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Development of a Multipurpose DNA Microarray for the Identification and Active Surveillance of Bacterial Pathogens

Institutions
London School of Hygiene and Tropical Medicine
Start date
2004
End date
2007
Objective
We will construct a DNA microarray to identify pathogens and monitor their genome flux, including the acquisition of antibiotic resistance genes. This active surveillance pathogen (ASP) microarray will provide an early warning system for newly emerging pathogens, particularly those that are highly virulent or antibiotic resistant.

The original version of the array, ASP1, will include specific sequences unique to each of 80 known pathogens. These sequences will identify test organisms, and provide positive controls to confirm DNA hybridisation has been successful. The array will also include 7600 sequences known to move between bacterial genomes, for example antibiotic resistance genes, pathogenicity islands, phage sequences and plasmids. These sequences will allow us to screen isolates? genomes and assess which of these determinants are present.

We will amplify sequences to be included on the microarray by PCR, using an in house software programme to design primers that amplify unique DNA fragments from genomic DNA, while avoiding cross-hybridising sequences. Amplicons will be 200 to 300 bp and designed to avoid extremes of G+C content. We will use a Microgrid II micro-gridding robot to print the resulting PCR products onto CMT-GAPSTM coated glass slides, in collaboration with the Bacterial Pathogen Microarray facility at St. George?s Hospital, with whom the applicant is co-manager.

We will validate the ASP1 microarray using DNA from 200 selected isolates from hospitals that serve large populations of Birmingham, East and South London. Baseline information about the genome content of these strains will enable us to develop an updated array, ASP2, which will be made available nationally for testing highly virulent or multidrug-resistant strains - identifying them as well as screening for the presence of mobile genetic elements. Monitoring strains throughout the course of an outbreak will provide information about the origin and mechanism of transfer of those elements.

We also hope to test the hypothesis that because antibiotic resistance genes are selected against in environments where they confer no advantage, multidrug-resistant bacteria are more likely to be highly virulent and/or transmissible and to have gained virulence determinants by lateral gene transfer.

The ASP2 microarray has several longer term applications, including providing information about the spread of antibiotic resistance in nosocomial settings and among animals fed antibiotics, as well as identifying bioterrorist agents that may have been genetically modified, for example by adding genes for toxins or antibiotic resistance.

Project number
G0300020
Categories
Antimicrobial Resistance
Bacterial Pathogens