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Open Access Methodology article

Development of multiple strain competitive index assays for Listeria monocytogenes using pIMC; a new site-specific integrative vector

Ian R Monk12, Pat G Casey2, Michael Cronin3, Cormac GM Gahan124 and Colin Hill12*

Author Affiliations

1 Department of Microbiology, University College Cork, Cork, Ireland

2 Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland

3 Department of Maths and Statistics, University College Cork, Cork, Ireland

4 Department of Pharmacy, University College Cork, Cork, Ireland

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BMC Microbiology 2008, 8:96  doi:10.1186/1471-2180-8-96

Published: 13 June 2008

Abstract

Background

The foodborne, gram-positive pathogen, Listeria monocytogenes, is capable of causing lethal infections in compromised individuals. In the post genomic era of L. monocytogenes research, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tag L. monocytogenes strains, with a particular emphasis on the development of multiple strain competitive index assays.

Results

We have constructed a new site-specific integrative vector, pIMC, based on pPL2, for the selection of L. monocytogenes from complex samples. The pIMC vector was further modified through the incorporation of IPTG inducible markers (antibiotic and phenotypic) to produce a suite of four vectors which allowed the discrimination of multiple strains from a single sample. We were able to perform murine infection studies with up to four EGDe isolates within a single mouse and showed that the tags did not impact upon growth rate or virulence. The system also allowed the identification of subtle differences in virulence between strains of L. monocytogenes commonly used in laboratory studies.

Conclusion

This study has developed a competitive index assay that can be broadly applied to all L. monocytogenes strains. Improved statistical robustness of the data was observed, resulting in fewer mice being required for virulence assays. The competitive index assays provide a powerful method to analyse the virulence or fitness of L. monocytogenes in complex biological samples.