component of the EPM [39], 8 of the genes encoded within the CPS cassette (SP_0346-SP_0360) were down-regulated during biofilm growth. Other virulence determinants with decreased expression included the type I pilus ancillary protein RrgC (SP_0464), pneumolysin (SP_1923), and choline binding protein PcpA (SP_2136). In contrast, Pneumococcal serine-rich repeat protein (PsrP; SP_1772) was the only established virulence determinant with enhanced expression during mature biofilm growth. PsrP is a host cell and intra-species bacterial adhesin previously shown to contribute to biofilm formation. PsrP is encoded within the pathogenicity island psrP-secY2A2 (SP_1755-SP_1772) along with 10 glycosyltransferases and 7 components of an alternate Sec translocase [11,26,40]. These accessory genes are putatively responsible for PsrP glycosylation and transport and, along with psrP, were significantly up-regulated during biofilm growth [41,42,43]. Not surprisingly, genes encoding stress-related chaper- onins and proteases were also enhanced. These included genes encoding GroEL (SP_1906), Class I heat shock proteins (SP_0516- SP_0517), a member of the universal stress protein family (SP_1996), thioredoxin (SP_1000) and 2 Clp proteases (SP_0338, SP_0820). Three genes involved in the high affinity phosphate transport system were also elevated (SP_2084-SP_2088), as were 3 genes involved in glycogen synthesis (SP_1121-SP_1123). Interest- ingly, genes encoding transposases (SP_0392, SP_0814, SP_0850, SP_1485, SP_1593, SP_1613) were also up-regulated suggesting they are sensitive and responsive to physiological stress. Finally, 18 hypothetical proteins were also enhanced, among which SP_1793 was found to be up-regulated as high as 45-fold. To verify our microarray results we performed qRT-PCR on 19 genes identified by the microarray as either down, no change, or up-regulated using RNA from each biological replicate (See methods; Table S3). We then determined the correlation coefficient between our microarray data with the results from the qRT-PCR. A strong positive correlation was observed with the correlation coefficients ranging from 0.66 to 0.94, except for a single 4 h replicate 1 at 0.59. Thus transcript levels as determined by microarray were reliable. Included among the genes tested by qRT-PCR were pspA (SP_0117) and spxB (SP_0730) that encode proteins responsible for complement resistance and production of hydrogen peroxide, respectively, and were confirmed as un- changed [44,45]. ply that encodes pneumolysin that was confirmed as reduced. Finally, psrP, the Clp protease SP_0338, and SP_1793, which were all enhanced. Unexpectedly cbpA (SP_2190), which encodes the pneumococcal adhesin Choline binding protein A (CbpA) [46,47,48], and nanA (SP_1693), the gene encoding neuraminidase A [49], were up-regulated during biofilm growth as measured by qRT-PCR. Likewise, rlrA (SP_0461) and rrgA (SP_0462) the type I pilus transcriptional regulator and the pilus adhesin protein, respectively, were down-regulated [50,51]. Presumably, the increased sensitivity of PCR versus detection of hybridized labeled cDNA during microarray analyses explains the uncovering of these additional differentially regulated genes by qRT-PCR. In summation, the microarray data was in agreement with studies that suggest biofilm bacteria are quiescent. Furthermore, these studies suggest that bacteria within a mature biofilm: 1) produce less capsular polysaccharide, 2) are undergoing consid- erable physiological stress, and 3) with exception to PsrP and possibly CbpA and Neuraminidase A, have reduced virulence determinant expression. Notably, as determined by the unchanged expression of genes involved with competence, we did not observe that pneumococci within biofilms were in a competent state. This was consistent with findings reported by Trappetti et al., showing Altered Virulence of Biofilm Pneumococci that deletion of ComD, which senses competence stimulating peptide, had no effect on pneumococcal biofilm formation in a continuous flow-through reactor [52]. Reduced capsule production by biofilm pneumococci Since deletion of capsule enhances bacterial adhesion but results in a complete loss of virulence [53], and because we observed a similar phenotype with biofilm pneumococci, we used a direct ELISA approach to measure total levels of CPS in TIGR4 cultures and determined that planktonic bacteria had a 3 to 5-fold greater amount of capsular polysaccharide than their biofilm derivatives (Figure 4A). We subsequently tested the ability of T4R, an unencapsulated derivative of TIGR4, and R6, an unrelated and un-encapsulated serotype 2 derivative that naturally lacks PsrP, to adhere to cells following either planktonic or biofilm culture (Figure 4B) [28,54]. For both strains tested, a less dramatic but persistent biofilm hyper-adhesive phenotype was observed, indicating that capsule levels played an important, but not complete, role in the observed hyper-adhesive phenotype. Thus, we confirmed that the capsule is down-regulated during biofilm growth and that its reduction most likely contributed to, but was not solely responsible for, the observed hyper-adhesive state. Biofilm formation selects for the transparent phenotype S. pneumoniae undergoes phase variation, alternating between a 1) transparent, low-capsule and high teichoic acid phase, and 2) an opaque, high-capsule and low teichoic acid state [34]. During nasopharyngeal colonization the majority of pneumococci are of the transparent phenotype, which has an enhanced ability to bind to host cells. This is due to reduced capsule, but also an increased amount of surface exposed ChoP, which binds to the host protein platelet-activating factor receptor (PAFr) on epithelial cells [27]. Importantly, C-reactive protein binds to bacterial ChoP, opson- izing the bacteria and promoting its phagocytosis [55]. We observed that colonies on plates grown from biofilm isolated pneumococci were consistently smaller than those used to seed the biofilm reactor. Upon close examination by trans-oblique illumination they were determined to more frequently belong to the transparent phenotype (Figure 5A,B). We determined that 69% of pneumococci isolated from a mature biofilm were transparent. In contrast, pneumococci in the cultures used to inoculate the biofilm reactor were predominantly of the opaque phenotype (63% opaque). We confirmed the transparent pheno- type by measuring teichoic acid levels. Using direct ELISA to calculate relative levels of teichoic acid in cell lysates, we observed a 3–4 fold increase in optical density of the developed ELISA for biofilm cultures (Figure 5C). This was confirmed by immunoblot for teichoic acids using TEPC-15, a mouse IgA monoclonal against ChoP (Figure 5D). Not only was a difference observed in the total amount of teichoic acid for biofilm pneumococci, but two additional, higher molecular weight teichoic acid polymers were also observed for biofilm cultures. Presence of these additional bands was consistent with published studies contrasting the two phenotypes [34]. Biofilm pneumococci modulate pneumolysin, PsrP, and CbpA production Following SDS-PAGE separation of equal protein amounts, we confirmed decreased production of pneumolysin by biofilm pneumococci as well as increased production of the bacterial adhesin CbpA which binds to laminin receptor and has been shown to be enhanced during the transparent phenotype (Figure 6) [27,34,56]. Immunodot blot was used to confirm enhanced PsrP PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e28738