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Polyethylenimines (PEIs), a group of polycationic molecules, are known to impair the outer membrane of Gram-negative bacteria and exhibit antimicrobial activity. The outer membrane of Gram-negative strains hinders the uptake of photosensitizer chlorophyllin. In this study, we report chlorophyllin and branched PEI combinations’ activity against Escherichia coli strains DH5α and RB791, Salmonella enterica sv. Typhimurium LT2, and Bacillus subtilis 168. The minimal bactericidal concentration (MBC) was determined by plating cells treated with different concentrations of PEI and chlorophyllin on agar and monitoring their growth after 24 h. All tested combinations of PEI and chlorophyllin were lethal for S. enterica after 240 min of incubation in light, whereas PEI alone (−1) was ineffective. In the darkness, complete inhibition was noted with a combination of ≥2.5 µg mL−1 chlorophyllin and 50 µg mL−1 PEI. If applied alone, PEI alone of ≥800 µg mL−1 of PEI was required to completely inactivate E. coli DH5α cells in light, whereas with ≥5 µg mL−1 chlorophyllin, only ≥100 µg mL−1 PEI was needed. No effect was detected in darkness with PEI alone. However, 1600 µg mL−1 PEI in combination with 2.5 µg mL−1 resulted in complete inactivation after 4 h dark incubation. PEI alone did not inhibit E. coli strain RB791, while cells were inactivated when treated with 10 µg mL−1 chlorophyllin in combination with ≥100 µg mL−1 (in light) or ≥800 µg mL−1 PEI (in darkness). Under illumination, B. subtilis was inactivated at all tested concentrations. In the darkness, 1 µg mL−1 chlorophyllin and 12.5 µg mL−1 PEI were lethal for B. subtilis. Overall, PEI can be used as an antimicrobial agent or potentiating agent for ameliorating the antimicrobial activity of chlorophyllin.

Colistin (polymyxin E) is a membrane-destabilizing antibiotic used against Gram-negative bacteria. We have recently reported that the outer membrane prevents the uptake of antibacterial chlorophyllin into Gram-negative cells. In this study, we used sub-toxic concentrations of colistin to weaken this barrier for a combination treatment of Escherichia coli and Salmonella enterica serovar Typhimurium with chlorophyllin. In the presence of 0.25 µg/mL colistin, chlorophyllin was able to inactivate both bacteria strains at concentrations of 5−10 mg/L for E. coli and 0.5−1 mg/L for S. Typhimurium, which showed a higher overall susceptibility to chlorophyllin treatment. In accordance with a previous study, chlorophyllin has proven antibacterial activity both as a photosensitizer, illuminated with 12 mW/cm2, and in darkness. Our data clearly confirmed the relevance of the outer membrane in protection against xenobiotics. Combination treatment with colistin broadens chlorophyllin’s application spectrum against Gram-negatives and gives rise to the assumption that chlorophyllin together with cell membrane-destabilizing substances may become a promising approach in bacteria control. Furthermore, we demonstrated that colistin acts as a door opener even for the photodynamic inactivation of colistin-resistant (mcr-1-positive) E. coli cells by chlorophyllin, which could help us to overcome this antimicrobial resistance.