopeptide, macrolides, and sulfonamides in all strains. A high tetracycline resistance level has been detected in strain M17PTZA496, whose genome analysis indicated the presence of the tet(S) gene and the multidrug and toxic compound extrusion (MATE) family efflux pump. Moreover, an in-depth genomic analysis revealed genomic islands and an integrative and mobilizable element (IME) in the proximity of the gene tet(S). However, despite the presence of a prophage, genomic islands, and IME, no horizontal gene transfer was detected to Lactobacillus delbrueckii subsp. lactis DSM 20355 and Lactobacillusrhamnosus GG during 24 h of skim milk fermentation, 2 weeks of refrigerated storage, and 4 h of simulated gastrointestinal transit.

5 citations

Journal ArticleDOI
TL;DR: FOS with fewer side effects than colistin, excellent tissue distribution and minimal side effects may be recommended in combination with meropenem, and FOS + MEM and Fos + AK are excellent colistIn sparing combinations against ST 231, ST-395 and ST-147 XDR and PDR K. pneumoniae.
Abstract: Fosfomycin has emerged as a very useful antimicrobial in management of extremely drug resistant (XDR) and pan drug resistant (PDR) Klebsiella pneumoniae. In this study, we assessed in-vitro synergy of colistin sparing combinations of fosfomycin (FOS) with meropenem (MEM), tigecycline (TGC) and amikacin (AK) against XDR and PDR Klebsiella pneumoniae. Method: Non-replicate fully characterised 18 clinical isolates of K. pneumoniae (15 XDR and 3 PDR strains) were subjected to in-vitro synergy testing by checkerboard and time kill assay. Combinations tested were FOS-MEM, FOS-TGC and FOS-AK with glucose-6-phosphate being incorporated in all runs.WGS was carried out on the Illumina next-generation sequencing platform. Results: FOS-MEM and FOS-AK both demonstrated excellent synergy against all PDRs and all but one XDR. Synergy led to lowering of MICs to susceptible breakpoints. FOS-TGC demonstrated antagonism. MLST-231 K. pneumoniae predominated (14), followed by ST-395 (3) and ST147 (1). Majority harboured OXA-232 (n = 15), while n = 2 carried NDM-1 type and n = 1 co-carried NDM-5 + OXA-232. Mortality was high in both ST-231 (57.1%) and ST-395 (66.6%). Synergy was observed despite widespread presence of resistance markers against aminoglycosides [aph(3′)-Ic, aacA4, and rmtf], beta-lactams [blaSHV-11, blaTEM-1b, blaCTX-M-15, and blaOXA-232], fosfomycin [fosA6 and fosA5] and presence of porin proteins OmpK37, OmpA and K. pneumoniae antibiotic efflux pumps Kpn F, H, G, and E. Conclusion: FOS + MEM and FOS + AK are excellent colistin sparing combinations against ST 231, ST-395 and ST-147 XDR and PDR K. pneumoniae. FOS with fewer side effects than colistin, excellent tissue distribution and minimal side effects may be recommended in combination with meropenem.

5 citations

Posted ContentDOI
02 Apr 2020-bioRxiv
TL;DR: Short-read MAGs are largely ineffective for the analysis of mobile genes, including those of public-health importance like AMR and VF genes, and it is proposed that microbiome researchers should instead primarily utilise unassembled short reads and/or long-read approaches to more accurately analyse metagenomic data.
Abstract: Motivation: Metagenomic methods have emerged as a key tool in public-health microbiology for surveillance of virulence and antimicrobial resistance (AMR) genes. However, metagenomic data, even when assembled, usually results in complex, mixed sets DNA sequence fragments rather than fully resolved individual genomes. Recently, metagenome-assembled genomes (MAGs) have emerged as a promising approach that groups sequences into bins that are likely derived from the same underlying genome. However, MAGs have not been well assessed for their ability to identify some of the key sequences of interest for infectious disease surveillance purposes: AMR and VFs associated with mobile genetic elements (MGEs) such as plasmids and genomic islands (GIs). We hypothesized that due to the different copy number and sequence composition of plasmids and GIs compared to core genome sequence, such sequences will be under-represented in MAG-based approaches. Results: To evaluate the impact of MAG recovery methods on recovery of AMR genes and MGEs, we generated a simulated metagenomic dataset comprised of 30 genomes with up to 16.65% of the chromosomal DNA consisting of GIs and 65 associated plasmids. MAGs were then recovered from this data using 12 different MAG pipelines and evaluated for recovery accuracies. Across all pipelines, 81.9-94.3% of chromosomes were recovered and binned. However, only 37.8-44.1% of GIs and 1.5-29.2% of plasmids were recovered and correctly binned at >50% coverage. In terms of AMR and VF genes associated with MGEs, 0-45% of GI-associated AMR genes and 0-16% of GI-associated VF genes were correctly assigned. More strikingly, 0% of plasmid-borne VF or AMR genes were recovered. This work shows that regardless of the MAG recovery approach used, plasmid and GI dominated sequences will disproportionately be left unbinned or incorrectly binned. From a public-health perspective, this means MAG approaches are less suited for analysis of mobile genes, especially key groups such as AMR and VF genes. This underlines the utility of read-based and long-read approaches to thoroughly evaluate the resistome in metagenomic data.

4 citations

Journal ArticleDOI
TL;DR: In this article, an optrA-carrying E. faecalis ST16 isolate was recovered from a river water sample in Switzerland and was subjected to comprehensive genotypic characterisation and analysis of the genetic environment of the oxazolidinone/phenicol resistance gene OptrA.
Abstract: Objectives The objective of this work was to characterise an optrA-carrying Enterococcus faecalis ST16 isolate recovered from a river water sample in Switzerland. Methods Linezolid-resistant E. faecalis F102 was recovered from surface water in Switzerland and was subjected to comprehensive genotypic characterisation and analysis of the genetic environment of the oxazolidinone/phenicol resistance gene optrA. Whole-genome sequencing (WGS) was performed to detect linezolid resistance mechanisms, including mutations in 23S rRNA and ribosomal protein genes as well as acquired resistance genes. The isolate was further characterised by multilocus sequence typing (MLST) and identification of virulence genes. Results WGS detected the presence of optrA identical to the original optrA gene from E. faecalis E349. Analysis of the genetic environment revealed the association of optrA with fexA and a Tn6674-like transposon in co-existence with spc and erm(A) resistance genes. Sequence alignment indicated that the genetic environment of optrA was identical to a Tn6674-like variant from E. faecalis previously isolated from diseased and healthy humans and food-producing animals in the Asia-Pacific region. Enterococcus faecalis F102 did not contain any mutations in 23S rRNA or in genes encoding ribosomal proteins L3, L4 and L22. A total of 14 other resistance genes and 16 virulence genes were detected. Enterococcus faecalis F102 was assigned in silico to ST16. Conclusion The spread of optrA-carrying E. faecalis ST16 with a high pathogenic potential in surface water is a worrisome aspect from a public-health perspective.

4 citations

Journal ArticleDOI
TL;DR: The presence of a plasmid in a multi-drug-resistant C. jejuni strain isolated from a gastroenteritis patient revealed an undescribed variant of the aadE-sat4-aphA-3 gene cluster, providing resistance to at least kanamycin and gentamycin.
Abstract: Campylobacter jejuni is a foodborne pathogen causing bacterial gastroenteritis, with the highest incidence reported in Europe. The prevalence of antibiotic resistance in C. jejuni, as well as in many other bacterial pathogens, has increased over the last few years. In this report, we describe the presence of a plasmid in a multi-drug-resistant C. jejuni strain isolated from a gastroenteritis patient. Mating experiments demonstrated the transference of this genetic element (pCjH01) among C. jejuni by plasmid conjugation. The pCjH01 plasmid was sequenced and assembled, revealing high similarity (97% identity) with pTet, a described tetracycline resistance encoding plasmid. pCjH01 (47.7 kb) is a mosaic plasmid composed of a pTet backbone that has acquired two discrete DNA regions. Remarkably, one of the acquired sequences carried an undescribed variant of the aadE-sat4-aphA-3 gene cluster, providing resistance to at least kanamycin and gentamycin. Aside from the antibiotic resistance genes, the cluster also carries genes coding for putative regulators, such as a sigma factor of the RNA polymerase and an antisigma factor. Homology searches suggest that Campylobacter exchanges genetic material with distant G-positive bacterial genera.

4 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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