•  refers to any E. coli strain that produces Shiga toxin (Stx)

•  inclusion in this category does not necessarily confirm pathogenicity in the absence of other virulence factors

•  includes enterohemorrhagic E. coli (EHEC) as a subset

•  E. coli O157:H7 is the EHEC prototype  

Shiga toxin (Stx) – main virulence factor of EHEC – ribosome-inactivating toxin – shuts down protein synthesis – causes cell death

(Kaper et al. 2004. Nat. Rev. Microbiol. 2:123-140):

contain the genes for •  Shiga toxin (stx) •  virulence factors that

cause A/E lesions — intimin (eae) — others

hemorrhagic  coli,s  

attaching-effacing (A/E) lesions

endothelial  necrosis  

FSIS deemed adulterated E. coli O157:H7-contaminated raw non-intact beef products and intact cuts that are to be further processed into non-intact products before being distributed for consumption.

On September 20, 2011, the USDA declared these 6 serogroups to be adulterants in non-intact, raw beef (Federal Register, Vol. 76, No. 182, 58157-58165).

Six  O  groups    comprising    6  serogroups  and  13  serotypes  are  responsible  for  ~71%  of  the  cases  of  non-­‐O157  STEC  infec,on  in  the  US.  

Brooks et al. 2005. J. Infect. Dis. 192:1422-9.

“Top  6”  “Big  6”  

STEC O157 Non-O157 STEC

Total

All illnesses 92,872 137,502 230,374

Foodborne illnesses

63,153 112,752 175,905 (76%)

Hospitalizations 2,138 271 2,409

Deaths 20 0 20

•  Long  Term  Goal:  

To  reduce  the  occurrence  and  public  health  risks  from  STEC-­‐8  [STEC  O26,  O111,  O103,  O121,  O45,  O145,  O157:H7/NM  (STEC-­‐7)  and  O104:H4]  in  beef  using  a  quan%ta%ve  microbial  risk  assessment  plaZorm.  

STEC-6 = “Top 6” STEC-7 = “Top 6” + O157:H7 STEC-8 = STEC-7 + O104:H4 (EHEC-6) (EHEC-7) (EHEC-8)

Pillar  1  -­‐  Live  ca)le  &  beef  producers  

Pillar  2  -­‐  Slaughter,  fabricate,  meat  processing  &  processors  

Pillar  3  -­‐  Retail  products,  food  service  &  consumers  

Obj  1  -­‐  STEC  detec,on:  -­‐  reagents,  sampling,  assays,  technology,  partnerships  

Obj  2  -­‐  STEC  biology-­‐  microbiology,  ecology,  epidemiology,  modifiable  risk,  best    targets  

Obj  3  -­‐  Interven,ons  for  STEC  risk  reduc,on:  value,  feasibility,  cost-­‐benefit,  impacts  

Obj  4  –  STEC  risk  analysis  -­‐  risk  assessment  (QMRA)  

Obj  5  -­‐  Beef  chain  STEC-­‐8  transla,onal  educa,on,  outreach  ,  and  evalua,on  

1) Develop STEC-8 resources: isolates, antigen and DNA detection reagents and kits

2) Develop economical qualitative and enumerative STEC-8 assays to inform risk assessment

3) Develop novel and rapid molecular technology for STEC-8 detection

4) Perform STEC-8 comparative diagnostic evaluation and test validation

5) Develop statistically-based sampling plans

•  Strain collection •  Enrichment broth •  Chromogenic agar •  Monoclonal Antibodies •  Conventional Multiplex PCR •  Multiplex Real-time Quantitative PCR •  Multiplex Oligonucleotide Ligated (MOL)-PCR •  Waveguide-based Optical Biosensor

Sta,onary  for  24  h  at  42°C  

6.5  

7  

7.5  

8  

8.5  

9  

9.5  

10  

10.5  

1   3   5   7   9   11   13   15   17   19   21   23   25   27   29   31   33   35   37   39   41   43   45   47   49   51   53   55  

CFU/m

l  (Log 1

0)  

Strain  

TSB-­‐NVRBT  

TSB  

•   55  of  55  strains  tested  had  reduced  growth        in  TSB-­‐NVRBT,  compared  to  TSB  •   2  strains  (*)  had  poor  growth  @42°C  •   Mean  CFU/ml  Log10  reduc,on  =  1.18          (P  =  6.65  x  10-­‐28,  paired  t-­‐test)          

*  

*Strain  #4  =  O26  (MT#10).  *Strain  #31  =  O157:H7  (S2006  #4).  

*  

Possé et al. J. Appl. Microbiol. 2008. 105:227–35.

 Gentry  L.  Lewis  

*

TSB   EC  mTSB  

Treatment Growth Log(CFU)

TSB 6.23b

mTSB 6.37ab

•  NalR or RifR STEC strains were inoculated into bovine fecal samples. •  Inoculum levels for each strain: 100, 1,000, 10,000 CFU/g •  Enriched stationary for 6 hours at 40°C. •  Treatments: E. coli broth (EC), trypticase soy broth (TSB) and trypticase soy broth with bile salts

[modified TSB (mTSB)]. •  Enumeration by serial dilution and spread plating on mPossé medium with appropriate antibiotics.

Zach  Stromberg  

CHROMagarTM STEC R&F®Non-O157 STEC Chromogenic Plating Medium

Means with unlike superscripts within a column are significantly different (P<0.05).

®  

n  =  38  n  =  79  

Values are means with 95% confidence limits in parentheses. Means with unlike superscripts within a column are significantly different (P<0.05).

®  

Modified Rainbow® O157 medium

Modified Possé medium

Colorless,  instead  of  blue-­‐purple  

Colorless,  instead  of  turquoise  

USMARC medium

Dark  blue-­‐violet,  instead  of  mauve  

Fischer,  Wijemanne,  Lewis,  Moxley  

Inoculum  Streak  Plate   10-­‐0   10-­‐1  

Inoculated  Bovine  Feces  with  106.8  CFU,  Dilu,on,  Spread  Plate  

mPossé2  CDC  90-­‐3128  O103:H2  

Medium,  Strain,    Serotype  

USMARC  DEC10B  O26:NM  

mRainbow®  

DA-­‐10  O26:NM  

10-­‐2   10-­‐3  

•  Crowding on plates prevents development of expected phenotype and hampers detection.

•  Dilution allows one to overcome the effect, but you may lose the STEC unless it is in very high number.

Medium  AnQbioQc  ConcentraQon  (mg/L)   Reference  

Novobiocin  Cefixime  Trihydrate  

Potassium  Tellurite  

Nal  or  Rif*  

Possé   8   0   2.5   0  J.  Appl.  Microbiol.  2008;105:227-­‐35  

mPossé1   0   0   0   40  or  100   None  

mPossé2   5   0   0.5   0  J.  Food  Prot.  

2013;76:192-­‐9†  

mPossé3   5   0.05   0.15   0  

FSIS  MLG5B.01  (11/4/2011)  through  current  [FSIS  MLG  5B.04  (10/1/2013)]‡  

*Nal = nalidixic acid, 40 mg/L; Rif = rifampicin, 100 mg/L †These antibiotics and concentrations were adopted from USMARC medium in this publication. ‡These antibiotics and concentrations were adopted from mRainbow® agar in these publications.

DetecQon  of  STEC  in  Samples  Inoculated  with  100  CFU  or  1,000  CFU  100  CFU  (32  strains;  1  strain/sample)   1,000  CFU  (32  strains;  1  strain/sample)  

Agar   CHROMagarTM  STEC  

Possé   mPossé  #2   mPossé  #3   CHROMagarTM  STEC  

Possé   mPossé  #2   mPossé  #3  

No.  Strains  Detected  

10   5   5   1   9   5   6   0  

Inoculated  Samples  (n=64)  

True  Posi,ve   False  Posi,ve   %  Posi,ve  

CHROMagar™  STEC   19   45   29.7C  

Possé   10   54   15.6AB  

mPossé  #2   11   53   17.2BC  

mPossé  #3   1   63   1.6A  

Pre

vale

nce

(%)

0.5  0.6  

40.8  0.0  

18.6  0.0  

4.5  0.0  

2.4  0.0  

49.1  0.2  

14.4  0.6  

100.0  4.0  

99.7  5.3  

Stromberg,    Lewis,  Cernicchiaro,  Renter,  Moxley  

61.2  1.6  

0

20

40

60

80

100

120

NeoSEEK

Culture

0.5  0.0  

1.2  0.0  

6.9  0.0  

0.2  0.0  

0.0  0.0  

2.3  0.0  

2.1  14.9  0.0  

Pre

vale

nce

(%)

28.7  

71.9  66.8  

60.1  

20.5  24.0  

Baumann,    Cernicchiaro,  Renter,  Bai,  Nagaraja,  Phebus  

0

10

20

30

40

50

60

70

80

NeoSEEK

Assurance GDS

Culture

10.8  

Developing  Discriminatory  Biomarker  Assays  for  Non-­‐O157  STEC  

Steve Graves Harshini Mukundan Loreen Lamoureux

 DiscriminaQng  between  STECs  and  other  E.  coli  is  difficult  

 Serotyping  STEC  is  problemaQc  due  to  cross  reacQvity  of  available  polyclonal  anQbodies  

 Leads  to  higher  degree  of  non-­‐specific  interacQons   Higher  occurrence  of  false  posiQves  

 Developing  monoclonal  anQbodies  with  higher  specificity  for  O-­‐anQgens  may  miQgate  the  shortcomings  of  commercial  anQsera  along  with  lowering  costs  

 AnQgens  are  not  available  for  selecQng  hybridomas  and  therefore  must  be  extracted,  purified,  characterized  and  tested  

Background  

 Seven  strains  of  non-­‐O157  STEC  were  cultured  and  lipopolysaccharides  extracted  via  hot  phenol  method  

 Ultra  centrifuga,on  to  remove  proteins  and  CTAB  precipita,on  to  remove  nucleic  acids  

 Weak  acid  hydrolysis  to  remove  lipid  A  group  from  the  whole  lipopolysaccharide  

AnQgen  ExtracQon  &  PurificaQon  

  O26   DEC10B     O111   0201  9611    O45   B8227-­‐C8     O121   MDCH-­‐4    O103   MT#80     O145   GS  G5578620    O104   TY-­‐2482  

1.5%  Ace,c  Acid  

100˚C,  4  hours  LPS  Extract  

AnQgen  CharacterizaQon:  Immunoblohng  Obtained  6  monoclonal  and  1  polyclonal  an,bodies  from  Abraxis  ALL  an,bodies  were  tested  again  ALL  LPS  crude  extracts,  O-­‐An,gens,  and  Lipid  A  isolates  Pink  highlight  represents  the  posi,ve  control  for  that  an,body  

AnQgen  CharacterizaQon:  Immunoblohng  

AnQgen  CharacterizaQon:  Immunoblohng  

AnQgen  CharacterizaQon:  Immunoblohng  

Ab   pAb  Lipid  A  

mAb  O157  

mAb  O26  

mAb  O45  

mAb  O103  

pAb  O104  

mAb  O111  

mAb  O121  

mAb  O145  

Ag   O111:B4   O157   O26   O45   O103   O104   O111   O121   O145  

AnQgen  CharacterizaQon:  Western  Blohng  

Waveguide  Assay  Plajorm  

hν

Evanescent  field  

~200  nm  

Waveguide  with  coupled  light  

Waveguide  is  func,onalized  with  lipid  bilayer  

Amphiphilic  biomarker  inserts  into  bilayer  

Detected  with  single  fluorescent  reporter  LPS  

Mukundan  et  al.  Sensors  2009,  9:5783  

DetecQon  of  LPS  with  Membrane  InserQon  

0  

5000  

10000  

15000  

20000  

25000  

625   675   725   775   825  

0  

500  

1000  

1500  

2000  

2500  

3000  

625   675   725   775   825  

LPS  Serotype  DetecQon  

O157    S:N  =  50.8  

O103    S:N  =  41  

O45        S:N  =  30.2  

O104    S:N  =  18.4  

O26        S:N  =  1.3  

Noise  

AnQgen:  200  µg/mL  LPS  serotype  as  specified  25  nM  pAb  LPS  O157-­‐af647  **LoD  =  7.55  µg/mL    O157  LPS  only  

AnQgen:  200  µg/mL  LPS  O111:B4  80  nM  pAb  LPS-­‐af647;  S:N  =  126  LoD  =  2.2  µg/mL    

RelaQ

ve  Fluorescen

ce  Units    

Wavelength  (nm)  

LPS  Membrane  InserQon  

RelaQ

ve  Fluorescen

ce  Units    

Wavelength  (nm)  

Signal  

Noise  

Acknowledgements  

This  project  was  supported,  in  part,  by  Agriculture  and  Food  Research  Ini,a,ve  

Compe,,ve  Grant  no.  2012-­‐68003-­‐30155  from  the  USDA  Na,onal  Ins,tute  of  Food  and  

Agriculture.  

Rod Moxley

Gabriel Montaño

Harshini Mukundan

Steve Graves

Rama Sakamuri Zach Stromberg

Basil Swanson

Afsheen Banisadr Priya Dighe

Aaron Anderson

Advisors  

7-­‐plex    STEC-­‐7  serogroups  

11-­‐plex  STEC-­‐7  +  stx1,  stx2,  eae  &  ehxA  

12-­‐plex    STEC-­‐8  +  stx1,  stx2,  eae  &  ehxA  

Paddock  et  al.  2012.  Vet.    Microbiol.  156:381-­‐8.  

Bai  et  al.  2012.    Foodborne  Pathog.  Dis.  9:541-­‐8  

O104  

100  bp  Marker  

O26  O157  

O145  

O111  

O121  

O103  

O45  

7  O-­‐types  

100  bp  Marker   100  bp  

Marker  

•  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

T.  G.  Nagaraja   Jianfa  Bai  

Fecal  sample  enriched  in  EC  broth  (6  h  at  40°C)  

IMS  aliquots  with  O26,  O45,  O103,  O111,  O121  or  O145  Beads  (1/serogroup)  

Spread  onto  mPossé2  agar,  1  IMS/plate  

Six  chromogenic  colonies/plate  (mauve,  blue,  purple,  or  green)  

7-­‐plex  PCR  on  pooled  colonies  (1  pool/plate;  six  7-­‐plex  PCRs/sample)  

11-­‐plex  PCR  on  individual  colonies  

(36  PCR/sample)  

2013 Summer feedlot study: n=576 fecal samples

Fecal samples

mqPCR Conventional PCR

Culture method

Positive by RT-PCR 547 445 422

Negative by RT-PCR

29 0 7

Samples positive for one or more serogroups

A. Deshpande, et al. J. Microbiol. Meth. 2010;80:155-163.

Alina Deshpande

Steven Graves

Travis Woods

•  Ligation based assay - uses microsphere arrays and flow cytometry

•  Direct detection of multiple nucleic acid signatures - unique sequences, single nucleotide polymorphisms (SNPs), insertions, deletions, repeats, etc.

•  Simultaneous pathogen detection and characterization •  Can be performed in large multiplexed formats with

relative ease •  Assay is easy to reconfigure and adapt to changing

needs of detection, with minimal increases in costs

Goal: Facilitate screening of sample simultaneously for STEC, include strain characterization Two multiplexed assays being developed •  11-plex assay for genetic sequence signatures of 8

STEC serogroups, stx1, stx2, and eae (Bai et al. 2012 Foodborne Pathog. Dis. 9:541)

•  Multiplex assay that targets 22 SNPs in O-antigen gene cluster to differentiate between STEC and Non-STEC serogroups present in samples [Bono et al. 2012. Appl. Environ. Microbiol. 78:6689.]

!"#$%&'(%#)$#)*+

,-.'/,01$)

,23'/,01$)

,456'/,01$)

,452'/,01$)

,444'/,01$)

,4-4'/,01$)

,423'/,01$)

,437'/,01$)

(&894'/,01$)

(&89-'/,01$)

:"%'/,01$)

,-.';<= 29 1 1 1 1 1 1 1 37 90 29,23';<= 1 32 1 1 1 1 1 1 40 2 35,456';<= 1 1 35 1 1 1 1 1 32 2 24,452';<= 1 2 1 12 1 1 1 2 1 41 7,444';<= 1 1 1 1 23 1 1 1 36 88 31,4-4';<= 1 1 1 1 1 19 1 1 18 57 15,423';<= 1 1 1 1 1 1 5 1 27 80 19,437';<= 1 1 1 1 1 1 1 29 20 64 9

Detection assay response profile (signal-to-noise ratio)

Actual ATCC serogroup response profile !"#$%&'

(%#)$#)*+,-.'

/,01$),23'

/,01$),456'

/,01$),452'

/,01$),444'

/,01$),4-4'

/,01$),423'

/,01$),437'

/,01$)(&894'

/,01$)(&89-'

/,01$) :"%'/,01$)

,-.';<=,23';<=,456';<=,452';<=,444';<=,4-4';<=,423';<=,437';<=

Serogroup/  virulence  gene

Moligo  design  for  unique  DNA  signatures  (Assay  1)

Moligo  design  for  (SNP)  signatures  (Assay  2)

Assay  1  protocol  opQmizaQon  (Singleplex)

Assay  2  protocol  opQmizaQon  (Singleplex)

Assay  1  MulQplex  tesQng

Assay  2  MulQplex  tesQng

O157 Completed Completed Completed Ongoing  –  1.  Tes,ng  on  100  

blinded  samples  from  Kansas  State  University  (KSU)  

2.  Development  of  automated  data  processing  algorithm  using  44  KSU  samples  as  training  set  

O26 Completed Completed Completed

O45 Completed Completed Completed

O103 Completed Completed Completed

O104 Completed Completed Completed O111 Completed Completed Completed

O121 Completed Completed Completed

O145 Completed Completed Completed

stx1 Completed Completed Completed

stx2 Completed Completed Completed

eae Completed Completed Completed

•  Complete blinded sample study

•  Test automated data processing algorithm

•  Evaluate sensitivity of assay using spiked samples provided by KSU

•  Develop SNP-based characterization assay