Development of a competitive index assay to evaluate the virulence of Listeria monocytogenes actA mutants during primary and secondary infection of mice

Abstract

We developed a competitive index assay for murine listeriosis that tests the virulence of Listeria monocytogenes strains in different organs and at various times postinoculation. Studies presented here demonstrate the reproducibility of this assay during primary and secondary infection of inbred and outbred mice. We verified the validity of this assay by performing competitive index analysis of a well-characterized strain of L. monocytogenes lacking the actA gene. In addition, we found that while L. monocytogenes strains unable to recruit vasodilator-stimulated phosphoprotein (VASP) to their surface exhibit a 10-fold virulence attenuation in the livers of naive animals, they display a 50-fold survival defect in the liver during secondary listeriosis.

FIG. 1

Competitive index analysis of the reference strain and wild-type 10403S L. monocytogenes. Groups of 3 to 10 naive or L. monocytogenes-immunized mice were infected with a 1:1 mixture of DP-L3903 and 10403S. CD-1 mice were sacrificed 48 h postinoculation, while BALB/c and C57BL/6 mice were euthanized after 60 h. A competitive index of 1 indicates that the two strains are proliferating equally in vivo. Abbreviations: S, spleen; L, liver. 1°, infection of naive mice; 2°, infection of L. monocytogenes-immunized mice; y axis, competitive index.

FIG. 2

Competitive index analysis during primary infection (A and B) or secondary infection (C and D) in the spleen (A and C) or liver (B and D). Groups of four or five naive or immunized BALB/c mice were infected with a 1:1 mixture of the reference strain (DP-L3903) and ΔActA3, ΔActA3-GG, or ΔActA6. All mice were sacrificed 60 h postinoculation. Average fold defect was calculated by dividing the number of reference strain CFU by the number of test strain CFU for each organ. Primary infection competitive indexes of ΔActA6 and ΔActA3-GG were significantly lower in livers than spleens, with two-sided P values of 0.0004 for ΔActA6 and 0.0002 for ΔActA3-GG by the Mann-Whitney test as calculated by MiniTab (State College, Pa.) software. Liver competitive indexes of ΔActA6 and ΔActA3-GG were significantly lower during secondary infection than during primary infection, with two-sided P values of 0.037 for ΔActA6 and 0.0048 for ΔActA3-GG. Symbols: ○, outlying value as determined by KaleidaGraph (Synergy Software, Reading, Pa.); ∗, underestimated values (1 or 0 erythromycin-sensitive colonies out of approximately 200 screened). y axis, competitive index.