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Listeria Risk: the Role of Whole Genomic Sequencing, South Africa outbreak case Jérôme COMBRISSON PhD, Steakexpert , 12 ème édition - 2018

ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

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Page 1: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

Listeria Risk: the Role of Whole

Genomic Sequencing, South Africa

outbreak case

Jérôme COMBRISSON PhD,

Steakexpert , 12ème édition - 2018

Page 2: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

Millions of DNA strands sequenced in parallel, yielding substantially more

throughput, in 1 single run

Next Generation Sequencing (NGS)

2Source : http://www.nature.com/nrg/journal/v11/n1/abs/nrg2626.html, oncode

"Next generation sequencing"

all the technologies/sequencing platforms developed since 2005

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3

NGS

Whole Genomic

Sequencing (WGS)

MetaSequencing

Metabarcoding

SOURCE : ACTEMIUM, SCIENCE PHOTO LIBRARY, ISTEMA-HUMANO.COM.AR

Next Generation Sequencing (NGS)

NGS can also be called ‘Post-Sanger sequencing technologies’. Covers mixed microbial

communities (MetaSequencing/Metabarcoding) and isolated micro-organisms (WGS)

identification & characterization.

Mixed microbial

communities

Isolated

micro-organisms

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4

Total Nucleic Acid Extraction

Bioinformatics sequence analysis

Food &

Environment

sample

Strain isolation Microorganism communities

DNA DNA

PCR

Shotgun

Genomics

Targeted

MetaGenomics

Pangenome CoreGenome Phylogenic

composition

WGS

Meta

barcodingsource : modified from Mayo et alSource: Antimicrob. Agents Chemother. February 2011 vol. 55 no. 2 623-630 Source : Feng Guo Scientific Reports volume5, Article number: 7637 (2015)

Next Generation Sequencing (NGS)

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Terminology

5

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Identification

Main identification/characterization

methods

6

Isolated

micro-organisms

Characterization Level 1

Characterization Level 2

...

Class

Order

Family

Genus

Species

Serotype

Type

Strain

Virulome

Resistome

Levels

Sources : Xiaomei Yan et al. Appl. Environ. Microbiol. 2012;78:6637-6642

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MICROBIOLOGICAL/BACTERIAL IDENTIFICATION:

Gives a genus & species name to a bacterial isolate.

Usual steps:

1) morphological characterization e.g. shape, size, color;

2) biochemical characterization e.g. acid production,

carbohydrate/amino acid/citrate utilization, starch/gelatin

hydrolysis;

3) physiological characterization e.g. salinity, T°, pH;

4) Molecular ID analysis e.g. 16S rRNA analysis, MALDI-TOF.

Main identification/characterization

methods : Isolated micro-organisms

7

...

Class

Order

Family

Genus

Species

Serotype

Type

Strain

Virulome

Resistome

Levels

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Main identification/characterization

methods : Isolated micro-organisms

8

Characterization Level 1

- Serotyping (Antibodies, Antigen reaction)- Molecular subtyping / Ribotyping (by Riboprinter®)

- pcrMLST

- PFGE

Characterization Level 2

WGS (MLST, coreMLST wgMLST, wgSNP)

...

Class

Order

Family

Genus

Species

Serotype

Type

Strain

Virulome

Resistome

Levels

MICROBIOLOGICAL/BACTERIAL characterization:

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In order to determine a microorganism's serotype, you must determine which antigens are found on their surface. The process of clumps forming when an antigen is mixed with its antibodies is known as agglutination

Serotyping : agglutination

9Source : http://jb.asm.org/content/185/18/5573/F6.large.jpg

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MLST

10Source : AppliedMaths

Multilocus sequence typing (MLST) is

a molecular typing technique

whereby a number of well chosen

housekeeping genes (loci) are

sequenced, usually in part.

In case seven housekeeping genes

are studied, each strain is thus

characterized by a profile of seven

allele numbers. The allelic

profiles can be considered as a

character set of 7 categorical

characters. MLST has been used

successfully to study population

genetics and reconstruct micro-

evolution of epidemic bacteria and

other micro-organisms.

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Whole Genome Sequencing (WGS)

Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's genome at a single time

Provide comprehensive details about the organisms: genes, plasmids, virulence factors, serotype, and resistance genes (e.g., antibiotic resistance, heavy metal resistance, stress resistance).

WGS analysis can look differences at the nucleotide level

Generation phylogenetic trees and determine the relationships among strains (strain typing).

Genomes assembled in a de novo fashion, which provides an unbiased construction of genome.

Complete sequencing the genome of bacterium, the deepest level of strain characterization possible.

Source: Teresa M. Bergholz et al. Applied and Environmental Microbiology 82(3) · November 2015Source : Leopold et al J. Clin. Microbiol. July 2014 vol. 52 no. 7 2365-2370

11Source: Antimicrob. Agents Chemother. February 2011 vol. 55 no. 2 623-630

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12

Whole Genome Sequencing (WGS)

Page 13: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

What are the advantages of WGS ?

SerotypeLineage MLST PFGE WGS

Brisse et al 2011

WGS is more efficient than other

methods such as PFGE

WGS is the deepest technology

the only one able to identify

bacteria at the clone level better determination of

relationship between strains

https://www.abmgood.com/marketing/knowledge_bas

e/next_generation_sequencing_WGS.php

13

Example : 6237/12 and IHV116260 are

indistinguishable by PFGE

but WGS

revealed that they are highly divergen (samples from

a Campylobacter jejuni outbreak in Europe in 2012 )

https://www.abmgood.com/marketing/knowledge_base/next_generation_sequen

cing_WGS.php

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L. monocytogenes world : level of

characterization

14 Source : http://objectifamille.over-blog.com, www.presse-citron.net, www.lextalk.com,

L. mono world

Lineage/Serotyping

(1st stage)

• Book • Chapter

<

• Text

<• wgSNP• wgMLST• MLST

CC/ST < <

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From MLST to SNP analysis level

15

wgSNP

wgSNP

(L. monocytogenes)

≈ 3 000 000 basepairs

wgMLST

wgMLST

(L. monocytogenes)

4797 loci< <(hqSNP)-based clustering pipelines

MLST

MLST (->CC/ST)

(L. monocytogenes)

7 housekeeping genes

7 MLST

• Book • Chapter

<• Text

<

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South Africa Listeriosis Outbreak

16

Prior to 2017, an average of 60 to 80

laboratory-confirmed listeriosis cases per

year (approximately 1 per week), were

reported in South Africa.

In July 2017, an increase in laboratory-

confirmed cases of listeriosis was reported

to National Institute for Communicable

Diseases (NICD) which was followed by

investigations into the reported increase

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South Africa Listeriosis Outbreak

17

On 05 December 2017,

the listeriosis outbreak

was declared by the

Minister of Health, Dr.

Aaron Motsoaledi.

from clinical isolates obtained from patients (patient blood), 9 sequence

types (ST) of Listeria monocytogenes were isolated and 91% were of

sequence type 6 (ST6).

It was concluded that time that this outbreak is driven by ST6 and are

highly genetically related.

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South Africa Listeriosis Outbreak

18

……..On Friday 12th January, nine children under the age of 5 years

presented to Chris Hani Baragwanath Hospital with febrile gastro-

enteritis…..

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School meal the first clue

On Friday 12th January

19

9 children under the age of

5 years presented to Chris

Hani Baragwanath Hospital

with febrile gastro-enteritis

The paediatrician suspected

foodborne disease, including

listeriosis, as a possible cause.

The environmental health practitioners

(EHPs) were informed and on the same

day visited the crèche……..

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School meal the first clue

On Friday 12th January

20

……, and obtained samples from two unrelated polony brands (manufactured by Enterprise and Rainbow Chicken Limited (RCL) respectively) and submitted these to the laboratory for testing.

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School meal the first clue

On Friday 12th January

21

…All 3 isolates belong to ST6 (Saturday 27th January)

Listeria monocytogenes

was isolated from stool

collected from one of the

ill children

…and from both of the polony specimens

collected from the crèche. These isolates

were sent to the NICD Centre for Enteric

Diseases for WGS

+

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South Africa Listeriosis Outbreak

22

The source of the outbreak was identified as ready-to-eat processed meat products

Listeriosis

A recall of affected products was initiated on 04 March 2018.

But ……

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School meal the first clue

On Friday 12th January

23

…All isolates belong to ST6 are from 1 company (TigerBrand)

Massive sampling on environment and on products by the health authorities in the 2

food companies to track the sources.

All isolates of L monocytogenes funded were analyzed by WGS

But ……

Page 24: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

The recall, difficulties and real

life…

24

The minister also told South Africans not to consume ready-to-eat

processed meat across the board due to the risk of cross-

contamination.

Such products could be cross-

contaminated in shops as they are often

stored next to polony

Page 25: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

ST6 in the world of L. monocytogenes

25Source : Radoshevich and Cossart, Nature Reviews Microbiology, 2018

Linage I , 4b

CC6 =

ST6 (7/7 alleles shared )

+ST80 (6/7 alleles shared )

+ ST81 (6/7 alleles shared )

Complex Clonal = Sequence Type

clustering with at least 6 alleles

shared

Page 26: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

From MLST to SNP analysis level

26

wgSNP

wgSNP

(L. monocytogenes)

≈ 3 000 000 basepairs

wgMLST

wgMLST

(L. monocytogenes)

4797 loci< <(hqSNP)-based clustering pipelines

MLST

MLST (->CC/ST)

(L. monocytogenes)

7 housekeeping genes

7 MLST

• Book • Chapter

<• Text

<

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wgSNP analysis level

27

Single Nucleotide Polymorphisms (SNPs) on whole

genomes sequences

wgSNP (L. monocytogenes) ≈ 3 000 000 basepairs

• Text

Source : AppliedMath

1 SNP ≠

3 SNP ≠

8 SNP ≠

Page 28: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

Info ST6 SA

28

A CB D

ID : L. mono WGSST A, B, C = ST6

wgSNP A, B, C ≤ 5 SNP

1 32 4

ID : L. mono WGS

ST 1, 2 = ST6

wgSNP

A, B, C,1,2 ≤ 7SNP*

1 SNP ≠

3 SNP ≠

8 SNP ≠

Source : *NCID

Page 29: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

In summary

29

January, 2017 to early june 2018 : 1049 laboratory-confirmed cases of listeriosis and 209 deaths.

590 clinical isolates have undergone WGS; 91% belong to the sequence type 6 (ST6) outbreak strain and the remainder are 16 different types

Mid may environnemental and food samples from production facility inspections for L. monocytogenesare tested with WGS

the NICD has deposited 10 representative ST6 sequences in the public GenBank - NCBI database

(source NICD

…2 food companies was suspected and affected by a massive

recall but……

…One truly and undoubtedly was the sources thanks to WGSanalysis……

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In summary

30(source NICD

…One truly and und²oubtedly one factory is the sources thanks

to WGS analysis……

The deadly strain ST6 bacteria was found in 26 places in the Tiger Brands Polokwane

Enterprise factory and on the outside of two rolls of polony.

Head of the Centre for Enteric Diseases at the National Institute for Communicable

Diseases (NICD) Dr Juno Thomas said: "The fact that we haven’t found the ST6 listeria

monocytogenes strain in retail Rainbow polony or in the RCL foods production facility

adds further strength to the findings that it is the Enterprise facility that is causing the

outbreak.“

Rainbow Chicken‚ whose polony was also recalled‚ still had other strains of listeria

bacteria in its polony and in its factory‚ which can make people sick and require

corrective action and monitoring‚ health spokesman Popo Maja said.

Page 31: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

Why is WGS useful?

Better knowledge of past events

(retrospective studies)

build comprehensive internal strain bank (strong

characterization of strains all along the food chain)

understand & solve recurring contamination

31

Outbreaks monitoring & Traceability

ingredient responsible in a multi-ingredient food

geographic regions of origin

link between illnesses and processing facilities

Safeguard food integrity & brand equity

prevention of food crisis through continuous &

efficient tracking (raw material, food processes,

environmental monitoring)

past

present

future

Page 32: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

« Despite the usefulness of PFGE in identifying clusters of listeriosis cases over the past few decades, the limited discriminatory power of PFGE can indicate that unrelated isolates are indistinguishable, thus leading to identifying and investigating false PFGE cluster-alerts. The increased discriminatory power of cgMLST enabled identification of such false cluster-alerts”

“On the basis of results of this prospective study, PFGE typing has been discontinued, and L. monocytogenes surveillance in France has relied on cgMLSTsince January 2017”.

“This procedure also refined epidemiologic investigations to include only phylogenetically closely related isolates, improved source identification, and facilitated epidemiologic investigations, enabling identification of more outbreaks at earlier stages”

Case Study: WGS for L.mono, France

32Source : Moura et al.: https://wwwnc.cdc.gov/eid/article/23/9/17-0336_article

Alexandra Moura Institut Pasteur, Paris

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« Compared with previous typing methods, WGS has a higher discriminatory power and the ability to determine genetic distance between isolates. The introduction of WGS for surveillance of food-borne infections has shown that it improves outbreak detection and facilitates outbreak investigations and likely helps reduce the number of infections”

Case Study: WGS in Denmark &

France

33Source : Susanne Schjorring et al uro Surveill. 2017;22(50):pii=17-00762. https://doi.org/10.2807/1560-7917.ES.2017.22.50.17-00762

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“The results also illustrate that when applying WGS analysis every outbreak should be considered in its own context and that there should not be a single universal cut off value for separating outbreak and background isolates. Analysis and interpretation of WGS clusters requires expert knowledge and collaborative input from bioinformaticians, epidemiologists and microbiologists”

“In terms of antimicrobial resistance there was remarkable low presence of tetracycline (<0.1%) and penicillin (1%) resistance genes

Resistance to detergents and antiseptics via efflux activity was significant with mechanisms detected at a prevalence approaching 20%

Closing gaps for performing a risk assessment on

L.mono in RTE foods

34Source : Eva Møller Nielsen et al : http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2017.EN-1151/pdf

LISEQ – WGS analysis of Listeria from food and human sources© European Food Safety Authority, 2017

Better knowledge may support & improve hygiene procedures

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WGS new golden standard !?!

35

Page 36: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

3636Source : AppliedMath

Source : AppliedMath

Page 37: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

NGS : Next Generation Sequencing

Summary for Food Industries

37

WGS MetaSeq

Strain

ID/characterization

Phylogeny

Strain Tracking

Outbreak Investigation

Virulome

Resistome

…..

37

Ecosystem Diversity

Food product spoilage ID

Ingredients monitoring

Factory Environment monitoring

Food shelf-life extension

….

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38

NGS

Whole Genomic

Sequencing (WGS)

MetaSequencing

Metabarcoding

SOURCE : ACTEMIUM, SCIENCE PHOTO LIBRARY, ISTEMA-HUMANO.COM.AR

Next Generation Sequencing (NGS)

NGS can also be called ‘Post-Sanger sequencing technologies’. Covers mixed microbial

communities (MetaSequencing/Metabarcoding) and isolated micro-organisms (WGS)

identification & characterization.

Mixed microbial

communities

Isolated

micro-organisms

Page 39: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

Impacts of spoilage flora

39

Impacts on consumer satisfaction (regarding food quality

regarding competitors)

Losses of confidence in the quality of the brand products (if

there are a lot of batch recalls)

Customer

Ecologic Issue

1.3 billion tones of food are thrown away per year and

generate 3.3 gigatons of CO2

Economic issue

Productions & cleaning costs, discard products

the loss of traditional customers

50% of the spoilage flora come from the processing

environment

Industry

(« Food wastage footprint, Impacts on natural resources » FAO, 2016)

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Spoilage: routes of transmission

40

[4]

Human

The spoilage comes from raw material, and human behavior.

Environment is a spoiler’s niche

Raw material & human = input channels

Potential resident bacterial communities

Process can drive the environment microbiota

All this microbiota can persists and spoils the product

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Metabarcoding 16S

41

Page 42: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

Modification of bacterial populations

metabarcoding on food samples

42

Date of purchase BBD+9 dBBD+2 d

unclassified

dominant: Pseudomonas 55%

subdominant : Lactococcus, Leuconostoc….

Page 43: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

Modification of bacterial populations

metabarcoding on food samples

43

Date of purchase BBD+9 dBBD+2 d

Page 44: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

Sequencing the Food Factory

44

Factory environment

Raw Material, products during the food process, finish product

Manufacturing incident

Page 45: ListeriaRisk: the Role of Whole Genomic Sequencing, South ... · Whole Genome Sequencing (WGS) Whole Genome Sequencing (WGS) determines the complete DNA sequence of an organism's

The production environment impacts

quality and food security

Acinetobacter

Brochothrix

Psychrobacter

Pseudomonas

Source addapted from De Filippis et al., 2013

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NGS : Next Generation Sequencing

Summary for Food Industries

47

WGS MetaSeq

Strain

ID/characterization

Phylogeny

Strain Tracking

Outbreak Investigation

Virulome

Resistome

…..

47

Ecosystem Diversity

Food product spoilage ID

Ingredients monitoring

Factory Environment monitoring

Food shelf-life extension

….

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NGS yes but with all data related !

48

Microbiology

• Cultural approaches

• Cytometry

Molecular

Biology

• ID (16S, …)

• WGS

• Metagenomic

Chimistry

Biochemistry

• Volatile compound profile

• Proteins product profile

Process Knowledg

e• Processing steps

Polyphasic &

complementary

Approaches

Complexe & multi data Data Management NEEDSSources : actemium.fr, allgraph.bid, http://elitetrack.com