Altered Virulence of Biofilm Pneumococci Figure 1. Visual characterization of S. pneumoniae mature biofilm development in vitro. Scanning electron microscipy images representative (n = 6) of mature S. pneumoniae (TIGR4) biofilms developed in vitro under once through conditions. Images of biofilm growth at 4 h (A), 12 h (B), 24 h (C) and 48 h (D) are depicted. Transmission electron microscopy image of a cross section of a mature pneumococcal biofilm grown for 48 h is shown (E). Note the demarcation of the outer matrix edge (black arrows), the presence of viable and dead pneumococci enveloped within the matrix, and the presence of surface exposed diplococci available for dispersal (within black box). (F) Tolerance to the killing effect of the designated antimicrobials was determined by measuring the concentration for which .103 CFU were viable after 5 h’s of incubation with the antibiotic. Wells were inoculated with 105 CFU/ml. Results from three independent experiments are shown. Statistical analysis was performed using a two-tailed Student’s t-test. doi:10.1371/journal.pone.0028738.g001 antibiotic resistance assays are performed over a 16–24 hour period, this was not done as the biofilm phenotype would be lost over this extended time-period and a shortened testing period has been accepted as a viable method for testing the biofilm phenotype [37]. In contrast to TIGR4 grown in a planktonic state, biofilm derived bacteria were highly tolerant to a 5 h exposure to erythromycin, clindamycin, and to a modest level of penicillin. Biofilm and planktonic bacteria were equally susceptible to cefazolin and vancomycin. Notably, the observed tolerence to protein translation inhibitors was consistent with other published biofilm studies [16,29]. Thus these experiments served as verification that our biofilm model was valid and that we would be examining mature biofilm bacteria in our subsequent experiments. Biofilm-derived bacteria are avirulent Having confirmed our ability to grow mature biofilms, we tested whether biofilm pneumococci were virulent when compared to their planktonic counterparts. Following intranasal challenge of mice with equivalent CFU of TIGR4, no difference between the biofilm and planktonic cultures could be discerned in nasal lavage counts (Figure 2A), or in their ability to form the previously described biofilm-like aggregates within the nasopharynx (n = 6/ cohort; data not shown) [11]. Importantly, only those mice that received planktonic pneumococci proceeded to develop bacter- emia and died. At 24, 72 and 120 h 4, 7, and 7 of the 9 mice infected intranasal with planktonic bacteria (or 44%, 77%, and 77% respectively) had either positive blood cultures or had succumbed to infection. In contrast, none of the biofilm-infected PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28738