STEC in Michigan- from shedding in cattle to human disease

Shannon Manning www.uni-heidelberg.de/zentral/ztl/grafiken_bilder/bilder/e-coli.jpg

Project goal: To identify factors important for STEC shedding

STEC ecology in Michigan

Collect samples in MI

Identify intestinal microbes that support or prevent

colonization

Disease prevention strategies in cattle

Examine associations with certain

immune cells and diseases

K. Murphy

Identify genotypic and phenotypic characteristics that facilitate colonization Characterize

STEC from wildlife reservoirs

2011-67005-30004

•  11 farms (May-Oct, 2011, 2012) •  Pen floors, manure

storage, water

•  1,195 animals were sampled •  RAJ swabs, fecal

grabs, blood

•  153 animals were followed (~2 weeks, 3 times) •  Fecal grabs, rumen,

tonsil swabs

Blood sample

RAJ swab

Environmental sampling

Fecal grab sample

Rumen fluid

Tonsil swab

Serotyping stx, eae profiling

Immunomagnetic separation (IMS) for O157

STEC CHROMagar™ SMAC

STEC CHROMagar™

E. coli broth,

GN broth

Genotyping and phenotypic assays

Pick up to 20 colonies/plate

qPCR for stx

Community DNA isolation and sequencing

•  Culture data from 1,195 animals

•  Overall, 175 (16%) animals were STEC+

•  Average herd prevalence varies •  By year (p<.0001)

•  10% (2011) vs. 23% (2012)

•  By farm type (p<.0007)

Dairy (13%) Beef (21%)

0

10

20

30

40

50

60

Freq

uenc

y (%

)

qPCR for stx found 70% of animals to be shedding STEC at >104 cfu/g

•  Non-O157 STEC predominated in Michigan cattle –  65 (75%) isolates were non-typeable

•  O157 STEC was found in only 22 (26%) animals

n = 86 animals from 5 herds Lindsey Ouellette

•  Non-O157 STEC infections have been increasing in Michigan since 2006

•  The less common serotypes were found in high frequency in Michigan •  At least 23 different STEC

serotypes were found

0 10 20 30 40 50 60 70 80 90

100

Freq

uenc

y (%

)

O157 (n=896) Non-O157 (n=398)

stx/eae profiling •  3 to 8 profiles per herd •  1 to 4 profiles per

animal –  16% of animals had >1

STEC profile •  12 animals at 4 visits

–  4 unique profiles

Are we underestimating the diversity?

Animal   O   stx1   stx2c   eae  

724   NT   -   -   +  

725   NT   -   -   +  

728   NT   -   +   -  

737   NT   -   -   +  

740   NT   -   +   -  

752   NT   -   +   -  

758   NT   -   -   +  

760   NT   -   +   -  

761   NT   -   +   -  

764   NT   -   +   -  

768   NT   -   +   -  

776   O103   +   -   +  

776 O26 + - + Same animal Chris Elkins, Dave Lacher,

Scott Jackson

•  Non-O157 STEC population is extremely diverse –  8 profiles detected in

the same 12 animals

Animal O H stx1 stx2c eae eae allele

724 145 28 - - + g1

725 NT 10 - - + i1

728 168 8 - + -

737 115 25 - - + g2

740 6 34 - + -

752 6 34 - + -

758 178 11 - - + b1

760 168 8 - + -

761 6 34 - + -

764 6 34 - + -

768 6 34 - + -

776 O103 2 + - + e1

776 O26 11 + - + b1

How many animals persistently shed the same STEC strain

over time?

O H stx eae O H stx O H stx

6 34 stx2

6 34 stx2

O26 11 stx1 b1

O26 11 stx1 b1

168 8 stx2

O26 11 stx1 b1 O157 7 stx1,2 6 34 stx2

O26 11 stx1 b1 168 8 stx2

168 8 stx2

Visit 1 Visit 2 Visit 3

6 34 stx2 O103 2 stx1 e1

6 34 stx2 O103 2 stx1 e1

6 34 stx2

168 8 stx2

6 34 stx2

6 34 stx2 O103 2 stx1 e1

6 34 stx2

Animal O H stx eae

724 145 28 g1

725 NT 10 i1

728 168 8 stx2

737 115 25 g2

740 6 34 stx2

751

752 6 34 stx2

753

758 178 11 b1

760 168 8 stx2

761 6 34 stx2

764 6 34 stx2

767

768 6 34 stx2

776 O103 2 stx1 e1

776 O26 11 stx1 b1

O H stx O H stx eae

4 persistent shedders, 4 acquisitions

3 persistent shedders, 6 acquisitions

Is persistence within a herd attributable to specific STEC

characteristics (e.g., biofilms)?

•  Crystal violet assays for 97 strains from animals in one herd (4 visits)

•  Significant variation in ability to form biofilms across strains

0

1

2

3

4

5

6

7

1721

8 17

251

1721

7 17

259

1721

4 17

220

1721

3 17

258

1726

3 17

245

1726

6 17

215

1723

1 17

216

1720

1 17

200

1724

4 17

171

1726

1 17

265

1718

2 17

264

1717

3 17

170

1717

4 17

181

1717

2 17

202

1725

4 17

190

1721

9 17

210

1723

5 17

212

1724

2 17

241

1723

6 17

225

1721

1 17

233

1726

0 17

177

1723

8 17

248

1724

7 17

246

1723

2 17

257

1725

5 17

206

1717

8 17

249

1719

1 17

256

1720

9 17

208

1725

0 17

262

1718

7 17

196

1723

0 17

183

1719

2 17

175

1719

7 17

204

1725

2 17

253

1719

9 17

194

1718

0 17

267

1717

6 17

203

1719

3 17

185

1718

4 17

179

1718

8 17

186

1723

4 17

239

1722

2 17

198

1722

1 17

226

1724

3 17

223

1718

9 17

240

1722

4 17

227

1723

7 17

228

1722

9 17

205

1719

5

OD

STEC strains evaluated

Mutagenesis studies are ongoing

Pallavi Singh

O H stx eae O H stx O H stx

6 34 stx2

6 34 stx2

O26 11 stx1 b1

O26 11 stx1 b1

168 8 stx2

O26 11 stx1 b1 O157 7 stx1,2 6 34 stx2

O26 11 stx1 b1 168 8 stx2

168 8 stx2

Visit 1 Visit 2 Visit 3

6 34 stx2 O103 2 stx1 e1

6 34 stx2 O103 2 stx1 e1

6 34 stx2

168 8 stx2

6 34 stx2

6 34 stx2 O103 2 stx1 e1

6 34 stx2

Animal O H stx eae

724 145 28 g1

725 NT 10 i1

728 168 8 stx2

737 115 25 g2

740 6 34 stx2

751

752 6 34 stx2

753

758 178 11 b1

760 168 8 stx2

761 6 34 stx2

764 6 34 stx2

767

768 6 34 stx2

776 O103 2 stx1 e1

776 O26 11 stx1 b1

O H stx O H stx eae

O6 and O103 STEC formed the strongest

biofilms in vitro

Is high STEC diversity in a herd mostly due to STEC transmission

or Stx-phage transmission?

Yi-AnYang

•  Stx-phage typing PCR-based RFLP assay –  Targets a 10-17 kb

variable regions in the Stx1-, Stx2- and Stx2c-phage genomes

–  Evaluated 86 STEC from animals across 4 herds

•  17 Phage types identified overall –  11 Stx1, 3 Stx2, 3

Stx2c

Stx1 phage types (PT)

Stx2a phage types

Stx2c phage types

PT1 PT9 PT14 PT7 PT3 PT11

PT4 Spinach PT12 PT13

PT10 Sakai PT15 PT16

PT6 PT6 PT5 Sakai PT17 PT2 PT8

• Distinct phage types in circulation among STEC in each herd –  One PT was

found in two herds

–  Deer sharing pasture with cattle in herd 9 shared a PT

Visit I No. Stx1 Stx2c O:H 4

5 PT4 NT

6

10

11 PT4 O6:H34

12

13 PT4 O6:H34

15

16 PT4 O6:H34 PT4 NT

18

20

22 PT3 - O26

Visit II Stx1 Stx2c O:H

PT2 - O103:H2 PT2 PT4 NT PT2 PT4 O103 PT2 - O103:H2

- PT4 NT PT2 PT4 NT

- PT4 NT

PT2 - O103:H2 - PT4 O6:H34 - PT4 O6:H34

Visit III Stx1 Stx2c O:H

- PT4 O6:H34

PT3 - O26

PT3 - O26 PT3 - O26 PT3 PT4 O157:H7

PT3 - O26 - PT4 O6:H34

PT3 - O26

PT3 - O26 PT3 -­‐ O26 PT3 - O26

- PT4 NT

Visit IV Stx1 Stx2c O:H PT3 - O26

- PT4 NT

- PT4 NT

- PT4 NT

- PT4 NT

PT3 - O26 - PT4 NT

Summary •  Non-O157 STEC predominated in Michigan cattle in

2011-2012 –  Beef farms had higher overall prevalence –  Non-O157 STEC have also increased in human infections

•  STEC acquisition occurs frequently over time –  Specific STEC types are more likely to persist within

herds •  Certain bacterial characteristics may promote persistence

•  The STEC population is more diverse than the Stx-phage population per herd –  Stx-phage transmission is important for the emergence of

novel STEC strains (e.g., O104:H4, O157:H7)

Future work

•  Analyses ongoing – Diversity analyses across herds – Association between bovine immune response and

STEC shedding – Characterization of the intestinal microbiome in 2

dairy vs. 2 beef herds •  High vs. low STEC herd prevalence •  Evaluation of animals who acquire/lose STEC over

time –  Identification of epidemiological factors important for

STEC acquisition

Funding:

2011-67005-30004

Acknowledgements

Lindsey Ouellette Pallavi Singh Katherine Jernigan Rebekah Mosci Samantha Wengert Akanksha Khare Scott Henderson J.D. Lehnert Davis Thomas Aaron Balogh Marion Tseng

Dan Grooms

Steven Rust Paul Bartlett

Cristina Venegas Paul Coussens

Yi-An Yang Bo Norby Julie Funk Robert Britton Linda Mansfield

Chris Elkins, Dave Lacher, Scott Jackson Jim Rudrik, Jim Collins

•  All 3 eae+, stx- were lost; no phage acquisition •  9 of the 15 (60%) animals acquired at least 1 new

STEC 4-8 weeks later –  5 (33%) animals acquired >1 STEC strain type

•  4 of the 8 STEC profiles were maintained among the 15 animals up to 8 weeks –  Are certain strains more likely to persist within a herd?

•  1 novel STEC type (O157) emerged in the animals over time –  What factors drive the emergence of new strain types?