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

Abstract

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.

Keywords: aPDT; alternative antibiotics; antimicrobial photodynamic therapy; chlorophyll; photosensitization.

Figure 1

Light emission spectrum for PRAKASA 300 W.

Figure 2

Overview of experimental procedures. (A) Chlorophyllin extraction from spinach. (B) Determination of chlorophyllin stability in light. (C) Experimental setup to test the effects of chlorophyllin on the growth and viability of different bacteria. (D) 96-well matrix plate layout for Colony-forming units (CFU) assays and the determination of minimum inhibitory chlorophyllin concentrations (MIC test). The final volume of each well was 200 µL. After inoculation, the plate was incubated at 37 °C in large plastic bags, saturated with water vapor. Parts of the figure were drawn by using pictures from Servier Medical Art, licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).

Figure 3

Effect of chlorophyllin on the early growth phase of (A) Escherichia coli DH5α, (B) Bacillus subtilis 168, and (C) Escherichia coli NR698. Liquid cultures of bacteria (initial cell number: 10⁶ cfu/mL) were incubated in Erlenmeyer flasks in standard LB medium (red) and in presence of a chlorophyllin concentration of 22 mg/L (green). Cells grew either illuminated with 12 mW/cm² (bright plots, upper row) or in darkness (grey plots, lower row). Depicted are measured values (circles) and fitted curves (solid lines) with corresponding 95% confidence limits (red and green areas). Dashed lines describe growth of example cultures in LB medium + MeOH/KOH (solvent of chlorophyllin).

Figure 4

Schematic presentation of evaluation of CFU ability after incubation to chlorophyllin. Differently dense liquid cultures of (A) Escherichia coli DH5α, (B) Bacillus subtilis 168, and (C) Escherichia coli NR698 were supplemented with different chlorophyllin concentrations between 0.1 and 25 mg/L. Cells grew in 96-well matrix plates either illuminated with 12 mW/cm² (A–C) or protected from light (D–F). Samples (2.5 µL) were drawn at different time points and transferred onto LB agar plates. After overnight incubation at 37 °C in the dark, colony growth was analyzed. Dot size quantifies colony growth. Original pictures of the agar plates can be found in Figure S1.

Figure 5

Influence of different chlorophyllin concentrations on the growth of (A) Escherichia coli DH5α, (B) Bacillus subtilis 168 and (C) Escherichia coli NR698 (initial cell number: ~10⁸ cfu/mL). Bacteria were cultured for 24 h at 37 °C exposed to light (12 mW/cm²; bright plots, upper row) or in darkness (grey plots, lower row). To determine the lower limit of efficacy, additional chlorophyllin concentrations were tested: (D) Escherichia coli DH5α, 20↑100 mg/L (E) Bacillus subtilis 168, 1.0↓0.01 mg/L and (F) Escherichia coli NR698, 1.0↓0.01 mg/L. Depicted are means (growth curves) together with corresponding 95% confidence limits (bar charts). Blue numbers indicate respective chlorophyllin concentrations. * p < 0.05 vs. control culture without chlorophyllin.

Figure 6

(A) Chemical structure of isolated chlorophyllin. (B) Alterations of chlorophyllin’s absorption spectrum in darkness and (C) exposed to light with 12 mW/cm². (D) Reduction of chlorophyllin concentration exposed to different light intensities. (E) Required light exposure times to sterilize an E. coli DH5α suspension culture initial cell number: ~10⁶ cfu/mL) with a light intensity of 12 mW/cm² using different chlorophyllin concentrations. (F) Required light exposure times to sterilize an E. coli DH5α suspension culture (initial cell number: ~10⁶ cfu/mL) with a chlorophyllin concentration of 25 mg/L and different light intensities. For 12.5% light intensity no sterilization was observed within 300 min. Depicted are means ± standard deviation.

Figure 7

Fluorescence microscopic images of (A) Escherichia coli DH5α and (B) Escherichia coli NR698 after exposure to chlorophyllin (red fluorescence). Assumed modes of action of chlorophyllin against Gram-positive and Gram-negative bacteria. (C) Chlorophyllin (Chl) molecules cannot pass the intact outer membrane of Gram-negative bacteria. (D) Chlorophyllin is degraded in light. Degradation products can enter both Gram-negative and Gram-positive cells. The red crosses indicate cell death.