Total RNA was isolated from 6, 12, 18, 24, and 30 h-old cultures of strains 17 and 17-2, and the relative expression levels of these genes were recorded by the strain using real-time RT-PCR. The expression levels of these genes were fluctuating in strain
17 but not in strain 17-2. Data are representative NU7026 of two independent experiments. dnaK: PINA1058; dnaJ: PINA1756; groEL: PINA1797; groES: PINA1798; clpB: PINA2006. Abscess induction in mice To examine the influence of the biofilm phenotype on pathogeniCity of P. intermedia, the abilities of strains 17 and 17-2 to induce abscesses in mice were compared. An injection of 500 μl of strain 17 at a concentration of 107 CFU/ml induced abscesses in mice (Fig. 8, left panel). In contrast, injection of a similar amount of strain 17-2 at the same growth phase did
not induce abscesses in mice. A much higher cell concentration (109 CFU/ml) of strain 17-2 was required to induce abscesses in mice (Fig 8, right panel). However, an injection of a similar concentration of strain 17 was lethal for mice (data not shown). Figure 8 Abscess induction Selleckchem PF-4708671 in mice. Abscess formation was induced when 0.5 ml of bacterial cell suspension (3 × 107 CFU/ml) of strain 17 was injected into the inguinal area of a mouse (left panels). In contrast, the subcutaneous injection of strain 17-2 (0.5 ml at a concentration of 107 and 108 CFU/ml) failed to induce an abscess in mice (right panels). Relatively small abscesses were induced when a
higher cell concentration of strain 17-2 (109 CFU/ml) was injected (right bottom panel). The data are from one of three independent experiments. Internalization of bacterial cells by human PMNLs In the phagocytosis experiments, strain 17 cells were rarely internalized, though many of these cells were bound to the cell surface of PMNLs (Fig. 9A). In contrast, strain 17-2 cells were readily Z-VAD-FMK internalized by PMNLs after 90 min incubation. Many of these bacteria were found in cytoplasmic Verteporfin solubility dmso vacuoles (Fig. 9B). Figure 9 Resistance of viscous material-producing strain 17 against the phagocytic activity of human neutrophils. Strain 17 cells were not internalized by neutrophils though many of these cells were bound to the cell surface of neutrophils (A, arrows). In contrast, viscous material non-producing strain 17-2 cells were internalized and the ingested bacteria appear to be enclosed within cytoplasmic vacuoles (B, asterisks). Bars = 2.8 μm. Gene expression profiles of strain 17 in biofilm in vitro We next attempted to compare gene expression patterns of strain 17 between in biofilm and in planktonic conditions in vitro. Total RNA was isolated from 12 h cultures of strain 17 on solid culture media as its biofilm-forming cells and liquid cultures as planktonic cells, respectively.