variation in virulence among clades of e. coli o157:h7 · national food safety & toxicology...
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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