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Tried • ABRES Finder – input amino acid sequence • ABRICATE – need to download and cannot on Labtop • ARDB – no longer maintained, last updated 2009 • ARG-ANNOT - DISCONTINUED • ARIBA - need to download and cannot on Labtop • NCBI AMRFinder – looked at it but difficult to navigate and see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/ tet(O/32/O) yes yes yes W) Tetracycline resistance resistance 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 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., 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 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 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. Please inform curator

tolC no yes antibiotic efflux acrB no yes antibiotic efflux Escherichia coli acrA no yes antibiotic efflux Escherichia coli ampH beta‐lactamase no yes antibiotic inactivation emrB no yes antibiotic efflux emrR no yes antibiotic efflux PmrF no yes antibiotic target alteration cpxA no yes antibiotic efflux mdtG no yes antibiotic efflux H‐NS no yes antibiotic efflux mdtE no yes antibiotic efflux antibiotic efflux, reduced permeability to marA no yes antibiotic mdtB no yes antibiotic efflux msbA no yes antibiotic efflux emrY no yes antibiotic efflux evgA no yes antibiotic efflux

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. Please inform curator This gene is intrinsic in E. coli. It may be a useful marker to tolC no yes antibiotic efflux acrB no yes antibioticverify efflux correct species identification. Escherichia coli acrA no yes antibiotic efflux Escherichia coli ampH beta‐lactamase no yes antibiotic inactivation emrB no yes antibiotic efflux emrR no yes antibiotic efflux PmrF no yes antibiotic target alteration cpxA no yes antibiotic efflux mdtG no yes antibiotic efflux H‐NS no yes antibiotic efflux mdtE no yes antibiotic efflux antibiotic efflux, reduced permeability to marA no yes antibiotic mdtB no yes antibiotic efflux msbA no yes antibiotic efflux emrY no yes antibiotic efflux evgA no yes antibiotic efflux

Date DTU Title Escherichia coli , isolate code 4 , CARD perfect hits 17, strict hits 38

How to report strict matches?

This really depends on the specific purpose of the analysis.

Date DTU Title Escherichia coli , isolate code 4 , ResFinder Chromosomal point mutations - Results

Date DTU Title Escherichia coli , isolate code 4 Are there differences between the results from different bioinformatics tools? If yes, how can you explain them? YES

• Possible explanation: - differences in database curation - different algorhitms of data handling…???

Date DTU Title Isolate 5 – Salmonella sp.

Date DTU Title 33 Group 5 and Species ID of the isolate: 15 - Salmonella sp. Strain 5

Which bioinformatics tools did you use and why? - PointFinder -CARD - ResFinder It was suggested us to use those. Some tool are specific for some microrganisms. Easier to use.

Which bioinformatics tools did you not use and why? - the remaining Not enough time to use more tools. Some tools are specific for some microrganisms (ex. MUBII-TB-DB). Some tools need expertise, to be downloaded and to work in command line.

Date DTU Title Observed resistance What are the results?Tools Predicted resistance phenotype phenotype ResFinder-3 AMR gene or mutation (90,60) CARD PointFinder blaTEM-1B/TEM-1 yes yes Beta-lactam resistance Alternate name; RblaTEM-1 Ampicillin R fosA7 yes yes resistance tet(A) yes yes Tetracycline resistance Tetracycline R sul1 yes yes Sulphonamide resistance sul3 yes yes Sulphonamide resistance aac(3)-VIa yes yes resistance Gentamicin R R (MIC of colistin does not always increase above mcr-9 yes yes ECOFF) aac(6')-Iaa yes no Aminoglycoside resistance Gentamicin R aac(6')-Ib3 yes no Aminoglycoside (Amikacin, Tobramycin) aac(6')-Ib-cr yes no Fluoroquinolone and aminoglycoside resistance Cipro S; Gentamicin R aadA1 yes no Aminoglycoside resistance golS no yes Antibiotic efflux Mar-A no yes Antibiotic efflux AAC(6')-Iy no yes Aminoglycoside antibiotic Gentamicin R H-NS no yes antibiotic efflux sdiA no yes antibiotic efflux MdtK no yes antibiotic efflux baeR no yes antibiotic efflux kdpE no yes aminoglycoside antibiotic, antibiotic efflux Gentamicin R Escherichia coli acrA no yes antibiotic efflux acrB no yes antibiotic efflux Escherichia coli ampH beta-lactamase no yes ampC-type beta-lactamase bacA no yes peptide antibiotic emrB no yes antibiotic efflux emrR no yes antibiotic efflux CRP no yes antibiotic efflux AAC(6')-Ib9 no yes aminoglycoside antibiotic Gentamicin R UhpT no yes fosfomycin GlpT no yes fosfomycin parC mutationDate DTU T57S Nalidixic Acid; Ciprofloxacin Cipro S; nalidixicTitle acid S Observed resistance What are the results?Tools Predicted resistance phenotype phenotype ResFinder-3 AMR gene or mutation (90,60) CARD PointFinder blaTEM-1B/TEM-1 yes yes Beta-lactam resistance Alternate name; RblaTEM-1 Ampicillin R fosA7 yes yes Fosfomycin resistance tet(A) yes yes Tetracycline resistance Tetracycline R sul1 yes yes Sulphonamide resistance sul3 yes yes Sulphonamide resistance aac(3)-VIa yes yes Aminoglycoside resistance Gentamicin R Colistin R (MIC of colistin does not always increase above mcr-9 yes yes ECOFF) aac(6')-Iaa yes no Aminoglycoside resistance Gentamicin R aac(6')-Ib3 yes no Aminoglycoside (Amikacin, Tobramycin) aac(6')-Ib-cr yes no Fluoroquinolone and aminoglycoside resistance Cipro S; Gentamicin R aadA1 yes no Aminoglycoside resistance golS no yes Antibiotic efflux Mar-AThis gene was not no100% identical yesto the reference gene in Antibiotic the database. efflux Only specific mutations in aac(6’)-Ib-cr are AAC(6')-Iy no yes Aminoglycoside antibiotic Gentamicin R H-NSassociated with ciprofloxacin no resistance yes thus finding a gene antibiotic that hasefflux <100% identity to the gene in the database may sdiA no yes antibiotic efflux MdtKlead to two hypothesis: no yes antibiotic efflux baeR no yes antibiotic efflux kdpE1. The aac(6’)-Ib-cr no variant detected yes is not associated with aminoglycoside ciprofloxacin antib resistanceiotic, antibiotic efflux Gentamicin R Escherichia coli acrA no yes antibiotic efflux acrB no yes antibiotic efflux Escherichia2. The colidatabase ampH does not contain all variants known to be associated with ciprofloxacin resistance beta-lactamase no yes ampC-type beta-lactamase bacAKnowing the ResFinder no database, yesI can tell you that in this peptide case antibiotic hypothesis 1 is the correct one. This is another emrB no yes antibiotic efflux emrRexample of the importance no of knowing yes the databases used antibiotic and of efflux communicating with colleagues as we all have CRP no yes antibiotic efflux AAC(6')-Ib9different expertise andno WGS-based yes detection of AMR requires aminoglycoside knowledge antibiotic of different disciplines Gentamicin R UhpT no yes fosfomycin GlpT no yes fosfomycin parC mutationDate DTU T57S Nalidixic Acid; Ciprofloxacin Cipro S; nalidixicTitle acid S Observed resistance What are the results?Tools Predicted resistance phenotype phenotype ResFinder-3 AMR gene or mutation (90,60) CARD PointFinder blaTEM-1B/TEM-1 yes yes Beta-lactam resistance Alternate name; RblaTEM-1 Ampicillin R fosA7 yes yes Fosfomycin resistance tet(A) yes yes Tetracycline resistance Tetracycline R sul1 yes yes Sulphonamide resistance sul3 yes yes Sulphonamide resistance aac(3)-VIa yes yes Aminoglycoside resistance Gentamicin R Colistin R (MIC of colistin does not always increase above mcr-9 yes yes ECOFF) aac(6')-Iaa yes no Aminoglycoside resistance Gentamicin R aac(6')-Ib3 yes no Aminoglycoside (Amikacin, Tobramycin) aac(6')-Ib-cr yes no Fluoroquinolone and aminoglycoside resistance Cipro S; Gentamicin R aadA1 yes no Aminoglycoside resistance golS no yes Antibiotic efflux Mar-A no yes Antibiotic efflux AAC(6')-Iy no yes Aminoglycoside antibiotic Gentamicin R H-NS no yes antibiotic efflux sdiA no yes antibiotic efflux MdtK no yes antibiotic efflux baeR no yes antibiotic efflux kdpE no yes aminoglycoside antibiotic, antibiotic efflux Gentamicin R Escherichia coli acrA no yes antibiotic efflux acrB no yes antibiotic efflux Escherichia coli ampH beta-lactamase no yes ampC-type beta-lactamase bacAThis apparent genotype-phenotype no yes discordance is due to the peptide fact antibiotic that parC T57S alone is not sufficient for nalidixic acid emrB no yes antibiotic efflux emrRand ciprofloxacin resistance, no but requires yes simultaneous occurrence antibiotic efflux of other mutations such as gyrA_S83Y.F.A or CRP no yes antibiotic efflux AAC(6')-Ib9gyrA_D87N.G.Y.K no yes aminoglycoside antibiotic Gentamicin R UhpT no yes fosfomycin GlpT no yes fosfomycin parC mutationDate DTU T57S Nalidixic Acid; Ciprofloxacin Cipro S; nalidixicTitle acid S Isolate 7 – Enterococcus faecium

Date DTU Title 38 Date DTU Title 39 Comment

Date DTU Title 40 Comment resistance is mediated by genes that are part of operons. In this case, vanB is the resistance gene and all the other van genes are components of the operon and only some of them are essential for vanB expression. Different databases deal differently with genes in operons (e.g. some databases include the genes one by one - for example vanH, vanB and vanX - and other databases include the full operon as one sequence – for example vanHBX)

Date DTU Title 41 Comment

Date DTU Title 42 Comment

Date DTU Title 43 Date DTU Title 44 Isolate 8 – Enterococcus faecium

Date DTU Title 45 Our experience with bioinformatic tools used Tools used Name of tool ResFinder3.2 CARD ResFinder4 Input file (fastq or fasta) fasta fasta fasta other observations you may want to report take a time RGI only, BLAST need one line - there is one finally or delete the row one contig table more - like conclusion, the second table is like from ResFinder 3.2, but there are information for point mutation also other observations you may want to report two types of results: aquired ARM graf interactive tool, we were or delete the row antimicrobial resistent result, confused due to a lot of data, which point mutation result are no so important

Reasons for not using the tool Predicted phenotype in one some genes are not present in this is the best one pack of results, sorted base predicted fenotype table antimicrobial resistance gene

Date DTU Title Enterococcus faecium Group no.: Isolate ID: 8 Tools Predicted resistance phenotypeObserved resistance phenotype AMR gene or ResFinder CARD mutation pbp5 yes Ampicillin R parC yes Nalidixic acid, Ciprofloxacin R gyrA yes Nalidixic acid, Ciprofloxacin R cat(pC221) 97,69% yes Phenicol resistance S aac(6')-Ii yes yes Warning: gene is missing from Notes file. Please inform R curator. aac(6')-aph(2'') yes no Aminoglycoside resistance R ant(6)-Ia yes no Aminoglycoside resistance R erm(B) yes yes Macrolide resistance R msr(C) 98,92 no Macrolide, Lincosamide and Streptogramin B resistance Macrolide R tet(M) yes yes Tetracycline resistance R VanHAX yes yes vancomycin R dfrF no yes not tested aad(6) no yes aminoglycoside antibiotic efmA no yes macrolide antibiotic; fluoroquinolone antibiotic; lincosamide antibiotic; fosfomycin; cephalosporin; glycylcycline; bicyclomycin; penam; nucleoside antibiotic; tetracycline antibiotic; peptide antibiotic; acridine dye; oxazolidinone antibiotic; antibiotic; diaminopyrimidine antibiotic; phenicol antibiotic; ; benzalkonium chloride; rhodamine; antibacterial free fatty acids; nitroimidazole antibiotic Note from Valeria: comments tetU no yes macrolide antibiotic; fluoroquinolone antibiotic; lincosamide antibiotic; fosfomycin; cephalosporin; glycylcycline; bicyclomycin; penam; nucleoside to the results in the next slide antibiotic; tetracycline antibiotic; peptide antibiotic; acridine dye; oxazolidinone antibiotic; rifamycin antibiotic; diaminopyrimidine antibiotic; phenicol antibiotic; isoniazid; benzalkonium chloride; rhodamine; antibacterial free fatty acids; Date DTU Title nitroimidazole antibiotic Enterococcus faecium Group no.: Isolate ID: 8 Tools Predicted resistance phenotypeObserved resistance phenotype AMR gene or ResFinder CARD mutation pbp5 yes Ampicillin R parC yes Nalidixic acid, Ciprofloxacin R gyrA yes Nalidixic acid, Ciprofloxacin R cat(pC221) 97,69% yes Phenicol resistance S aac(6')-Ii yes yes Warning: gene is missing from Notes file. Please inform R curator. aac(6')-aph(2'') yesNote the no genotype-phenotype Aminoglycoside discordance forresistance phenicol resistance: R ant(6)-Ia yes no Aminoglycoside resistance R erm(B) yesgenotypically yes we would predict resistance Macrolide resistance based on cat(pC221) presence R msr(C) 98,92 no Macrolide, Lincosamide and Streptogramin B resistance Macrolide R tet(M) yeswhereas yes phenotypically the isolate Tetracycline was chloramphenicol resistance susceptible. However, R VanHAX yesthe cat yes gene was <100% identical to vancomycin the gene in the database, maybe the R dfrF no yes trimethoprim not tested aad(6) nomutation(s) yes inserted a stop codon aminoglycoside and the gene antibiotic was not translated? Prediction efmA no yes macrolide antibiotic; fluoroquinolone antibiotic; lincosamide antibiotic; of phenotype basedfosfomycin; on detection cephalosporin; of glycylcycl genesine; with bicyclomycin; <100% penam; identity nucleoside to reference antibiotic; tetracycline antibiotic; peptide antibiotic; acridine dye; oxazolidinone genes may be problematic,antibiotic; rifamycin however antibiotic; diaminop detectionyrimidine of antibiotic; such phenicol genes antibiotic; may still be isoniazid; benzalkonium chloride; rhodamine; antibacterial free fatty acids; nitroimidazole antibiotic tetU norelevant yes for epidemiologicalmacrolide antibiotic; purposes fluoroquinolone antibiotic; lincosamide antibiotic; fosfomycin; cephalosporin; glycylcycline; bicyclomycin; penam; nucleoside antibiotic; tetracycline antibiotic; peptide antibiotic; acridine dye; oxazolidinone antibiotic; rifamycin antibiotic; diaminopyrimidine antibiotic; phenicol antibiotic; isoniazid; benzalkonium chloride; rhodamine; antibacterial free fatty acids; Date DTU Title nitroimidazole antibiotic Enterococcus faecium Group no.: Isolate ID: 8 Tools Predicted resistance phenotypeObserved resistance phenotype AMR gene or ResFinder CARD mutation No phenotype for aac(6’)-aph(2’’) was tested (it should confer pbp5 yes Ampicillin R parC yesgentamicin, amikacin and tobramycin Nalidixic acid, resistance) Ciprofloxacin so we cannot R gyrA yesconclude on genotype-phenotype Nalidixic concordance acid, Ciprofloxacin for this gene! R cat(pC221) 97,69% yes Phenicol resistance S aac(6')-Ii yes yes Warning: gene is missing from Notes file. Please inform R curator. aac(6')-aph(2'') yes no Aminoglycoside resistance R ant(6)-Ia yes no Aminoglycoside resistance R erm(B) yes yes Macrolide resistance R msr(C) 98,92 no Macrolide, Lincosamide and Streptogramin B resistance Macrolide R tet(M) yes yes Tetracycline resistance R VanHAX yes yes vancomycin R dfrF no yes trimethoprim not tested aad(6) no yes aminoglycoside antibiotic efmA no yes macrolide antibiotic; fluoroquinolone antibiotic; lincosamide antibiotic; fosfomycin; cephalosporin; glycylcycline; bicyclomycin; penam; nucleoside antibiotic; tetracycline antibiotic; peptide antibiotic; acridine dye; oxazolidinone antibiotic; rifamycin antibiotic; diaminopyrimidine antibiotic; phenicol antibiotic; isoniazid; benzalkonium chloride; rhodamine; antibacterial free fatty acids; nitroimidazole antibiotic tetU no yes macrolide antibiotic; fluoroquinolone antibiotic; lincosamide antibiotic; fosfomycin; cephalosporin; glycylcycline; bicyclomycin; penam; nucleoside antibiotic; tetracycline antibiotic; peptide antibiotic; acridine dye; oxazolidinone antibiotic; rifamycin antibiotic; diaminopyrimidine antibiotic; phenicol antibiotic; isoniazid; benzalkonium chloride; rhodamine; antibacterial free fatty acids; Date DTU Title nitroimidazole antibiotic Enterococcus faecium Group no.: Isolate ID: 8 Tools Predicted resistance phenotypeObserved resistance phenotype AMR gene or ResFinder CARD mutation pbp5 yes Ampicillin R parC yes Nalidixic acid, Ciprofloxacin R gyrA yes Nalidixic acid, Ciprofloxacin R cat(pC221) 97,69% yes Phenicol resistance S aac(6')-Ii yes yes Warning: gene is missing from Notes file. Please inform R curator. aac(6')-aph(2'') yes no Aminoglycoside resistance R ant(6)-Ia yes no Aminoglycoside resistance R erm(B) yesNo phenotype yes for aac(6’)-Ii was Macrolide tested (it resistance should confer amikacin and R msr(C) 98,92 no Macrolide, Lincosamide and Streptogramin B resistance Macrolide R tet(M) yestobramycin yes resistance) so wecannot Tetracycline conclude resistance on genotype- R VanHAX yes yes vancomycin R dfrF nophenotype yes concordance for this gene! trimethoprim not tested aad(6) no yes aminoglycoside antibiotic efmA no yes macrolide antibiotic; fluoroquinolone antibiotic; lincosamide antibiotic; fosfomycin; cephalosporin; glycylcycline; bicyclomycin; penam; nucleoside antibiotic; tetracycline antibiotic; peptide antibiotic; acridine dye; oxazolidinone antibiotic; rifamycin antibiotic; diaminopyrimidine antibiotic; phenicol antibiotic; isoniazid; benzalkonium chloride; rhodamine; antibacterial free fatty acids; nitroimidazole antibiotic tetU no yes macrolide antibiotic; fluoroquinolone antibiotic; lincosamide antibiotic; fosfomycin; cephalosporin; glycylcycline; bicyclomycin; penam; nucleoside antibiotic; tetracycline antibiotic; peptide antibiotic; acridine dye; oxazolidinone antibiotic; rifamycin antibiotic; diaminopyrimidine antibiotic; phenicol antibiotic; isoniazid; benzalkonium chloride; rhodamine; antibacterial free fatty acids; Date DTU Title nitroimidazole antibiotic Isolate 9 – Staphylococcus aureus

Date DTU Title 51 Exercise 1 Isolate 9 Staphylococcus aureus Group 12

Date DTU Title Tools used

Resfinder kmerResistanc Name of tool CARD 3.2 e

Input file (fastq or fasta) FASTA FASTA FASTA

We used theese tools because we are not experienced

Date DTU Title Results Observed resistance Tools Predicted resistance phenotype phenotype AMR gene or ResFind KmerRes mutation CARD er 3.2 istance mgrA yes no Multi-class (Efflux pump) norA yes no Multi-class (Efflux pump) mepR yes no fluroquinolone, glycylcycline, tetracycline arlR yes no Multi-class (Efflux pump) arlS yes no Multi-class (Efflux pump) DHA-1 yes no beta-lactam mecA yes yes yes penam, betalactam blaZ yes yes yes penam, betalactam erythromycin, ermB yes yes yes macrolide, lincosamide, streptogramin clindamycin AAC (6')-le-APH(2'')- la yes yes yes aminoglycoside gentamicin gyrA yes no fluoroquinolone parC yes no fluroquinolone Date DTU Title Phenotypic resistance

cefoxitin clindamycin erythromycin fusidic acid gentamicin linezolid tetracycline

MIC: MIC: MIC: MIC: 1000000.0 1000000.0 > 1000000.0 > 1000000.0 > MIC: 0.0 > MIC: 0.0 > > 4.0 0.25 1.0 MIC: 0.0 > 0.5 2.0 4.0 MIC: 0.0 > 0.032 1.0 - Resistant to cefoxitin, clindamycin, erythromycin, gentamicin

Genotype-phenotype discordance:

- Tetracycline resistance not detected (not a typical tet-resistance gene. Efflux pumps is more general)

- Isolate not tested to fluoroqinolones

Date DTU Title Conclusion

• Card detects more AMR-genes than the other tools • Genotypic tools detects resistance that might not be expressed. Phenotypic tools only detects what is in the panel.

Date DTU Title Isolate 10 – Staphylococcus aureus

Date DTU Title 57 Group 2, 20 (Sophie, Ryan, Rosemarie) Isolate 10: Staphylococcus aureus • Genotype (ResFinder + CARD):

Group no.: 2 Isolate ID: 10

Tools Predicted resistance phenotype Observed resistance phenotype AMR gene or mutation Tool name Tool name Tool name Tool name blaZ (96% ident. Card) ResFinder3 Card (e.g. , Ampicillin, , …) not tested FosB Card not listed (Fosfomycin) not tested DHA‐1Card , cefoxitin cefoxitin suseptible diverse efflux mechanisms (e.g. mgrA, SanorA, arlS….) Card

• Phenotype (disk diffusion): S to all

Date DTU Title This clearly shows one of the big adavantages Isolate 10: Staphylococcus aureus of WGS: the WGS data need to be produced only once and then many tests can be run on Genotype vs. Phenotype: them • ResFinder and Card identified beta-lactam resistance (blaZ) – blaZ resistance does not confer resistance to cefoxitin but to penicillins, these were not tested by disk difussion. – Even if we test phenotypic resistance to penicillin family of antibiotics, it may not be expressed and testing should preferably be carried out after induction of expression of blaZ. – Isolate 10 is a MSSA, not a MRSA according to the Cefoxitin screening test ResFinder vs. CARD: • Card identified additionally FosB and blaDHA-1 genes. • Knowledge on the involvement of fos genes in fosfomycin resistance is still limited. If of clinical relevance fosfomycin resistance could be tested penotypically • Relevance of DHA-1(blaDHA-1) detection in S. aureus? • CARD identifies many efflux pumps that are likely not expressed or do not confer phenotypic resistance, they are also hard to test phenotypically. Additional info MSSA (spaTyper, SCCmecTyper): • spa t280, ST25, MSSA (no mecA, no mecC), probably community- or hospital associated, not livestock.

Date DTU Title I would not expect to find this gene in S. Isolate 10: Staphylococcus aureus aureus. In cases like this, the sequence from the database needs to be retrived to verify if it Genotype vs. Phenotype: is the correct one • ResFinder and Card identified beta-lactam resistance (blaZ) – blaZ resistance does not confer resistance to cefoxitin but to penicillins, these were not tested by disk difussion. – Even if we test phenotypic resistance to penicillin family of antibiotics, it may not be expressed and testing should preferably be carried out after induction of expression of blaZ. – Isolate 10 is a MSSA, not a MRSA according to the Cefoxitin screening test ResFinder vs. CARD: • Card identified additionally FosB and blaDHA-1 genes. • Knowledge on the involvement of fos genes in fosfomycin resistance is still limited. If of clinical relevance fosfomycin resistance could be tested penotypically • Relevance of DHA-1(blaDHA-1) detection in S. aureus? • CARD identifies many efflux pumps that are likely not expressed or do not confer phenotypic resistance, they are also hard to test phenotypically. Additional info MSSA (spaTyper, SCCmecTyper): • spa t280, ST25, MSSA (no mecA, no mecC), probably community- or hospital associated, not livestock.

Date DTU Title Some comments about Exercise 1

Date DTU Title Reading the ResFinder-3 output

ALIGNMENTS

100% ID 97% ID 100% ID 95% ID 100% lenght 100% lenght 70% lenght 70% lenght

Date DTU Title 62 Reading the ResFinder-4 output

BASED ON ECOFF!

WHY ‘NO RESISTANCE’ instead of susceptible?

???

Chromosomal mutations

AMR genes

Date https://cge.cbs.dtu.dk/services/ResFinder-4.0/DTU Title 63 Exercise 1 - thoughts

• Know the database you are using especially for questioning negative results (i.e. absence of AMR genes)

• Some knowledge of AMR genes is needed to fully understand the results – communication with colleagues across disciplines is essential!

Date DTU Title 64 Exercise 2 – Chasing MGEs

WGS data for exercise 2Target AMR gene Contig lenght Contig lenght Contig lenght

11, 16 blaCTX‐M‐55 22683 3980 38C2, 87C1 blaCTX‐M‐14 86178 2080 42C1, 67C2 blaCTX‐M‐27 10640 4271 3, 20, 66C3 blaCTX‐M‐15 4992 5415 15151 9, 10 blaCTX‐M‐55 and mcr‐1 4907 19891 Isolate_67, Isolate_81 blaCTX‐M‐14 Isolate_75, 2274‐3 blaCTX‐M‐14 C00003477, C00003590 blaCMY‐2 C00003585, C00003588 blaCMY‐2 4, 5 blaCMY‐2 19770 69411

Date DTU Title 65 Group presentations

Only group 11 and 6 & 16 presented their results. Here their presentations are provided

followed by the solutions provided by the course organizer.

Date DTU Title 66 Exercise 2

Group 11 Camille Thougaard Vester, Mattias Myrenås

Background Samples 11 (IncQ1) and 16 (IncF) Target gene blaCTX-M-55 Two E. coli isolates from pigs at the same farm sampled

Question Did blaCTX-M-55 transfer between E. coli from the two animals?

Strategy To follow the lab protocol step by step

Date DTU Title Contig analysis • We found the contig with the gene of interest in both data sets, using BLASTn, comparing our gene to our sequence assemblies We found that the gene was in full length in both data sets

• We extracted the contigs with the gene and used BLASTn against GenBank (nr/nt) Isolate 11: Only chromosomal hits on first page of results. Few hits with 100% coverage. Isolate 16: Mostly plasmid hits on first page of results but also a few chromosomal hits. Many hits with 100% coverage. Common plasmid?

• We analysed the ORFs in the contigs, using Artemis. BLASTp found the gene.

Date DTU Title ISfinder – results, top four

Date DTU Title ISfinder - results

Date DTU Title BLASTn comparing gene from isolate 11 to gene from isolate 16 (excerpt)

Date DTU Title 8. BLASTn gene against plasmids in file from Valeria

• 10 plasmids in Excel file contained blaCTX-M-55 • No good match using BLAST

Date DTU Title Conclusion

• We believe the blaCTX-M-55 did not transfer between the pigs

Date DTU Title Date DTU Title 74 Date DTU Title 75 Date DTU Title 76 An alignment like this one may suggest two scenarios: 1. The assembly in the query was wrong 2. The query contains a novel genetic arrangement that Is not present in any sequence on NCBI Date DTU Title 77 Date DTU Title 78 Date DTU Title 79 Date DTU Title 80 Something odd here The ORF indicated by the arrow cannot be blaCTX-M-14 since it is too long

Date DTU Title 81 Date DTU Title 82 Figure made using Easyfig

Date DTU Title 83 WGS data for exercise 2Target AMR gene Contig lenght Contig lenght Contig lenght

11, 16 blaCTX‐M‐55 22683 3980

The long contig is likely on chromosome whereas it is not possible to infer genomic location of the short contig. Different spaceDate betweenDTU ISEcp1 and blaCTX-M-55 does not support transfer of the AMR gene between the strains Title 84 WGS data for exercise 2Target AMR gene Contig lenght Contig lenght Contig lenght

38C2, 87C1 blaCTX‐M‐14 86178 2080

The long contig is likely on chromosome whereas it is not possible to infer genomic location of the short contig. We cannot excludeDate DTU transfer of the AMR gene between the strains, followed by some genetic rearrangement Title 85 WGS data for exercise 2Target AMR gene Contig lenght Contig lenght Contig lenght

42C1, 67C2 blaCTX‐M‐27 10640 4271

42C1

67C2

The long contig is likely on plasmid (repA of IncFII in the contig) whereas it is not possible to infer genomic location of the short contig. We cannot excludeDate DTU transfer of the AMR gene between the strains, followed by some genetic rearrangement. Title 86 WGS data for exercise 2Target AMR gene Contig lenght Contig lenght Contig lenght 3, 20, 66C3 blaCTX‐M‐15 4992 5415 15151

Not possible to infer genomic location of the contigs We cannot excludeDate DTU transfer of the AMR gene between the strains, followed by some genetic rearrangement. Title 87 WGS data for exercise 2Target AMR gene Contig lenght Contig lenght Contig lenght

9, 10 blaCTX‐M‐55 and mcr‐1 4907 19891

mcr-1 likely on IncF plasmid and no genetic linkage between mcr-1 and blaCTX-M-15

We cannotDate excludeDTU transfer of the AMR gene between the strains. Title 88 WGS data for exercise 2Target AMR gene Contig lenght Contig lenght Contig lenght

Isolate_67, Isolate_81 blaCTX‐M‐14

blaCTX-M-14 on IncK plasmids but very different genetic context and different blaCTX-M-14 variant. No transfer of the AMR gene between the strains.

Date DTU Title 89 WGS data for exercise 2Target AMR gene Contig lenght Contig lenght Contig lenght

Isolate_75, 2274‐3 blaCTX‐M‐14

blaCTX-M-14 on IncF plasmids and similar genetic context. We cannot exclude transfer of the AMR gene between the strains, followed by some genetic rearrangement.

Date DTU Title 90 WGS data for exercise 2Target AMR gene Contig lenght Contig lenght Contig lenght

C00003477, C00003590 blaCMY‐2

blaCMY-2 on IncI1 plasmids and identical genetic context. The plasmids are nearly identical and we cannot exclude transfer of the AMR plasmid between the strains, followed by some genetic rearrangement. See also Hansen et al. https://www.ncbi.nlm.nih.gov/pubmed/27235431

Date DTU Title 91 WGS data for exercise 2Target AMR gene Contig lenght Contig lenght Contig lenght

C00003585, C00003588 blaCMY‐2

blaCMY-2 on IncIK plasmids and identical genetic context. The plasmids are nearly identical and we cannot exclude transfer of the AMR plasmid between the strains, followed byDate someDTU genetic rearrangement. See also Hansen et al. https://www.ncbi.nlm.nih.gov/pubmed/27235431Title 92 WGS data for exercise 2Target AMR gene Contig lenght Contig lenght Contig lenght

4, 5 blaCMY‐2 19770 69411

blaCMY-2 likely on chromosome in both isolates. Slightly different genetic context - we cannot exclude transfer of the AMR gene plasmid between the strains, followed by some genetic rearrangement.

Date DTU Title 93 Exercise 2 - thoughts

• There is notable genetic rearrangement around acquired AMR genes

• Reconstruction of the genetic context of AMR genes from WGS data generated by short read technologies is time-consuming and not always accurate

• Analysis of the genetic context of an AMR gene can give significant information but such information will never be conclusive without epidemiological data

Date DTU Title 94 COURSE EVALUATION

Please follow this link: https://www.surveymonkey.com/r/EURL_AR_TC_2019

Date DTU Title 95 Please remember that presentations from all training courses are available at www.eurl-ar.eu

Date DTU Title 96