Genomics in research on verocytoxigenic E. coli

Pascal Delaquis

Summerland Research and Development Centre

Agriculture and Agri-Food Canada

Summerland, BC

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

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 …

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

Outbreaks: other causes

Outbreak vs sporadic infections

• Rates of sporadic infection in Canada?

• Some estimates suggest 50% or more

Rates of infection with VTEC

BCCDC, 2014.

Catford et al 2014. Int Food Risk Anal J 4:21

Indirect (?)

VTEC transmission

Direct

Indirect

Carriers

Reservoirs

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)

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

Prevalence, phenotypic and genotypic characteristics of

VTEC in surface waters, Fraser Valley of BC.

VTEC prevalence in the Lower Fraser, Sumas, Nicomekl and Serpentine River

watersheds. Samples were collected over one year.

HGMF Immunoblot method

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.

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

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

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

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)

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)

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

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

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

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:

Thank you for your attention!