Construction of a multiple fluorescence labelling system for use in co-invasion studies of Listeria monocytogenes

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. CONCLUSION: 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

Oxygen restriction increases the infective potential of in Caco-2 cells and in guinea pigs-2

<p><b>Copyright information:</b></p><p>Taken from "Oxygen restriction increases the infective potential of in Caco-2 cells and in guinea pigs"</p><p>http://www.biomedcentral.com/1471-2180/7/55</p><p>BMC Microbiology 2007;7():55-55.</p><p>Published online 14 Jun 2007</p><p>PMCID:PMC1899506.</p><p></p>-2 cells grown to selected densities. Counts were normalized to a concentration of 10per ml in the bacterial cultures prior to infection. Each bar represents an average of three different experiments. Error bars designate standard deviations. OD600 refers to optical density at 600 nm

Oxygen restriction increases the infective potential of in Caco-2 cells and in guinea pigs-1

<p><b>Copyright information:</b></p><p>Taken from "Oxygen restriction increases the infective potential of in Caco-2 cells and in guinea pigs"</p><p>http://www.biomedcentral.com/1471-2180/7/55</p><p>BMC Microbiology 2007;7():55-55.</p><p>Published online 14 Jun 2007</p><p>PMCID:PMC1899506.</p><p></p> oxygen-restricted (closed symbols), fluorescence-labeled bacteria. (A): Animals dosed with monocultures of CFP-labeled bacteria. (B): Integrated presentation of data from C and D. (C): Animals dosed with a mixture of CFP-labeled, un-restricted and YFP-labeled, oxygen-restricted . (D): Animals were dosed with a mixture of YFP-labeled, un-restricted CFP-labeled, oxygen-restricted . All animals were dosed at Day 0 and again at Day 1. Each data point in panel A and B represents the average of samples from 12 animals, while each data point in panel C and D represents the average of samples from 6 animals. Error bars designate standard errors of the means

Oxygen restriction increases the infective potential of in Caco-2 cells and in guinea pigs-0

<p><b>Copyright information:</b></p><p>Taken from "Oxygen restriction increases the infective potential of in Caco-2 cells and in guinea pigs"</p><p>http://www.biomedcentral.com/1471-2180/7/55</p><p>BMC Microbiology 2007;7():55-55.</p><p>Published online 14 Jun 2007</p><p>PMCID:PMC1899506.</p><p></p>-2 cells grown to selected densities. Counts were normalized to a concentration of 10per ml in the bacterial cultures prior to infection. Each bar represents an average of three different experiments. Error bars designate standard deviations. OD600 refers to optical density at 600 nm