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CFSAN Regulatory Codes: XI.A
CFSAN Program Priority Codes: 1.4b
Start Date: 10/01/99    Completion Date: 09/30/02

Statement of Research Problem:
Enterohemorrhagic Escherichia coli (EHEC) of serotype O157:H7 are phenotypically distinct from typical E. coli, in that they do not ferment sorbitol and do not exhibit glucuronidase (GUD) activity. They are also easily identified by their ability to production Shiga toxins (Stx) and by the presence of the somatic O157 and the flagellar H7 antigens. As a result, these traits are extensively utilized in methods used to isolate and detect O157:H7 in foods. However, new variant strains of EHEC, particularly O157:H7, are being isolated more frequently from foods, animals and humans worldwide. These include sorbitol-fermenting variants, strains that exhibit GUD, non-motile, toxigenic variants; serological variants; toxin non-producing strains, etc. These variants carry most of the trait EHEC virulence markers, so are potentially pathogenic and some have already been implicated in illness; however, due to changes in trait genotypic and phenotypic markers, EHEC variants elude detection by current testing methods.

Statement of Project Objective(s):
Genetically characterize pathogenic EHEC variants for trait markers and virulence genes and determine the factors causing the expression or non-expression of trait markers in these EHEC strains. These studies will provide information to account for the emergence of these variant strains by correlating the position of these variants in the existing evolutionary model for O157:H7 (Feng, et al, JID, 1998) and also identify new genetic or phenotypic markers that can be used to test for these emerging pathogenic variants. This work relates directly to the objectives of developing improved technology for detecting emerging food-borne pathogens.

Anticipated Impact on FDA Regulatory Program:


1. The likely impact will be a more complete accounting of major hazards in the food supply, leading to a more thorough and rapid application of control measures.

Project Priority Changes During FY2000: None

Administrative Liaison(s): Peter Feng: 202/205-4518

Research Personnel:


Name	Office/Division	FTE [00, 01, 02]	Component
Peter Feng	OPDFB/DMS	1.0, 1.0, 1.0	1
Steve Monday	OPDFB/DMS	1.0, 1.0, 1.0	1
	Total FTE:	2.0, 2.0, 2.0


Collaborators:Dr. Thomas S. Whittam, Penn State Univ.; Dr. Tae Takeda, Tokyo, Japan; Dr. Bala Swaminathan, CDC, Atlanta; Dr. Scott A. Minnich, Univ. Idaho; Dr. Christine Vernozy, Lyon, France.

Component 1 Objectives:
To characterize the following phenotypic variants of O157:H7 and other EHEC serotypes and identify new markers for developing detection assays.


1. O rough strains of O157:H7 to determine the cause for absence of O157 antigen expression.
   
2. Determine the causative factors for absence of GUD activity in O157:H7.
   
3. Determine the causative factors for non-motility in Stx-producing O157 strains from Germany.
   
4. Characterize an EHEC isolate that caused an outbreak of HC.
   
5. Characterize Stx-producing and non-producing isogens of O157:H7.
Component 1 FY 2000 Deliverables:

1. Characterization of a Stx producing and non-producing O157:H7 isogen isolated from HC in Japan.
   
2. Characterization of Stx-producing, non-motile variants of O157:H7 serotype.
   
3. Evaluate the effectiveness of an assay to detect EHEC variants.
   
4. Initiate characterization of other EHEC variants.
Component 1 FY 2000 Progress:

1. O157:H7 carry uidA gene for GUD; but has no enzyme activity. Studies showed that a double G insertion in the uidA results in a premature terminal codon that causes absence of GUD in O157:H7.
   
2. Developed multiplex PCR that detects 5 trait genes in EHEC. Markers: a uidA mutation for O157:H7 and its NM, toxigenic variants; gamma eaeA of EHEC1 clonal group (O157:H7); Stx1, Stx2 and both clusters of hemolysin (ehxA) variants that are present in most Stx-producing E. coli.
   
3. Genetic analysis of O104:H21 that caused a HC outbreak showed that it produced Stx2; but had no eaeA derivatives. It had enterohemolysin but the ehxA sequence was not typical of O157:H7; but instead, resembled that of Stx-producing, cluster II variants.
   
4. Genetic and PFGE clonal analysis showed that a Stx non-producing strain of O157:H7 was derived from its Stx-producing parent by simultaneous loss of both toxin-endcoding phages.
Technical Barriers to Meeting Component 1 Objectives or Deliverables: none listed
Component 1 FY 2001 Deliverables:

1. Analyze Stx-producing, non-motile variants of O157:H- to determine cause for absence of motility.
   
2. Determine prevalence of the double G insertion in uidA gene of other pathogenic E. coli and bacteria
   
3. Prepare manuscript on uidA mutations in O157:H7.
   
4. Prepare manuscript on Stx-producing and non-producing O157:H7 isogens.
   
5. Examine other pathogenic E.coli for the presence of cluster I or II ehxA genes.
Component 1 FY 2002 Deliverables:

1. Characterize absence of O157 antigen expression in rough variants.
   
2. Prepare manuscript on cause of non-motility in O157:H- variants from Germany.
   
3. Refine the evolutionary model for emergence of O157:H7, by including data on variants.

Vernozy-Roland, C., Feng, P., Montet, M.-P., Ray-Gueniot, S., Villard, L., Boutrand-Loei, S., Bavai, C., Mazuy, C., and Atrache, V. 2000. Detection of Escherichia coli O157:H7 in heifers' faecal samples with an automated immuno-concentration system. Lett. Appl. Microbiol. 30:217-222.

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Hypertext updated by dav 2001-OCT-02