Microbial Genomics

Molecular Technologies to detect pathogens

Illness due to microbiological contamination of foods is of major concern to consumers, retailers and manufacturers alike. Traditional plating methods for the detection of pathogenic bacteria from foods are extremely time-consuming. This has lead to the development of molecular techniques for the rapid, quantitative and specific detection of pathogens contaminating food and water.

Polymerase chain reaction (PCR) is a method of detecting and amplifying specific DNA sequences. At the Ashtown Food Research Centre, Teagasc this technique has been applied to the detection of food-borne pathogens by the amplification of genes that are synonymous with a particular organism. The use of Real-Time PCR, which uses fluorescence to detect the products of the PCR reaction in real time, and immunomagnetic separation (IMS) have greatly increased the specificity and speed of this approach.

Pulse field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) techniques are being used to detect and differentiate bacterial species responsible for food poisoning in Ireland, such as verocytotoxic E. coli (VTEC) and Campylobacter spp. This information is being used at the AFRC to establish a database on profiles of pathogens isolated from Irish foods.

Antisense RNA technology, which uses single stranded nucleic acids to target the messenger RNA of essential genes and inhibit protein expression, is being examined as a method of killing E. coli O157:H7 and other VTEC serogroups. Methods to improve the uptake and efficacy of antisense molecules will also be developed. (geraldine.duffy@teagasc.ie,) or (kaye.burgess@teagasc.ie)

Regulation of the acid tolerance response in Salmonella species and its effect on dietary control measures in cattle and pigs.

Agricultural animals are widely recognized as reservoirs of human enteric pathogens. These pathogens are shed in the faeces, which in turn serves as the primary source for contamination of various food products. In theory, Salmonella carriage in bovine animals should be restricted to the rumen as any bacterial pathogens entering the abomasum should be killed by the harsh acidic environment (pH 2 – 3). However, Salmonella survive passage through the bovine abomasum.

While the buffering capacity of ingested foods may contribute to this phenomenon, it is generally agreed that survival is primarily due to an acid tolerance response (ATR), triggered by mildly acidic conditions (pH 4-5). In bovine animals, these mildly acidic conditions are created by volatile fatty acids (VFAs) (propionic, acetic, butyric and lactic acid) produced by the fermentative action of lactic acid bacteria (LAB) in the rumen. This project will achieve a fundamental understanding of Salmonella ATR (genes involved and the factors which turn these on), which is essential, not just to control animal shedding of the pathogen, but also to address a range of other food safety concerns, eg. product formulation, pathogen hardening/toughening, virulence, survival in the human stomach and infection. (declan.bolton@teagasc.ie)

Virulence gene transfer in verocytotoxigenic E. coli

Verocytotoxigenic Escherichia coli (VTEC) is a serious pathogen of significant public health concern worldwide. Infection is usually characterised by bloody diarrhoea and can be life threatening due to the subsequent development of haemolytic-uremic syndrome which is mediated by verocytotoxins (VTs), also known as Shiga toxins (stx). The genes for these toxins are encoded in the genomes of mobile genetic elements called lamboid bacteriophages. The fact that verotoxin genes are encoded on a bacteriophage provides major potential for the spread of these genes among E. coli and for the emergence of new VTEC types. VTEC strains are strongly associated with cattle and this project focuses on the potential effect that environmental factors encountered in the bovine rumen have on the transfer of virulence factor genes from VTEC strains to other VTEC and non VTEC strains. (kaye.burgess@teagasc.ie) or (geraldine.duffy@teagasc.ie)