PMC4840421_01
PCR analysis of C144.66. PCR analysis of strains MNZ11 (cpsA-, pspK+) D39 (cpsA+, pspK-), and C144.66 (cpsA-, pspK+), respectively, for cpsA (lanes 2-4) and pspK (lanes 5-7). Lane 1 is a 1-Kb DNA ladder.
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Polymerase chain reaction analysis compared to an encapsulated strain showed that C144.66 lacked the highly conserved first gene in the capsule locus, cpsA (Fig. 1).
Flow cytometry for the presence of PspK. For analysis 1 x 107 log phase pneumococci were incubated with a mouse anti-PspK antiserum. A biotinylated anti-mouse secondary antibody was detected by streptavidin conjugated to Alex Flour 488. A representative histogram is shown. The negative control is MNZ85 which lacks PspK.
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Expression of PspK was confirmed through Western blot (data not shown), and flow cytometry comparing C144.66 to a known PspK positive strain (Fig. 2).
Colonization and ascension of C144.66 in a murine model. After intranasal inoculation of C144.66, colonization was achieved in all mice. Ascension into the middle ear occurred in two of the five mice. The dash line is the lower limit of detection.
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All mice were found to have nasopharyngeal colonization by C144.66; additionally, C144.66 was found to have ascended to the middle ear in a subset of the mice (Fig. 3).