the contribution of typing methods to risk assessment ...€¦ · next generation sequencing ngs...
TRANSCRIPT
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The contribution of typing
methods to risk assessment
- the case of
Vero cytotoxin- (Shiga toxin-)
producing
E. coli (VTEC/STEC)
Flemming Scheutz
WHO Collaborating Centre for Reference and Research on
Escherichia and Klebsiella
Foodborne bacterial infections
Microbiology & Infection control STATENS SERUM INSTITUT
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Haemolytic uraemic syndrome
HUS • Acute renal failure • Microangiopathic haemolytic anaemia (Intravascular haemolysis and destruction of the red blood cells) • Thrombocytopenia (Drop in the blood platelet count)
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Once upon a time….
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Non-O157s now share the scene with O157
from cases of HUS
O26:H11 in Italy (stx2a highly virulent)
O80:H2 in France (stx2a, stx2a2d, stx2c2d, stx2d, eae ξ)
O104:H4 in the Republic of Georgia
O73:H18 in Spain
O91:H21 in Spain
O59:H19 EAEC in Argentina
O174:H8, H21 and H28 in Argentina
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vtx1
and/or
vtx2 eae
VTEC
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Stx subtypes and variants
Stx/VT1: 3 subtypes; 13
variants
VT2: 7 subtypes; 93
variants
47 vtx1 and vtx2 238 out of
285 valid sequences in GenBank
a
Stx/VT1
d
c
VT2 d
b g c
a e
f
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HUS & vtx subtypes
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HUS &
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HUS;
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HUS associated VTEC
= HUSEC
and to a lesser degree
vtx2d in eae negative
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Siphoviridae
Podoviridae
vtx1 and vtx2 bacteriophages
vtx1
vtx2
Courtesy of Maite Muniesa
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Diversity: Morphology
1b 12 13b 16b
1a 4 13a 16a
Courtesy of Maite Muniesa
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AEM 2013
62% of 100 fecal samples from 100 healthy individuals showing no enteric
symptoms carried Stx phages, with an average value of 2.6 x 104
Stx phages/g
Bacteriophages in healthy humans
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Prevalence of VTEC in healthy humans
including food handlers or workers in daycare
centers during 2010-2012 in Japan
Healthy carriers Diarrhoeal cases
84.2 2.1
/100,000 /100,000
Tomoko Morita-Ishihara et al. IASR 2015
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Stx phages in food
Minced Beef
0
1
2
3
4
5
6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36Salad
0
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Minced beef
Salad lo
g10 s
tx G
C. 25/g
log
10 s
tx G
C. 25/g
Sample nº
At least 50 % of the Stx phages in
these samples were infectious
102-105 Stx phages / 25 g minced beef. 100% of the samples
102-103 Stx phages / 25 g fresh salad. 69 % of the samples
Food
Microbiological criteria EU
regulation
Imamovic & Muniesa, AEM. 2011
Able to propagate in host strains
Bar: qPCR
*: + end point PCR
Barcelona area has a low
incidence of VTEC
infections
Courtesy of Maite Muniesa
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Courtesy of Stefano Morabito
:H10
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Next generation sequencing
NGS or
Whole genome sequencing
WGS
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18
2324 E. coli genomes – Panseq SNP phylogeny (ML)
O157
O121
O113
O145
O26
Courtesy of Chad Laing
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Acknowledgments
19 Application of WGS to outbreak investigation of gastrointestinal pathogens
1,075 strains of STEC O157 from clinical and animal isolates from England,
Northern Ireland, Wales and Scotland collected from 1985 to 2014
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Application of WGS to outbreak investigation of gastrointestinal pathogens
Maximum clade credibility
tree of 530 ∆25 SNP
representatives of O157:H7
= Family Tree of O157:H7
using ∆25 SNP addresses
SNP = Single Nucleotide
Polymorphism
Tim Dallman et al. Microbial Genomics 2015
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http://benfry.com/zipdecode/
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Application of WGS to outbreak investigation of gastrointestinal pathogens
175 years
~1840
150 years
400 years
The ancestral O157:H7 state is stx2c
(lineage II)
Early rapid diversification
Shiga toxin complement and insertion
sites are relatively stable
Stx2a is relatively recent and has been
acquired ~60 years ago on multiple
occasions
Courtesy of Tim Dallman
YEARS
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Courtesy of Tim Dallman
O157:H7 has become more pathogenic to humans
over its evolution
Strong association with stx2a and severe disease
YEARS
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24 Application of WGS to outbreak investigation of gastrointestinal pathogens
Uneven Diversity of O157
54 ∆250 Clusters
All 3 lineages have one ∆250 cluster
that comprises > 50% of isolates of that
lineage These 3 ∆250 clusters contains 84% of UK
Cattle isolates
∆250 clusters with <5 isolates
associated with foreign travel or
imported food
Supports Global Dispersion then Regional Expansion
Courtesy of Tim Dallman
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25 Courtesy of Tim Dallman
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The implicated O157
strain with the SNP
address 18.35.397.765
had a strong NW
geographical signal
including some possible
environmental exposures
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Once upon a time….
These days: Outbreaks:
• Watercress
Risk factors:
• Swimming
• Contact with ruminants
• Contact with another person with diarrhoea
• Visiting farms
• Steaks and ground beef
• Travel to Africa (Turkey and Marocco)
Host factors:
• Antaacids
• Cardiovascular disease
• Gastrointestinal infection
• Hispanic ethnicity (in the US)
Hamburgers =
ground beef
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Microbiological risk assessment (MRA)
(i) hazard identification:
• vtx/stx subtyping by PCR or WGS
(ii) hazard characterization:
• WGS of bacteriophages and their hosts;
• WGS assignment of host SNP addresses
(iii) exposure assessment &
(iv) risk characterisation
• WGS & EPIDEMIOLOGY
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ACKNOWLEDGEMENTS
• Tim Dallman, Public Health England
• Chad Laing; Public Health Agency of
Canada
• Stefano Morabito; Istituto Superiore di
Sanità, Rome
• Maite Muniesa; University of Barcelona
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THANK you for your attention