Detection and Characterization of Non-O157 STEC

Pina M. FratamicoUSDA, Agricultural Research ServiceEastern Regional Research Center

600 E. Mermaid LaneWyndmoor, PA 19038

pina.fratamico@ars.usda.gov

Development of efficient and practical methods for producing arbuscular mycorrhizal fungi: CRIS 010

David Douds – Lead Scientist

Development of methods for the on-farm production and utilization of AM fungus inoculum

Explore the role of AM fungi in carbon sequestration in organically farmed soils

Study the physiology and signaling/recognition events of the symbiosis

Microbial Communities and Interactions and their Impact on Food Safety : CRIS 58

George Paoli– Lead Scientist

Areas of Study

Mechanisms of biofilm formation in foodborne pathogens and the role of biofilms in pathogen persistence with emphasis on non-O157 STECs (Uhlich, Chen, Paoli, Irwin)

Examine the role of quorum sensing of microorganisms in food environments with emphasis on non-O157 STEC (Paoli, Uhlich, Chen Irwin)

Persistence and transmission of antimicrobial resistant bacteria in microbial ecosystems (Chen, Irwin, Paoli, Uhlich)

Microbial communities associated with food and food processing environments (Irwin, Chen, Paoli, Uhlich)

George PaoliChandi Wijey

Chin-Yi ChenTerri Strobaugh

Gaylen UhlichBryan Cottrell

Peter IrwinLy Nguyen

Detection and Typing of Foodborne Pathogens: CRIS 62

Andrew Gehring– Lead Scientist

Areas of Study

Rapid and effective means to separate and concentrate pathogens from food matrices (Brewster, Gehring, Bhaduri, He, Paoli)

Examine environmental factors and microbiological culture conditions affecting genotypes or phenotypes that are important for virulence, isolation, or detection of foodborne pathogens (Bhaduri, Gehring)

Protein- and nucleic acid-based methods for the multiplexed detection and characterization of foodborne pathogens (He, Gehring, Brewster)

Typing methods for pathogens of concern to associated food regulatory agencies (Brewster, Gehring, He, Paoli)

Saumya Bhaduri Jeff Brewster Andy Gehring Yiping He George PaoliKenyetta Chaney John Minutolo Joe Uknalis Sue Reed

Molecular characterization of pathogens and their responses to environmental factors: CRIS 60

Pina Fratamico – Lead Scientist

Shiga toxin-producing E. coli

P. Fratamico, L. Bagi, A. Abdul-Wakeel

Stress responses, comparative genomics, detection, molecular serotyping

Listeria monocytogenes

Yanhong Liu, Amy Ream

Stress responses, comparative genomics

Campylobacter spp.

Nereus “Jack” Gunther, Jonnee Almond

Characterization, intrinsic/extrinsic stress

Computational biologist – Xianghe Yan

New Project: Genomic and proteomic analyses of foodborne pathogens

Sequencing of the E. coli O antigen gene clusters: serogroup-specific PCR assays

E. coli chromosomeJUMPStart gnd

O antigen gene cluster

ca. 8-15 kilobase pairs

O26, O113: DebRoy et al. 2004. Appl. Environ. Microbiol. 70:1830-1832.

O45, O55: DebRoy et al. 2005. . Appl. Environ. Microbiol. 71:4919-4924.

O104: Wang et al., 2000. Gene 270:231-236.

O103: Fratamico et al. 2005. Can. J. Microbiol. 51:515-22

O121: Fratamico et al. 2003. J. Clin. Microbiol. 41:3379-3383.

O145: Fratamico et al. 2009. Foodborne Path. Dis. 6:605-611

Liu, Y. and Fratamico, P. 2006. Escherichia coli O antigen typing using

DNA microarrays. Mol. Cell Probes 20:239-244.

etc.

Sequence O-antigen gene cluster

Annotate genes, identify genes which are suitable targets for PCR assays

wzx (O antigen flippase), wzy (O antigen polymerase)

Non-O157 STEC

1994: O157:H7 nationally-reportable, adulterant in beef

2000: non-O157 STEC nationally-reportable

Cases reported to FoodNet increasing

2000-2006 4-fold increase

Brooks et al. 2005. JID. 192:1422

1983-2002: 71% of infections - 6 non-O157 STEC serogroups

O26: 22% O111: 16% O103: 12%

O121: 9% O45: 7% O145: 5%

Non-O157 declared as adulterants?

Petition to FSIS (Marler Clark law firm)

Senator Kirsten Gillibrand proposed new legislation to declare non-O157 serogroups as adulterants in beef

WASHINGTON, August 28, 2010 - Cargill Meat Solutions Corp., a Wyalusing, Pa. establishment, is recalling approximately 8,500 pounds of ground beef products that may be contaminated with E. coliO26, the U.S. Department of Agriculture's Food Safety

and Inspection Service (FSIS) announced today.

http://www.foodpoisonjournal.com/

E. coli O26 outbreak

3 illnesses: Maine, New York

Collaboration between USDA ARS and FSIS

Develop a method to detect and isolate STEC O157 and the top non-O157 STEC from ground beef, trim, and environmental surfaces

Target virulence genes and O-group-specific genes

Detection and isolation of non-O157 STEC

Sample enrichment

TaqMan-based real-time PCR assay targeting stx and eae genes

stx1-stx2, eae, internal amplification control (IC)

Samples positive for eae and stx are subjected to multiplex PCR assay targeting the wzx gene of E. coliO26, O45, O103, O111, O121, and O145

O26 wzx, O111 wzx, IC

O45 wzx, O121 wzx, IC

O103 wzx, O145 wzx, IC

Enrichment (Possé et al. 2008. J. Appl. Microbiol. 105:227-35)

TSB with novobiocin without novobiocin

E. coli O26 9.40 CFU/ml 9.51 CFU/ml

E. coli O111 7.76 CFU/ml 9.32 CFU/ml

E. coli O111 6.33 CFU/ml 9.38 CFU/ml

25 g ground beef – inoculate with ca. 2-20 CFU STEC

mTSB

225 ml TSB - add vancomycin and cefsulodin Stomacher - 1 min 6 h enrichment at 37°C add rifampicin, bile salts, and potassium tellurite

18 h at 42°C

Real-time multiplex PCR assays

DNA extraction from 1 ml of enrichment

stx1-stx2, eae, internal amplification control (IC)

stx1 - probe (TexRed)

stx2 - probe (TexRed)

eae - probe (FAM)

IC - probe (Cy5) 16S RNA, pUC19-pMB1rep

O26 wzx (FAM), O111 wzx (TexRed), IC

O145 wzx (FAM), O103 wzx (TexRed), IC

O45 wzx (FAM), O121 wzx (TexRed), IC

Detection limit: 104 CFU/ml – ca. 50 CFU/PCR

OmniMixTM HS

lyophilized

reagent beads

Detection and isolation of non-O157 STEC--Immunomagnetic separation (IMS)

Dynabeads® EPEC/VTEC O26

Dynabeads® EPEC/VTEC O103

Dynabeads® EPEC/VTEC O111

Dynabeads® EPEC/VTEC O145

IMS for E. coli O121 and E. coli O45

Polyclonal anti-E. coli O121 and anti-E. coli O45 typing serum isolation of IgG link to biotin coat streptavidin-coupled Dynabeads®

Incubate 20 μl of beads with 1 ml of enrichment for 10 min wash plate onto Rainbow® O157 Agar (K-tellurite+novobiocin)

Confirmation of presumptive isolates using PCR

Isolation of non-O157 STEC on Rainbow

Agar O157

E. coli

serogroup

Colony color on

Rainbow Agar

O157

No. colonies confirmed

as correct serogroup/No.

colonies picked

Plated

IMS without IMS

O26 Purple 15/21 7/12

O45Gray-purple, purple, light

magenta

23/27 14/22

O103 Gray, purple, blue-purple 18/20 10/12

O111 Gray-blue, gray 8/44 8/27

O121 Magenta, purple 22/27 12/24

O145 Gray-purple, blue-purple 17/20 9/10

IMS

Dynabeads® EPEC/VTEC O111: lower capture

rate compared to other STEC Dynabeads

(Verstraete et al. 2010. Vet. Microbiol. 145:106-112)

Captivate beads (Lab M Ltd., Manchester, UK)

Higher recovery O111 and O145 using Captivate beads compared to Dynabeads

Soon available in North America through Neutec Group

IMS beads for STEC O45 and O121 not available

FSIS Microbiology Laboratory Guidebook: MLG 5B.00

Detection and isolation of non-O157 Shiga toxin-producing E. coli (STEC) strains from meat products (effective Oct. 1, 2010)

mTSB+novobiocin and casamino acids

Evaluation of improved enrichment media

TSB, BPW with selective agents

15-22 h enrichment time

FSIS E. coli O157:H7 and non-O157 STEC methods

O157:H7

Day 1: Samples collected from plant

Day 2: Enrichment for 15-22 h

Day 3: PCR screen, O157 IMS, plating

Day 4: Agglutination test, plating onto SBA

Day 5: O157, H7 latex agglutination or O157 wzy / fliCh7 PCR, toxin ELISA or stxPCR, biochemical confirmation

FSIS E. coli O157:H7 and non-O157 STEC methods (con’t)

Non-O157 STEC Day 1: Samples collected from plant

Day 2: Enrichment for 15-22 h

Day 3: PCR screen for stx-eae, O-group PCR, O-group-specific IMS, plating

Day 4: Pick colonies and plate on SBA

Day 5: Confirm colonies using PCR and/or latex beads, plate onto SBA

Day 6: Biochemical tests for confirmation

Plating media for isolation of STEC

Rainbow Agar O157, washed sheep blood agar +mitomycin (Sugiyama et al. 2001. Lett. Appl. Microbiol. 33:193-95), CHROMagar O157—others?

Chromocult agar with cefixime, cefsulodin, vancomycin

Chromocult not available in US

Confirmation of presumptive positive colonies

Real-time multiplex PCR targeting stx, eae, and O-antigen genes

Latex agglutination

Preparation of latex beads

Purify IgG from polyclonal typing antisera

+

Latex beadsIgG reactive to O groups

Agglutination of presumptive positive colonies

Other collaborations related to non-O157 STEC methods

Pall/GeneSystems – GeneDisc STEC and E. coliO157

bioMerieux immunoconcentration strips (VIDAS)

DuPont/Qualicon tableted reagents

Acknowledgments

Lori Bagi (ARS)

Chitrita DebRoy (Pennsylvania State University)

Marjorie Medina (ARS)

Weilin Shelver (ARS)

William Cray (FSIS)

Neelam Narang (FSIS)

Brad Garman (ARS)

Thank you