B11001: Toxin production by non-culturable verocytotoxigenic Escherichia coli (VTEC)
Thursday 2 June 2005
This research project aims to gain a better understanding of the means and mechanisms of toxin production by non-culturable verocytotoxigenic Escherichia coli.
Background
This project will study environmental factors that influence the production of verotoxin (VT) by VTEC. Emphasis will be on the capacity of non-culturable cells of VTEC to produce VT since such organisms, which may remain undetected by conventional methodologies, may remain pathogenic and infective. New methodologies for the detection and evaluation of non-culturable VTEC cells will be the main objective of this project.
Research Approach
The main objectives are to:
- Establish well-defined non-culturable populations of VTEC
- Determine the capacity of cells within these populations to express ß-galactosidase (lacZ) in response to exogenous stimuli
- Study the production of verotoxins (VT) by non-culturable populations of VTEC
- Construct reporter-fusions of verotoxin (VT) genes with the Green Fluorescent Protein (GFP) and/or lacZ in order to determine Shiga-like toxin (SLT) production at the single bacterial cell level
- Use the reporter-fusions to study VT production in specialised environments including food and faecal microcosms
- Study the effects of external influences such as heat-stress and antibiotics on VT production in non-culturable cells.
Results and findings
In this project two areas of current microbiological concern were addressed. The biology of an important pathogen, Escherichia coli O157 and related strains, and the role that non-culturable forms of this group of organisms might play in human disease. In this context, non-culturable cells are of particular concern because they constitute a potential source of infection and continuing damage that would not be detected by conventional culture methods. The studies concentrated on one important damaging factor produced by VTEC, the protein referred to as type 2 verotoxin (VT2).
- The project developed a genetically modified reporter strain of VTEC that produced the marker enzyme ß-galactosidase in place of VT2 (chromosomal stx2AB::lacZ transcriptional fusion). The modified reporter strain was used to screen a wide variety of conditions that might influence toxin production and aspects of production in non-culturable cells.
- There was clear evidence that the marker enzyme was produced in response to a form of DNA damage that is associated with a repair process in bacteria known as the SOS response. Since several widely used antibiotics are known to be SOS inducers, the effects of these agents were also studied.
- Quinolone antimicrobials were found to increase local production of the marker enzyme to 100-fold over baseline. Trimethoprim was also found to be a potent inducer. However there was no direct evidence that these events occur in human infections. Other antibiotics, including furazolidone, sulphamethoxazole and some ß-lactam agents, were found to have minor inducing activity.
- In VTEC strains the gene encoding VT2 is carried on a bacteriophage (a virus affecting bacteria). The SOS response in these strains not only activates toxin production, it also promotes dissemination of the toxin-encoding bacteriophage. This study confirmed that the SOS-inducing antibiotics ofloxacin and trimethoprim could enhance bacteriophage production and therefore potentially enhance dissemination of the VT genes.
- Studies on carbon, nitrogen and chloramphenicol stressed populations of VTEC showed that non-culturable cells make some biologically active toxin. The levels involved were very low. It does appear that SOS-induced toxin production is lethal to the host bacterium so that the cells responsible for induced levels of toxin are non-culturable after induction.
Dissemination information
The final report is available from the FSA Library and Information centre. To obtain a copy, please contact the Enquiry Desk,
Dr Elsie Widdowson Library and 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