Exercise 1 – AMR tools Group presentations NOTE: This material is only intended for the EURL-AR training course 2019 participants as a follow-up to the discussions we had in the classroom and any use outside its scope is not under the responsibility of the EURL-AR Date DTU Title 1 Before we start… • It was on purpose not to give too detailed instructions about the tools of Exercise 1 and to let you look at the different tools and trigger your curiosity (last year we went step by step – please look at the manual from last year/send me an email if you would like to receive it) • Do not be frustrated if you did not reach the end of the exercises – it was not meant to happen for all, however each step represented a potential new knowledge gained and you should focus on what you have learned Date DTU Title 2 Isolate 1 – Campylobacter jejuni Date DTU Title 3 Exercise 1 Group 1 & 19: Daniel, Mike and Deirdre Species ID: Isolate number 1: Campylobacter jejuni Date DTU Title QC Fasta/Fastq • FastQC (free tool) –2.3 million reads, trimmed data all close to Q30 • Bbmap (free tool) – showed quality of assembly N50: 333 kb, Contigs: 13, Longest contig 646 kb, total number bps 1.7 Mb • Species finder (CGE): Campylobacter jejuni Conclusion: Species correct and data of good quality Note the freely available tools for estimating sequence quality Date DTU Title Bioinformatics tools/not used and why Successfully used • ResFinder-3 –user friendly, familiar with, reputable • ResFinder-4 - user friendly, familiar with, reputable • CARD – user friendly Tried • ABRES Finder – input amino acid sequence • ABRICATE – need to download and cannot on Labtop • ARDB – no longer maintained, last updated 2009 • ARG-ANNOT - DISCONTINUED • ARIBA - need to download and cannot on Labtop • NCBI AMRFinder – looked at it but difficult to navigate and see how to upload files • Noradab – could not upload files, had to copy the fasta sequence Date DTU Title Results Observed Predicted resistance resistance STRAIN 1 Tools phenotype phenotype RESFIND RESFIND RESFIND AMR gene or ER 3.2 ER 3.2 ER 4.0 mutation FASTA FASTQ FASTA CARD blaOXA-461 yes yes yes yes Natural gene in Campy Intrinsic resistance? tet(W/N/ Tetracycline tet(O/32/O) yes yes yes W) Tetracycline resistance resistance Ciprofloxacin Ciprofloxacin gyrA p.T86I (known) yes yes yes yes resistance resistance 4 unknown mutations in gyrA yes yes no 14 unknown mutations in 23S yes yes no ResFinder 4.0 – submitted fastq files but job failed Date DTU Title Date DTU Title Date DTU Title ResFinder 3.2 Fasta Date DTU Title Difference between Resfinder and Card Date DTU Title ResFinder vs CARD % Identity CARD ResFinder tet(W/N/W) 68.34 tet(O/32/O) 100 Conclusion: • If using CARD only Tet resistance could have been missed/unsure how to interpret • Output from CARD very fast • CARD showed gyrA present, not 100% (99.5%) • CARD does not show unknown mutations • Information provided by ResFinder more complete Date DTU Title Phenotypic results for isolate 1 ciprofloxacin erythromycin gentamicin nalidixic acid streptomycin tetracycline MIC: 16.0 > 0.5 MIC: <=1.0 > 4.0 MIC: 0.5 > 2.0 MIC: 8.0 > 16.0 MIC: 1.0 > 4.0 MIC: 32.0 > 1.0 Note that there seems to be genotype-phenotype discordance for nalidixic acid (the isolate is genotypically resistant (gyrA T86I) and phenotypically susceptible). However the nalidixic acid MIC measured phenotypically is close to the ECOFF and the isolate is also ciprofloxacin-resistant thus the observed discordance is likely due to limitations in the broth microdilution method in which a two-fold dilution difference between repeated measurements is considered acceptable. Date DTU Title Does the predicted phenotype match the phenotype measured in the laboratory? Yes • Phenotype measured in the Laboratory: CIP and TET resistant • Predicted phenotype based on bioinformatics analysis: Ciprofloxacin and Tetracycline resistance Date DTU Title Isolate 2 – Campylobacter jejuni Date DTU Title 15 Group 9 Isolate 2 Campylobacter jejuni • ResFinder 4 and 3.2, CARD and Kmer Resistance 2.2. • Reasons: is curated, updated regularly and used in many recent research works. CARD is having chromosomal mutations, more information can be generated. • Differences in the results are observed between different bioinformatics tools, • Reasons: different pipelines, quality parameters for selection might differ % of coverage and identity, FASTA Vs FASTQ data, different assemblers, some are not including the efflux pumps Note that ResFinder also includes PointFinder for detection of chromosomal mutations but the user needs to actively select this option in the front page Date DTU Title There is genotype-phenotype discordance for tetracycline since CARD Resfinder 4 ResFinder 3.2 Kmer genotypic detection of tetO would lead to prediction of AMR gene or (Fasta (FASTQ/FAS (FASTQ/FA (FASTA/FAST mutation ) TA) STA) tetracyclineQ) resistance whereas Predicted the phenotypic R test yielded a OXA460 yes No/no Yes/yestetracyclineYes/yes susceptible isolate.cephalosporin, However, tetO Beta-lactamhad <100% identity to the gene in the databaseMulticlass thus we(including cannot exclude macrolide fluoroquinolone, mutation(s) leading to stop codonaminoglycoside, and thus lack of tetO cmeR yes No/no No/no expression.No/no This could be easilycephalosporin, investigated bytetracycline) analyzing the tetO yes No/yes Yes/yes Yes/yes tetracycline Campylobacter jejuni sequence although this ‘manual’ analysis would not be very gyrA conferring easily applicable when analyzing large datasets. Furthermore, resistance to fluoroquinolones yes No/yes Yes/yesthe tetracyclineNo/no MIC measuredfluoroquinolone phenotypically is close to the ECOFF the broth microdilution method has limitations by which a two-fold dilution difference between repeated measurements is Phenotipic considered acceptable Isolate ciprofloxacin erythromycin gentamicin nalidixic acid streptomycin tetracycline 2 MIC: 16.0 > 0.5 MIC: <=1.0 > 4.0 MIC: 0.25 > 2.0 MIC: >64.0 > 16.0 MIC: 0.5 > 4.0 MIC: <=0.5 > 1.0 This discrepancy is unusual and will be further examined by ResFinder 4 developers Date DTU Title Isolate 3 – Escherichia coli Date DTU Title 18 GROUPS 3 and 17 Exercise 1 Date DTU Title • Isolate Nr. 3 • Escherichia coli • TOOLS USED ResFinder (usually used, reference method for comparison) – FASTA, FASTQ CARD (suggested one) – FASTA • Differences between FASTA and FASTQ input in the same tool but it didn’t change the predicted phenotype • Different output – table, or .txt file you need to figure out a way to open Date DTU Title RESULTS • CARD gave lot more hits mainly in antibiotic efflux mechanisms – CARD has more data WHY? • different approach – acquired or intrinsic, plasmid or chromosomal, functional or not • Different nomenclature (ampC – not normaly expressed in the chromosome except for a promotor mutation, expressed only on plasmid) • Difference in phenotype prediction between tools: CARD gave hits we cannot predict the phenotype with ResFinder gives hits only with a predictable phenotype • Both give mutated genes Date DTU Title RESULTS • CARD gave lot more hits mainly in antibiotic efflux mechanisms – CARD has more data WHY? • different approach – acquired or intrinsic, plasmid or chromosomal, functional or not • Different nomenclature (ampC – not normaly expressed in the chromosome except for a promotor ampC is a chromosomal gene normally repressed in E. coli. Chromosoaml mutation, expressed only on plasmid) mutations can lead to removal of such repression and expression of the chromosomal ampC that encodes an AmpC-type beta-lactamase. • Difference in phenotype prediction between tools: CARD gave hits we cannot predictOf the note, phenotype there are withmany genes other than chromosomal ampC that can encode AmpC-type beta-lactamases and they are usually plasmid- ResFinder gives hits only with a predictablemediated in phenotype E. coli • Both give mutated genes Date DTU Title RESULTS • CARD gave lot more hits mainly in antibiotic efflux mechanisms – CARD has more data WHY? • different approach – acquired or intrinsic, plasmid or chromosomal, functional or not • Different nomenclature (ampC – not normaly expressed in the chromosome except for a promotor mutation, expressed only on plasmid) • Difference in phenotype prediction between tools: CARD gave hits we cannot predict the phenotype with ResFinder gives hits only with a predictable phenotype • Both give mutated genes Date DTU Title CONCORDANCE • The predicted and measured phenotypes basicaly matched • CARD found other genes but could not predict the phenotype using them • Active or inactive genes, selective pressure, mutations Date DTU Title Isolate 4 – Escherichia coli Date DTU Title 25 Exploring the jungle of AMR detection tool Groups 7, 13 Exercise 1 • Specie ID isolate Escherichia coli, isolate code 4 • Which bioinformatics tools did you use and why? As a beginners => tools recommended in manual: ResFinder and CARD Date DTU Title Please note: even when using the same database, different Groups 7, 13 tools may give different results because: - The algorithm used by different tools is different Exercise 1 - It often happens that updates made in one database are not immediately captured by all tools using such database • Which bioinformatics tools did you not use and why? dedicated to another specific species e.g. Staphylococcus - SCCmecFinder-1.2 Tools based on the same databases as those that we used Those that require access account Date DTU Title Escherichia coli, isolate code 4 depending on used tool results differed Tools used Predicted resistance phenotype Observed resistance phenotype AMR gene/mutation ResFinder‐3CARD blaTEM‐1B yes yes (perfect match) Ampicillin, Cefotaxime,Ceftazidime Ampicillin, Cefotaxime,Ceftazidime tet(A) yes (99,92% identity) yes (strict match) Tetracycline Tetracycline R (MIC: >64.0 > 8.0) ampC promoter n.42C>T yes no Ampicillin, Cefotaxime, Ceftazidime Ampicillin, Cefotaxime, Ceftazidime gyrA p.D87N GAC‐>AAC yes no Ciprofloxacin, Nalidix acid Ciprofloxacin, Nalidix acid yes => Warning: gene is missing from Notes mdf(A) no Macrolide not provided, not tested? file.