Broad-Spectrum Antimicrobial Effects of Photocatalysis Using Titanium Dioxide Nanoparticles Are Strongly Potentiated by Addition of Potassium Iodide

Abstract

Photocatalysis describes the excitation of titanium dioxide nanoparticles (a wide-band gap semiconductor) by UVA light to produce reactive oxygen species (ROS) that can destroy many organic molecules. This photocatalysis process is used for environmental remediation, while antimicrobial photocatalysis can kill many classes of microorganisms and can be used to sterilize water and surfaces and possibly to treat infections. Here we show that addition of the nontoxic inorganic salt potassium iodide to TiO2 (P25) excited by UVA potentiated the killing of Gram-positive bacteria, Gram-negative bacteria, and fungi by up to 6 logs. The microbial killing depended on the concentration of TiO2, the fluence of UVA light, and the concentration of KI (the best effect was at 100 mM). There was formation of long-lived antimicrobial species (probably hypoiodite and iodine) in the reaction mixture (detected by adding bacteria after light), but short-lived antibacterial reactive species (bacteria present during light) produced more killing. Fluorescent probes for ROS (hydroxyl radical and singlet oxygen) were quenched by iodide. Tri-iodide (which has a peak at 350 nm and a blue product with starch) was produced by TiO2-UVA-KI but was much reduced when methicillin-resistant Staphylococcus aureus (MRSA) cells were also present. The model tyrosine substrate N-acetyl tyrosine ethyl ester was iodinated in a light dose-dependent manner. We conclude that UVA-excited TiO2 in the presence of iodide produces reactive iodine intermediates during illumination that kill microbial cells and long-lived oxidized iodine products that kill after light has ended.

FIG 1

KI potentiates the killing of Gram-positive MRSA by TiO₂-UVA in a dose-dependent manner. Bacteria (10⁸) cells/ml) were stirred in a suspension of 1 mM (A) or 5 mM (B) TiO₂ containing the specified concentrations of KI while being illuminated with UVA. At various times, aliquots were withdrawn for determination of CFU. Values are means from 3 repetitions, and error bars are standard deviations (SD).

FIG 2

KI potentiates the killing of Gram-negative E. coli by TiO₂-UVA in a dose-dependent manner. Bacteria (10⁸ cells/ml) were stirred in a suspension of 1 mM (A) or 5 mM (B) TiO₂ containing the specified concentrations of KI while being illuminated with UVA. At various times, aliquots were withdrawn for determination of CFU. Values are means from 3 repetitions, and error bars are SD.

FIG 3

KI potentiates the killing of the fungal yeast C. albicans by TiO₂-UVA in a dose-dependent manner. Yeast cells (10⁷ cells/ml) were stirred in a suspension of TiO₂ (10 mM) containing the specified concentrations of KI while being illuminated with UVA. At various times, aliquots were withdrawn for determination of CFU. Values are means from 3 repetitions, and error bars are SD.

FIG 4

Transmission electron microscopy of MRSA cells. (A) Control; (B) 1 mM TiO₂ and 40 J/cm² UVA; (C) 1 mM TiO₂, 10 mM KI, and 40 J/cm² UVA. Insets, higher magnification showing the cell membrane.

FIG 5

Transmission electron microscopy of E. coli cells. (A) Control; (B) 1 mM TiO₂ and 40 J/cm² UVA; (C) 1 mM TiO₂, 10 mM KI, and 40 J/cm² UVA. Insets, higher magnification showing the cell membrane.

FIG 6

Effects of adding bacteria after light. (A) MRSA cells were added 5 min after cessation of illumination of TiO₂ under the indicated conditions. (B) MRSA cells were added at different times after cessation of illumination.

FIG 7

Activation of ROS-specific fluorescent probes by photoactivated TiO₂. Probe (5 μM) solution containing TiO₂ (0.1 or 1 mM) was illuminated with UVA light (0 to 8 J/cm2) in the presence of KI (1 or 10 mM). Fluorescence was measured in a plate reader after each aliquot of light was applied. (A) HPF; (B) SOSG. Values are means for 6 wells, and error bars are SD.

FIG 8

Mechanistic chemical assays. (A) Fluence-dependent increase in absorption (620 nm) of the blue iodine-starch inclusion complex by TiO₂-KI-UVA. (B) Tri-iodide absorption (352 nm) generated by TiO₂-KI-UVA is reduced in the presence of MRSA bacterial cells. (C) Fluence-dependent increase in iodination of N-acetyl-tyrosine ethyl ester in the presence of TiO₂-KI-UVA as measured by LC-MS.