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


CONCLUSIONS
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.
Department of Microbiology, University College Cork, Cork, Ireland. ianmonknz@gmail.com
Ian R
IR
Monk
Pat G
PG
Casey
Michael
M
Cronin
Cormac Gm
CG
Gahan
Colin
C
Hill
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D005260:Female; D005822:Genetic Vectors / Q000235:genetics*; D008089:Listeria monocytogenes / Q000145:classification / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity*; D008088:Listeriosis / Q000382:microbiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D014774:Virulence