Variation in virulence among clades of E. coli O157:H7

Shannon Manning, Ph.D.Microbial Evolution Laboratory National Food Safety & Toxicology Center Michigan State UniversityEast Lansing, Michigan

April 17, 2008

E. coli O157:H7

• Emerged in 1982• Associated with hamburger

outbreak• Clinical illness:

• Bloody and non-bloody diarhhea• Hemolytic uremic syndrome (HUS)

• Transmitted in contaminated food and water– Low infectious dose (~100 cells)

• Reservoir in dairy and beef cattle

Image: www.uni-heidelberg.de/zentral/ztl/grafiken_bilder/bilder/e-coli.jpg

O157 outbreaks

• 350 US outbreaks(1982-2002)

• 8,598 cases– 1,493 (17.4%)

hospitalizations– 354 (4.1%) HUS

• Rate declined over time

– 40 (0.5%) deaths

Rangel et al. Emerg Infect Dis 2005;11:603

• Lettuce (Nov.-Dec.)– 71 cases in 5 states

– 53 (75%) hospitalized– 8 (11%) HUS

2006 O157 outbreaks

• Spinach (Aug.-Sept.)– 204 cases in 26 states

– 104 (51%) hospitalized– 31 (15%) HUS– 3 (1%) deaths

Avg. HUS rate =13% (3.2X> rate for 350 outbreaks*)

Avg. hospitalization = 63%(3.7X > rate for 350 outbreaks*)

*Rangel et al. Emerg Infect Dis 2005;11:603

Characterizing the pathogen

• PulseNet data revealed that the 2 outbreaks represented unique PFGE patterns

• Multilocus sequence typing reveals a lack of diversity– Nollen et al. J Clin Microbiol 2003;41:675

• Single nucleotide polymorphism (SNP) analysis to test in closely related O157 strains– Index genetic differences between strains to elucidate

evolution– Identify pathogenic clones

SNP genotyping strategy

David Alland and Alifiya MotiwalaGlobal Phylogeny of Mycobacterium tuberculosis

based on Single Nucleotide Polymorphism (SNP) Analysis: Insights into Tuberculosis

Evolution

Division of Infectious Disease,University of Medicine and Dentistry of New Jersey

(UMDNJ), New Jersey

Filliol et al. J. Bacteriol 2006;188: 759Work supported by NIH R01-AI049352

David Alland and Manzour Hazbón

Global phylogeny of M. tuberculosis

• Based on 212 SNPs– Resolved the 327 isolates

into 182 STs• SNP-lineages supported

the three “major genetic” groups

• Shown to be a successful strategy for resolving closely related genotypes

Filliol et al. J. Bacteriol 2006;188: 759

SNP analysis of O157 strains

Zhang et al Genomic Research 2006;16:757-67

• CDC collaboration (Wei Zhang, Bala Swaminathan)• Comparative genome

sequencing microarrays

• 11 strains (2 GUD+)• 906 SNPs in 523

chromosomal genes• Phylogenetic analysis

separated strains based on allelic differences at codons

SNP typing method

• 96 SNP loci in 83 genes– 52 non-synonymous sites– 43 synonymous sites– uidA insertion

• Real time PCR assays were devised to detect nucleotide substitutions at each locus• Performed by David

Alland’s lab at UMDNJ

Manning et al. PNAS 2008;105:4868

Strain collection• Epidemiologically

relevant population– 444 O157 strains

from Michigan during 2001-2006*

• 53% female• 44% <18 years• 68% sporadic

• 95 additional O157 strains and close relatives

*Manning et al. Emerg Infect Dis 2007;13:318

James RudrikPatricia SomselSteve Dietrich

Robbie Madera

Genetic diversity of O157

• The nucleotide diversity (pi) across 96 SNP loci ranged from 0 (for 2 monomorphic loci) to 0.50 for 25 loci.

• The average pi for the 96 loci was 0.212 ± 0.199.– Strains differed on average

at 20% of locin=403 O157 strains and close relatives

Manning et al. PNAS 2008;105:4868

Genetic relatedness of 403 E. coli O157 strains and relatives

AncestralClade

O55:H7

• The minimum evolution algorithm resolved 39 SNP genotypes (SGs)

• The consensus tree has 9 clades with >70% bootstrap confidence values based on 1,000 replicates

Manning et al. PNAS 2008;105:4868

Phylogenetic network

• Neighbor-net algorithm for all 528 strains applied to 48 parsimoniously informative sites

• The central group is connected by multiple paths – Suggests recombination or

recurrent mutation has contributed to divergence

• Clades at the end of branches with no evidence of conflicting phylogenetic signals– Indicates that these lineages are

diverging without evidence of recombination.

Manning et al. PNAS 2008;105:4868

Genotype distribution

Clade SGs SG geographic range Date(s) Freq. (%)

1 1-2 Japan, USA 1996-2001 3 (0.6)

2 3-11 Japan, USA, Australia 1998-2006 249 (47.1)

3 12-18 Japan, USA, Canada, Australia 1982-2004 57 (10.8)

4 19-20 USA 2002-2003 10 (1.9)

5 21-22 USA 2002-2006 3 (0.6)

NA USA 2002 1 (0.2)

6 24-27 USA, Australia 1998-2006 17 (3.2)

7 28-29 USA, Canada 1987-2006 38 (7.2)

8 30-34 USA, UK, Germany, Argentina 1984-2006 135 (25.5)

9 35-39 USA, Germany 1979-2004 16 (3.0)

• Several clades have an inter- continental distribution• 4 unique

to North America

• Clades 2,8 predominate in Michigan and elsewhere

Clinical characteristics vary by clades

Logistic regression adjusted for: bloody diarrhea, diarrhea, age, gender, HUS, abdominal pain, body aches, chills, and Manning et al. PNAS 2008;105:4868

Clade frequencies over time

Manning et al. PNAS 2008;105:4868

• A signifiicant increase in clade 8 since 2002 (P<0.0001)

Common O157 outbreaks

Strain YearClade (SG) Outbreak No. cases

No. (%) hosp.

No. (%) HUS

Sakai 1996 1 (1) Radish sprouts, Sakai, Japan 5,000-12,680 398-425

(3-5)0-122 (0-3)

93-111 1993 2 (9) Hamburger, Northwest U.S. 583 171

(29)41 (7)

EDL-933 1982 3 (12) Hamburger, MI and OR 47 33

(70)0

(0)

TW14359 2006 8 (30) Spinach, Western U.S. 204 104

(51)31

(15)

TW14588 2006 8 (30) Lettuce, Eastern U.S. 71 53

(75)8

(11)

350 O157 outbreaks in the USA (1982-2002)* 8,598 1,493 (17)

354 (4)

Manning et al. PNAS 2008;105:4868*Rangel et al. Emerg Infect Dis 2005;11:603

Phylogenetic network

2006Spinach, Lettuce

1982Hamburger

Genome strain (EDL-933)

1993Hamburger

1996Sakai

Genome strain

Conserved genes vary between clades

• 454 pyrosequencing of spinach outbreak strain

• Analyzed the number of highly conserved genes (n=2,741) among 3 genome strains

• Sakai and EDL-933 genomes are more similar to each other in gene content and nucleotide sequence identity

What makes clade 8 so virulent?

Shiga toxins (Stx)• Two major types - Stx1

and Stx2– Subtypes 2c, 2d

• Transferred between strains in nature by phages

Electron micrograph of CsCl-purified phage 2851 particles. Bar = 100 nm.

Strauch et al. Infect. Immun. 2004; 72:7030

Spinach genome has the Stx2c phage

Stx subtyping

• Multiplex PCR was used to identify if stx2c was present• PCR cannot distinguish

between stx2 and stx2c• Amplified a larger product

(1152 bp), digested with FokI followed by gel electrophoresis

2c2 2

2,2c2

Stx genes by clade

• The distribution of stx genotypes differs by clade– stx2 predominated

overall– stx1 absent from

several clades– Most clade 2 strains

had stx1,2

Stx genes by clade

• stx2c has a non- random distribution

• Clade 8 strains significantly (P<0.0001) more likely to have both stx2 and stx2c

Manning et al. PNAS 2008;105:4868

stx2c- positive

stx2c-negative

Does having stx2 and stx2c in clade 8 strains enhance virulence?

• Not all clade 8 strains have both stx2 and stx2c– The presence of both alone cannot be solely

responsible– But, perhaps stx2c is lost easily limiting detection

• Identified clade 8 strains from early 1980s with stx2c (strain E32511 SG-31)– Suggests the hypothesis that the emergent

population has acquired novel elements critical for virulence

Conclusions• SNP genotyping can be used to subtype O157

strains– 32 SNPs delineate all 39 SGs

• O157 lineages vary in their ability to cause severe disease

• The clade 8 lineage– is associated with HUS– has increased over time in Michigan– has contributed to 2 outbreaks with high frequency of

severe disease– more commonly contains both Stx2 and Stx2c

Future work

• Assess unique characteristics of emergentclade 8 subpopulation

• Evaluate O157 strains from the bovine reservoir• Investigate whether clade 8 strains differ in

transmission patterns or ability to survive in certain foods

• Expand surveillance efforts to assess genotype distributions from other states and assess epidemiologic relationships – CT, ID, MA, MI, MN, NC, OR, UT, WI

Acknowledgements

MSU - Thomas Whittam, PhD– David Lacher, PhD– Jan Mladonicky– Lindsey Ouellette– Cody Springman– Katie Schaeffer

UMDNJ– David Alland, MD– Alifiya Motiwala, PhD

CDC– Bala Swaminathan, PhD– Eija Hyytia-Trees, PhD

MDCH– Stephen Dietrich– Robbie Madera– James Rudrik, PhD– Patricia Somsel, DrPH

IL Institute of Technology– Wei Zhang, PhDNIH– Food and Waterborne

Diseases Integrated Research Network Contract # N01-AI30058 (Whittam, PI)

O157:H7 stepwise evolutionary model Feng et al 2007 EID 13:1701