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Develop a Novel Molecular Typing Method for Comparison of Foodborne Pathogens Using VTEC as a Model Organism

Institutions
Laboratory of the Government Chemist, UK
Start date
1999
End date
2001
Objective

The overall objective of the work was to carry out molecular analyses to identify polymorphic DNA markers that reproducibly represent specific traits in foodborne VTEC, to then establish and assess the framework of a working database to catalogue both DNA markers and known phenotypic characteristics:

  • Identify and catalogue representative strains with respect to such characteristics as geographical origin, time of isolation, biological/environmental origin, toxin production, phage type and serogroup.
  • Produce, through empirical determination, a robust, reproducible fAFLP protocol with defined limitations.
  • Define the influence of extra-chromosomal DNA-derived markers on fAFLP profiles.
  • Maximise sensitivity of the fAFLP technique (with respect to distinguishing closely related strains by identifying unique polymorphic markers).
  • Identify reproducible polymorphic DNA markers and by comparison with known characteristics and statistical analysis assign specific traits to markers and define identification categories for database framework.
  • Challenge and assess system by a 2-centre analysis of a minimum of 20 blind samples.
More information

Bacterial pathogens such as verocytotoxigenic Escherichia coli (VTEC), Salmonella and Campylobacter are a major cause of human infection, and present a significant public health problem.

The ability to unequivocally identify and characterise such foodborne pathogens accurately and rapidly would permit more effective outbreak tracing and facilitate improved epidemiological monitoring.

The aim of this project has been to evaluate the use of a molecular method, fluorescent Amplified Fragment Length Polymorphism (fAFLP) profiling for the characterisation of foodborne pathogens.

VTEC has been used as a model organism, as in recent years a number of serious outbreaks of E. coli O157 have occurred, highlighting the need for improved typing techniques.

Although the incidence of VTEC infection is relatively low in comparison with other enteric pathogens, the potentially severe consequences of infection and the low infective dose has made verocytotoxin-producing E. coli a pathogen of major clinical importance.

Human infection can occur as a result of consumption of contaminated food or water, by direct person-to-person transmission or direct contact with animals carrying the organisms, or environments contaminated with faeces from infected animals or people.

To control the spread of disease, identifying the source of infection is of critical importance.

Current typing methods routinely utilised to identify VTEC isolates lack discrimination, have poor reproducibility or require experienced operators to obtain consistent and meaningful results.

The fAFLP method reproducibly generates a detailed profile consisting of a number of accurately sized DNA fragments, by selective amplification of a total digest of the bacterial DNA. This effectively produces a &#39unique fingerprint&#39 for the bacterial strain.

Profiles produced from bacterial isolates can then be compared by computer analysis to assess the relatedness of strains.

The method is robust, and produces precise typing information through the use of internal controls and automated data analysis.

Find more about this project and other FSA food safety-related projects at the Food Standards Agency Research webpage.

Funding Source
Food Standards Agency
Project number
B01014
Categories
Escherichia coli
Bacterial Pathogens