Tapianen et al., Camilli et al., and ourselves, have found no correlation between the ability of isolates to form robust biofilms in vitro and virulence potential in humans and mice [12,20,21]. Thus, experiments directly testing the virulence potential of pneumo- coccal biofilms are needed to confirm or disprove their role during IPD. In this study, we show that biofilm pneumococci are capable of colonizing the nasopharynx yet unable to cause invasive disease. We show this to be in part the result of altered production of capsular polysaccharide (CPS) [22], pneumolysin [23], cell wall phosphorylcholine (ChoP) [24], Choline binding protein A (CbpA) [25], and Pneumococcal serine-rich repeat protein (PsrP) [26]. Our findings suggest a limited role for biofilms during IPD and provide information on how biofilm pneumococci might modulate their interactions with the host during nasopharyngeal coloniza- tion to support long-term quiescent colonization. Importantly, due to altered virulence determinant production by biofilm pneumo- cocci, our findings have important implications towards the selection of protein antigens for any next-generation vaccine against S. pneumoniae. Methods Bacterial strains and growth conditions Streptococcus pneumoniae serotype 4, strain TIGR4, T4R its unencapsulated derivative, T4 DpsrP a psrP deficient mutant, R6 an un-encapsulated serotype 2 laboratory strain, A66.1 a serotype 3 isolate, and all the clinical isolates used in this study have been previously described [22,27,28]. Bacterial strains were grown on tryptic soy blood agar plates (Remel, USA) at 37uC in 5% CO2. For planktonic growth, Todd Hewitt Broth (THB) was inoculated with overnight plate cultures and grown to mid-logarithmic phase (OD620 = 0.5; ,1.06108 CFU/ml) using normal culture condi- tions. Mature S. pneumoniae biofilms were grown under once- through flow conditions using a once-through biofilm line reactor, as previously described [11]. Briefly, planktonic seed cultures were used to inoculate 1 meter long silicone tubing (0.89 mm internal diameter, Cole Parmer Inc.). Bacteria in the line were allowed to attach for 2 h after which the flow rate of media was adjusted to 0.035 ml/min. Bacterial biofilms were grown for up to 2 days at 37uC in 5% CO2. Biofilm-derived bacteria were harvested from the line by pinching the tube along its entire length, thereby removing the bacterial cells. Frozen stocks of both biofilm and planktonic derived bacteria were made in THB containing 12% glycerol (vol/vol) and stored at 280uC. For animal experiments with biofilm-derived planktonic pneu- mococci, glass test tubes containing THB were inoculated with biofilm pneumococci at 105 colony forming units (CFU)/ml. Similar to the planktonic cultures, at mid-logarithmic growth phase frozen stocks were created and stored. In all instances viable bacterial CFU counts were determined by thawing aliquots, and plating serial dilutions. Scanning and transmission electron microscopy images of mature biofilms Following growth of biofilms within the biofilm reactor lines, the line containing the biofilms were cut in half to expose the lumen, fixed for 2 h with 2.5% glutaraldehyde in PBS, and then rinsed twice for 3 min in 0.1 M phosphate buffer (pH 7.4). Samples were submerged in 1% osmium tetroxide diluted in Zetterquist’s Buffer for 30 min then washed with the same buffer for 2 min. This was followed by stepwise dehydration with ethanol (i.e. 70%, 95%, and 100%); the first two steps for 15 min, the last for 30 min. Samples were treated with hexamethyldisilizane for 5 min prior to drying Altered Virulence of Biofilm Pneumococci in a desiccator overnight. The next day samples were sputter coated with gold palladium and viewed with a JEOL-6610 scanning electron microscope [11]. For transmission electron microscopy, biofilm derived bacteria grown under once through conditions as above were harvested at the indicated time points and fixed with a fixation solution containing 4% formaldehyde and 1% glutaraldehyde for 1 h. Following several washes the samples were then dehydrated with a graded series of acetone (10, 30, 50, 70, 90, and 100%) on ice for 15 min for each step and embedded within acrylic resin. Ultrathin sections of samples were cut with a diamond knife, and placed onto Formvar-coated copper grids (300 mesh). Counterstaining of the sections was performed with 4% aqueous uranyl acetate for 5 min. After air-drying, samples were examined with a JEOL 100CX transmission electron microscope. Antimicrobial susceptibility of biofilm and planktonic bacteria The effect of various antimicrobials was tested on biofilm and planktonic bacteria in 96-well flat bottom plates using a modified version of the standard microdilution assay including: erythromy- cin (0.015–32 mg/ml), clindamycin (0.015–32 mg/ml), penicillin (0.03–8 mg/ml), cefazolin (0.03–8 mg/ml), and vancomycin (0.06– 4 mg/ml) [29]. Briefly, planktonic or biofilm derived bacteria were suspended in 10 ml of 0.85% saline and directly inoculated into 96 well plates containing a pre-diluted antibiotic in 100 ml of Cation- adjusted Mueller-Hinton Broth enriched with 3% lysed horse blood such that the final titer was 105 CFU/ml. The plates were then incubated at 37uC for 5 h. Following incubation the content of each well was diluted and plated onto blood agar plates for colony counting. Bacterial susceptibility was determined by measuring the concentration for which ,103 CFU/ml were thereafter viable. All experiments were performed in triplicate and the results are expressed as the average values. Virulence studies in mice All animal experimentation were conducted following the National Institutes for Health guidelines for housing and care of laboratory animals. Animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at The University of Texas Health Science Center at San Antonio; protocol number 09022-34. Female BALB/cJ mice (The Jackson Laboratories) of 5 to 6 week of age were anesthetized with 2.5% isoflurane and infected with either planktonic, biofilm, or biofilm-derived planktonic pneumococci suspended in PBS. For intranasal challenge (n = 10/cohort), each mouse was infected drop wise into the left nare with 106 CFU in 25 ml of PBS. On days 1, 3, and 5 bacterial titers in the nasopharynx and blood were determined by nasopharyngeal lavage with 10 ml saline or collection of blood from the tail vein and plating of serial dilutions, respectively. For intratracheal challenge (n = 6–9/cohort) 105 CFU in 100 ml PBS was instilled into the lungs by forced aspiration; aspiration was induced by gently pulling the tongue of anesthetized mice outward, placing the bacterial suspension in the throat, and covering the nostrils. Mice were sacrificed 24 h post-challenge and bacterial titers in the lungs and blood determined. Bacterial titer in the lungs was determined by plating serial dilutions of lung homogenates and normalized per gram of total tissue. Finally, for intraperitoneal challenge (n = 6–7/cohort), mice were injected with 104 CFU in 100 ml PBS using a 27-gauge needle. Bacterial titers in the blood were determined at 24 h post challenge by plating of serial dilutions of blood collected from the tail vein. PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28738