Fluorinated galactose analogues inhibit Streptococcus pneumoniae biofilm formation

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, causing invasive diseases such as pneumonia, sepsis and meningitis, but also less severe localised infections like otitis media. To enable pneumococcal survival in the nutritionally challenging nasopharyngeal niche, it possesses an extensive repertoire of genes dedicated to the utilisation of a wide range of carbohydrates. Galactose, the predominant carbohydrate found in the respiratory tract, is important in pneumococcal colonisation and virulence, and therefore represents an attractive target for the development of new treatments. In this study we investigated the potential of fluorinated galactose analogues as a strategy for preventing and treating pneumococcal biofilms. The analogues were tested against planktonic pneumococci, and their effect on in vitrobiofilms assessed through biomass and viability measurements with validation through confocal and scanning electron microscopy. Their effect on adhesion of pneumococcus to Detroit562 pharyngeal epithelial cells and cytotoxicity was also assessed. Treatment with up to 2% w/v galactose fluorinated at the C-3 (Gal-3F) and C-6 (Gal-6F) positions had no effect on final planktonic growth after 24 h within glucose-rich BHI medium, however, a reduction in mid-exponential growth rate was observed with 2% Gal-3F. Correlating with the transition towards biofilm formation involving a switch to galactose metabolism, 1-2% w/v Gal-3F successfully inhibited biofilm formation for four out of five strains tested; however, no activity was observed against 48-hour established biofilms and there was no inhibition of epithelial adhesion. The equivalent concentrations of Gal-6F had no effect on pneumococcal biofilms, suggesting that the site of fluorination is important in activity. The inhibition of biofilm formation by Gal-3F confirms that targeting galactose metabolism is a potentially viable strategy for treating pneumococcal infection, and that the rational design of fluorinated glycomimetics offers an alternative approach to targeting pneumococcal biofilms.