Altered Virulence of Biofilm Pneumococci Figure 3. Biofilm-derived pneumococci have an enhanced ability to adhere to host cells. (A) Comparison of the adhesive properties of planktonic bacteria and biofilm-derived S. pneumoniae TIGR4 and T4DpsrP to A549 cells and Detroit 562 cells in vitro. (B) Bacterial adhesion assays using a panel of invasive clinical isolates of S. pneumoniae unrelated to TIGR4. Values are expressed as fold-increase adhesion relative to the planktonic counterparts. All experiments were performed in triplicate and repeated independently at least three times. Statistical analysis was performed using a two-tailed Student’s t-test. Single asterisks denotes P,0.001, double asterisks denotes P,0.00001. doi:10.1371/journal.pone.0028738.g003 Despite degradation, sufficient amounts of RNA could be isolated from the 24 and 48 h time points for efficient Cy3 and Cy5 labeling and subsequent microarray gene expression analyses using S. pneumoniae version 8 microarrays obtained from the NIAID Pathogen Functional Genomics Research Center (http://pfgrc. org). Nonetheless, to ensure that our microarray results were robust we processed 2–3 (3 replicates for 4 and 12 h time points, 2 replicates for 24 and 48 h time points) independent RNA samples for each time point (biological replicates) and performed dye- flipped hybridizations (technical replicates) for each sample, except for the 48 h time point where no technical replicates could be performed due to limiting amounts of RNA material. Stringent rules for hybridized signal strength and required minimum number of usable data points per time point were applied prior to the calculation of average expression levels and P-values (see methods). Thus in many instances, and in particular at later time points, data on transcript levels of some pneumococcal genes are not presented (labeled as ‘‘NA’’ in Tables S1, S2, and S3). During transition from planktonic to biofilm growth we observed changes in TIGR4 gene transcription that encompassed almost all aspects of pneumococcal cell biology. The majority of genes with differential expression were down-regulated with significantly lower RNA levels for 40, 62, 32, and 62 genes at 4, 12, 24, and 48 h, respectively (Table S1). In contrast, 16, 40, 14, and 8 genes were determined to have enhanced transcription, at the same time points, respectively (Table S2). In total, a surprisingly small number of genes were observed to have altered transcription during biofilm growth. When compared to plank- tonic culture, 6.2% of the 1674 TIGR4 genes tested for by the microarray were determined to be significantly altered after 12 h of biofilm culture, the time point with the greatest number of differentially expressed genes. A complete list of the genes spotted on the microarray and their expression during bioflm growth is provided through the Gene Expression Omnibus (GEO, http:// www.ncbi.nlm.nih.gov/geo/) accession number GSE26976. Val- idation of the microarray results was also performed by qRT-PCR (Figure S3). Consistent with the concept that biofilms are in a quiescent state [38], 32 genes encoding either ribosomal proteins or translation initiation and elongation factors were down-regulated during biofilm growth. This was accompanied by a reduction in 8 genes encoding the ATP synthase machinery (SP_ 1506- SP_1514) and 9 other genes, including the Fab operon, involved in fatty acid metabolism and phospholipid biosynthesis (SP_0415-SP_0427). Other indicators that biofilm bacteria were in an inert state included decreased expression of the cell division gene ftsZ (SP_1666), decreased expression of assorted Sec pre-protein translocase components (SP_0230, SP_1702,SP_2029), and re- duced expression of over 32 conserved hypothetical or hypothet- ical proteins. Despite an established role for capsule as a major PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e28738