AFM evaluation of mophologic and tomographical alterations in Escherichia coli after PDT

Abstract

In antimicrobial PDT (photodynamic therapy), bacterial cells inactivation is strongly dependent on the interaction of the photosensitizer with the cell envelope. The aim of this work was to evaluate, by atomic force microscopy (AFM), the alterations caused by red laser irradiation on a Gram negative microorganism (Escherichia coli) using the methylene blue as a photosensitizer. Cells culture were grown until a stationary phase to reach a concentration of approximately 108 cells/mL allowing the production of extracellular slime in a biofilm-like structure. The cells including the extracellular matrix were spread on a clean glass coverslip, and their structures observed using a Shimadzu SPM 9500J3 AFM operating in non contact dynamic mode. Subsequently, a water solution of methylene blue at 60 µM was applied over the same cells. After waiting a pre-irradiation time of three minutes, the cells were illuminated with a diode laser (λ=660 nm, output power 40 mW, fluence 180 J/cm2, beam diameter 0,04 cm2) for three minutes. AFM images of the treated cells were then obtained. A second set of experiments was performed with fewer cells per area, without extracellular slime, and other laser conditions (10 min of irradiation at 600J/cm2). The results showed alterations on cellular scaffold markedly dependent on the number of cells, fluence, and the presence of extracellular slime. The slime is targeted by the photosensitizer, and after irradiation a destruction of the matrix was observed. When fewer cells were evaluated with higher fluence, the destruction was much more evident. The AFMimagessuggested rupture of the cellular membrane and cellular fragments were observed. These preliminary findings indicate that the combination of a sensitizer and a specific wavelength can lead to the loss of the bacterial surface integrity, and depending on the studied parameters dramatic changes of the cell shape can be detected by AFM. Therefore, AFM seems to be a powerful tool to investigate parameters associated with photodestruction of microorganisms.