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Detection and Characterization of Non-O157 STEC
Pina M. FratamicoUSDA, Agricultural Research ServiceEastern Regional Research Center
600 E. Mermaid LaneWyndmoor, PA 19038
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)