Comparison of Detection Methods of non-O157 STEC€¦ · Comparison of Detection Methods of non-O157 STEC Mick Bosilevac, PhD USDA -Agricultural Research Service U.S. Meat Animal

Comparison of Detection Methods

of non-O157 STEC

Comparison of Detection Methods

of non-O157 STEC

Mick Bosilevac, PhDMick Bosilevac, PhDMick Bosilevac, PhD

USDA - Agricultural Research Service

U.S. Meat Animal Research Center

Meat Safety and Quality Research Unit

Clay Center, Nebraska

Mick Bosilevac, PhD

USDA - Agricultural Research Service

U.S. Meat Animal Research Center

Meat Safety and Quality Research Unit

Clay Center, Nebraska

USDA is an equal opportunity provider and employer

Use of product names by USDA implies no approval to the exclusion of others that may also be suitable

17 June, 2014

Shiga Toxin ProducingShiga Toxin Producing

Escherichia coliEscherichia coli(O157:H7 and non(O157:H7 and non--O157)O157)

•• Shiga toxin producing Shiga toxin producing E. coliE. coli live in the intestines of ruminant animals live in the intestines of ruminant animals

such as cattle, goats, and sheep.such as cattle, goats, and sheep.

•• These These E. coliE. coli generally do not make the animals sick, and other kinds of generally do not make the animals sick, and other kinds of •• These These E. coliE. coli generally do not make the animals sick, and other kinds of generally do not make the animals sick, and other kinds of

animals, like birds and wild pigs can animals, like birds and wild pigs can spread spread the the E. coliE. coli through the through the

environment to things such as environment to things such as produce and water sources.produce and water sources.

•• The major source for human illnesses are attributed to cattle.The major source for human illnesses are attributed to cattle.

•• An infected person may have severe stomach cramps, fever, vomiting, An infected person may have severe stomach cramps, fever, vomiting,

and bloody diarrhea. Most people and bloody diarrhea. Most people recover within recover within 5 to 7 days. However 5 to 7 days. However

some infections can become severe and lifesome infections can become severe and life--threatening. threatening.

•• YYoung oung children and the elderly are more likely to develop severe illness children and the elderly are more likely to develop severe illness

and hemolytic uremic syndrome (HUS) than others, but even healthy and hemolytic uremic syndrome (HUS) than others, but even healthy

young young adults can become seriously ill.adults can become seriously ill.



•• The CDC estimates that there are approximately 176,000 foodborne The CDC estimates that there are approximately 176,000 foodborne

illnesses associated with STEC annually in the U.S.illnesses associated with STEC annually in the U.S.

•• E. coli E. coli O157:H7 is responsible for approximately 63,000 (36%) of the O157:H7 is responsible for approximately 63,000 (36%) of the



•• E. coli E. coli O157:H7 is responsible for approximately 63,000 (36%) of the O157:H7 is responsible for approximately 63,000 (36%) of the

foodborne STEC illnesses. foodborne STEC illnesses.

•• The remainder of the illnesses associated with STEC (113,000 or 64%) are The remainder of the illnesses associated with STEC (113,000 or 64%) are

caused by noncaused by non--O157 STEC. O157 STEC.

•• 70 to 80 percent of confirmed non70 to 80 percent of confirmed non--O157 STEC illnesses are caused by six O157 STEC illnesses are caused by six

STEC STEC serogroupsserogroups –– O26, O45, O103, O111, O121, and O145.O26, O45, O103, O111, O121, and O145.

•• These illnesses can be as sever to those caused by These illnesses can be as sever to those caused by E. coli E. coli O157:H7.O157:H7.

•• In the U.S, at least one outbreak and several sporadic illnesses from nonIn the U.S, at least one outbreak and several sporadic illnesses from non--

O157 STEC O157 STEC serogroupsserogroups have been associated with ground beef products. have been associated with ground beef products.

•• Because of the public health concern regarding the nonBecause of the public health concern regarding the non--O157 STEC O157 STEC

serogroupsserogroups, in 2011, FSIS announced , in 2011, FSIS announced its its intent to declare intent to declare the six the six nonnon--



serogroupsserogroups, in 2011, FSIS announced , in 2011, FSIS announced its its intent to declare intent to declare the six the six nonnon--

O157 STECs (O26, O45, O103, O111, O121, and O145) adulterants in O157 STECs (O26, O45, O103, O111, O121, and O145) adulterants in

nonnon--intact raw beef products and product intact raw beef products and product components. components.

•• On On June 4, 2012, FSIS began testing beef June 4, 2012, FSIS began testing beef trimmings for trimmings for the six nonthe six non--

O157 STEC in addition to O157 STEC in addition to E. coli E. coli O157:H7O157:H7..

•• At that time there were no commercial methods for nonAt that time there were no commercial methods for non--O157 STEC O157 STEC

that had been AOAC approved, so the FSIS granted Letters of No that had been AOAC approved, so the FSIS granted Letters of No

Objection Objection to to testing methods on a casetesting methods on a case--byby--case case basis basis so beef so beef

processors had supporting processors had supporting documentation regarding the reliability of documentation regarding the reliability of

nonnon--O157 STEC O157 STEC verification verification testing results.testing results.

NoNo--Objection Letters Issued for Objection Letters Issued for

NonNon--O157 STEC Test O157 STEC Test MethodsMethods

Log NumberLog Number Company NameCompany Name Method NameMethod Name

1212--SMPSMP--08480848--NN--AA Biocontrol SystemsBiocontrol Systems Assurance GDS Top 7 STEC (eae) methodAssurance GDS Top 7 STEC (eae) method

1212--SMPSMP--08490849--NN--AA BiocontrolBiocontrol SystemsSystems Assurance GDS MPX Top 7 STEC methodAssurance GDS MPX Top 7 STEC method

1212--SMPSMP--08500850--NN--AA IEH LaboratoriesIEH LaboratoriesIEH NonIEH Non--O157 STEC detection and O157 STEC detection and

identification methodidentification method

http://www.fsis.usda.gov/wps/portal/fsis/topics/regulatory-compliance/new-technologies/summary-table-of-nols-non-o157-stec-test-methods

1212--SMPSMP--08500850--NN--AA IEH LaboratoriesIEH Laboratoriesidentification methodidentification method

1212--SMPSMP--08540854--NN--AA Life TechnologiesLife TechnologiesRapidFinderRapidFinder STEC STEC Screening and Screening and

Confirmation Assays for Beef ProductsConfirmation Assays for Beef Products

1212--SMPSMP--08550855--NN--AA Neogen CorporationNeogen CorporationNeoSeekNeoSeek Approach Approach to STEC Detection to STEC Detection

Identification Identification (confirmatory test)(confirmatory test)

1212--SMPSMP--08580858--NN--AA BioBio--Rad LaboratoriesRad LaboratoriesiQiQ CheckCheck VirXVirX andand

iQiQ CheckCheck SerOSerO STEC test methodsSTEC test methods

1212--SMPSMP--08600860--NN--AA DuPont QualiconDuPont Qualicon BAX® System RealBAX® System Real--Time PCR STEC SuiteTime PCR STEC Suite

1212--SMPSMP--09140914--NN--AA Vivione Biosciences, Inc.Vivione Biosciences, Inc. RapidRapid--B nonB non--0157 STEC test kit0157 STEC test kit

1212--SMPSMP--09260926--NN--AA Pall CorporationPall Corporation GeneDiscGeneDisc® Top 6 STEC test kit® Top 6 STEC test kit

Foodborne Pathogen Test Kits Validated by Foodborne Pathogen Test Kits Validated by Independent OrganizationsIndependent Organizations

Method NameMethod Name ManufacturerManufacturer External ValidationExternal Validation

GeneDiscGeneDisc STECSTEC Pall Pall GeneDiscGeneDisc TechnologiesTechnologies AOACAOAC--RI # 021103RI # 021103

GeneDiscGeneDisc STEC Top 6STEC Top 6 Pall Pall GeneDiscGeneDisc TechnologiesTechnologies AOACAOAC--RI # 021106RI # 021106

iQiQ--Check™ STEC Check™ STEC VirXVirX andandiQiQ--Check™ STEC Check™ STEC SerOSerO

BioBio--Rad LaboratoriesRad Laboratories AOACAOAC--RI # 121203RI # 121203

GeneDiscGeneDisc STEC & STEC & SalmonellaSalmonella Pall Pall GeneDiscGeneDisc TechnologiesTechnologies AOACAOAC--RI # 021105RI # 021105

http://www.fsis.usda.gov/wps/wcm/connect/909c8279-6865-424d-ab7a-e1f165646c63/Validated-Test-Kit-Spreadsheet.xls?MOD=AJPERES

GeneDiscGeneDisc STEC & STEC & SalmonellaSalmonella Pall Pall GeneDiscGeneDisc TechnologiesTechnologies AOACAOAC--RI # 021105RI # 021105

IEHIEH E. coliE. coli O157 and STEC with O157 and STEC with IntiminIntimin Test SystemTest System

IEH LaboratoriesIEH Laboratories AOACAOAC--RI # 100701RI # 100701

BAX® System RealBAX® System Real--Time PCR Suite Time PCR Suite for detecting nonfor detecting non--O157:H7 STECO157:H7 STEC

DuPont Nutrition & Health DuPont Nutrition & Health DiagnosticsDiagnostics

AOACAOAC--RI # 091301RI # 091301

Assurance GDS MPX Top 7 STECAssurance GDS MPX Top 7 STEC BioControlBioControl Systems Systems AOACAOAC--RI # 071301RI # 071301

Atlas STEC EG2 Combo Detection Atlas STEC EG2 Combo Detection AssayAssay

Roka Bioscience, Roka Bioscience, IncInc AOACAOAC--RI # 011402RI # 011402

GeneDiscGeneDisc Plate STEC Top 7Plate STEC Top 7 Pall Pall GeneDiscGeneDisc TechnologiesTechnologies AOACAOAC--RI # 031401RI # 031401

Genes for Shiga toxins Genes for Shiga toxins

((stxstx), intimin (), intimin (eaeeae), and O), and O--

group (group (wzxwzx, , wzywzy) are easy ) are easy

Genes for Shiga toxins Genes for Shiga toxins

((stxstx), intimin (), intimin (eaeeae), and O), and O--


Detection methods for non-O157 STECDetection methods for non-O157 STEC

The FSIS MLG method targets stx, eae and O-group (wzx, wzy) genes using PCRThe FSIS MLG method targets stx, eae and O-group (wzx, wzy) genes using PCR

STEC: Targets for detectionSTEC: Targets for detectionSTEC: Targets for detectionSTEC: Targets for detection

"Escherichia coli (E. coli) with "Escherichia coli (E. coli) with PiliPili and Flagella"and Flagella"MrMr AngAng Li. National University of Singapore (Singapore) Li. National University of Singapore (Singapore) "Escherichia coli (E. coli) with "Escherichia coli (E. coli) with PiliPili and Flagella"and Flagella"MrMr AngAng Li. National University of Singapore (Singapore) Li. National University of Singapore (Singapore)


targets for PCR detection targets for PCR detection

that can indicate the that can indicate the

presence of an EHEC.presence of an EHEC.


targets for PCR detection targets for PCR detection

that can indicate the that can indicate the

presence of an EHEC.presence of an EHEC.

PCR = stx+, eae+, O26+PCR = stx+, eae+, O26+

oor these targets can indicate r these targets can indicate

the presence of two or three the presence of two or three

separate separate E. coli E. coli thatthat are not are not

adulterant EHEC,adulterant EHEC,

oor these targets can indicate r these targets can indicate

the presence of two or three the presence of two or three

separate separate E. coli E. coli thatthat are not are not

adulterant EHEC,adulterant EHEC,




"Escherichia coli (E. coli) with "Escherichia coli (E. coli) with PiliPili and Flagella"and Flagella"MrMr AngAng Li. National University of Singapore (Singapore) Li. National University of Singapore (Singapore) "Escherichia coli (E. coli) with "Escherichia coli (E. coli) with PiliPili and Flagella"and Flagella"MrMr AngAng Li. National University of Singapore (Singapore) Li. National University of Singapore (Singapore)

adulterant EHEC,adulterant EHEC,adulterant EHEC,adulterant EHEC,

because PCR cannot because PCR cannot

discriminate cells that discriminate cells that

contain all three targets contain all three targets

from cells that do not.from cells that do not.

because PCR cannot because PCR cannot

discriminate cells that discriminate cells that

contain all three targets contain all three targets

from cells that do not.from cells that do not.

PCR = stx+, eae+, O26+PCR = stx+, eae+, O26+




“Potential positive” = stx + eae + any Top6 O-group“Potential positive” = stx + eae + any Top6 O-group

stx1/stx2+stx1/stx2+stx1/stx2+

eae+

n=815

eae+

n=815

eae+

n=815

n=232n=232

n=261n=261n=437n=437

n=232

n=261n=437

****** ***** *




n = 3,972n = 3,972n = 3,972

stx1/stx2+

n=959

stx1/stx2+

n=959

stx1/stx2+

n=959

n=815n=815n=815

O-group+

n=753

O-group+

n=753

O-group+

n=753

n=107n=107n=139n=139

n=183n=183

n=324n=324

n=107n=139

n=183

n=324

n=415n=415

= Top6 EHEC isolate= Top6 EHEC isolate

** **

****

* * = Top6 EHEC isolate

* *

**

*

Detection MethodsDetection Methods

Commercial KitsCommercial Kits

-- BAXBAX

-- BioControlBioControl GDSGDS

-- Pall Pall GeneDiscGeneDisc

STEC Detection Methods ISTEC Detection Methods I

Referral LabReferral Lab

-- IEHIEH

-- NeoSeekNeoSeek

Official MethodsOfficial Methods

-- FSIS MLGFSIS MLG

-- US MARC US MARC confirmationconfirmation

•• Comparison using inoculated materialsComparison using inoculated materials

-- Individual Methods as per manufacturerIndividual Methods as per manufacturer

-- One enrichment tested by all One enrichment tested by all methodsmethods

•• Comparison using 500 Comparison using 500 “natural” “natural” enrichmentsenrichments

Objective: To identify strengths and weaknesses of commercial Shiga toxin-producing E coli detection methods and kits in a side by side fashion.

– FSIS MLG 5B STEC Detection

– DuPont Qualicon BAX® System STEC Suite


– DuPont Qualicon BAX® System STEC Suite

– BioControl Systems Assurance GDS Top STEC

– Pall GeneDisc System for non-O157 STEC

– IEH Laboratories

– Neogen NeoSEEK

– US Meat Animal Research Center STEC detection and isolation protocols

Three part evaluationThree part evaluation•• Two inoculation studiesTwo inoculation studies

1.1. Each method/system used according to its protocol Each method/system used according to its protocol

(medias, incubation temperatures & times, material)(medias, incubation temperatures & times, material)


(medias, incubation temperatures & times, material)(medias, incubation temperatures & times, material)

2.2. Each method/system used to test the same TSB Each method/system used to test the same TSB

enrichments (ground beef varying weight/volume/time)enrichments (ground beef varying weight/volume/time)

•• One set of “natural” samplesOne set of “natural” samples

3.3. Each method/system used to on 500 enrichments from a Each method/system used to on 500 enrichments from a

regional service labregional service lab

Part Part 1 1 -- Inoculation Inoculation studiesstudies

Each method/system used according to recommended guidelines. Either package Each method/system used according to recommended guidelines. Either package

insert, AOAC validation document, or personal communication with manufacturer.insert, AOAC validation document, or personal communication with manufacturer.

Three of each Top6 EHEC used (in samples <200g), or Two of each Top6 EHEC used (in Three of each Top6 EHEC used (in samples <200g), or Two of each Top6 EHEC used (in

samples >200g) all inoculated at low CFU/sample, plus one negative (nonsamples >200g) all inoculated at low CFU/sample, plus one negative (non--inoculated) inoculated)

for each set examined.for each set examined.


OO stxstx eaeeae Ave CFUAve CFU

2626 11 ++ 2.22.2

4545 11 ++ 2.22.2

103103 11 ++ 3.03.0

111111 1,21,2 ++ 1.81.8

121121 22 ++ 3.03.0

145145 1,21,2 ++ 5.25.2

All enrichments confirmed to be inoculated by Culture Isolation. Results are presented All enrichments confirmed to be inoculated by Culture Isolation. Results are presented

as number of Culture Isolation results (positive and negative) properly identified.as number of Culture Isolation results (positive and negative) properly identified.

MediaMedia

BPWBPW

mTSBmTSB

mTBSnovomTBSnovo88

mTSBnovomTSBnovo22

mEHECmEHEC

prewarmedprewarmed to to

42C or 46C42C or 46C

IncubationIncubation

temperature & timetemperature & time

41C or 42C41C or 42C

9h to 20h9h to 20h

MaterialsMaterials

Trim (TRM)Trim (TRM)

150g, 375g150g, 375g

Ground Beef (GB) Ground Beef (GB)

25g, 325g25g, 325g

SampleSample Medium (42C)Medium (42C) TimeTime TempTemp

MLGMLG 375g TRM375g TRM 1L mTSBnovo1L mTSBnovo88 18h18h 42C42C

BAXBAX 325g GB325g GB 1L 1L mTSBnovomTSBnovo22 12h12h 41C41C

(46C)(46C) 20h20h 41C41C

GDSGDS 325g GB325g GB 1L 1L mEHECmEHEC 10h10h 42C42C

Results correctResults correct

nn No.No. %%

1313 1010 7777

88 77 8888

1313 1212 9292

1313 1313 100100

Part Part 1 1 -- Inoculation Inoculation studiesstudiesSTEC Detection Methods ISTEC Detection Methods I

GDSGDS 325g GB325g GB 1L 1L mEHECmEHEC 10h10h 42C42C

18h18h 42C42C

375g TRM375g TRM 1L 1L mEHECmEHEC 10h10h 42C42C

18h18h 42C42C

PallPall 25g GB25g GB 225mL BPW225mL BPW 18h18h 42C42C

375g TRM375g TRM 1.5L BPW1.5L BPW 18h18h 42C42C

IEHIEH 150g TRM150g TRM 150mL IEH150mL IEH 9h9h 42C42C

NeoSEEKNeoSEEK 325g GB325g GB 1L 1L mTSBmTSB 16h16h 42C42C

1313 1313 100100

1313 55 3838

1313 1313 100100

1313 1313 100100

1919 1818 9595

1313 1313 100100

1919 1919 100100

1313 1313 100100

Each method/system used to detect Top6 EHEC from same ground beef enrichment.Each method/system used to detect Top6 EHEC from same ground beef enrichment.Enrichments varied in sample size, volume of Enrichments varied in sample size, volume of mTSBmTSB used and time enriched at 42C.used and time enriched at 42C.

Three of each Top6 EHEC used at low CFU/sample, plus one negative (nonThree of each Top6 EHEC used at low CFU/sample, plus one negative (non--inoculated) for each set examined. Repeated using two of each in 325g samples due inoculated) for each set examined. Repeated using two of each in 325g samples due to FedEx shipping failure.to FedEx shipping failure.

A group of 34 challenge A group of 34 challenge E. E. coli previously isolated from ground beef (noncoli previously isolated from ground beef (non--Top6 Top6


SampleSample Medium (42C)Medium (42C) TimeTime TempTemp

25g GB25g GB 225mL 225mL mTSBmTSB 8h8h 42C42C

65g GB65g GB 585mL 585mL mTSBmTSB 12h12h 42C42C

325g GB325g GB 1L mTSB1L mTSB 16h16h 42C42C

A group of 34 challenge A group of 34 challenge E. E. coli previously isolated from ground beef (noncoli previously isolated from ground beef (non--Top6 Top6 EHEC/Top6 nonEHEC/Top6 non--EHEC/other) inoculated at higher CFU into 25g sample conditions.EHEC/other) inoculated at higher CFU into 25g sample conditions.

All enrichments confirmed to be inoculated by Culture Isolation and comparison All enrichments confirmed to be inoculated by Culture Isolation and comparison back to the initial strain that was used. Results are presented as number of Culture back to the initial strain that was used. Results are presented as number of Culture Isolation results properly identified.Isolation results properly identified.

OO stxstx eaeeae CFUCFU OO stxstx eaeeae CFUCFU

1515 22 ++ 4040 121121 11 -- 3030

2626 -- ++ 180180 121121 11 -- 3030

4545 -- ++ 4040 121121 22 -- 2222

5555 -- ++ 1414 121121 22 -- 1616

OO stxstx eaeeae Ave CFUAve CFU

2626 11 ++ 2.12.1

4545 11 ++ 1.91.9

103103 11 ++ 2.52.5

111111 1,21,2 ++ 2.82.8

Isolates used in second round of inoculation studies.Isolates used in second round of inoculation studies.

Top6 EHECTop6 EHEC Challenge strainsChallenge strains


5555 -- ++ 1414 121121 22 -- 1616

8484 11 ++ 2323 153153 11 ++ 8080

9191 11 -- 8080 157157 1,21,2 ++ 77

9191 22 -- 150150 163163 1,21,2 -- 3636

9191 22 -- 2929 172172 22 ++ 2020

9191 22 -- 2525 174174 22 -- 2727

9191 1,21,2 -- 2121 untunt 22 -- 3030

9191 1,21,2 -- 3333 untunt 22 -- 100100

9898 11 ++ 5050 untunt 22 -- 200200

103103 -- ++ 6060 untunt 22 -- 200200

103103 -- ++ 140140 untunt 22 ++ 2626

113113 22 -- 180180 untunt -- ++ 3131

113113 22 -- 4141 untunt 1,21,2 -- 2020

113113 1,21,2 -- 2121 untunt -- -- 150150

111111 1,21,2 ++ 2.82.8

121121 22 ++ 3.53.5

145145 1,21,2 ++ 2.92.9

Top6 EHECTop6 EHEC

Sample SetSample Set nn MLGMLG BAXBAX GDSGDS PallPall IEHIEH NeoSEEKNeoSEEK

25g GB25g GB 1919

65g GB65g GB 1919

325g GB325g GB 1919

11 (58%)11 (58%)

17 (89%)17 (89%)

14 (74%)14 (74%)

19 (100%)19 (100%)

19 (100%)19 (100%)

14 (74%)14 (74%)

18 (95%)18 (95%)

18 (95%)18 (95%)

16 (84%)16 (84%)

19 (100%)19 (100%)

19 (100%)19 (100%)

19 (100%)19 (100%)

18 (95%)18 (95%)

18 (95%)18 (95%)

nrnr

19 (100%)19 (100%)

19 (100%)19 (100%)

nrnr


325g GB325g GB 1919

1313

Challenge StrainsChallenge Strains

25g GB25g GB 3535

over all percent over all percent

correct:correct:

14 (74%)14 (74%)

10 (77%)10 (77%)

34 (97%)34 (97%)

8282

14 (74%)14 (74%)

7 (54%)7 (54%)

35 (100%)35 (100%)

9090

16 (84%)16 (84%)

5 (38%)5 (38%)

30 (86%)30 (86%)

8383

19 (100%)19 (100%)

13 (100%)13 (100%)

29 (83%)29 (83%)

9494

nrnr

13 (100%)13 (100%)

35 (100%)35 (100%)

9898

nrnr

13 (100%)13 (100%)

35 (100%)35 (100%)

100100

•• Each method/system used on 500 enrichments Each method/system used on 500 enrichments

collected from a regional service labcollected from a regional service lab

–– beef trim, rapid enrichment media, 42C, 8beef trim, rapid enrichment media, 42C, 8--10h10h

–– 10mL poured off for our use and analyzed (or the DNA 10mL poured off for our use and analyzed (or the DNA

lysis/extract prepared) the same daylysis/extract prepared) the same day

Part Part 3 3 –– Natural SamplesNatural SamplesSTEC Detection Methods ISTEC Detection Methods I

lysis/extract prepared) the same daylysis/extract prepared) the same day

•• Any sample found positive for Any sample found positive for stxstx and and eae eae by one by one

or more methods taken into culture confirmationor more methods taken into culture confirmation

–– One EHECOne EHEC--O26 confirmed in the 500 enrichmentsO26 confirmed in the 500 enrichments

–– Another 73 enrichments contained a mixtures of Another 73 enrichments contained a mixtures of stxstx++

E.E. colicoli, , eaeeae++ E.E. coli coli and and E. coli E. coli of Top6 O groups of Top6 O groups ((stxstx--, , eaeeae--))

nn=135=135

Culture IsolationCulture Isolation

EHECEHECSTECSTEC

((stxstx++))

EPECEPEC

((eaeeae++))

Top6 OTop6 O

((stxstx++,, eaeeae++))

77

Number of Methods Number of Methods

that identified a that identified a

sample assample as

Potential PositivePotential Positive

22 11 11 11 11


77

66

55

44

33

22

11

22

44

66

1414

2121

3636

5252

11 11 11 11

22 22 33

33 33 55

22 66 88

77 33 1313

99 44 77

55 44 33

Number of Natural samples found Reactive and Potential Positive by each Number of Natural samples found Reactive and Potential Positive by each

method with types of method with types of E. coli E. coli of isolated from Potential Positive samples.of isolated from Potential Positive samples.

MLGMLG BAXBAX GDSGDS PallPall IEHIEH NeoSeekNeoSeek USMARCUSMARC

Screen/ReactiveScreen/Reactive

Confirm/Confirm/

4848 8383 3838 8383 3232 103103 585870


Confirm/Confirm/

Potential positivePotential positive

EHECEHEC

STECSTEC

EPECEPEC

Top6 OTop6 O

no isolateno isolate

3333

11

55

1010

1414

66

5252

11

1515

1212

3232

88

77

11

22

33

55

11

6868

11

1717

1515

2525

1515

1414

11

33

55

77

44

77

11

11

22

11

44

2424

11

44

55

1010

1010

44

1

10

10

21

3

Analysis Analysis

software software

improvedimproved

ConclusionsConclusions

•• All methods examined detect Top6 EHEC as well as, or All methods examined detect Top6 EHEC as well as, or

better than the reference MLG methodbetter than the reference MLG method

•• Inoculation studiesInoculation studies

–– Each system should be run as described by its manufacturerEach system should be run as described by its manufacturer


–– Each system should be run as described by its manufacturerEach system should be run as described by its manufacturer

–– Changes to media showed limitations in some methodsChanges to media showed limitations in some methods

•• “Natural” samples“Natural” samples

–– Show agreement on true positivesShow agreement on true positives

–– Considerable disagreement on negative or nonConsiderable disagreement on negative or non--confirmable confirmable

samplessamples

–– The number of Potential Positive samples identified that The number of Potential Positive samples identified that

appear to be “false positive” ranges from 86appear to be “false positive” ranges from 86--98%98%

Intimin subtype : serotypeIntimin subtype : serotypeIntimin subtype : serotypeIntimin subtype : serotype

• Particular serogroups or O-

groups only carry particular

intimin (eae) subtypes.

• Particular serogroups or O-

groups only carry particular

intimin (eae) subtypes.

Improved targets for STEC detectionImproved targets for STEC detectionImproved targets for STEC detectionImproved targets for STEC detection

intimin (eae) subtypes.intimin (eae) subtypes.

"Escherichia coli (E. coli) with "Escherichia coli (E. coli) with PiliPili and Flagella"and Flagella"MrMr AngAng Li. National University of Singapore (Singapore) Li. National University of Singapore (Singapore) "Escherichia coli (E. coli) with "Escherichia coli (E. coli) with PiliPili and Flagella"and Flagella"MrMr AngAng Li. National University of Singapore (Singapore) Li. National University of Singapore (Singapore) "Escherichia coli (E. coli) with "Escherichia coli (E. coli) with PiliPili and Flagella"and Flagella"MrMr AngAng Li. National University of Singapore (Singapore) Li. National University of Singapore (Singapore)

EHEC OEHEC O--groupgroup eaeeae

Ο26Ο26 ββΟ145,Ο145, Ο157Ο157 γγΟ45, Ο103, Ο121Ο45, Ο103, Ο121 εεΟ111Ο111 θ(γθ(γ22))

O Group gene SNP’s : EHECO Group gene SNP’s : EHECO Group gene SNP’s : EHECO Group gene SNP’s : EHEC

•• Sequencing of Sequencing of E. coli E. coli genomes genomes

has identified single nucleotide has identified single nucleotide

polymorphisms (SNP’s) that polymorphisms (SNP’s) that


polymorphisms (SNP’s) that polymorphisms (SNP’s) that

correlate with EHEC lineage.correlate with EHEC lineage.

•• These SNP’s are detected by These SNP’s are detected by

PCR and Mass Spec analysis of PCR and Mass Spec analysis of

the PCR product. the PCR product.

•• Thus EHEC and nonThus EHEC and non--EHEC of the EHEC of the

same Osame O--group can be group can be

distinguished from one another.distinguished from one another.


•• NeoSEEKNeoSEEK analysis uses analysis uses the the

relationship between relationship between eaeeae

subtype and subtype and serogroupserogroup with with


subtype and subtype and serogroupserogroup with with

EHEC OEHEC O--group SNP to provide a group SNP to provide a

“molecular culture confirmation”.“molecular culture confirmation”.

•• Example: in a sample with Example: in a sample with stxstx

+ eae+ eae ββ + O26 STEC+ O26 STECSNPSNP = = POSPOS

+ eae+ eae εε + O45 + O45 nonSTECnonSTECSNPSNP = = NEGNEG


Correlating Correlating eaeeae subtype and O groupsubtype and O group

reduces the number of potential positive samples reduces the number of potential positive samples ((stxstx++, , eaeeae++, O group, O group++))

Correlating Correlating eaeeae subtype and O groupsubtype and O group

reduces the number of potential positive samples reduces the number of potential positive samples ((stxstx++, , eaeeae++, O group, O group++))

n =

stx+ =

stx+ eae+ =

n =

stx+ =

stx+ eae+ =

500 (100%)

165 (33.0%)

83 (16.6%)

500 (100%)

165 (33.0%)

83 (16.6%)

500 (100%)

197 (39.4%)

103 (20.6%)

500 (100%)

197 (39.4%)

103 (20.6%)stx eae =

stx+ eae+ O group+ =

stx+ eae subtype:O group =

stx eae =

stx+ eae+ O group+ =

stx+ eae subtype:O group =

83 (16.6%)

67 (13.4%)

83 (16.6%)

67 (13.4%)

103 (20.6%)

76 (15.2%)

103 (20.6%)

76 (15.2%)

O group subdivisions =O group subdivisions = 7 (1.4%)7 (1.4%)

31 (6.2%)31 (6.2%) 49 (9.8%)49 (9.8%)

The natural sample containing the

EHEC-O26 was identified by both methods.

The natural sample containing the

EHEC-O26 was identified by both methods.

Evolution of Enterohemorrhagic

Escherichia coli Hemolysin Plasmids and

the Locus for Enterocyte Effacement in

Shiga Toxin-Producing E. coli










Boerlin et al., I&I 66:2553Boerlin et al., I&I 66:2553Boerlin et al., I&I 66:2553

Natural samples from across the U.S.Natural samples from across the U.S.

400 ground beef and 150 beef trim enrichments400 ground beef and 150 beef trim enrichments

FSIS MLGFSIS MLG

BAXBAX

BioradBiorad

Pall Pall GeneDiscGeneDisc

325g samples enriched in 1L mTSB+novo325g samples enriched in 1L mTSB+novo88..

STEC Detection Methods IISTEC Detection Methods II

Pall Pall GeneDiscGeneDisc

NeoSeekNeoSeek

RokaRoka

2 EHEC 2 EHEC (O26 & O157(O26 & O157) ) 5 EPEC 5 EPEC (eae(eae++, stx, stx--))

10 STEC10 STEC 3 3 EE. coli . coli of Top6 O groupof Top6 O group

All samples positive for All samples positive for eaeeae and and stxstx by one or more methods by one or more methods

((nn=27) were subjected to thorough rounds of culture =27) were subjected to thorough rounds of culture

isolation that identified in 14 of the samples:isolation that identified in 14 of the samples:

Number of Number of 400 ground beef and 150 beef 400 ground beef and 150 beef trim samples found Reactive and Potential Positive trim samples found Reactive and Potential Positive

by each method with types of by each method with types of E. coli E. coli of isolated from Potential Positive samples.of isolated from Potential Positive samples.

MLGMLG BAXBAX BioBio--RadRad PallPall NeoSeekNeoSeek RokaRoka


Confirm/Confirm/

99 1616 1313 1212

(9)(9)

44 1212

STEC Detection Methods IISTEC Detection Methods II

Confirm/Confirm/


EHEC (2)EHEC (2)

STEC (10)STEC (10)

EPECEPEC (5)(5)

Top6 Top6 O (3)O (3)


77

11

44

22

11

22

66

22

55

11

22

00

44

22

44

00

22

00

(9)(9)

22

22

22

00

11

00

44

22

33

00

11

11

1212

22

77

33

11

66

Analyses Analyses of FSIS of FSIS Beef BrothsBeef Broths

FSIS has FSIS has been providing ARS enrichment broths from their been providing ARS enrichment broths from their

Microbiological Testing Program for Escherichia coli O157:H7 Microbiological Testing Program for Escherichia coli O157:H7

and nonand non--O157 Shiga toxinO157 Shiga toxin--producing Escherichia coli (STECproducing Escherichia coli (STEC))

-- O157:H7 screen positiveO157:H7 screen positive

-- STEC screen positive (STEC screen positive (stxstx+/+/eaeeae+) O+) O--groupgroup--

STEC Detection Methods IIISTEC Detection Methods III

-- STEC screen positive (STEC screen positive (stxstx+/+/eaeeae+) O+) O--groupgroup--

-- STEC screen negative STEC screen negative ((stxstx+/+/eaeeae--) ) ((stxstx--//eaeeae+)+)

-- Matched negative broth for each of the aboveMatched negative broth for each of the above

Assigned an identifier & data recorded

Aliquoted (x8)

Various DNA lysis prepared

365 have been fully processed

Each FSIS broth is tested with BAXEach FSIS broth is tested with BAX--O157 O157

screen + STEC screen/confirmation.screen + STEC screen/confirmation.

Other STEC detection methods are also Other STEC detection methods are also

performed for comparison.performed for comparison.

BAXBAX--STEC STEC



performed for comparison.performed for comparison.

GDS Top7 STEC, GDS Top7 STEC, GeneDiscGeneDisc STEC,STEC,

BioRadBioRad VirXVirX, etc…, etc…

Any FSIS broths that are Potential Positive Any FSIS broths that are Potential Positive

go forward to culture isolation.go forward to culture isolation.

Suspect colonies are screened and Suspect colonies are screened and

isolates are serotyped and characterized isolates are serotyped and characterized

for virulence factors.for virulence factors.

IMS &IMS &

CultureCulture

isolation isolation

Other STEC Other STEC

detection detection

methodsmethods



==365 FSIS Broths processed.365 FSIS Broths processed.

MLG 5B is now equivalent to the BAX real time STEC suiteMLG 5B is now equivalent to the BAX real time STEC suiteMLG 5B is now equivalent to the BAX real time STEC suiteMLG 5B is now equivalent to the BAX real time STEC suite

BioBio--Rad Rad iQiQ checkcheck NeogenNeogen ANSR ANSR LifeTechnologiesLifeTechnologies RapidFinderRapidFinder

GenediscGenedisc SystemSystem BioControlBioControl GDSGDS Roka ATLASRoka ATLAS



•• 365 FSIS Broths processed.365 FSIS Broths processed.

•• 161 found reactive (161 found reactive (stxstx++//eaeeae++) by one or more methods.) by one or more methods.

•• 144 identified as Potential Positive by one or more methods.144 identified as Potential Positive by one or more methods.

•• Culture isolation has identified 78 samples that contain: EHEC, Culture isolation has identified 78 samples that contain: EHEC,

STEC, EPEC and STEC, EPEC and E. coli E. coli of target of target serogroupsserogroups..STEC, EPEC and STEC, EPEC and E. coli E. coli of target of target serogroupsserogroups..

–– 28 contain EHEC28 contain EHEC--O157:H7O157:H7

–– 1 contains EHEC1 contains EHEC--O157:H7 and EHECO157:H7 and EHEC--O45O45

–– 3 contain EHEC3 contain EHEC--O103O103

–– 1 contains EHEC1 contains EHEC--O26O26

–– 2 contain EHEC of other 2 contain EHEC of other serogroupsserogroups (O5 and O74)(O5 and O74)

–– The remainder of the samples (n=43) contain: 27 STEC, 12 EPEC and The remainder of the samples (n=43) contain: 27 STEC, 12 EPEC and

32 32 E. coli E. coli of target of target serogroupsserogroups..

BAXBAX BioBio--RadRad ANSRANSR LifeTechLifeTech GDSGDS PallPall RokaRoka


Confirm/Confirm/

123123 7979 8181 103103 100100

6767

9191



Confirm/Confirm/


EHEC (35,EHEC (35,29 O157/7 non29 O157/7 non--O157O157))

STEC (27)STEC (27)

EPECEPEC (12)(12)

Top6 Top6 O (32)O (32)


100100

20/520/5

2424

88

2828

3939

ndnd

19/319/3

2222

66

2222

3030

3434

11/111/1

77

22

66

1111

7272

25/425/4

1919

88

1616

2222

4747

17/317/3

1111

77

1313

1313

6767

19/319/3

1010

77

1717

2727

8383

22/522/5

2020

99

2626

2828

•• Continued sideContinued side--byby--side comparison of STEC detection methods side comparison of STEC detection methods

does not result in a clear winner.does not result in a clear winner.

•• Methods that rely solely on the presence of Methods that rely solely on the presence of stxstx, , eaeeae and Oand O--

group identify an unacceptable number of Potential Positive group identify an unacceptable number of Potential Positive

samples.samples.

STEC Detection Methods II & IIISTEC Detection Methods II & IIIConclusionsConclusions

samples.samples.

•• Of concern is the large number of False Negatives observed in Of concern is the large number of False Negatives observed in

the comparison performed within the FSIS broths. the comparison performed within the FSIS broths.

-- No method managed to identify all 5 TopNo method managed to identify all 5 Top--6 EHEC samples6 EHEC samples..

•• Methods that use multiple targets or targets uniquely Methods that use multiple targets or targets uniquely

characteristic of EHEC may offer the best option, but more characteristic of EHEC may offer the best option, but more

reliable targets reliable targets are needed. As are methods that can more are needed. As are methods that can more

accurately detect the cells of interest.accurately detect the cells of interest.

•• Sequencing the genome of Sequencing the genome of EE. coli . coli

O157:H7 O157:H7 identified genomic identified genomic

islands unique to islands unique to E. coli E. coli O157:H7 O157:H7

compared to compared to E. coli E. coli K12.K12.

•• Termed OTermed O--islands (OI) these islands (OI) these

portions of the genome turned portions of the genome turned

out to posses a number of out to posses a number of

STEC: Improved targets for detectionSTEC: Improved targets for detectionSTEC: Improved targets for detectionSTEC: Improved targets for detection

Nature 409, 529-533(25 January 2001)Nature 409, 529-533(25 January 2001)

Outer circle shows the distribution of islands.

Blue: shared co-linear backbone.

Red: position of EDL933-specific sequences (O-islands).

Green: MG1655-specific sequences (K-islands).

Tan: O-islands and K-islands at the same locations in the backbone.

Purple: Hypervariable regions.

Outer circle shows the distribution of islands.

Blue: shared co-linear backbone.

Red: position of EDL933-specific sequences (O-islands).

Green: MG1655-specific sequences (K-islands).

Tan: O-islands and K-islands at the same locations in the backbone.

Purple: Hypervariable regions.

out to posses a number of out to posses a number of

virulence related genes.virulence related genes.

•• OIOI--57, OI57, OI--71, and OI71, and OI--122 122 in in

particular contain genes that particular contain genes that

correlate with EHEC.correlate with EHEC.-- nlenle genes; genes; nleBnleB, , nleFnleF, etc…, etc…

-- Z2098, Z2099Z2098, Z2099

-- espKespK

Prevalence of alternative targets for detection of Prevalence of alternative targets for detection of

EHEC in enrichments with and without EHEC in enrichments with and without stxstx

Prevalence of alternative targets for detection of Prevalence of alternative targets for detection of

EHEC in enrichments with and without EHEC in enrichments with and without stxstx

marker

% prevalence in enrichment

All samples(n=253)

STEC(stx pos)

no-STEC(stx neg)

stx1 51.8 60.1 0.0

stx2 81.4 94.5 0.0

chuA 98.0 99.1 91.4

eae 82.6 85.3 65.7

ehx 79.4 85.3 42.9

•• 253 beef carcass samples were 253 beef carcass samples were screened using PCR for screened using PCR for stx1stx1, , stx2stx2, , eaeeae and 14 different and 14 different genes associated with genes associated with increased virulence of EHEC.increased virulence of EHEC.

•• Then prevalence rates in Then prevalence rates in

marker


All samples(n=253)

STEC(stx pos)

no-STEC(stx neg)

stx1 51.8 60.1 0.0

stx2 81.4 94.5 0.0

chuA 98.0 99.1 91.4

eae 82.6 85.3 65.7

ehx 79.4 85.3 42.9

marker


All samples(n=253)

STEC(stx pos)

no-STEC(stx neg)

stx1 51.8

stx2 81.4

chuA 98.0

eae 82.6

ehx 79.4

marker


All samples(n=253)

STEC(stx pos)

no-STEC(stx neg)

stx1stx2

chuAeaeehxehx 79.4 85.3 42.9

esp K1 27.7 29.4 17.1

fyuA 97.6 98.6 91.4

G5-2 13.4 14.7 5.7

Ibe 57.7 60.1 42.9

irp2 97.2 99.1 85.7

nleB 75.1 80.7 40.0

nleC 19.0 21.6 2.9

nleF 50.2 52.8 34.3

pch D 34.8 36.7 22.9

Q 933 18.6 21.6 0.0

subA 31.2 35.8 2.9

tra T 100.0 100.0 100.0

•• Then prevalence rates in Then prevalence rates in enrichments with and without enrichments with and without stx were examined. Some stx were examined. Some markers showed varying markers showed varying degrees of correlation with degrees of correlation with stxstx..

•• All All stxstx++ enrichments were enrichments were cultured and EHEC, STEC and cultured and EHEC, STEC and EPEC were isolated.EPEC were isolated.

ehx 79.4 85.3 42.9

esp K1 27.7 29.4 17.1

fyuA 97.6 98.6 91.4

G5-2 13.4 14.7 5.7

Ibe 57.7 60.1 42.9

irp2 97.2 99.1 85.7

nleB 75.1 80.7 40.0

nleC 19.0 21.6 2.9

nleF 50.2 52.8 34.3

pch D 34.8 36.7 22.9

Q 933 18.6 21.6 0.0

subA 31.2 35.8 2.9

tra T 100.0 100.0 100.0

ehx 79.4

esp K1 27.7

fyuA 97.6

G5-2 13.4

Ibe 57.7

irp2 97.2

nleB 75.1

nleC 19.0

nleF 50.2

pch D 34.8

Q 933 18.6

subA 31.2

tra T 100.0

ehxesp K1fyuAG5-2Ibeirp2nleBnleCnleF

pch DQ 933subAtra T

Number and types of Number and types of E. E. colicoli isolatedisolated

from beef carcass swab enrichments withfrom beef carcass swab enrichments with

increasing increasing number of screening number of screening markersmarkers

Number and types of Number and types of E. E. colicoli isolatedisolated

from beef carcass swab enrichments withfrom beef carcass swab enrichments with

increasing increasing number of screening number of screening markersmarkers

15

20

25

30

Isol

ates

EHEC

STEC

EPEC

n

•• Enrichments Enrichments that contained between 10 and 15 virulence markers yielded increasing proportions of that contained between 10 and 15 virulence markers yielded increasing proportions of

EHEC and EHEC and decreasing proportions of decreasing proportions of STEC.STEC.

EHECSTEC

EPECn

0

5

10

4 5 6 7 8 9 10 11 12 13 14 15 16 17

Isol

ates

Number of virulence markers present in enrichment

Improving on sensitivity for detectionImproving on sensitivity for detection

of nonof non--O157 STEC?O157 STEC?

The latest test methods that have come into

our lab for evaluation.

Recombinant bacteriophage tail

fibers from EHEC specific phage

may offer increased performance in

term of specificity and sensitivity to

concentrate the target EHEC before

placing in a detection assay or

plating for isolation

A flow cytometric approach that

interrogates each individual cell

within an enrichment across

parameters of size, shape, internal

and external immuno- and

molecular targets simultaneously

and in real time.

Comparison of Detection Methods of non-O157 STEC€¦ · Comparison of Detection Methods of non-O157 STEC Mick Bosilevac, PhD USDA -Agricultural Research Service U.S. Meat Animal

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