real world applications of whole genome sequencing · short introduction to me • molecular...
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Real world applications
of
whole genome sequencing
Henrik Hasman
Bacteria, parasites and fungi
Statens Serum Institut, Denmark
REAL WORLDAPPLICATIONS
…or application of WGS for outbreak detection, national
surveillance and research at
The Reference Laboratory for Antimicrobial Resistance
Karin Sixhøj Pedersen, lab tech
Frank Hansen, lab tech
Louise Roer, PhD
Anette M. Hammerum, PhD
Henrik Hasman, PhD
Short introduction to me
• Molecular microbiologist
• Employed at DTU FOOD until 2015 working with bacterial typing and AMR
• Involved in the CGE project
• Co-organizer of a WGS course for clinical microbiologists
• Co-developer of ResFinder, PlasmidFinder, FimTyper, VirulenceFinder…..
• Employed at SSI working with bacterial typing and AMR.
Timeline for introduction to
WGS in relation to AMR at SSI:
• 2013: Pilot projects (A. baumannii) using Whole-genome sequencing
• 2014-: WGS of ESBL-producing E. coli from bloodstream infections
• 2014-: WGS of carbapenemase producing bacteria
• 2015-: WGS for all clinical vancomycin resistant enterococci
• 2015-: Colistin resistant bacteria
CARBAPENEM RESISTANCE
Carbapenems are among the last antibiotics for treatment of infections
with multi-drug resistant bacteria (e.g. ESBL-producing E. coli or
Klebsiella pneumoniae)
Carbapenem resistant bacteria are often multi-resistant – can only be
treated with few (colistin and tigecyklin) or no antibiotics
Carbapenemases:
➢Serin carbapenemases :
✓ KPC, GES-2, -4, -5, -6, -8,NMC, SME, IMI-1, -2
➢ Metallo-beta-lactamases:
✓ IMP, VIM, SPM-1, GIM-1, SIM-1, AIM-1, KHM-1, NDM,
DIM-1, TMB-1
➢ Oxacillinases:
✓OXA-23-gr, OXA-24-gr, OXA-58-gr, OXA-143, OXA-
48-gr
➢K. pneumoniae➢ E. coli➢ other Enterobacteriaceae➢ P. aeruginosa➢ A. baumannii
➢ A. baumannii➢ Enterobacteriaceae
Red: Found in DK
RAPID GLOBAL SPREAD OF NDM-1
EnterobacteriaceaeK. pneumoniae, E. coli, K. oxytoca, E. cloacae, Proteus spp.M. morganii, C. freundii, Providencia spp. + + + +
Pseudomonas spp., A. baumannii + + + +
USA
NorwayUK
France
PakistanSingapore
Sweden
Austria
Slovenia
GermanyCanada
Italy
Finland
Spain
Holland
India
Belgium
Bangladesh
Denmark
AustraliaKenya
China
Japan
Malaysia
Taiwan➢Hospitalization:
✓India & Pakistan ✓(Balkan-region)
➢ Nosocomial transmission
Oman
➢ 2010: 13 European countries
Patient #1 at Aalborg hospital
80 year old male.
Produced carbapenem resistant Citrobacter freundii isolated from urine
on October 31. 2012
And a carbapenem resistant Citrobacter freundii isolated from fecal swap
upon readmission on November 4. 2013
PCR identified the NDM-1 gene
The isolates were subjected to WGS at SSI
Bioinformatic tools I
MLST, resistance genes and plasmid replicons
Citrobacter freundii ST18
blaNDM-1
blaCMY-6 like
blaCMY-79 like
blaDHA-1’
blaOXA-1
blaTEM-1b
strA
strB
aac(6')-Ib-cr
aadA5
rmtCcatA1
sul1
sul2
dfrA17
IncFIB
IncA/C2
IncHI2
IncQ1
IncHI2A
Outbreak of NDM-1
31th Oct 2012 C. freundii (urine)*NDM-1 IncA/C plasmid, ST18
3rd Apr 2013 C. freundii (urine)*NDM-1,IncA/C plasmid, ST18
2nd Aug 2013 C. freundii (blood)*NDM-1, IncA/C plasmid, ST18
23rd Oct 2013 C. freundii (urine)NDM-1, IncA/C plasmid, ST18
Patient 1 (80 yrs) Patient 2 (83 yrs) Patient 3 (63 yrs) Patient 4 (66 yrs)
? ? ??
2012-2017: 16 patients
No travel activity recorded
4th Nov 2013 (re-admission)C. freundii (faecal swab)NDM-1IncA/C plasmids, ST18
21st May 2013C. freundii (anal fissure)*NDM-1,IncA/C plasmid, ST18
K. pneumoniae (anal fissure)NDM-1
4th Nov 2013C. freundii (faecal swab)NDM-1 IncA/C plasmid, ST18
K. pneumoniae (faecal swab)NDM-1
18th Dec 2013K. pneumoniae (sputum)^NDM-1
2nd Jan 2014 C. freundii (faecal swab)NDM-1 IncA/C plasmid, ST18
K. pneumoniae (faecal sw.)NDM
E. coli (faecal swab)NDM-1
NDM-1 outbreak – initial outbreak
Ridom SeqSphere cgMLST analysis (1701 genes)
44 allelesNCBI
China
Non-blaNDM-1
<8 alleles
NCBI RefSeq + DK genome data
Patient #2
blaNDM-1, blaCMY-6 like, blaCMY-79 like, blaDHA-1’, blaOXA-1, blaTEM-1b
strA, strB, aac(6')-Ib-cr, aadA5, rmtC, catA1, sul1, sul2, dfrA17
IncFIB, IncA/C2, IncHI2, IncQ1, IncHI2A
Citrobacter freundii ST18
blaNDM-1,blaCMY-6 like, blaDHA-1’, blaOXA-1, blaTEM-1b, blaSHV-11, blaCTX-M-15
strA, strB, aac(6')-Ib-cr, aac(3)-IIa, rmtC, fosA,catB3 sul1, sul2, qnrB66,tetA, dfrA14
IncFII(K), IncA/C2, IncFIB(K)
Klebsiella pneumoniae ST392
Patient #2
blaNDM-1, blaCMY-6 like, blaCMY-79 like, blaDHA-1’, blaOXA-1, blaTEM-1b
strA, strB, aac(6')-Ib-cr, aadA5, rmtC, catA1, sul1, sul2, dfrA17
IncFIB, IncA/C2, IncHI2, IncQ1, IncHI2A
Citrobacter freundii ST18
blaNDM-1, blaCMY-6 like, blaDHA-1’, blaOXA-1, blaTEM-1b, blaSHV-11, blaCTX-M-15
strA, strB, aac(6')-Ib-cr, aac(3)-IIa, rmtC, fosA,catB3 sul1, sul2, qnrB66,tetA, dfrA14
IncFII(K), IncA/C2, IncFIB(K)
Klebsiella pneumoniae ST392
Illumina assembly data (CLC Genomic WB v9.0.1)
IncA/C2
39120 bp
23 x coverage
blaNDM-1
13230 bp
22 x coverage
Plasmid purification and llumina
assembly data
blaNDM-1
25121 bp
46 x coverage
IncA/C2
123070 bp
44 x coverage
• No direct link between blaNDM-1 and the IncA/C2 replicon
• PCR linking ended in a wild-goose chase
Single Molecule Real Time (SMRT)
Pacific Biosciences Oxford Nanopore MinION
1500 $Assembly using
SPADes
(mixed assembly using
also MiSeq data)
Assembly using CANU
and proofreading with MiSeq
Single Molecule Real Time (SMRT)
IncA/C2
blaNDM-1
aacA4
aac(6’)Ib-cr
rmtC
strAstrB
sul1
blaOXA-1
blaCMY-6
BLAST atlas to pT1 (server.gview.ca)
NATIONAL SURVEILLANCE OF
VANCOMYCIN RESISTANT
ENTEROCOCCUS FAECIUM
ENTEROCOCCI❖Enterococci belong to the normal gastrointestinal flora of human and animals
❖Enterococci extremely resistant to heat and can survives for extended periods on dry surfaces
❖Enterococci are a common cause of hospital-acquired infections
❖The most frequent infections caused by enterococci are: Urinary tract infections, bacteraemia and endocarditis
❖E. faecalis has in the past causes the most human infections; however infections caused by E. faecium have become more frequent during the last decade
❖Treatment: Combination of an aminoglycoside (gentamicin) and a cell-wall-active agent (penicillin or vancomycin)
VANCOMYCIN RESISTANT
ENTEROCOCCI
▪ Many genes: vanA, vanB, vanC, vanD, vanE, vanG, vanL, vanM, vanN and (vanO)
▪ vanA and vanB are most common
▪ The vanA gene is typically located on transposons of the Tn1546 type
Tn1546
WGS of VRE at SSI
In 2014:
- Isolates from bloodstream infections (n=32)
2015, 2016 and 2017:
- All VRE from infections (n=375 for 2015) and (n=436 for 2016)
Clinical VRE (2005-2015)
0
50
100
150
200
250
300
350
400
450
500
2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016
No.
of is
ola
tes
E. faecalis vanB
E. faecalis vanA
E. faecium vanB
E. faecium vanA
n = 375
n = 303
n = 436
Distribution of vanA E. faecium 2015
0
20
40
60
80
100
120
89% of the isolates are from Zealand
VRE
VRE
VRE
VRE
VRE
Total
ST-117
ST-1196
ST-1197
ST-1198
ST-1199
ST-1200
ST-1201
ST-18
ST-192
ST-203
ST-80
Unknown ST
MLST based on 7 genes divided into 12 STs
STs for the 372 E. faecium from 2015
Only few ST203 before 2015
ST203 (51%)
ST80 (33%)
ST117 (10%)
SeqSphere (cgMLST) for E. faecium
• In 2016, sub-typning of VRE faecium was done
using SeqSphere
• Comparison of 1423 genes
• Easy to communicate as a number (CT859)
• Easy to compare with other isolates in the
database (including international isolates)
• cgMLST for E. faecium are very similar to data
from SNP-trees
cgMLST tree (34 types) for 372 VRE faecium
Total
• ST203
• CT859 (51%)
• Only one isolates
with another CT
• ST80 into 12
cluster typer
Comparison of 1423 gener
ST203-CT859
0
10
20
30
40
50
60
70
• CT859 have been spread to Sweden and the Farroe Islands
VRE
VRE
VRE
VRE
VRE
CONCLUSION
WGS and SeqSphere are useful for typing of E. faecium isolates
VRE faecium clones are spread across hospitals and different regions in
Denmark
Fast clonal shifts
- ST203-CT859 only few isolates in 2014, but 51% in 2015 and 65% in
2016.
0
100
200
300
400
500N
o.
of is
ola
tes
E. faecalis vanB
E. faecalis vanA
E. faecium vanB
E. faecium vanA
Total
14
15
16
20
24
46
859
860
861
862
863
Colistin resistance (mcr-1 and mcr-2)
Colistin can be used for treatment of infections caused to multi-resistant
bacteria (carbapenemase-producing E. coli and K. pneumoniae)
Colistin are used for both humans and animals
Plasmid mediated colistin resistance (mcr-1) described in China in
November 2015
>mcr-1_1_KP347127
ATGATGCAGCATACTTCTGTGTGGTACCGACGCTCGGTCAGTCCGTTTGTTCTTGTGGCGAGTGTTGCCGTTTTCTTGACCGCGACCGCCAATCTTACCTTTTTTGATAAAATCAGCCAAACCTATCCCATCGC
GGACAATCTCGGCTTTGTGCTGACGATCGCTGTCGTGCTCTTTGGCGCGATGCTACTGATCACCACGCTGTTATCATCGTATCGCTATGTGCTAAAGCCTGTGTTGATTTTGCTATTAATCATGGGCGCGGTGA
CCAGTTATTTTACTGACACTTATGGCACGGTCTATGATACGACCATGCTCCAAAATGCCCTACAGACCGACCAAGCCGAGACCAAGGATCTATTAAACGCAGCGTTTATCATGCGTATCATTGGTTTGGGTGTG
CTACCAAGTTTGCTTGTGGCTTTTGTTAAGGTGGATTATCCGACTTGGGGCAAGGGTTTGATGCGCCGATTGGGCTTGATCGTGGCAAGTCTTGCGCTGATTTTACTGCCTGTGGTGGCGTTCAGCAGTCATTA
TGCCAGTTTCTTTCGCGTGCATAAGCCGCTGCGTAGCTATGTCAATCCGATCATGCCAATCTACTCGGTGGGTAAGCTTGCCAGTATTGAGTATAAAAAAGCCAGTGCGCCAAAAGATACCATTTATCACGCCA
AAGACGCGGTACAAGCAACCAAGCCTGATATGCGTAAGCCACGCCTAGTGGTGTTCGTCGTCGGTGAGACGGCACGCGCCGATCATGTCAGCTTCAATGGCTATGAGCGCGATACTTTCCCACAGCTTGCCAAG
ATCGATGGCGTGACCAATTTTAGCAATGTCACATCGTGCGGCACATCGACGGCGTATTCTGTGCCGTGTATGTTCAGCTATCTGGGCGCGGATGAGTATGATGTCGATACCGCCAAATACCAAGAAAATGTGCT
GGATACGCTGGATCGCTTGGGCGTAAGTATCTTGTGGCGTGATAATAATTCGGACTCAAAAGGCGTGATGGATAAGCTGCCAAAAGCGCAATTTGCCGATTATAAATCCGCGACCAACAACGCCATCTGCAACA
CCAATCCTTATAACGAATGCCGCGATGTCGGTATGCTCGTTGGCTTAGATGACTTTGTCGCTGCCAATAACGGCAAAGATATGCTGATCATGCTGCACCAAATGGGCAATCACGGGCCTGCGTATTTTAAGCGA
TATGATGAAAAGTTTGCCAAATTCACGCCAGTGTGTGAAGGTAATGAGCTTGCCAAGTGCGAACATCAGTCCTTGATCAATGCTTATGACAATGCCTTGCTTGCCACCGATGATTTCATCGCTCAAAGTATCCA
GTGGCTGCAGACGCACAGCAATGCCTATGATGTCTCAATGCTGTATGTCAGCGATCATGGCGAAAGTCTGGGTGAGAACGGTGTCTATCTACATGGTATGCCAAATGCCTTTGCACCAAAAGAACAGCGCAGTG
TGCCTGCATTTTTCTGGACGGATAAGCAAACTGGCATCACGCCAATGGCAACCGATACCGTCCTGACCCATGACGCGATCACGCCGACATTATTAAAGCTGTTTGATGTCACCGCGGACAAAGTCAAAGACCGC
ACCGCATTCATCCGCTGA
RAPID RE-ANALYSIS
(FROM LAB TO LAP-TOP)
Nov. 18. 2015(Lancet Infect Dis)
Nov. 23. 2015(Genbank)
Nov. 24. 2015(ResFinder)
Nov. 26. 2015(NGS data scanned)
Nov. 30. 2015(Contamination?)
Dec. 2. 2015(1. draft MS)
Dec. 07. 2015(Resequencing)
Dec. 10. 2015(online in Eurosurveillance)
pHNSHP45 carrying the mcr-1 gene
MCR-1 POSITIVE ISOLATES
REVERSE GENOTYPIC SCREENING
Positive phenotype
(unknown genotype)
Negative phenotype
REVERSE GENOTYPIC SCREENING
Gene(s) specific
To new phenotype
MCR-1 EXAMPLE
Positive
phenotype
Negative
phenotype
Ten colistin sensitive E. coli isolates
from pigs in Denmark
MCR-1 EXAMPLE
Positive
phenotype
Negative
phenotype
MCR-1 EXAMPLE
Positive
phenotype
Negative
phenotype
MCR-1 EXAMPLE
Positive
phenotype2605 bp
mcr-1
Thank you
To all my colleagues at Statens Serum Institut
To all my former colleagues at DTU
To my co-workers from the Danish Departments of Clinical Microbiology
This work was supported by the Danish Ministryof Health and Prevention
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