discrepancy is the accumulation of dead bacteria and their proteins in a mature biofilm. This would be an inseparable and confounding factor in any proteomic analysis of mature biofilms. In summary, we observed a dramatic enhancement in the ability of biofilm pneumococci to attach to host cells as well as a dramatic reduction in their ability to cause invasive disease. Notably, biofilm pneumococci colonized the nasopharynx normally. As the vast majority of S. pneumoniae do not cause invasive disease, it is most likely that these biofilm related changes occur so as to facilitate long-term colonization of the nasopharynx rather than promote development of invasive disease. Therefore, and based on the available information, we suggest that the ability to form robust biofilms is not required for virulence, but instead contributes towards long-term colonization and transmission of the pneumococcus. Supporting Information Figure S1 Quantification of viable and dead cells during mature pneumococcal biofilm development. (A) Repre- sentative Transmission Electron Microscopy (TEM) images of biofilm bacteria at designated time points; white bars represent 2 microns. Note that viable cells are electron dense, while dead cells appear as lighter images or ‘‘ghosts’’. (B) Enumeration of viable and dead cells during mature biofilm development. Percentages of viable and dead pneumococci as determined by cell counts from six images per indicated time point. (EPS) Figure S2 Assessment of the quality of isolated bacterial RNA from mature pneumococcal biofilms. (A) Formalde- hyde gels demonstrating the quality of RNA samples taken from S. pneumoniae biofilms grown under once-through conditions at the designated time points. (B) RNA profiles of samples collected from biofilms above using the Agilent 2100 Bioanalyzer. Note the considerable amount of degradation of RNAs at the later time points. (EPS) References 1. WHO (1999) Pneumococcal vaccines. WHO position paper. Wkly Epidemiol Rec 74: 177–183. 2. Lexau CA, Lynfield R, Danila R, Pilishvili T, Facklam R, et al. (2005) Changing epidemiology of invasive pneumococcal disease among older adults in the era of pediatric pneumococcal conjugate vaccine. JAMA 294: 2043–2051. 3. Overturf G, Powars D (1980) Infections in sickle cell anemia: pathogenesis and control. Tex Rep Biol Med 40: 283–292. 4. Wong WY, Overturf GD, Powars DR (1992) Infection caused by Streptococcus pneumoniae in children with sickle cell disease: epidemiology, immunologic mechanisms, prophylaxis, and vaccination. Clin Infect Dis 14: 1124–1136. 5. O’Brien KL, Wolfson LJ, Watt JP, Henkle E, Deloria-Knoll M, et al. (2009) Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. Lancet 374: 893–902. 6. Roush SW, Murphy TV (2007) Historical comparisons of morbidity and mortality for vaccine-preventable diseases in the United States. JAMA 298: 2155–2163. 7. Maruyama T, Gabazza EC, Morser J, Takagi T, D’Alessandro-Gabazza C, et al. (2010) Community-acquired pneumonia and nursing home-acquired pneumonia in the very elderly patients. Respir Med 104: 584–592. 8. Hall-Stoodley L, Hu FZ, Gieseke A, Nistico L, Nguyen D, et al. (2006) Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media. JAMA 296: 202–211. 9. Hoa M, Tomovic S, Nistico L, Hall-Stoodley L, Stoodley P, et al. (2009) Identification of adenoid biofilms with middle ear pathogens in otitis-prone children utilizing SEM and FISH. Int J Pediatr Otorhinolaryngol 73: 1242–1248. 10. Reid SD, Hong W, Dew KE, Winn DR, Pang B, et al. (2009) Streptococcus pneumoniae forms surface-attached communities in the middle ear of experimen- tally infected chinchillas. J Infect Dis 199: 786–794. 11. Sanchez CJ, Shivshankar P, Stol K, Trakhtenbroit S, Sullam PM, et al. (2010) The pneumococcal serine-rich repeat protein is an intra-species bacterial adhesin that promotes bacterial aggregation in vivo and in biofilms. PLoS Pathog 6. Altered Virulence of Biofilm Pneumococci Figure S3 Validation of microarray studies. RNA levels obtained with microarrays (X-axis) and qRT-PCR (y-axis) are in good agreement. The qRT-PCR DCt values (Livak and Schmitt- gen, 2001) (y-axis) are compared to the log2 transformation of microarray query/reference ratios (x-axis) for each biological replicate (2–3) of each time point (4–48 h). Log2 values are used to obtain a linear correlation. Strong correlation coefficients are observed, ranging from 0.66 to 0.94, except for at the 4 h replicate 1 at 0.59. (EPS) Table S1 Genes with significantly decreased expression in at least one time point during biofilm growth. (DOCX) Table S2 Genes with significantly increased expression in at least one time point during biofilm growth. (DOCX) Table S3 Relative expression of S. pneumoniae genes following biofilm growth versus planktonic cultures as determined by microarray and qRT-PCR DCT values. (DOCX) Acknowledgments We thank Dr. Elaine Tuomanen at St. Jude Children’s Research Hospital for the gift of antibodies against CbpA. We also thank Ms. Barbara Hunter at the University of Texas Health Science Center Electron Microscopy Core for her assistance in processing samples for analyses. Author Contributions Conceived and designed the experiments: CJS JHJ HT CJO. Performed the experiments: CJS NK AL PS JCDH. Analyzed the data: CJS NK HT CJO. Contributed reagents/materials/analysis tools: JHJ HT CJO. Wrote the paper: CJS HT CJO. 12. Camilli R, Pantosti A, Baldassarri L (2010) Contribution of serotype and genetic background to biofilm formation by Streptococcus pneumoniae. Eur J Clin Microbiol Infect Dis 30: 97–102. 13. Allegrucci M, Sauer K (2007) Characterization of colony morphology variants isolated from Streptococcus pneumoniae biofilms. J Bacteriol 189: 2030–2038. 14. Oggioni MR, Trappetti C, Kadioglu A, Cassone M, Iannelli F, et al. (2006) Switch from planktonic to sessile life: a major event in pneumococcal pathogenesis. Mol Microbiol 61: 1196–1210. 15. Parker D, Soong G, Planet P, Brower J, Ratner AJ, et al. (2009) The NanA neuraminidase of Streptococcus pneumoniae is involved in biofilm formation. Infect Immun 77: 3722–3730. 16. del Prado G, Ruiz V, Naves P, Rodriguez-Cerrato V, Soriano F, et al. (2010) Biofilm formation by Streptococcus pneumoniae strains and effects of human serum albumin, ibuprofen, N-acetyl-l-cysteine, amoxicillin, erythromycin, and levo- floxacin. Diagn Microbiol Infect Dis 67: 311–318. 17. Munoz-Elias EJ, Marcano J, Camilli A (2008) Isolation of Streptococcus pneumoniae biofilm mutants and their characterization during nasopharyngeal colonization. Infect Immun 76: 5049–5061. 18. Trappetti C, Kadioglu A, Carter M, Hayre J, Iannelli F, et al. (2009) Sialic acid: a preventable signal for pneumococcal biofilm formation, colonization, and invasion of the host. J Infect Dis 199: 1497–1505. 19. Trappetti C, Ogunniyi AD, Oggioni MR, Paton JC (2011) Extracellular Matrix Formation Enhances the Ability of Streptococcus pneumoniae to Cause Invasive Disease. PloS one 6: e19844. 20. Lizcano A, Chin T, Sauer K, Tuomanen EI, Orihuela CJ (2010) Early biofilm formation on microtiter plates is not correlated with the invasive disease potential of Streptococcus pneumoniae. Microb Pathog 48: 124–130. 21. Tapiainen T, Kujala T, Kaijalainen T, Ikaheimo I, Saukkoriipi A, et al. (2010) Biofilm formation by Streptococcus pneumoniae isolates from paediatric patients. APMIS 118: 255–260. 22. Briles DE, Crain MJ, Gray BM, Forman C, Yother J (1992) Strong association between capsular type and virulence for mice among human isolates of Streptococcus pneumoniae. Infect Immun 60: 111–116. PLoS ONE | www.plosone.org 12 December 2011 | Volume 6 | Issue 12 | e28738