transparent pneumococci present in our biofilms facilitated the opsonophagocytosis of the attached opaque bacteria. This would suggest that naturally occurring mixed biofilms are avirulent. Alternatively, is our use of a continuous flow reactor for mature biofilm development; Trappetti et al. used a static biofilm model. In separate studies both Trappetti et al. and ourselves found that that the use of different biofilm models resulted in variable phenotypes [11,52]. Finally, is our use of the TIGR4 strain of S. pneumoniae whereas Trappetti et al. used a 19F clinical isolate. Despite our observation of considerable EPM surrounding the electron dense and presumably viable bacteria, biofilm pneumo- cocci were determined to be hyper-adhesive, suggesting that in addition to a loss in capsule and increased ChoP, CbpA, and PsrP protein levels by pneumococci, the EPM may also contain adhesive elements. This possibility is also supported by findings by Trappetti et al., which determined that opaque sessile (i.e biofilm) pneumococci adhere to A549 and Detroit 562 cells better than transparent sessile pneumococci. The latter was unexpected as the transparent phenotype is associated with increased expression of CbpA and ChoP and transparent planktonic pneumococci have been shown to adhere to cells in an enhanced manner [24,34,64]. It is for this latter reason that we believe mice challenged with biofilm-derived planktonic pneumococci, which would be mostly transparent, developed pneumonia but were unable to cause bloodstream infection. Importantly, our observa- tion of enhanced biofilm adhesion by numerous strains and enhanced PsrP, ChoP, and CbpA production indicates that the hyper-adhesive phenotype is a pan-pneumococcal biofilm property that is multi-factorial, involving numerous components along with the production of EPM. In an effort to develop a working model that coalesces the published data with our own, we propose that the selective death of opaque pneumococci might be occurring during biofilm formation. Opaque cell death would provide a mechanism for the release of DNA and other components that are known make up the EPM. It would also provide an explanation for the high numbers of dead bacteria observed in our biofilms as well as our recovery of predominantly transparent pneumococci from mature biofilms. In context of in vivo transmission our model implies that the opaque variant would be responsible for formation of the EPM in the nasopharynx and thereby confer in vivo persistence, whereas the transparent variant, which is better able to attach to cells and colonize na¨ıve animals [34], would remain available in greater numbers for spread to the next host and gain from the enhanced adhesive capacity of the surrounding EPM. In the next host, partial reversion to the opaque variant would t also be necessary to reform a biofilm. Thus, studies are warranted to ascertain if differential cell death dependent on phase-variation occurs within biofilms and to test its impact on transmission. The observed tolerance of biofilm pneumococci to antimicro- bials was in agreement with previously published studies [29], moreover, was indicative that we were in fact examining mature biofilms. Importantly, biofilm pneumococci remained susceptible to cell wall acting antimicrobials suggesting that maintenance of the cell wall remained a critical function during the quiescent state. Ours is the most comprehensive analysis of pneumococcal gene expression during biofilm growth to date. However, our gene expression data reflects the biases of our biofilm model which includes biofilm-related changes in the ratio of opaque and transparent pneumococci as well as increasing amounts of remnant mRNA from dead bacteria. This most likely explains why our microarray results do not exactly match previous studies that explore differences between opaque and transparent pneu- mococci [19,65]. Altered Virulence of Biofilm Pneumococci We determined that biofilm bacteria down-regulated .50 genes involved in protein translation, the ATP synthase machinery, fatty acid metabolism, phospholipid synthesis, and replication. A strong reduction in capsule operon cassette expression was observed consistent with a previous study by Moscoso et al., which showed that cps3A, the first gene in the capsule operon cassette was down- regulated during biofilm production [63]. Previously, for serotype 3 strains, a non-phase variable deletion within the capsule operon cassette resulting in a rough mutant has been shown to occur and contribute towards biofilm formation [66]. A reduction in capsule would serve to expose surface components such as adhesins and facilitate attachment. This notion is supported by our previous findings with PsrP, where a version of the protein unable to extend past the capsule layer failed to mediate adhesion, as well studies as completed by Munoz-Elias et al., that found use of an unencapsulated strain facilitated the identification of genes involved in biofilm formation in vitro [17,26]. Concomitantly, a reduction in capsule would reduce the virulence potential of individual pneumococci. As indicated, the observed reduction in the physiological state of bacteria may also contribute to their attenuated phenotype. Metabolically inert bacteria would take longer to adapt to hostile host environments such as the lower respiratory tract and produce the necessary determinants required for survival such as pneumolysin. Along this line, our observation that biofilm pneumococci down-regulate pneumolysin allows for speculation that biofilm pneumococci stop producing factors that elicit a strong inflammatory response during biofilm formation within the nasopharynx. Presumably, this would promote long-term coloni- zation by modulating the immune response. This concept is supported by the finding that invasive serotypes of S. pneumoniae colonize the nasopharynx for a shorter duration than non-invasive serotypes [67]. The reduction in pneumolysin and PcpA levels during biofilm growth also suggests that immunization with pneumolysin or PcpA would also have a modest effect against colonization but might still protect against disseminated (i.e. planktonic) disease. In contrast, antibodies against CbpA and PsrP, which are up-regulated during biofilm growth, might deter nasopharyngeal colonization and thereby promote species re- placement, such as with Staphylococcus aureus, in immunized individuals. As such the differential production of protein vaccine candidates during biofilm versus planktonic growth should be an important consideration in the design of any future protein vaccine against S. pneumoniae or other bacterial pathogens [68]. Of note this concept is consistent with findings by Oggioni et al., showing altered pneumococcal virulence gene expression occurred during sessile bacterial growth on fixed surfaces versus planktonic [14]. Finally, the enhanced expression of psrP and its accessory proteins was in agreement with our previous studies that showed PsrP contributes to robust biofilm formation [26]. While unconfirmed microarray and qRT-PCR data implies that the type I pilus might be down regulated during biofilm production; this would be surprising as the pilus of Group A Streptococci and Group B Streptococci have been shown to play an important role in biofilm formation [69,70]. Of note, microarrays did not reveal enhanced cbpA expression, however, it was determined by qRT- PCR and immunoblot that CbpA levels were increased. This emphasizes the necessity for validation of RNA data with protein studies and raises the possibility that other determinants are also altered. Thus a proteomics approach is warranted to address this gap. Of note, our microarray findings are in stark contrast to those by Allegrucci et al., who found a dramatic increase in the number of detectable biofilm proteins when examining 2-dimensional gels of a serotype 3 isolate [13]. A possible explanation for this PLoS ONE | www.plosone.org 11 December 2011 | Volume 6 | Issue 12 | e28738