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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.

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