PCR- and Culture-based

Methods for Detection of

STEC-7 in Cattle Feces

Detection Methods for

STEC-7 in Cattle Feces

Real-Time PCR

Traditional

(End-Pont) PCR

Culture

method

Objective 1-STEC-7 Detection: Assays

Develop and validate assays

Traditional PCR 7-plex

STEC-7 serogroups

11-plex STEC-7 + stx1, stx2,

eae, and ehxA

12-plex STE- 8 + stx1, stx2,

eae, and ehxA

Paddock et al., 2012 Bai et al., 2012 Bai et al., 2010

6-plex rfbE, fliCH7, stx1,

stx2, eae, and ehxA

Xiaorong

Shi

Multiplex Real-Time PCR • Three sets of RT-PCR to detect/quantify STEC-7

and three virulence genes (stx1, stx2 and eae):

• Assay 1: O157 (rfbEO157, stx1, stx2 and eae)

• Assay 2: O26, O103, and O111 (wzxO26,

wzxO103, and wzxO111)

• Assay 3: O45, O121, and O145 (wzxO45,

wbqEO121, wbqFO121 and wzxO145)

• Sensitivity of the assays in feces:

– 104 to 105 before enrichment

– 101 to 102 after enrichment in EC broth for 6

hours at 40 C

Culture Method for O157

Fecal sample enriched in Escherichia

coli broth (6 h at 40 C)

IMS with O157 Beads

CT-SMAC Medium

Six sorbitol-negative colonies

Latex agglutination and Indole tests

6-plex PCR (rfbE, fliCH7, stx1, stx2, eae, and ehxA)

Culture Method for Non-O157

Fecal sample enriched in EC broth

(6 h at 40 C)

IMS with O26, O45, O103, O111, O121,

and O145 Beads

Modified Possé Medium

Six chromogenic colonies

(Mauve, Blue, Purple, or Green)

O26 IMS

O45 IMS

O103 IMS

O111 IMS

O121 IMS

O145 IMS

Culture Method for Non-O157

Fecal sample enriched in Escherichia

coli broth (6 h at 40 C)

IMS with O26, O45, O103, O111,

O121, and O145 Beads

Modified Possé Medium

Six chromogenic colonies

7-plex PCR on pooled colonies

(Six PCR/sample)

11-plex PCR on individual colonies

(36 PCR/sample)

Detection Methods for

STEC-7 in Cattle Feces

• Three methods: Traditional

PCR, RT-PCR, and Culture

Method.

• Summer of 2013 (Jun to Aug).

• Total of 576 fecal samples.

Pragathi

Shridhar

Lance

Noll

Diana

Dewsbury

Traditional PCR vs.

Culture Method

Method O26 O45 O103 O111 O121 O145 O157

PCR 256

(44.4)

103

(17.9)

326

(56.6)

5

(0.9)

132

(22.9)

11

(1.9)

315

(54.7)

Culture 131

(22.7)

96

(16.7)

347

(60.2)

1

(0.2)

13

(2.3)

17

(3.0)

247

(42.9)

Fecal samples: n=576

Enriched in EC broth for 6 h at 40 C

O26

Lance Noll

Traditional PCR vs. Culture Method

Sero-

groups

Total

positivesa

% positive of the total

positive

PCR only Culture only

O26 52.3 56.5 15.0

O45 26.6 37.3 32.7

O103 74.7 19.3 24.2

O157 70.8 39.5 22.8 Samples positive by PCR and/or culture method

Lance Noll

PCR vs. Culture Method for E. coli O157

Fecal samples RT-PCR Traditional

PCR

Culture

method

Positive by

RT-PCR (n=100) 100 27 48

Negative by

RT-PCR (n=100) 0 0 21

2011 Summer feedlot study: n=278 fecal samples

Lance Noll

2013 Summer feedlot study: n=576 fecal samples

Fecal samples RT-PCR Traditional

PCR

Culture

method

Positive by

RT-PCR

526

(91%) 410 394

Negative by

RT-PCR

50

(9%) 0 11

Assay 1: O26, O103, and O111

RT-PCR vs. Culture Method for Non-O157 E. coli

Pragathi Shridhar

2013 Summer feedlot study: n=576 fecal samples

Fecal samples RT-PCR Traditional

PCR

Culture

method

Positive by

RT-PCR

426

(74%) 218 106

Negative by

RT-PCR

150

(26%) 0 9

Assay 2: O45, O121, and O145

RT-PCR vs. Culture Method for Non-O157 E. coli

Pragathi Shridhar

Specificity of IMS Beads?

Sero-

groups

No. of samples positive with IMS beads (%)

O26 O45 O103 O111 O121 O145

O26 98 (17.0)

18 (3.1)

20 (0.9)

22 (3.8)

22 (3.8)

12 (2.1)

O103 128

(22.2)

118

(20.5)

216

(37.5)

99

(17.2)

117

(20.3)

110

(19.1)

2013 Summer feedlot study: n=576 fecal samples

Lance Noll

Specificity of IMS Beads

2013 Summer feedlot study: n=576 fecal samples

Serogroups

No. of samples positive with IMS

beads (%)

O26 O103 Other

beads Total

O26 98 (17.0)

33 (5.7)

131 (22.7)

O103 216

(37.5)

131

(22.7)

347

(60.2)

Lance Noll

Can IMS beads be pooled?

• Individual vs. Pooled beads?

Method No. of serogroups

1 2 3 4 5

PCR 29.3 26.7 26.2 14.1 3.9

Culture 45.3 37.0 13.7 3.1 0.8

Fecal samples: n=576

PCR positive: n=484

Culture positive: n=481

Lance Noll

Can IMS beads be pooled?

• Individual beads vs. pool of 3, 4, or 6 beads

• Feces spiked with pure cultures or fecal

samples from feedlot and dairy cattle.

IMS beads O26 O45 O111 O103 O121 O145

Individual 39 11 24 66 4 13

Pooled

O26+O45+O111 46 10 25

O103+O121+O145 66 7 8

Fecal samples: n=96

Cody Baumgartner

Summary • Traditional PCR: Major application is in

the identification of putative STEC isolates.

• Culture method: Requires further refinement

– Specificity of the beads?

– Pooling of beads?

• Real-time PCR: Most sensitive method for detection. However, there are PCR-negative samples that are positive by culture method.

Acknowledgement • Collaborators

– Dr. Jianfa Bai – Dr. Natalia Cernicchiaro – Dr. Dave Renter

• Graduate Students – Charley Cull – Diana Dewsbury – Lance Noll – Zac Paddock – Pragathi Shridhar

• Lab technicians – Xiaorong Shi – Neil Wallace

• Interns – Cody Baumgartner – David Hanks – Rachel Henderson – Melisa Juby – Diane Larson – Shelby Miller – Joy Maus – Chris Siepker – Emily Sloan – Megan Spencer

Acknowledgement

This project was supported by an Agriculture

and Food Research Initiative Competitive

Grant no. 2012-68003-30155 from the USDA

National Institute of Food and Agriculture.