title: Construction of a multiple fluorescence labelling system for use in co-invasion studies of Listeria monocytogenes.
abstract: BACKGROUND
  Existing virulence models are often difficult to apply for quantitative comparison of invasion potentials of Listeria monocytogenes. Well-to-well variation between cell-line based in vitro assays is practically unavoidable, and variation between individual animals is the cause of large deviations in the observed capacity for infection when animal models are used. One way to circumvent this problem is to carry out virulence studies as competition assays between 2 or more strains. This, however, requires invasion-neutral markers that enable easy discrimination between the different strains.
  
  
  RESULTS
  A fluorescent marker system, allowing visualization and identification of single L. monocytogenes cells as well as colonies in a non-destructive manner, was developed. Five different fluorescent labels are available, and allowed simultaneous visual discrimination between three differently labelled strains at the single cell level by use of fluorescence microscopy. More than 90% of the L. monocytogenes host cells maintained the fluorescence tags for 40 generations. The fluorescence tags did not alter the invasive capacity of the L. monocytogenes cells in a traditional Caco-2 cell invasion assay, and visual discrimination between invaded bacteria carrying different fluorescent labels inside the cells was possible.
  
  
  CONCLUSIONS
  The constructed fluorescent marker system is stable, easy to use, does not affect the virulence of L. monocytogenes in Caco-2 cell assays, and allows discrimination between differently labelled bacteria after internalization in these cells.
authors.affiliation: Danish Institute for Food and Veterinary Research, Department of Microbiological Food Safety, Mørkhøj Bygade 19, 2860 Søborg, Denmark. jeban@dfvf.dk
authors.forename: Jens B
authors.initials: JB
authors.lastname: Andersen
authors.forename: Bent B
authors.initials: BB
authors.lastname: Roldgaard
authors.forename: Ariel B
authors.initials: AB
authors.lastname: Lindner
authors.forename: Bjarke B
authors.initials: BB
authors.lastname: Christensen
authors.forename: Tine R
authors.initials: TR
authors.lastname: Licht
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D018938:Caco-2 Cells / Q000382:microbiology; D005453:Fluorescence; D006801:Humans; D008089:Listeria monocytogenes / Q000145:classification / Q000472:pathogenicity*; D008088:Listeriosis / Q000382:microbiology* / Q000503:physiopathology; D013194:Staining and Labeling; D014774:Virulence
chemical_list: D001426:Bacterial Proteins