Genomic epidemiology of bacterial pathogens

Sylvain Brisse

Institut Pasteur, Microbial Evolutionary Genomics Unit

&

CNRS, UMR 3525, Paris, France

Genotype VirulenceResistance

Microbiological diagnostic

Identification

Epidemiology

Genotype Virulence elementsResistance elements

Genome sequencing: ‘all in one technology’

Sequencing Data analysisSample

DNA extraction

Library preparation

Real-time genome sequencing?

5 days

Microbes know no borders…

• Standardized genotyping strategy

• Sequence databases & common nomenclature

Nomenclature & knowledge

database

Provenance data

& genomes

Isolates

databases

Act locally, think globally

Confidentiality of epi data Standardized approach

Core genome << Pangenome

Touchon et al., 2009

39%UPEC

O157:H7

K12

Escherichia coli

Genomic diversity inside bacterial species

Core genome

~2,000

All genes

(pangenome)

~20,000

families

~11,500

IS excluded

~10,000

Genomic island

Phages

Transposons, IS

ICE

Plasmids

...

Core genome MLST

Step 1. Define core genome

Step 2. Define variation at

core genes1

1

2

3

3

Step 3. Define allelic profiles

Whole-genome genotyping (cgMLST)

Contig 1 Contig 3Contig 2

Gene 1 …. Gene 2000

~ 2000 core genes

BLAST

~5 days

~30 seconds /strain

~ few weeks

~30 seconds /strain

Core-genome based genotyping and classification

Gene 1 …. Gene 2000

Strain A

Strain B

Strain C

time

Expanding clone

Founder

genotype

Ubiquitous

• Human and animal carriage, plants, water, environment

(re)-emergence as a community pathogen

• Severe pneumonia; meningitis: ~20-40% mortality rate

• Pyogenic liver abscess; endophthalmitis (1980’s - )

Hypervirulent K. pneumoniae (hvKP)

Emergence as a multidrug resistant pathogen

• 5 - 8% nosocomial infections Europe / USA

• Urinary, respiratory and bloodstream infections

• Outbreaks

Multidrug resistant (MDR) strains

Klebsiella pneumoniae

Global emergence of Kp carbapenem resistance

Woodford et al., 2011; Nordmann et al., 2011

• KPC

USA 1996; now global

ST258: predominant clone

• NDM-1:

Sweden / India (2008)

Diversity of clones, incl. ST14

Highly MDR (ESBLs; aminoglycosides)

• OXA-48, VIM, …

• Few remaining drugs available

• Death rate > 50%

• ‘urgent threat’ (CDC 2013)

• Colistin, tygecycline resistance

• Need to monitor,

understand and control

emergence

• Local and global levels

MLST-based K. pneumoniae population structure

• Recognition of ST258 as emerging KPC

producer

• Lack of clear-cut clonal structure

ST23

ST258

http://www.pasteur.fr/mlst

> 2200 STs

> 2500 isolates

> 200 submitters

Diancourt et al 2005 JCM

(core-genome MLST, 694 genes)

Genome-wide definition of K. pneumoniae clonal groups

Bialek, Criscuolo et al 2014 EID

• Clear-cut clonal structure

• Cut-off value of 100 mismatches (out of 694) for

delimitation of clonal groups

1000 700CG23

CG258

CG23

Emergence of K. pneumoniae clonal groups

KPC-producing CG258

Israel

Italy

USA

Finland

Poland

Greece

Israel

USA

mainly

Bowers et al 2015 PLoS One

20121994

Virulence factors

known variants

Sequence

database

Toxins

Capsular genes

Adhesins

Secretion systems

…

β-lactams Aminoglycosides Quinolones

Antimicrobial resistance genes

known variants

Sequence

database

β-lactamases

• Class A (TEM, SHV, CTX-M, KPC)

• Class B (NDM, VIM)

• Class C (AmpC)

• Class D (OXA)

• Inactivating enzymes

(AAC, ANT, APH)

• 16S rRNA methylases

(ArmA, Rmt)

• Target changes (GyrAB, ParCE)

• Inactivating enzymes(AAC(6’)-Ib-cr)

• Target protection (Qnr)

• Efflux pumps (OqxAB)

CG23

CG258

Virulence

genes

Gene content breakdown by clonal group

Beta-lactams AminoglycosidesQuinolones

Resistance

genes

blaKPC blaOXA

Bialek, Criscuolo et al., 2014

rmpA

735 genomes

36 genomes

635 genomes

300 genomes

83 genomes

1748 core genes1695 genomes

Listeria global initiative

L. monocytogenes

global population structure

A. Moura;

Listeria NRC &

Lecuit’s unit

• Most intra-outbreak pairs < 7 mismatches

• 7 : best Dunn’s clustering index

Cut-off value to define cgMLST types (CT) : 7

Defining a threshold for cgMLST ‘types’

Listeria monocytogenes population structure

Lineage

Sublineage

cgMLST type

• More precise definition of clustered cases

• More relevant epidemiological investigations

NRC

Core genome MLST discrimination >> PFGE

cgMLST types

• Listeria isolates sent to NRC

• ‘real-time’ genomic sequencing

• Detection of clusters

• Epidemiological investigation

• Remove contaminated productsSingle PFGE type

International cgMLST ‘types’

• No epidemiological link found upon retrospective analysis

• Long-term stability of CTs

• Surveillance at global scale

http://bigsdb.web.pasteur.fr/listeria

Public database for L. monocytogenes cgMLST nomenclature

BIGSdb: Bacterial Genomes Isolates Database

Jolley & Maiden 2010

Acknowledgements: Klebsiella

Microbial Evolutionary Genomics

Suzanne BIALEK, Alexis CRISCUOLO, Virginie PASSET, Marie TOUCHON,

Eduardo ROCHA

Center for information Technology & Communication Center, Institut Pasteur

Louis JONES, Emmanuel QUEVILLON

Paris Hospitals (AP-HP)

Dominique DECRE, Guillaume ARLET, Marie-Hélène NICOLAS-CHANOINE

Enteric Bacterial Pathogens, Institut Pasteur

Simon LE HELLO

MaGe/ MicroScope team, Genoscope, CEA, Evry

Zoé ROUY, Claudine MEDIGUE

Oxford University

Keith JOLLEY, Martin MAIDEN

Biology of Infection Unit & National Reference Center for Listeria, Institut Pasteur

Hélène DIEYE, Morgane LAVINA, Pierre THOUVENOT, Alexandre LECLERCQ, Marc LECUIT

Microbial Evolutionary Genomics, Institut Pasteur

Alexandra MOURA, Mylène MAURY, Marie TOUCHON, Alexis CRISCUOLO, Eduardo ROCHA, Sylvain BRISSE

Center for information Technology, Institut Pasteur

Louis JONES, Emmanuel QUEVILLON

Applied Maths, Belgium

Hannes POUSEELE, Bruno POT

CDC, Atlanta, USA

Peter GERNER-SMIDT, Cheryl TARR

Heather CARLETON, Lee KATZ, Zuzana

KUCEROVA, Steven STROIKA, John

BESSER

SSI, Denmark

Jonas LARSSON, Eva NIELSEN

Public Health England

Tim DALLMAN, Kathie GRANT

PHAC, CanadaAleisha REIMER, Matthew WALKER, Celine NADON

Oxford University, UKKeith JOLLEY

Funding:

Acknowledgements: Listeria

Vincent ENOUF