Streptococcus pneumoniae in Biofilms Are Unable to Cause Invasive Disease Due to Altered Virulence Determinant Production Carlos J. Sanchez1, Nikhil Kumar2, Anel Lizcano 1 , Pooja Shivshankar1, Julie C. Dunning Hotopp 2 , James H. Jorgensen1,3, Herve´ Tettelin2, Carlos J. Orihuela1* 1 Department of Microbiology and Immunology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America, 2 Department of Microbiology and Immunology, Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 3 Department of Pathology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America Abstract It is unclear whether Streptococcus pneumoniae in biofilms are virulent and contribute to development of invasive pneumococcal disease (IPD). Using electron microscopy we confirmed the development of mature pneumococcal biofilms in a continuous-flow-through line model and determined that biofilm formation occurred in discrete stages with mature biofilms composed primarily of dead pneumococci. Challenge of mice with equal colony forming units of biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Biofilm pneumococci of numerous serotypes were hyper-adhesive and bound to A549 type II pneumocytes and Detroit 562 pharyngeal epithelial cells at levels 2 to 11-fold greater than planktonic counterparts. Using genomic microarrays we examined the pneumococcal transcriptome and determined that during biofilm formation S. pneumoniae down-regulated genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide (CPS) production, and virulence. We confirmed these changes by measuring CPS by ELISA and immunoblotting for the toxin pneumolysin and the bacterial adhesins phosphorylcholine (ChoP), choline-binding protein A (CbpA), and Pneumococcal serine-rich repeat protein (PsrP). We conclude that biofilm pneumococci were avirulent due to reduced CPS and pneumolysin production along with increased ChoP, which is known to bind C-reactive protein and is opsonizing. Likewise, biofilm pneumococci were hyper-adhesive due to selection for the transparent phase variant, reduced CPS, and enhanced production of PsrP, CbpA, and ChoP. These studies suggest that biofilms do not directly contribute to development of IPD and may instead confer a quiescent mode of growth during colonization. Citation: Sanchez CJ, Kumar N, Lizcano A, Shivshankar P, Dunning Hotopp JC, et al. (2011) Streptococcus pneumoniae in Biofilms Are Unable to Cause Invasive Disease Due to Altered Virulence Determinant Production. PLoS ONE 6(12): e28738. doi:10.1371/journal.pone.0028738 Editor: Indranil Biswas, University of Kansas Medical Center, United States of America Received June 8, 2011; Accepted November 14, 2011; Published December 8, 2011 Copyright: 2011 Sanchez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health grants DE14318 to CJS and AI078972 to CJO. Work completed by NK, JCDH and HT was supported by University of Maryland internal funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: orihuela@uthscsa.edu Introduction Streptococcus pneumoniae (the pneumococcus) is a leading cause of otitis media, community-acquired pneumonia, sepsis and menin- gitis. S. pneumoniae typically colonizes the human nasopharynx asymptomatically with invasive pneumococcal disease (IPD) occurring as a result of dissemination to, and bacterial replication at, normally sterile sites including the middle ear, lungs, and bloodstream. IPD is opportunistic in nature and primarily occurs in infants, the elderly, and those with underlying medical conditions [1,2,3,4]. Worldwide the pneumococcus is responsible for more than 14.5 million episodes of IPD annually and up to 11% of all deaths in children [5,6]. Notably, in individuals .65 years of age the case-fatality rate for IPD can be as high as 30% [7]. Thus pneumococcal infections are a major medical problem for both children and the elderly. S. pneumoniae biofilm formation has been shown to occur in humans during nasopharyngeal colonization and recurrent otitis media. Pneumococcal biofilms have been detected in human sinus mucosa biopsies, resected adenoids from individuals with tonsil- litis, and biofilms have been observed within tympanostomy tubes collected from children with chronic otitis media [8,9]. Fulfilling Koch’s postulates, biofilms and biofilm-like pneumococcal aggre- gates have been observed in the middle ears of experimentally infected chinchillas as well as bronchial and nasal lavage fluids taken from the nasopharynx of infected mice, respectively [10,11]. Thus biofilm formation is a naturally occurring, if not yet fully understood, biological mechanism for S. pneumoniae. During the past 10 years considerable effort has gone towards dissecting the molecular mechanisms underlying biofilm develop- ment in vitro and its recalcitrance to antimicrobial therapy in vivo [12,13,14,15,16]. Importantly, and despite these considerable findings, whether biofilm formation contributes towards the development of IPD remains unclear. For example, studies by Munoz-Elisa et al., Parker et al., and Trappetti et al., indicate that genes required for robust biofilm formation in vitro are important for nasopharyngeal colonization and in some instances progression towards lung disease [15,17,18,19]. In contrast, studies by PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28738