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Listeria Risk: the Role of Whole
Genomic Sequencing, South Africa
outbreak case
Jérôme COMBRISSON PhD,
Steakexpert , 12ème édition - 2018
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
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
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)
Terminology
5
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
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
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:
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
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.
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
12
Whole Genome Sequencing (WGS)
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
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 < <
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
<
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
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.
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…..
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……..
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.
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
+
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 ……
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 ……
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
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
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
<
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 ≠
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
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……
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.
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
« 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
« 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
“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
WGS new golden standard !?!
35
3636Source : AppliedMath
Source : AppliedMath
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
….
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
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)
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
Metabarcoding 16S
41
Modification of bacterial populations
metabarcoding on food samples
42
Date of purchase BBD+9 dBBD+2 d
unclassified
dominant: Pseudomonas 55%
subdominant : Lactococcus, Leuconostoc….
Modification of bacterial populations
metabarcoding on food samples
43
Date of purchase BBD+9 dBBD+2 d
Sequencing the Food Factory
44
Factory environment
Raw Material, products during the food process, finish product
Manufacturing incident
The production environment impacts
quality and food security
Acinetobacter
Brochothrix
Psychrobacter
Pseudomonas
Source addapted from De Filippis et al., 2013
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
….
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