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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

    http://www.nature.com/nrg/journal/v11/n1/abs/nrg2626.html

  • 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

    barcoding source : 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

    9 Source : 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 2015 Source : Leopold et al J. Clin. Microbiol. July 2014 vol. 52 no. 7 2365-2370

    11 Source: 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

    https://www.abmgood.com/marketing/knowledge_base/next_generation_sequencing_WGS.php https://www.abmgood.com/marketing/knowledge_base/next_generation_sequencing_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