Quantitative mapping of chlorhexidine in natural river biofilms

Soft X-ray scanning transmission X-ray microscopy has been applied to map chlorhexidine, a ubiquitous antimicrobial agent, relative to major biochemical components (proteins, lipids, polysaccharides, Ca2+, K+, CO32−) in natural river biofilms. For the first time, bio-accumulation of chlorhexidine in diatoms has been observed unambiguously. The quantitative results show that chlorhexidine bioaccumulated extensively in lipid-rich regions of diatoms and bacteria. Confocal laser scanning microscopy was used to document changes in the biofilm community. The bioaccumulation provides a significant entry point for chlorhexidine into the aquatic food chain. It results in modification of the biofilm community and it impacts the photosynthetic and protozoan species in particular. X-ray microscopy mapping at high spatial resolution is shown to be a powerful tool for studies of antimicrobial agents in the environment.