Bio and Microbiology Measurements

as Support of Safety of Foods,

Production, Trade and Quality of Life
Dr. Denise OSullivan
Simposio de Metrologa

Science

for a safer world

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Health is our current focus

Cancer
Infectious diseases
Genetics
Surrogate Biomarkers
Single cell

Characterisation of nucleic acids

Nucleic acid

Type
Sequence
Quantity
Quality
Modifications

Molecular Biology Capability

Targeted Nucleic Acid Analysis
Polymerase chain reaction (PCR)
Quantitative PCR
Digital PCR
High-throughput PCR
Isothermal amplification methods
Global Nucleic Acid Analysis
Spectrophotometry
Fluorometry
Electrophoresis (Capillary)
Sequencing
Sanger sequencing
Next generation sequencing

Absolute DNA Quantification Digital PCR

qPCR 1 20 l reactions

Split
sample by
dilution

dPCR 20 1 l
reactions

Infectious diseases
Work packages
1. Higher order methods
2. Measurement challenges
associated with emerging
methodologies.
3. Near-patient testing
4. Development of a
reference measurement
framework to improve
current clinical approaches

Model diseases
. Human cytomegalovirus,
Influenza, Tuberculosis and
chronic obstructive
pulmonary disease

INFECT-MET : Standards for

infectious disease diagnostics

Next generation sequencing

Kahvejian et al., Nat Biotech (2008)

Next generation sequencing

Tuberculosis
Investigating rare mutation detection in the development of
drug resistance for MDR TB (deep sequencing)
Assessment of whole genome sequencing for detection of
novel drug resistance mutations and epidemiological
analysis (WGS)
Technologies are being evaluated for technical sensitivity,
specificity and repeatability

Introduction
Molecular Microbial Profiling

Microbial Molecular Profiling

Microbial Molecular Profiling characterises the structure
of bacterial communities and their dynamics in the
environment
Early molecular profiling studies revealed that there are
large groups of micro-organisms that cannot be cultured
and thus cannot be sequenced
Metagenomics (the study of metagenomes) can analyse
the culturable and unculturable micro-organisms in an
environment

Metagenomics for microbial analysis

Metagenomics is the study of metagenomes
A metagenome is the microbial genetic material present
within an environmental sample

The Human Microbiome Project

-Oral (1010) bacteria

-Skin (1012) bacteria
-Intestine (1014) bacteria
Microbiomes play a crucial role in
health
Implicated in many diseases
Microbiome analysis will define:
How they affect health and
disease
Role in Diagnostics/Prognostic

Human
Human
microbiome
microbiome
8,000,000+
8,000,000+
genes
genes

Human
genome
23,000 genes
11 >360 times

Molecular quantification of microbes

Used clinically for prognostic monitoring
Important for defining accurate limit of detection for
microbial identification
Therefore for traceable molecular measurement
(quantitative and ID) there is a need to investigate:
Quantitative methods
Control material development and requirements

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The Complications
High level of complexity of information offered by
microbial molecular profiling methods
Poses a considerable challenge during analytical
standardisation
Disparate methods could cause problems
So how can control materials help?

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A Control Material for Microbial

Molecular Profiling

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Previous Work

The Metagenomic Control Material (MCM)

A Prototype Reference Material

Mix of DNA from 10 bacterial species prepared

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Microbial Metagenomics
Mass based

Method A

Method B
Neisseria meningitidis
Streptococcus pneumoniae
Staphylococcus aureus
Klebsiella pneumoniae
Streptococcus pyogenes
Streptococcus agalactiae
Enterococcus faecalis
Escherichia coli
Pseudomonas aeruginosa
Acinetobacter baumannii

Study
Aim: to perform a comprehensive investigation into the
inherent error in profiling microbial communities
Use the control material (metagenomic control material
MCM) for community analysis which was already
prepared
Characterise control material using two independent
methods dPCR and fluorescence
Can use the MCM to investigate errors when performing
different sequencing strategies (amplicon v shotgun)
Compare the relative abundances of the component
organisms of the MCM as determined by dPCR,
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fluorescence and next generation sequencing
methods

Relative copy number, expressed as % of

MCM

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Relative copy number, expressed as % of

MCM

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Conclusions
dPCR offers a simple method for providing confidence
when measuring control materials
All sequencing methods performed comparably
Good agreement between sequencing methods and
dPCR (WGS demonstrating very high reproducibility)
Any discrepancy between abundances were in general <
3 fold
Care should be taken when comparing small differences;
need to apply control material
Provides a foundation for future work looking at microbial
abundances in a community
Future work looking at impact of extraction 21methods

Profiling Food Spoilage

Microorganisms

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Microorganisms in Food Spoilage

Microorganisms, and their by-products can be harmful in
food
Traditional methods can be slow, laborious and often
inconsistent
Microbial profiling methods could be used to understand
the complex communities in foodstuffs and which
microorganisms are involved
how changes in the microbiota as whole could indicate
food spoilage conditions
how the shelf life could be improved through rapid ID of
spoilage bacteria
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Test Cases
Gammon samples stored at +4oC and -20oC for several
weeks
Freeze-dried chicken samples (LGC Standards)
Perform 16S rDNA sequencing on the Roche GS Junior

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Conclusions from Test Cases

Performed microbial profiling of food samples to identify
food spoilage agents
Require more sequencing depth to determine rarer and
under-represented species
16S rDNA strategy was not able to identify down to
species level
Different storage conditions resulted in differences in
profiles
Need to develop strategy for the identification of moulds
and yeasts
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Further Impact of Molecular

Methods on Microbial Quantification

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Molecular vs Culture
P Q01.1 Report on the first pilot study- Establishing
Comparability for Microbial Culture Quantitation (NMIA)
Dr. Dean Clarke, NMIA

Morpeth et al ISPPD 2012

gDNA materials
Available from IRMM for
E. coli O157, (strain EDL 933)
L. monocytogenes (strain 4B, NCTC 11994)

Available from NIM China for

E.coli O157 (Strain EDL 931, ATCC 35150)
L. monocytogenes (Strain Li 23,ATCC 19114)

Positive control for PCR reactions for diagnostics PCRs

Provided lyophilised (Nominal g value and uncertainty
provided)

Considerations for molecular quantification

Chromosomal size
E. coli = ~5 Mb
L. monocytogenes = ~2.9 Mb

Gene dosage
Replication
Multicopy genes (16S rDNA)

Epigenetics
Methylation

Bacterial replication

www.pasteur.fr
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C A

B
A

C
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Chromosomal Targets
E. coli has large genome
Target four regions

Ori, sequence closer to the origin of replication

dif , sequence close to the termination site
uidA, diagnostic sequence used by LGC
16S rDNA, multi copy sequence (7 in the strains under
investigation)

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rrsH

B
rrs

E
rrs

E. coli gDNA
or
iC

sA
rr

rrs

0kb
520kb

4680kb

1040kb

4160kb

Escherichia coli CFT073

5231428 bp

rrsD

1560kb

3640kb

2080kb

3120kb

rrsG

2600kb

E. coli CFT073

di
f
ui
dA
Clockwise
= Strand +
Anticlockwise
= Strand -

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http://www.ncbi.nlm.nih.gov/nucleotide/26111730?report=genbank&log$=nuclalign&blast_rank=74&RID=KW9A1N24016
http://www.ecogene.org/old/genemap/map.php?search_topic=132

Questions?
How does chromosomal location effect result (qPCR,
dPCR)
Is this similar between
Strains
Preparations/Laboratories

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E. coli gDNA
Three different E. coli strains
O157:H7, (strain EDL 933). Supplied by IRMM
O157:H7, (strain EDL 931), Supplied by NIMC
CFT073, Supplied by ATCC

Prepared by 3 different laboratories (China, US,

Belgium)
Re-suspended at LGC
Quantified by fluorescence
Same amount of each extract measured by dPCR

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E. coli gDNA

3000
2500
2000

Estimated copies

O157:
933

1500
1000
500
0
oriC

dif

uidA

16S

Target gene

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E. coli gDNA

3000
2500
2000

Estimated copies

CFT0
73

1500
1000

500
0
oriC

dif

uidA

16S

Target gene

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E. coli gDNA

600
500
400

Mean Est

CFT0
73

300
200
100
0
oriC

dif

uidA

16S

Target gene

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Absolute Quantification
Concordance between assays is very impressive
dPCR has the potential to improve microbial molecular
measurement
Do we need to assign a value to gDNA extracts?
Other things to consider

Different cultures (strains/growth stages etc.)

Matrices
Organisms
Assay specificity

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How much precision is necessary?

What are we aiming for with regards to precision
Viral load testing decisions rarely made on differences
that are < 1 log
What about limit of detection?

Other considerations
How do we standardise
qPCR/amplicon sequencing/shotgun?
Different laboratories?

If we produce a reference material what do we trace it to:

Colony forming units?
DNA gram weight?
Genome equivalents?

Just what does 100 bacterial genomes mean to:

Microbe viability ?
Microbe infectivity?
Microbe toxicity?

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Acknowledgements
LGC
Jim Huggett, Carole Foy, Tan Temisak, Nicholas Redshaw
University of Exeter
David Studholme, Thomas Laver
St. Georges University of London
Philip Butcher, Ken Laing
Great Ormond Street Hospital
Kathryn Harris
National Measurement Institute China
Lianhua Dong

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Thanks for listening

denise.osullivan@lgcgroup.com

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