An Integrated View on Listeria Genomics and Virulence

Sylvain Brisse

Institut Pasteur

Listeria monocytogenes is everywhere, and

infects humans through food

• ~ 300 human cases/year in France (1,600 in the USA)

• Severity of invasive forms (hospitalization rate around 100%)

• Mortality rate of 20 to 30 % (up to 45% in case of CNS infection)

• Strict control procedures in food industry

Listeria monocytogenes invasive infections

• CNS infection

• Bacteremia

• Materno-neonatal infection

Objectives

Population structure

Strain nomenclatures

Databases

Genome dynamics

Phenotypic diversity

Link genotype-phenotype

Applications: diagnostic, epidemiology, risk assessment…

1

2

3

L. monocytogenes strain typing methods

• Serotyping

• Pulsed-field gel electrophoresis (PFGE)

• Ribotyping

• Amplified Fragment Length Polymorphism (AFLP)

• Multilocus sequence typing (MLST)

• Multi-virulence sequence typing (MvLST)

• Multilocus VNTR Analysis (MLVA)

• Single Nucleotide Polymorphisms (SNP)

• Genome sequencing

L. monocytogenes: four phylogenetic lineages,

of which two are epidemiologically important

4b, 1/2b

1/2a,1/2c

I

II

III

IV

Tsai et al., 2011

Den Bakker et al., 2010

Piffaretti et al., 1989

Konstantinidis & Tiedje 2005

DNA-DNA reassociation

ANI

I

II

III

IV

4.99%

5.3%

7.57%

Average Nucleotide Identity

L. monocytogenes lineages ≈ distinct genomic species

ANI among L. monocytogenes lineages:

Lineage I

4b

1/2b

1/2b

CC1

CC4

CC2

CC3

3b

7 Ragon et al., 2008

Lineage II

1/2a

1/2a

1/2a

EGDe (1/2a)

1/2c

CC7

CC101

CC9

Major L. monocytogenes serotypes ≈ lineages

abcZ bglA cat dapE dat ldh lhkA

3 1 1 1 3 1 3

3 1 1 1 3 1 2 3

1 2 1 7 22 39 7 2

3 1 1 1 3 1 3 1

Alleles Sequence type (ST)

1

1

2

3

3

•Allele • Sequence of one gene (lhkA, 480 bp)

Listeria monocytogenes Multilocus Sequence Typing

www.pasteur.fr/mlst

~3,500 entries, > 1000 STs

> 60 labs

Common language / strains Salcedo et al. 2003

Ragon et al. 2008

MLST: major L. monocytogenes clones

Lineage I

Lineage II

Ragon et al 2008

Chenal-Francisque et al 2011

Haase et al 2014

…

9 14

15

3

9

10

4

1

12

10 10

5

7 5

7

2

7

7

10

1

10

1

13

3

3

12

1

3

14

12

2 3

12

14

3

1

3

8

10

5

9

10

America (n = 77)

Europe (n = 156)

North Africa

(n = 33)

Asia / Middle

East / Oceania

(n = 30)

(No significant Fst)

Chenal-Francisque et al., 2011

MLST clones: Everything is Everywhere CC1

CC2

CC3

Lin. I, others

CC9

Lin. II, others

L. monocytogenes MLST clones are old Haase et al., Env. Microbiol. 2014

CC9

CC11

(ECIII)

CC8 (ECV)

CC7 (ECVII)

CC121 CC101

CC26

CC21

CC155

CC199

CC177

CC16

CC18

CC19 CC193

CC20

CC37

CC2 (ECIa or ECIV)

CC1 (ECI)

CC4

CC3

CC5

(ECVI)

CC59

CC6 (ECII)

CC315

CC195

CC90

ST558

FSLJ1-175

FSLJ1-194 FSLJ2-064

L028

EGD-e

FSLN3-165

10403S

EGD

08-5578, Canada, 2008 TS4/F.6854, USA 1989 J0161, USA 2000 G6054, USA 1994 CLIP 80459, France 1999 H7550, USA 1998-99 J1735, USA, 2002 J0211, USA 2000-2001 Scott A, USA, 1983 Aureli97, Italy 1987 TS38/L.3306, UK & Ireland 1987-89 TS27/L.4738, Canada 1981 TS60/L.4486b, Switzerland, 1983-87 F2365, California, 1986-1987

Outbreak strains:

111

17

1

62

1

2

3

4

> 4

Cantinelli et al., 2013

See also:

Kathariou & coll.

Knabel & coll.

Lineage II

Lineage I

‘Epidemic clones’ ≈ CCs

IIa

IIb

IIc

IVb

Food isolates

2006-2013

Clinical isolates

2006-2013

Food isolates

2006-2013

Clinical isolates

2006-2013

Lineage II

Lineage I

Lineages Serogroups MLST clones

Lineage I

Lineage II

Distribution in food vs. clinical samples

Data from French National Reference Center

NRC

Quantification of MLST clones in sources

2584 clinical

4049 food

6633 isolates

PFGE ApaI, AscI

CC1 CC4 CC6

2005 – 2013

NRC + NRL isolates

M. Maury

Unequal distribution of clones in food and clinical sources

Maury, Tsai et al. Nat Genet 2016

Clinical-associated clones are hypervirulent

Maury*, Tsai* et al. Nat Gen 2016

811 infected patients, immunosuppressive comorbidities

(MonaLisa study, C. Charlier) 6 633 isolates, invasive infections

R2 = 0.9995, P < 0.03 Coll. lab

M. Lecuit

LLS LIPI-1

CC2

CC1

CC6

CC4

CC9

CC31

CC121

Truncation Internal deletion

Absent Present / full length

ST13 CC193

Lineage II

Lineage I

0.01

Variation of conserved virulence factors

Maury*, Tsai* et al. Nat Gen 2016

FA

M000548

F

AM

000450

FA

M003296

FA

M004348

F

AM

003164

FA

M003292

FA

M002725

F

AM

002828

7.0

21.7

29.3

58.2

2.6

0.0

40.7

16.0

20.3

32.2

25.0

35.1

35.9

68.2

46.2

45.0

31.0

52.2

45.2

41.9

37.1

42.5

71.3

58.1

61.5

50.9

65.6

0.01

FA

M002728

FA

M002730

F

AM

002729

FA

M002925

FA

M003293

F

AM

003294

FA

M004349

FA

M003297

F

AM

003298

FA

M003299

FA

M000549

F

AM

001910

F

AM

002113

F

AM

002114

F

AM

002292

F

AM

002293

FA

M003315

FA

M002768

FA

M002772

FA

M004135

FA

M004139

FA

M004150

FA

M004151

FA

M004152

FA

M004153

FA

M004154

FA

M002808

FA

M004136

FA

M004137

FA

M004138

FA

M002769

FA

M002770

FA

M003316

FA

M003317

FA

M003318

20

Present

Absent

Lineage II

Lineage I

Clin

ica

l fr

equ

ency

104 genomes

Novel putative virulence factors

Maury*, Tsai* et al.

Nat Gen 2016

21 Listeria pathogenicity island 4 (LIPI-4)

CC4 (LM09-00558)

1 kb CC4∆PTS

Maury*, Tsai* et al. Nat Gen 2016

Biodiversity of Listeria monocytogenes

• Hypervirulent clones

• Novel putative virulence factors

• First CNS-associated virulence factor

Nat Genet. March 2016

Epidemiology

Diagnostics

Global diversity

Population structure

Ecology (patho)physiology

Integration of public health and biological research

Chenal-Francisque, Maury et al., J Clin Microbiol, 2015

Clonogrouping: rapid multiplex PCR assay

to identify major clones of L. monocytogenes

95.6% of correct identification at clone level

From: foodNetwork@ecdc.org

Subject: type CT131 Denmark outbreak

Body: Have you observed this type in your country?

Talking the same language:

How can we share nomenclatures of strains?

1. Define genotypic groups and nomenclature designations

2. Develop a nomenclature database

3. Provide on-line genotyping tools

Core-genome genotyping (cgMLST)

Contig 1 Contig 3 Contig 2

Gene 1 …. Gene 2000

~ 2000 core genes

BLAST

~5 days

~30 seconds /strain

~30 seconds /strain

~ few weeks

Standard

codes

A nomenclature proposal for L. monocytogenes strains

Lineage

Sublineage

CT

Lineage - Sublineage – ST – cgMLST type (CT)

LII – SL7 – ST7 – CT932

Nomenclature

database

Provenance data

& genomes

Isolates

databases

Share nomenclature, not genomes or provenance data

Confidentiality of data Standard nomenclature

BIGSdb

Jolley & Maiden 2010

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

• L. monocytogenes genotypic categories can be defined at

different phylogenetic depths

• Integration of epidemiological surveillance and fundamental

biology research provides novel insights on pathophysiology,

ecology

• Standard genotyping strategy and public database for strain

nomenclature: to be agreed upon

• L. monocytogenes clones are everywhere; International

transmission both at evolutionary and at epidemiological

timescales

• Need for internationally coordinated surveillance of

L. monocytogenes

Conclusions

Acknowledgements

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, Canada Aleisha REIMER, Matthew WALKER, Celine NADON

Oxford University, UK Keith JOLLEY

Funding:

Microbial Evolutionary Genomics, Institut Pasteur

Alexandra MOURA, Mylène MAURY, Marie TOUCHON

Eduardo ROCHA

Center for information Technology, Institut Pasteur

Louis JONES, Emmanuel QUEVILLON

C3BI/HubB

Alexis CRISCUOLO

Julien GUGLIELMINI

Elise LARSONNEUR

Biology of Infection Unit

& National Ref. Center for Listeria,

Institut Pasteur

Yu-Huan TSAI

Vivivane CHENAL-FRANCISQUE

Caroline CHARLIER

Alexandra MOURA

Alexandre LECLERCQ

Marc LECUIT