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Listen to this siteWednesday 25 June 2003
This research project aims to develop a detection method for Enteroaggregative E. coli that can be used to screen clinical, food and environmental samples without the need to refer them to a specialist laboratory.
Study Duration : May 2002 to May 2004
Contractor : Health Protection Agency
The Enteroaggregative E. coli (EAEC) are a diverse group of the enterovirulent E. coli and cause Infectious Intestinal Disease (IID) in humans. A Department of Health funded study of IID in England (the IID Study) demonstrated that EAggEC are a significant cause of IID in humans. EAEC were isolated from 5.1% of cases presenting to GPs and 2.9% of cases in the community.
Our knowledge of the pathways by which EAEC are transmitted to humans is limited by the lack of widely available methods for detecting these bacteria. Detection of EAEC in clinical or food samples is currently limited to specialist laboratories that conduct cell adhesion assays or use gene probes to detect the aggregative adhesion phenotype. Work was needed to develop a detection method for EAEC that can be used to screen clinical, food and environmental samples without the need to refer them to a specialist laboratory for testing.
The project aims to develop a multiplex PCR method able to detect EAEC strains from patient faecal material and food samples.
The project relates to human disease caused by a form of enteric bacterium called Escherichia coli ( E. coli ) that causes chronic diarrhoea and is particularly important as a cause of illness in young children living in certain developing countries, and a potential cause of travellers� diarrhoea for those visiting countries where these bacteria are endemic. The group of E. coli which cause this chronic diarrhoeal disease is only poorly understood, a fact that has been largely due to our lack of an easy method for identifying these bacteria. The current test involves mixing bacteria with laboratory-grown human tumour (HEp-2) cells and seeing how the bacteria stick to the HEp-2 cells. The E. coli of interest stick to the cells in a specific formation and this pattern has been termed �stacked-brick� since it resembles bricks in a wall. The bacteria forming this pattern have been termed enteroaggregative E. coli or (EAggEC). The test used to determine the pattern of sticking to HEp-2 cells is expensive and time consuming, and can only be performed in specialised laboratories. As a consequence, there is a need for a simplified test that could be used by diagnostic laboratories to enable EAggEC to be identified and so increase our understanding of the true incidence of illness due to EAggEC.
The ability of bacteria to cause human disease relies on them having a range of properties, which enables them to survive in the gut of a person, become attached to the intestinal wall and cause diarrhoeal disease. All these properties are carried on the bacterial genes and it is possible to locate which parts of the genes are responsible for the various steps involved in causing disease. Detecting the genes that enable bacteria to adhere in the �stacked brick� pattern can provide information as to whether or not a given bacterium is an EAggEC.
Strains of EAggEC stick to human cells in a �stacked-brick� pattern and it is thought that this �sticking� process is also used by these bacteria when adhering to human intestinal cells, preventing them from being dislodged when intestinal contents pass through a person. However, there are situations when the bacteria need to �let go� of the intestinal cells and when this is required the bacteria produce a protein which enables EAggEC to �unstick� themselves from the intestinal cells and become dispersed. Appropriately this protein is called �dispersin�. The idea was to see if a test could be developed to detect the dispersin protein.
Rationale and Objectives
The aim of the project was to develop simple, but sensitive tests for the detection of EAggEC. A collection of EAggEC, held by the Laboratory of Enteric Pathogens (LEP) at the Health Protection Agency, was used as a basis for the research and collaborators also helped in isolating new strains of EAggEC by sending samples of patients� faeces to the LEP for screening for EAggEC. An investigation was also carried out into the way these bacteria behaved whilst growing in laboratory media, with the aim of examining if EAggEC could be identified by using defined growth conditions. Finally, any promising tests were evaluated by testing samples of foodstuffs and normal human faeces, which had been artificially contaminated with EAggEC. Once identified, a simplified means of detecting EAggEC would enable the rapid diagnosis of patients with a diarrhoeal illness caused by EAggEC. The same test could be used to analyse foods for EAggEC, ensuring the safety of foodstuffs, particularly those that are consumed raw.
Outcome/ Key results Obtained
Tests were developed which could detect specific genes carried by EAggEC. The 140 Strains of EAggEC, from the collection held by the LEP, were readily detected by the methods used. Using patients� faecal samples, submitted by collaborators at HPA Leeds, the tests successfully identified strains of EAggEC in 11 of 12 patients, illustrating that the procedure performed well in the presence of normal faecal bacteria. This was investigated further using normal human faeces with EAggEC bacteria added to them. Once again, the tests were able to identify EAggEC in a complex mix of faecal bacteria. The procedures used were also able to detect strains of EAggEC which had been used to artificially contaminate foodstuffs such as lettuce.
The EAggEC were not found to have unusual growth patterns and so a growth medium specific for detecting EAggEC was not identified. With respect to studies involving dispersin, purified dispersin was obtained and used to prepare specific antibodies in a rabbit. Tests were developed to successfully identify dispersin being made by EAggEC but a simple method that could be applied in a routine laboratory was not identified.
Strains of EAggEC were used to �spike� mineral water, minced beef, cooked ham, milk, lettuce, and bean sprouts and these bacteria were found to remain viable and could be detected readily; however, naturally occurring components in minced beef were found to hinder the detection of EAggEC in this product.
Final report is available from the Agency's Information centre.
To obtain a copy, please contact the Enquiry Desk, Information Services, Food Standards Agency (tel: 020 7276 8181/8182 or email:
infocentre@foodstandards.gsi.gov.uk
).
Contact
: For any enquiries concerning this research project, please contact the relevant Programme contact or email:
science@foodstandards.gsi.gov.uk
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