What an Escherichia coli Mutant Can Teach Us About the Antibacterial Effect of Chlorophyllin

Due to the increasing development of antibiotic resistances in recent years, scientists search intensely for new methods to control bacteria. Photodynamic treatment with porphyrins such as chlorophyll derivatives is one of the most promising methods to handle bacterial infestation, but their use is dependent on illumination and they seem to be more effective against Gram-positive bacteria than against Gram-negatives. In this study, we tested chlorophyllin against three bacterial model strains, the Gram-positive Bacillus subtilis 168, the Gram-negative Escherichia coli DH5α and E. coli strain NR698 which has a deficient outer membrane, simulating a Gram-negative “without” its outer membrane. Illuminated with a standardized light intensity of 12 mW/cm2, B. subtilis showed high sensitivity already at low chlorophyllin concentrations (≤105 cfu/mL: ≤0.1 mg/L, 106–108 cfu/mL: 0.5 mg/L), whereas E. coli DH5α was less sensitive (≤105 cfu/mL: 2.5 mg/L, 106 cfu/mL: 5 mg/L, 107–108 cfu/mL: ineffective at ≤25 mg/L chlorophyllin). E. coli NR698 was almost as sensitive as B. subtilis against chlorophyllin, pointing out that the outer membrane plays a significant role in protection against photodynamic chlorophyllin impacts. Interestingly, E. coli NR698 and B. subtilis can also be inactivated by chlorophyllin in darkness, indicating a second, light-independent mode of action. Thus, chlorophyllin seems to be more than a photosensitizer, and a promising substance for the control of bacteria, which deserves further investigation.