genomics in research on verocytoxigenic e. coli · 11/7/2016 · waterborne outbreaks...
TRANSCRIPT
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Genomics in research on verocytoxigenic E. coli
Pascal Delaquis
Summerland Research and Development Centre
Agriculture and Agri-Food Canada
Summerland, BC
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Verocytotoxin-producing E. coli (VTEC): pathogenesis
• Cellular necrosis in the intestines contributing to hemorrhagic colitis
• Necrosis of kidney cells: hemolytic uremic syndrome (HUS)
• Can also bind to cells in the central nervous system
• More than 300 additional virulence factors identified to date in E.coli
Glycolipid
(globotriaosylceramide)
Gb3 receptor
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VTEC serotypes
• Serotyping scheme is based on somatic (O) and flagellar (H) antigens
• More than 200 serotypes have been reported to produce verocytotoxins
• The most “notorious” serotype is O157:H7 …
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Waterborne outbreaks
• Walkerton, 2000
• E. coli O157:H7
• 2,321 illnesses
• 7 deaths
• 27 HUS cases
Food-borne outbreaks
1993 Jack in the Box E. coli outbreak
732 people infected with Escherichia coli O157:H7
originating from undercooked beef patties in hamburgers
The majority of the victims were children aged under 10-
years old. Four children died and 178 other victims were
left with permanent injury including kidney and brain
damage
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Outbreaks: other causes
Outbreak vs sporadic infections
• Rates of sporadic infection in Canada?
• Some estimates suggest 50% or more
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Rates of infection with VTEC
BCCDC, 2014.
Catford et al 2014. Int Food Risk Anal J 4:21
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Indirect (?)
VTEC transmission
Direct
Indirect
Carriers
Reservoirs
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Limiting the burden of infectious disease:
• Identify exposure sources
• Define route (s) of transmission
• Implement measures to reduce exposure
VTEC: challenges
• Gaps in the quantity and quality of data (eg. prevalence of relevant
pathotypes)
• Complexity of environments along potential transmission
• Complex biology of E. coli – not easy to isolate, discriminate from other
E. coli, characterize (eg. pathotype)
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Genomics Research and Development Initiative (GRDI)
Relevant research questions:
• Can genomic data be used to extract molecular signatures to identify
VTEC strains that are persistent, contributing to the burden of disease ?
• What are the most common exposure sources (water, food, environment)?
• Which strains/genome types specifically are contributing to the burden of
human disease in Canada ?
Applied objectives:
• Information to improve farm, food and water safety policies that will
reduce the burden of VTEC infections in Canada
• Information for the assessment of risks associated with surface waters
used for irrigation in the Fraser Valley
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Prevalence, phenotypic and genotypic characteristics of
VTEC in surface waters, Fraser Valley of BC.
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VTEC prevalence in the Lower Fraser, Sumas, Nicomekl and Serpentine River
watersheds. Samples were collected over one year.
HGMF Immunoblot method
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Watershed No. of samples analyzed Prevalence rate (%)
Water
Lower Fraser 65 9.2
Sumas River 97 21.6
Nicomekl River 86 23.2
Serpentine River 82 19.5
Total: 330 Overall rate: 19.1
Sediments
Sumas 21 23.8
VTEC prevalence rates in four Fraser Valley watersheds. Samples were
collected monthly in 5 sampling sites in each watershed, sediment samples
on 5 separate occasions over a period of one year.
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Serology of VTEC recovered from surface waters and sediments
(4.2%)O157:H7, O157:NM
(13.6%)O26, O103,
O111
(82.2%)31 Other serotypes
O5:NM, O6:H10, O8:H9, O8:H19, O22:H8, O69:H11, O76:H19, O84:H2, O98:NM, O113:H21, O116:H25, O128:H2, O130:H8, O136:H12, O136:H16, O141ac:H8, O146:H8, O151:H12, O156:H25, O163:NM, O163:H19, O165:NM, O165:H25, O168:H8, O174:H8, O174:H21, O177:NM, O182(O109):H5, OR:NM, OR:H21, O?:H19
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Prevalence data and phenotypic characterization provided
evidence of:
• Seasonal variation and geographic differences in prevalence
• Diversity in VTEC populations, notably non-O157 serogroups
…but no information about:
• Relatedness, potential pathogenicity
Approach: whole genome sequencing and analysis
• Sequence 50 isolates (short read - Illumina Miseq)
• Analysis to compare isolates from different sources; isolates that have
caused disease
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Approximate maximum likelihood tree deduced from genome wide single
nucleotide polymorphisms and presence/absence matrix for virulence factor
genes in the genomes of 50 VTEC isolated from water and 16 outbreak isolates
Lower Fraser watershed, 49 days apart
Sumas River watershed
Serpentine River watershed
Nicomekl River watershed
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Observations derived from whole genome analysis:
• Evidence that some strains are recurrent within sampling
sites/watersheds. Persistence in the environment, or continuous release
from point sources?
• Showed that some VTEC isolated from surface waters and sediments in
the Fraser Valley share complements of virulence factor genes with
strains that have caused outbreaks
Implications:
• Signals a potential risk to public health
• Caution: the presence of specific virulence factors or combinations
thereof do not predict the potential of VTEC to cause human disease
with 100% certainty (at this time)
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Additional information derived genomic analysis
• Serotypes for all VTEC isolates correctly predicted
• Antibiotic resistance
0
10
20
30
40
50
60
70
80
90
100
% R
esis
tan
ce
Antibiotic
Resistant
Intermediate
Susceptible
FFC: florfenicol1
TET: tetracycline2
STR: streptomycin2
BCN: kanamycin2
AMP: ampicillin2
TIO: ceftofiur3
AMX: amoxicillin3
Medium1, high2, very
high3 importance in
human medicine (Health
Canada)
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Isolate Resistance phenotype Resistance genes detected with
ResFinder
296 O111:NM AMPR, STRR, SXTR, TETR, AMXI,
FFCIaadA, blaTEM-1b, drfA8, dfrA12, mphA,
strA, strB, sul1, sul2, tetA, tetB
298 O111:NM AMPR, BCNR, STRR, TETR, FFCR,
AMXI, TIOIaph(3')-1a, blaTEM-1b, strA, strB, sul2,
tetA
299 O111:H8 AMPR, TIOR, BCNR, STRR, TETR,
AMXI, FFCIaph(3')-1a, blaTEM-1b, strA, strB, sul2,
tetA
356 O69:H11
AMPR, STRR, SXTR, TETR, AMXI,
FFCI blaTEM-1b, dfrA8, strA, strB, sul2, tetB
373 O165:H25
BCNR, STRR, TETR, FFCR, AMPI,
TIOI aph(3')-1c, strA, strB, tetB
374 O165:NM BCNR, STRR, TETR, FFCR, AMPI aph(3')-1c, strA, strB, tetB
385 O111:NM AMXR, AMPR, TIOR, FOXR, STRR,
SXTR, CHLR, TETR, FFCR, CROI,
GENI, BCNI
aadA1, aadA2, aac(3)-Vla, blaCMY-2,
dfrA12, floR, mphA, strB, strA, sul1, sul2,
tetA
391 O103:H2 STRR, SXTR, CHLR, TETR, AMPI,
FFCIaadA1, catA1, dfrA1, strA, strB, sul1,
sul2, tetB
Antibiotic resistance phenotypes and resistance genes detected in the
genomes of multidrug resistant VTEC isolated from water in the Fraser Valley
Aminoglycoside
resistance,
streptomycin
β-lactam
resistance,
ampicillin,
amoxicillin
β-lactam
resistance,
ceftofiur
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Approximate maximum likelihood tree and presence/absence matrix for
antibiotic resistance genes common to the genomes of 50 VTEC isolated from
water and 106 clinical isolates from Canada
Resistance
geneAntibiotic
Statistically over-
represented in
water isolates?
aac N
aadA Aminoglycosides - STR Y
aph N
blaCMY_2 N
blaCTX_M N
blaTEM N
catA N
dfrA Sulfa (Trimethoprim – TMP) Y
floR N
gyrA83 N
mphA Macrolides - ERY Y
strA N
strB N
sul1 Sulfa (Sulfamethoxazole – SMZ) Y
sul2 N
tetA N
tetB Tetracyclines Y
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Genomics: impact on VTEC research
• Identification and description of:
reservoirs
vectors
transmission routes
in natural & agricultural environments, food chains
(on regional, national and international scales)
• Discovery of traits that influence the survival/growth of
VTEC in the environment and in foods
• Development of far more “informed” risk assessments due
to vastly improved hazard characterization
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Drs. Kevin Allen and Jessica Chen, Stephanie Nadya, Food, Nutrition and Health
Program, Faculty of Land and Food Systems, the University of British Columbia,
Vancouver, B.C.
Dr. Roger Johnson, Kim Ziebell, National Microbiology Laboratory @ Guelph, Guelph, ON
Drs. Chad Laing and Victor Gannon, Laboratory for Foodborne Zoonoses, Lethbridge, AB
Drs. Susan Bach, Summerland Research Center, Summerland, BC
Dr. Ed Topp, London Research Centre, London, ON
Collaborators:
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Thank you for your attention!