animals had detectable bacteria in the blood at any time tested. This difference in virulence potential was verified by intratracheal challenge of mice (Figure 2B). All mice receiving planktonic bacteria developed pneumonia and bacteremia, whereas the majority of the mice infected with biofilm derived bacteria had low levels of bacteria in their lungs and no detectable levels of bacteria in the blood. The biofilm-infected mice all successfully cleared the infection. Finally, 24 h after intraperitoneal challenge, 43% of the mice challenged with planktonic bacteria had died, whereas all of the biofilm bacteria remained alive (Figure 2C). Thus, despite challenge with equal bacterial titers as determined by CFU, mice infected with biofilm-derived pneumococci cleared infection, indicating that the biofilm phenotype was attenuated for invasive disease but not for colonization. Biofilm pneumococci are hyper-adhesive We tested whether the decreased virulence of biofilm bacteria was due to an inability to bind epithelial cells. Biofilm-derived bacteria had a 9-fold and 12-fold greater ability to adhere to A549 and Detroit 562 cells versus their planktonic counterparts, respectively (Figure 3A). Using clinical isolates of 3 additional Altered Virulence of Biofilm Pneumococci serotypes (i.e. 6A, 14 and 15), we confirmed that the hyper- adhesive biofilm state occurred in both a serotype and strain- independent manner to both cell lines (Figure 3B). Thus, an inability to attach to host cells was not responsible for the attenuated virulence of biofilm pneumococci. Temporal biofilm-related changes in gene expression To determine why biofilm pneumococci were avirulent we compared pneumococcal gene expression after 4, 12, 24 and 48 h of biofilm growth versus that of planktonic exponential phase cultures using microarrays. RNA isolated from our exponential phase TIGR4 seed cultures and after 4 or 12 h of biofilm growth could be obtained at high yields and in an intact state. In contrast, samples collected from the 24 and 48 h biofilms demonstrated substantial and escalating levels of total RNA degradation (Figure S2). The levels of degraded RNA were consistent with the increasing numbers of dead bacteria observed by transmission electron microscopy at these time points (Figure S1). Thus this suggested that the degraded RNA was most likely isolated from the dead or dying pneumococci found within these biofilms. Figure 2. Biofilm-derived pneumococci are unable to cause invasive disease. Bacterial titers were measured for individual 5-week old female BALB/cJ mice infected with either planktonic (dark circles), biofilm-derived (white circles), or biofilm-derived planktonic (grey circles) S. pneumoniae TIGR4 via intranasal (106 CFU; n = 10) (A), intratracheal (105 CFU; n = 6–8) (B), or intraperitoneal (104 CFU n = 6–8) (C) routes. For intranasal challenge, nasal lavages and blood were collected at 24, 72, and 120 h post-infection. For intratracheal and intraperitoneal challenge, samples were collected at 24 h post-infection. Horizontal bars represent the median value. Statistical analysis was performed using a two-tailed Student’s t-test. doi:10.1371/journal.pone.0028738.g002 PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e28738