The Comprehensive Antibiotic Resistance …rami-ngs.org/.../The-comprehensive-antibiotic-resistance-database_a...The Comprehensive Antibiotic Resistance Database ... • An essential

The Comprehensive Antibiotic Resistance Database A Platform for Antimicrobial Resistance Surveillance

Andrew G. McArthur, Ph.D.

Michael G. DeGroote Institute for Infectious Disease Research

McMaster University, Hamilton, Ontario, Canada

arpcard.mcmaster.ca

Global AMR Crisis

Which resistance genes are present?

Which genes are moving around?

Which pose a threat?

NDM-1 metallo-beta-lactamase

Promise of Molecular Surveillance


retrospective & survey

DNA sequencing

clinical DNA

sequencing


PHAC’s ‘One Health’ Concept

Wright. 2010. Expert Opin. Drug Discov. 5:779-788.

A Wicked Problem

• No single solution – but critical demand for action

• Very complex – requires the combined expertise of many individuals,

groups, and disciplines

• Working in ‘silos’ without collaboration does not solve wicked problems

• Solutions require a very comprehensive and interdisciplinary strategy

• AMR gene surveillance a key component

A Wicked Problem

A Wicked Problem

genes and

pathogens we are

tracking

A Wicked Problem

genes and

pathogens we are

tracking

characterized genes

we don’t routinely

track; variants of

known genes

A Wicked Problem

genes and

pathogens we are

tracking

characterized genes

we don’t routinely

track; variants of

known genes

emergent threats


DNA sequencing

prediction of

phenotype &

antibiogram

comparison to reference

sequences

prediction of

resistome

Comprehensive Antibiotic Resistance Database

arpcard.mcmaster.ca


• High quality reference data on the molecular basis of AMR – expert

curation.

• Organized by the Antibiotic Resistance Ontology (ARO), a theoretical

framework for organizing antibiotic resistance information.

• Breadth of data – AMR via horizontal gene transfer (HGT) +

comprehensive mutation data for genome-based AMR.

• Advanced analytics – predicts resistome based on both sequence

similarity and mutant detection.

• Discovery – development of methods for detection of new variants and

emergent threats

• Growth – constantly curated resource + building algorithms for

detection of AMR mechanisms not examined by other databases (rRNA

mutations, van clusters, etc.)


• A controlled vocabulary for the codification of drugs, targets, resistance

genes, and mechanisms.

• Resistance terms are linked together by a set of relationships.

• Allows for computation over a network of curated AMR knowledge.

• An essential step towards the development of standards for data sharing

among research teams.

• Development to date has been a scrum-style accumulation of ontology

terms and metadata

• Now moving to systematic approaches to ARO development

• ESKAPE pathogens

• Formal ontology structures

• Computer-assisted literature curation

Antibiotic Resistance Ontology

Antibiotic Resistance Ontology

Detection Model Ontology

DNA sequencing

prediction of

phenotype &

antibiogram

comparison to reference

sequences

prediction of

resistome

detection models &

parameters


• protein homolog model

• protein variant model

• rRNA mutation model

• absent protein homolog model – resistance by absence

• gene order model – functional glycopeptide resistance clusters

• multiple gene mutations model – resistance by co-mutation

• regulatory models – efflux upregulation by mutations in promoters


• protein homolog model

• reference sequence

• BLASTP cut-off


• protein variant model

• reference sequence (often sensitive wildtype)

• BLASTP cut-off

• Mutations – SNPs, indels, etc.


• gene order meta-model

• secondary interpretation of resistome predictions

• a step towards prediction of phenotype

vanR vanS vanH vanA vanX vanY vanZ

functional VanA operon meta-model

vanR

vanS

vanH

vanA

vanX

vanY

vanZ

vanC

vanXYc

vanT

vanBc

vanDc

Gen

om

e sequ

ence

etc

RGI

3458 Ontology Terms, 3228 Reference Sequences, 703 SNPs, 1979

Publications, 2263 AMR Detection Models

• McArthur et al. 2013. The

Comprehensive Antibiotic Resistance

Database. Antimicrobial Agents and

Chemotherapy, 57, 3348-3357.

• Cited 158 times since publication

• Data used for genome analysis

• Data used to build AMR models

(e.g. ResFams)

• Data used to enrich a database

(e.g. IslandViewer, ARG-

ANNOT)

• RGI use to predict resistome

• Averaging ~3000 visits per month

and ~300 RGI analyses per month

1

List of antibiotic resistance genes curated in the CARD as of February 2015. Each entry provides

citation, molecular sequence, protein structure, mechanism, and Antibiotic Resistance Ontology (ARO)

classification details. * There a large number of antibiotic efflux pump proteins, see the CARD for the

complete list. Efflux pumps are cross-referenced to the antibiotic molecules exported. aminocoumarins

aminocoumarin resistant DNA topoisomerases

aminocoumarin resistant GyrB, ParE, ParY

aminoglycosides aminoglycoside acetyltransferases

AAC(1), AAC(2'), AAC(3), AAC(6')

aminoglycoside nucleotidyltransferases

ANT(2''), ANT(3''), ANT(4'), ANT(6), ANT(9)

aminoglycoside phosphotransferases

APH(2''), APH(3''), APH(3'), APH(4), APH(6),

APH(7''), APH(9)

16S ribosomal RNA methyltransferases

ArmA, RmtA, RmtB, RmtC, Sgm, etc.

b-lactams

class A b-lactamases

AER, BLA1, CTX-M, KPC, SHV, TEM, etc.

class B (metallo-) b-lactamases

BlaB, CcrA, IMP, NDM, VIM, etc.

class C b-lactamases

ACT, AmpC, CMY, LAT, PDC, etc.

class D b-lactamases

OXA b-lactamases

methicillin resistant PBP2

mecA, mecC

mutant porins protein conferring antibiotic resistance

antibiotic resistant Omp36, OmpF, PIB (por)

genes modulating b-lactam resistance

bla (blaI, blaR1) and mec (mecI, mecR1) operons

chloramphenicol chloramphenicol acetyltransferase (cat)

chloramphenicol phosphotransferase

chloramphenicol/florfenicol export proteins

cmlA, floR, MexEF-OprN, AcrAB-TolC, etc.

elfamycins elfamycin resistant elongation factor Tu

facT

ethambutol ethambutol resistant arabinosyltransferase (EmbB)

fluoroquinolones

fluoroquinolone acetyltransferase

fluoroquinolone resistant DNA topoisomerases

fluoroquinolone resistant GyrA, GyrB, ParC

quinolone resistance proteins (Qnr)

fosfomycin fosfomycin phosphotransferases

FomA, FomB, FosC, FosC2

fosfomycin thiol transferases

FosA, FosB, FosX

fosfomycin resistant murA

fusidic acid fusidic acid esterase fusH

glycopeptides bleomycin resistance protein (BRP)

teicoplanin resistance protein vanJ

glycopeptdie resistance clusters

VanA, VanB, VanC, VanD, etc.

lincosamides Cfr 23S ribosomal RNA methyltransferase

clbA, clbB, clbC

Erm 23S ribosomal RNA methyltransferases

ErmA, ErmB, Erm(31), etc.

lincosamide nucleotidyltransferase (Lin)

linezolid Cfr 23S ribosomal RNA methyltransferase

lipopeptide antibiotics polymyxin resistance genes arnA, rosA, rosB

daptomycin resistance liaFSR

antibiotic resistant cardiolipin synthetase (cls)

lipopeptide resistant beta-subunit of RNA polymerase

macrolides Cfr 23S ribosomal RNA methyltransferase



non-Erm 23S ribosomal RNA methyltransferase

rlmA(II), chrB, myrA, tlrB

macrolide esterases

EreA, EreB

macrolide glycosyltransferases

GimA, Mgt, Ole

macrolide phosphotransferases (MPH)

MPH(2')-I, MPH(2')-II, MPH-C

mupirocin mupirocin resistant isoleucyl-tRNA synthetases

MupA, MupB

peptide antibiotics integral membrane protein MprF

bactitracin resistance genes bacA, bcrC

non-Erm 23S ribosomal RNA methyltransferase tsnr

viomycin phosphotransferase

phenicol

Cfr 23S ribosomal RNA methyltransferase

chloramphenicol/florfenicol resistance protein (cmlA)

rifampin

rifampin ADP-ribosyltransferase (Arr)

rifampin glycosyltransferase

rifampin monooxygenase

rifampin phosphotransferase

rifampin resistance RNA polymerase-binding proteins

DnaA, RbpA

rifampin resistant beta-subunit of RNA polymerase (RpoB)

streptogramins Cfr 23S ribosomal RNA methyltransferase



streptogramin Vgb lyase

Vat acetyltransferase

streptothricin streptothricin acetyltransferase (sat)

sulfonamides sulfonamide resistant dihydropteroate synthases

Sul1, Sul2, Sul3, sulfonamide resistant FolP

tetracyclines mutant porin PIB (por) with reduced permeability

tetracycline inactivation enzyme TetX

tetracycline resistance ribosomal protection proteins

TetM, TetO, TetQ, Tet32, Tet36, etc.

trimethoprim

trimethoprim resistant dihydrofolate reductase dfr

tunicamycin tunicamycin binding protein tmrB

efflux pumps conferring antibiotic resistance * ATP-binding cassette (ABC) antibiotic efflux pumps

major facilitator superfamily (MFS) antibiotic efflux pumps

multidrug & toxic compound extrusion (MATE) transporters

resistance-nodulation-cell division (RND) efflux pumps

small multidrug resistance (SMR) antibiotic efflux pumps

genes modulating antibiotic efflux adeR, acrR, baeSR, mexR, phoPQ, mtrR, etc.

perfect match

screening

novel emergent

AMR genes

novel functional

variants

dedicated resistance genes;

often plasmid-borne

resistance by mutation;

predominantly genomic

whole genome sequencing

(WGS); whole AMR genes

whole community sequencing

(metagenomics); partial gene sequencing

intrinsic resistance;

regulatory

Analyzing the Resistome

Resistance Gene Identifier

perfect

strict

loose / discovery

matches reference

sequence

similarity within

model

similarity outside

of model

“known known”

“known unknown”

“unknown unknown”


McMaster University Hospital – clinical MDR Klebsiella pneumoniae isolate








• RGI designed for whole genomes or assembly contigs

• BLAST-based homlogy analysis + SNP mapping

• Thus effective based on CARD’s canonical reference sequences

• Is CARD data effective for metagenomics analysis?

• Active area of RGI development

• CARD’s canonical reference sequences may under-represent nucleotide

sequence diversity for AMR genes ‘in the wild’

• Burrows-Wheeler Transform read mapping to CARD:

• has a false negative rate due to missing sequence diversity in CARD

• needs to incorporate SNP screening for total resistome prediction


Freschi et al. 2015. Frontiers in Microbiology 6:01036.


Freschi et al. 2015. Frontiers in Microbiology 6:01036.


• RGI has false positives for a Brazilian epidemic strain of Pseudomonas aeruginosa

• Not a failure of the RGI algorithms, but instead a gap in CARD curation

• blaSPM-1 included in the Antibiotic Resistance Ontology but models not yet

added

• Byciclomycin resistance not yet curated into CARD, including the bcr-1 gene

CARD

human curation

AMR detection model with reference sequence, cut-offs, and parameters

• Reference sequence must be published and have GenBank accession

• Publication must have clear evidence of resistance

CARD

human curation




CARD*Shark text mining algorithms to prioritize and

triage AMR literature

CARD

human curation

Wild*CARD algorithms




New distribution of CARD

reference sequences among

pathogens, genomes, plasmids

Detection of sequence variants

that have been published and

have GenBank accessions

Detection of novel sequence

variants among pathogens

Downloads

• Ontology OBO, JSON, tab-delimited files

• Antibiotic Resistance Ontology

• Detection Model Ontology

• NCBI Taxon Ontology

• CARD Detection Models

• JSON format

• Mutation catalogs

• Nucleotide & Protein Sequences

• FASTA by model type (i.e. homolog screening versus mutant screening)

• ARO classification and index files

• Stand-alone, command line Resistance Gene Identifier software

• Commercial use?

• With a license or collaborative agreement

Frequently Asked Questions

• What data can be included? Can I add unpublished data?

• Only peered reviewed, published data that is also associated with a

GenBank accession can be included in the CARD. SNP and other

mutations not known from clinical settings are currently excluded.

• How do I find a list of all resistance genes in a particular organism?

• CARD is based on reference gene sequences, so does not fully annotate

genomes.

• For intrinsic resistance genes for which resistance is conferred by specific

mutations, does CARD include all known mutant sequences?

• CARD only maintains a complete list of all resistance SNPs relative to a

reference sequence, which may either be a reported mutant sequence or a

wild-type sequence. As such, it is important that SNP mapping be included

in analysis of any genes that require mutation to confer resistance.

Frequently Asked Questions

• How are Minimum Inhibitory Concentration (MIC) data curated?

• The CARD does not yet curate MIC data directly, but instead records the

resistance profile of resistance genes, e.g. beta-lactamases

confers_resistance_to beta-lactams.

• Can CARD and the RGI accurately predict antibiogram?

• Curation of confers_resistance_to_drug relationships for accurate

prediction of antibiogram is currently inconsistent throughout the CARD

and our RGI software is focussed primarily upon accurate prediction of

resistome, not antibiogram.

• Does the Resistance Gene Identifier (RGI) work for metagenomics data?

• Currently the RGI does not analyze metagenomics data, outside of a simple

BLASTX algorithm (with SNP screening) available as a beta-test feature.

The default RGI behaviour attempts to predict complete open reading

frames (ORFs) from submitted nucleotide data, which will fail for short

metagenomic reads.

Challenges

• Biocuration of reference data is difficult to fund, but without gold standard

reference data the power of NGS erodes

• AMR genomic data is never static – unlike traditional biocuration, we are not

annotating a static genome – the target is always moving

• Sequencing technology is getting smaller and cheaper – we will be collecting

increasing amounts, even at the level of clinic or farm – we need a far-reaching

data storage and sharing paradigm

• metagenomics data is difficult and computationally costly – we are currently

only analyzing the easiest subsets of AMR mechanisms; mutation often ignored

• Easy to use translational tools increasingly needed!

What we are not currently collecting

• Minimum inhibitory dose (MIC) – GenBank is planning MIC collection

• Pathogen and AMR gene prevalence

• Poor curation of data on plasmids and other aspects of the mobilome

• Environmental and clinical metadata

Thanks!

Amos Raphenya, Brian Alcock, Biren Dave, Kara Tsang, Briony Lago

Justin Jia

Arjun Sharma

Pearl Guo

(UWaterloo)

Sachin Doshi Tariq Elsayegh

(Royal College of

Surgeons in Ireland)

Daim Sardar

Thanks!

amr.mcmaster.ca

Arjun Sharma

• Dr. Gerry Wright (McMaster) and the CARD Consortium

• GenEpiO Consortium

• CARD & RGI Beta-testers!

• McMaster University & Cisco Systems Canada, Inc.

• Sightline Innovation, Inc.

• National Microbiology Laboratory, Canada – IRIDA Surveillance Project

• United States Department of Agriculture – Agricultural Research Service

• National Center for Biotechnology Information (NCBI, USA)

• bioMérieux France, Inc.

• Pseudomonas Consortia – Dr. Roger C. Lévesque, Laval University

• IslandViewer Database – Dr. Fiona Brinkman, UBC

• Dr. Laura Piddock (UK) – efflux in the ARO & CARD

• β- lactamase nomenclature and informatics consortium

• Many more…

Thanks!

Documents

The Comprehensive Antibiotic Resistance …rami-ngs.org/.../The-comprehensive-antibiotic-resistance-database_a...The Comprehensive Antibiotic Resistance Database ... • An essential