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.
Shiga Toxin ProducingShiga Toxin Producing
Escherichia coliEscherichia coli(O157:H7 and non(O157:H7 and non--O157)O157)
•• 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
Shiga Toxin ProducingShiga Toxin Producing
Escherichia coliEscherichia coli(O157:H7 and non(O157:H7 and non--O157)O157)
•• 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--
Shiga Toxin ProducingShiga Toxin Producing
Escherichia coliEscherichia coli(O157:H7 and non(O157:H7 and non--O157)O157)
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--
group (group (wzxwzx, , wzywzy) are easy ) are easy
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)
group (group (wzxwzx, , wzywzy) are easy ) are easy
targets for PCR detection targets for PCR detection
that can indicate the that can indicate the
presence of an EHEC.presence of an EHEC.
group (group (wzxwzx, , wzywzy) are easy ) are easy
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,
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)
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+
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
“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
****** ***** *
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
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
STEC Detection Methods ISTEC Detection Methods I
– 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)
STEC Detection Methods ISTEC Detection Methods I
(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.
STEC Detection Methods ISTEC Detection Methods I
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
Part Part 2 2 -- Inoculation Inoculation studiesstudiesSTEC Detection Methods ISTEC Detection Methods I
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
Part Part 2 2 -- Inoculation Inoculation studiesstudiesSTEC Detection Methods ISTEC Detection Methods I
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
Part Part 2 2 -- Inoculation Inoculation studiesstudiesSTEC Detection Methods ISTEC Detection Methods I
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
Part Part 3 3 –– Natural SamplesNatural SamplesSTEC Detection Methods ISTEC Detection Methods I
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
Part Part 3 3 –– Natural SamplesNatural SamplesSTEC Detection Methods ISTEC Detection Methods I
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
STEC Detection Methods ISTEC Detection Methods I
–– 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
Improved targets for STEC detectionImproved targets for STEC detectionImproved targets for STEC detectionImproved targets for STEC detection
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.
"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)
•• NeoSEEKNeoSEEK analysis uses analysis uses the the
relationship between relationship between eaeeae
subtype and subtype and serogroupserogroup with with
Improved targets for STEC detectionImproved targets for STEC detectionImproved targets for STEC detectionImproved targets for STEC detection
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
"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)
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
Evolution of Enterohemorrhagic
Escherichia coli Hemolysin Plasmids and
the Locus for Enterocyte Effacement in
Shiga Toxin-Producing E. coli
Evolution of Enterohemorrhagic
Escherichia coli Hemolysin Plasmids and
the Locus for Enterocyte Effacement in
Shiga Toxin-Producing E. coli
Improved targets for STEC detectionImproved targets for STEC detectionImproved targets for STEC detectionImproved targets for STEC detection
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
Screen/ReactiveScreen/Reactive
Confirm/Confirm/
99 1616 1313 1212
(9)(9)
44 1212
STEC Detection Methods IISTEC Detection Methods II
Confirm/Confirm/
Potential positivePotential positive
EHEC (2)EHEC (2)
STEC (10)STEC (10)
EPECEPEC (5)(5)
Top6 Top6 O (3)O (3)
no isolateno isolate
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
Analyses Analyses of FSIS of FSIS Beef BrothsBeef Broths
STEC Detection Methods IIISTEC Detection Methods III
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
Analyses Analyses of FSIS of FSIS Beef BrothsBeef Broths
STEC Detection Methods IIISTEC Detection Methods III
==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
Analyses Analyses of FSIS of FSIS Beef BrothsBeef Broths
STEC Detection Methods IIISTEC Detection Methods III
•• 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
Screen/ReactiveScreen/Reactive
Confirm/Confirm/
123123 7979 8181 103103 100100
6767
9191
Analyses Analyses of FSIS of FSIS Beef BrothsBeef Broths
STEC Detection Methods IIISTEC Detection Methods III
Confirm/Confirm/
Potential positivePotential positive
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)
no isolateno isolate
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
% 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
marker
% prevalence in enrichment
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
% prevalence in enrichment
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.