23. Rubins JB, Charboneau D, Paton JC, Mitchell TJ, Andrew PW, et al. (1995) Dual function of pneumolysin in the early pathogenesis of murine pneumococcal pneumonia. J Clin Invest 95: 142–150. 24. Weiser JN, Markiewicz Z, Tuomanen EI, Wani JH (1996) Relationship between phase variation in colony morphology, intrastrain variation in cell wall physiology, and nasopharyngeal colonization by Streptococcus pneumoniae. Infect Immun 64: 2240–2245. 25. Orihuela CJ, Gao G, Francis KP, Yu J, Tuomanen EI (2004) Tissue-specific contributions of pneumococcal virulence factors to pathogenesis. J Infect Dis 190: 1661–1669. 26. Shivshankar P, Sanchez C, Rose LF, Orihuela CJ (2009) The Streptococcus pneumoniae adhesin PsrP binds to Keratin 10 on lung cells. Mol Microbiol 73: 663–679. 27. Cundell DR, Gerard C, Idanpaan-Heikkila I, Tuomanen EI, Gerard NP (1996) PAf receptor anchors Streptococcus pneumoniae to activated human endothelial cells. Adv Exp Med Biol 416: 89–94. 28. Hoskins J, Alborn WE, Jr., Arnold J, Blaszczak LC, Burgett S, et al. (2001) Genome of the bacterium Streptococcus pneumoniae strain R6. J Bacteriol 183: 5709–5717. 29. Garcia-Castillo M, Morosini MI, Valverde A, Almaraz F, Baquero F, et al. (2007) Differences in biofilm development and antibiotic susceptibility among Streptococcus pneumoniae isolates from cystic fibrosis samples and blood cultures. J Antimicrob Chemother 59: 301–304. 30. Rose L, Shivshankar P, Hinojosa E, Rodriguez A, Sanchez CJ, et al. (2008) Antibodies against PsrP, a novel Streptococcus pneumoniae adhesin, block adhesion and protect mice against pneumococcal challenge. J Infect Dis 198: 375–383. 31. Jiang SM, Ishmael N, Hotopp JD, Puliti M, Tissi L, et al. (2008) Variation in the Group B Streptococcus CsrRS regulon and effects on pathogenicity. J Bacteriol 190: 1956–1965. 32. Saeed AI, Sharov V, White J, Li J, Liang W, et al. (2003) TM4: a free, open- source system for microarray data management and analysis. Biotechniques 34: 374–378. 33. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408. 34. Kim JO, Weiser JN (1998) Association of intrastrain phase variation in quantity of capsular polysaccharide and teichoic acid with the virulence of Streptococcus pneumoniae. J Infect Dis 177: 368–377. 35. Stewart PS, Costerton JW (2001) Antibiotic resistance of bacteria in biofilms. Lancet 358: 135–138. 36. Stewart PS (2002) Mechanisms of antibiotic resistance in bacterial biofilms. Int J Med Microbiol 292: 107–113. 37. Allison KR, Brynildsen MP, Collins JJ (2011) Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature 473: 216–220. 38. Gilbert P, Maira-Litran T, McBain AJ, Rickard AH, Whyte FW (2002) The physiology and collective recalcitrance of microbial biofilm communities. Adv Microb Physiol 46: 202–256. 39. Hall-Stoodley L, Nistico L, Sambanthamoorthy K, Dice B, Nguyen D, et al. (2008) Characterization of biofilm matrix, degradation by DNase treatment and evidence of capsule downregulation in Streptococcus pneumoniae clinical isolates. BMC Microbiol 8: 173. 40. Obert C, Sublett J, Kaushal D, Hinojosa E, Barton T, et al. (2006) Identification of a candidate Streptococcus pneumoniae core genome and regions of diversity correlated with invasive pneumococcal disease. Infect Immun 74: 4766–4777. 41. Bensing BA, Gibson BW, Sullam PM (2004) The Streptococcus gordonii platelet binding protein GspB undergoes glycosylation independently of export. J Bacteriol 186: 638–645. 42. Bensing BA, Sullam PM (2002) An accessory sec locus of Streptococcus gordonii is required for export of the surface protein GspB and for normal levels of binding to human platelets. Mol Microbiol 44: 1081–1094. 43. Bensing BA, Takamatsu D, Sullam PM (2005) Determinants of the streptococcal surface glycoprotein GspB that facilitate export by the accessory Sec system. Mol Microbiol 58: 1468–1481. 44. Spellerberg B, Cundell DR, Sandros J, Pearce BJ, Idanpaan-Heikkila I, et al. (1996) Pyruvate oxidase, as a determinant of virulence in Streptococcus pneumoniae. Mol Microbiol 19: 803–813. 45. Ren B, Szalai AJ, Thomas O, Hollingshead SK, Briles DE (2003) Both family 1 and family 2 PspA proteins can inhibit complement deposition and confer virulence to a capsular serotype 3 strain of Streptococcus pneumoniae. Infect Immun 71: 75–85. 46. Orihuela CJ, Mahdavi J, Thornton J, Mann B, Wooldridge KG, et al. (2009) Laminin receptor initiates bacterial contact with the blood brain barrier in experimental meningitis models. J Clin Invest 119: 1638–1646. Altered Virulence of Biofilm Pneumococci 47. Zhang JR, Mostov KE, Lamm ME, Nanno M, Shimida S, et al. (2000) The polymeric immunoglobulin receptor translocates pneumococci across human nasopharyngeal epithelial cells. Cell 102: 827–837. 48. Rosenow C, Ryan P, Weiser JN, Johnson S, Fontan P, et al. (1997) Contribution of novel choline-binding proteins to adherence, colonization and immunoge- nicity of Streptococcus pneumoniae. Mol Microbiol 25: 819–829. 49. Camara M, Boulnois GJ, Andrew PW, Mitchell TJ (1994) A neuraminidase from Streptococcus pneumoniae has the features of a surface protein. Infect Immun 62: 3688–3695. 50. Moschioni M, Emolo C, Biagini M, Maccari S, Pansegrau W, et al. (2010) The two variants of the Streptococcus pneumoniae pilus 1 RrgA adhesin retain the same function and elicit cross-protection in vivo. Infect Immun 78: 5033–5042. 51. Hava DL, Hemsley CJ, Camilli A (2003) Transcriptional regulation in the Streptococcus pneumoniae rlrA pathogenicity islet by RlrA. J Bacteriol 185: 413–421. 52. Trappetti C, Gualdi L, Di Meola L, Jain P, Korir CC, et al. (2011) The impact of the competence quorum sensing system on Streptococcus pneumoniae biofilms varies depending on the experimental model. BMC Microbiol 11: 75. 53. Hammerschmidt S, Wolff S, Hocke A, Rosseau S, Muller E, et al. (2005) Illustration of pneumococcal polysaccharide capsule during adherence and invasion of epithelial cells. Infect Immun 73: 4653–4667. 54. Gosink KK, Mann ER, Guglielmo C, Tuomanen EI, Masure HR (2000) Role of novel choline binding proteins in virulence of Streptococcus pneumoniae. Infect Immun 68: 5690–5695. 55. Casey R, Newcombe J, McFadden J, Bodman-Smith KB (2008) The acute- phase reactant C-reactive protein binds to phosphorylcholine-expressing Neisseria meningitidis and increases uptake by human phagocytes. Infect Immun 76: 1298–1304. 56. Li-Korotky HS, Lo CY, Banks JM (2010) Interaction of pneumococcal phase variation, host and pressure/gas composition: virulence expression of NanA, HylA, PspA and CbpA in simulated otitis media. Microb Pathog 49: 204–210. 57. Ramirez P, Ferrer M, Torres A (2007) Prevention measures for ventilator- associated pneumonia: a new focus on the endotracheal tube. Curr Opin Infect Dis 20: 190–197. 58. Heo SM, Haase EM, Lesse AJ, Gill SR, Scannapieco FA (2008) Genetic relationships between respiratory pathogens isolated from dental plaque and bronchoalveolar lavage fluid from patients in the intensive care unit undergoing mechanical ventilation. Clin Infect Dis 47: 1562–1570. 59. Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a common cause of persistent infections. Science 284: 1318–1322. 60. Cundell DR, Gerard NP, Gerard C, Idanpaan-Heikkila I, Tuomanen EI (1995) Streptococcus pneumoniae anchor to activated human cells by the receptor for platelet-activating factor. Nature 377: 435–438. 61. Tong HH, James M, Grants I, Liu X, Shi G, et al. (2001) Comparison of structural changes of cell surface carbohydrates in the eustachian tube epithelium of chinchillas infected with a Streptococcus pneumoniae neuraminidase- deficient mutant or its isogenic parent strain. Microb Pathog 31: 309–317. 62. Glover DT, Hollingshead SK, Briles DE (2008) Streptococcus pneumoniae surface protein PcpA elicits protection against lung infection and fatal sepsis. Infect Immun 76: 2767–2776. 63. Moscoso M, Garcia E, Lopez R (2006) Biofilm formation by Streptococcus pneumoniae: role of choline, extracellular DNA, and capsular polysaccharide in microbial accretion. J Bacteriol 188: 7785–7795. 64. Weiser JN, Austrian R, Sreenivasan PK, Masure HR (1994) Phase variation in pneumococcal opacity: relationship between colonial morphology and nasopha- ryngeal colonization. Infect Immun 62: 2582–2589. 65. King SJ, Hippe KR, Gould JM, Bae D, Peterson S, et al. (2004) Phase variable desialylation of host proteins that bind to Streptococcus pneumoniae in vivo and protect the airway. Mol Microbiol 54: 159–171. 66. Allegrucci M, Sauer K (2008) Formation of Streptococcus pneumoniae non-phase- variable colony variants is due to increased mutation frequency present under biofilm growth conditions. J Bacteriol 190: 6330–6339. 67. Crook DW, Brueggemann AB, Sleeman KL, Peto TEA (2004) Pneumococcal Carriage. In: Tuomanen EI, Mitchell TJ, Morrison DA, Spratt BG, eds. The Pneumococcus. Washington D.C.: ASM Presss. pp 136–147. 68. Harro JM, Peters BM, O’May GA, Archer N, Kerns P, et al. (2010) Vaccine development in Staphylococcus aureus: taking the biofilm phenotype into consideration. FEMS Immunol Med Microbiol 59: 306–323. 69. Konto-Ghiorghi Y, Mairey E, Mallet A, Dumenil G, Caliot E, et al. (2009) Dual role for pilus in adherence to epithelial cells and biofilm formation in Streptococcus agalactiae. PLoS Pathog 5: e1000422. 70. Manetti AG, Zingaretti C, Falugi F, Capo S, Bombaci M, et al. (2007) Streptococcus pyogenes pili promote pharyngeal cell adhesion and biofilm formation. Mol Microbiol 64: 968–983. PLoS ONE | www.plosone.org 13 December 2011 | Volume 6 | Issue 12 | e28738