Effect of 5-azacytidine on in vitro biofilm formation of Streptococcus pneumoniae.

Streptococcus pneumoniae is a gram-positive bacterium that causes otitis media, pneumonia, meningitis and sepsis in young children and the elderly. Previous studies reported that pneumococci in different diseases do not behave as planktonic cells, but predominantly show characteristics of a biofilm. In this study we examine the effect of 5-azacytidine on S. pneumoniae, particularly on biofilm formation and investigate the gene expression involved in synthesis of autoinducer-2, competence and DNA repair. The effect of 5-aza on in vitro biofilm formation was studied by the crystal violet microtiter plate method. The S. pneumoniae biofilms were grown with different concentration of 5-azacytidine (15-500 μm), at variable time intervals and the inhibition percentages were calculated. The effects of 5-aza on the morphology of biofilms were analyzed by scanning electron microscope. The relative quantification of 11 genes of biofilms grown with 5-aza involved in autoinducer-2 synthesis, competence and DNA repair was carried out by real-time RT-PCR with respect to biofilms grown without 5-aza. The crystal violet microtiter assay detected a significant inhibitory effect of 5-aza on in vitro biofilm formation, at concentration that did not inhibited planktonic cell growth. The SEM analysis demonstrated thin and disrupted biofilms, without micro-colonies in the samples treated with 5-aza, while these structures were present in the biofilms grown without 5-aza. The relative quantification of gene expression of 5-aza biofilms showed a significant down regulation of genes involved in the methionine and homocysteine recycling pathway which produces quorum sensing molecule autoinducer-2 as by-products. A significant decrease in the expressions of luxS, metK, pfs and cmK was detected. In conclusion, 5-aza inhibits in vitro biofilm formation and decreases the expression of luxS, pfs and metK, which are involved in the synthesis of autoinducer-2 as by-products of the methionine recycling pathway. The inhibitory effect of 5-aza may be either due to down regulation of pfs, luxS and metK or due to accumulation of the toxic substrate of pfs, luxS and metK genes.