Time-dose reciprocity mechanism for the inactivation of Escherichia coli explained by a stochastic process with two inactivation effects

Abstract

There is a great demand for developing and demonstrating novel disinfection technologies for protection against various pathogenic viruses and bacteria. In this context, ultraviolet (UV) irradiation offers an effective and convenient method for the inactivation of pathogenic microorganisms. The quantitative evaluation of the efficacy of UV sterilization relies on the simple time-dose reciprocity law proposed by Bunsen-Roscoe. However, the inactivation rate constants reported in the literature vary widely, even at the same dose and wavelength of irradiation. Thus, it is likely that the physical mechanism of UV inactivation cannot be described by the simple time-dose reciprocity law but requires a secondary inactivation process, which must be identified to clarify the scientific basis. In this paper, we conducted a UV inactivation experiment with Escherichia coli at the same dose but with different irradiances and irradiation durations, varying the irradiance by two to three orders of magnitude. We showed that the efficacy of inactivation obtained by UV-light emitting diode irradiation differs significantly by one order of magnitude at the same dose but different irradiances at a fixed wavelength. To explain this, we constructed a stochastic model introducing a second inactivation rate, such as that due to reactive oxygen species (ROS) that contribute to DNA and/or protein damage, together with the fluence-based UV inactivation rate. By solving the differential equations based on this model, the efficacy of inactivation as a function of the irradiance and irradiation duration under the same UV dose conditions was clearly elucidated. The proposed model clearly shows that at least two inactivation rates are involved in UV inactivation, where the generally used UV inactivation rate does not depend on the irradiance, but the inactivation rate due to ROS does depend on the irradiance. We conclude that the UV inactivation results obtained to date were simply fitted by one inactivation rate that superimposed these two inactivation rates. The effectiveness of long-term UV irradiation at a low irradiance but the same dose provides useful information for future disinfection technologies such as the disinfection of large spaces, for example, hospital rooms using UV light, because it can reduce the radiation dose and its risk to the human body.

Figure 1

Optical setup of the UV inactivation system and emission spectrum of UV-LEDs. (a) Optical setup of the UV-LED inactivation system. (b) Emission spectra of 265 nm, 280 nm, and 308 nm UV-LEDs. These LEDs exhibited peak wavelength emissions at 266.5 nm, 280.6 nm, and 308.8 nm, respectively, with full-width at half-maximum bandwidths of 11.1 nm, 11.5 nm, and 12.0 nm. (c) Photograph of an E. coli bacterial sample irradiated by the UV-LED in an ultrasonic bath. An E. coli bacterial sample without UV irradiation was also placed in the ultrasonic bath as a control sample to account for the inactivation caused by ultrasonic vibrations.

Figure 2

Experimental plots of the inactivation ratio [Log(N/N₀)] for various irradiation durations (and various irradiances) at doses of 10 mJ/cm² (red circles) and 5 mJ/cm² (blue circles) and irradiation wavelengths of (a) 265 nm, (b) 280 nm, and (c) 308 nm. The red or blue line represents the theoretically fitted inactivation ratio as a function of irradiation duration at a constant dose (red: 10 mJ/cm², blue: 5 mJ/cm²) but different irradiance conditions. (d) Determination of Γ₁ by the initial slope of the curve for 265 nm and 10 mJ/cm² results, where the green curve is described by Γ₁ = 2 × 10⁻⁴ cm³/s, the red curve is described by Γ₁ = 2 × 10⁻³ cm³/s, and the blue curve is described by Γ₁ = 2 × 10⁻² cm³/s. (e) Determination of Γ₂ by the tail slope of the curve for 265 nm and 10 mJ/cm² results, where the green curve is described by Γ₂ = 0.07 cm²/mJ, the red curve is described by Γ₂ = 0.7 cm²/mJ, and the blue curve is described by Γ₂ = 7.0 cm²/mJ. (f) Determination of Γ₄ by the tail height of the curve for 265 nm and 10 mJ/cm² results, where the green curve is described by Γ₄ = 2.8 cm³/s, the red curve is described by Γ₄ = 28 cm³/s (red circles are experimental results), and the blue curve is described by Γ₄ = 280 cm³/s.

Figure 3

Quantitative model describing DNA damage processes. UV radiation directly causes DNA damage by the formation of thymine dimers or the generation of ROS radicals (red circles) at bacteria by UV radiation, which damages DNA. The rates of DNA damage are described as follows: Γ₀ (cm²/mJ): UV radiation directly causes DNA damage by the formation of thymine dimers, Γ₁ (cm³/s): ROS radicals damage DNA, Γ₂ (cm²/mJ): ROS radicals are generated at the bacteria by UV radiation, Γ₃ (s⁻¹): lifetime of ROS radicals, and Γ₄ (cm³/s): mutual destruction of ROS radicals.

Figure 4

(a) Temporal behaviour of R(t) obtained at 0.01 mW/cm² with 1000 s (red curve) or 10 mW/cm² with 1 s (blue curve in the inset) at the irradiation wavelength of 265 nm. (b) Total amount of ROS as a function of irradiation duration at the same dose (265 nm, 10 mJ/cm²).