Real world applications

of

whole genome sequencing

Henrik Hasman

Bacteria, parasites and fungi

Statens Serum Institut, Denmark

REAL WORLD APPLICATIONS

…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

Søren Overballe-Petersen, 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

rmtC

catA1

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)

? ? ??

2012-2018: 18 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

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 UniCycler

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

“OUTBREAK”

“What’s in a name? That which we call a rose

by any other name would smell as sweet”Romeo and Juliet (II, ii, 1-2)

Unfortunately, it’s not that simple, when we talk about outbreaks…..

Outbreak identification(core genome analysis by SNP)

SNP identification

AATTCCAGGTTTTTAAC

....ATCGAATTCCGGGTTTTTAACCGGATCGTACGATCGGGAAA....

ATTCCAGGTTTTTAAGCG

TTCCAGCTTTTTAAGCGGA

TCCAGGTTTTTAACCGGATC

CCAGGTTTTTAACCGGATCGT

Reference genome

1) All reads have a different base than the reference = HQ SNP

1

2) Single reads have a different base than the rest = method noise

2

3) Some reads have a different base than the rest = recombination or contamination?

3

Core genome typing

Core (reference) genome

Textbook – A clone is:

”a group of genotypic identical isolates descending from a common ancestor as part of a direct chain of replication”

A more realistic definition:

”the word clone will be used to denote bacterial cultures isolated independently from different sources, in different locations, and perhaps at different times, but showing so many identical phenotypic and genotypic traits that the most likely explanation for this identity is a common origin”

(Ørskov & Ørskov, 1983)

Clone theory 101

The Chromosomograph…an evolutionary clock!

E. coli phylotypes

The Chromosomograph…an evolutionary clock!

• Randomly generated across the chromosome over time (“The mutation rate”)

• …but influenced by external factors…

Diversity

Advanced clone theoryClonal drift

TimeHost

jumpTreatment

regimeEnvironment

Immune

system

• The more discriminatory a typing method is, the more difficult it will be for it to

accommodate biological variation caused by clonal drift over time (stability issues).

• On top of this, all typing methods will add methodological variation

(repeatability and reproducibility issues) thus blurring the picture even more.

OUTBREAK

Single source

Short time span

“Contaminated dish”

“Single infected patient”

Single source – local spread

Long time span

“Hospital or regional outbreak”

OUTBREAK

Single source

Long time span

“Contaminated processing plant / industry”

“Long-term colonized patient / healthcare worker”

International source

Long time span

“Imported food source”

“Travel-related outbreak”Harris et al., 2012. The Lancet

OUTBREAK

Single source

Long time span

“Contaminated processing plant / industry”

“Long-term colonized patient / healthcare worker”

International source

Long time span

“Imported food source”

“Travel-related outbreak”

Sequence based typing

• MLST

• cgMLST / SNP

• Presence/absence of genes and mobile elements

…..often a combination of the above is used to study outbreaks.

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 Ministry of Health and Prevention