Photoreactivation of Escherichia coli after low- or medium-pressure UV disinfection determined by an endonuclease sensitive site assay

Abstract

Photoreactivation of Escherichia coli after inactivation by a low-pressure (LP) UV lamp (254 nm), by a medium-pressure (MP) UV lamp (220 to 580 nm), or by a filtered medium-pressure (MPF) UV lamp (300 to 580 nm) was investigated. An endonuclease sensitive site (ESS) assay was used to determine the number of UV-induced pyrimidine dimers in the genomic DNA of E. coli, while a conventional cultivation assay was used to investigate the colony-forming ability (CFA) of E. coli. In photoreactivation experiments, more than 80% of the pyrimidine dimers induced by LP or MPF UV irradiation were repaired, while almost no repair of dimers was observed after MP UV exposure. The CFA ratios of E. coli recovered so that they were equivalent to 0.9-, 2.3-, and 1.7-log inactivation after 3-log inactivation by LP, MP, and MPF UV irradiation, respectively. Photorepair treatment of DNA in vitro suggested that among the MP UV emissions, wavelengths of 220 to 300 nm reduced the subsequent photorepair of ESS, possibly by causing a disorder in endogenous photolyase, an enzyme specific for photoreactivation. On the other hand, the MP UV irradiation at wavelengths between 300 and 580 nm was observed to play an important role in reducing the subsequent recovery of CFA by inducing damage other than damage to pyrimidine dimers. Therefore, it was found that inactivating light at a broad range of wavelengths effectively reduced subsequent photoreactivation, which could be an advantage that MP UV irradiation has over conventional LP UV irradiation.

FIG. 1.

Gel images for ESS assays of E. coli during exposure to LP, MP, or MPF UV lamps. (A) Exposure to LP UV. Lanes 1 and 2, standard markers; lane 3, no UV; lanes 4 to 6, UV doses of 1.9, 3.8, and 5.7 mJ · cm⁻², respectively. (B) Exposure to MP UV. Lanes 1 and 6, standard marker; lane 2, no UV; lanes 3 to 5, UV doses of 2.1, 4.2, and 6.3 mJ · cm⁻², respectively. (C) Exposure to MPF UV. Lane 1, standard marker; lane 2, no UV; lanes 3 to 5, UV doses of 1.8, 3.6, and 5.4 mJ · cm⁻², respectively.

FIG. 2.

Numbers of ESS in E. coli after exposure to an LP UV lamp (♦), an MP UV lamp (▵), or an MPF UV lamp (○). The data are the results of five independent exposures to each type of lamp.

FIG. 3.

CFA ratios for E. coli after exposure to an LP UV lamp (♦), an MP UV lamp (▵), or an MPF UV lamp (○). The data are the results of five independent exposures to each type of lamp.

FIG. 4.

Relationships between the numbers of ESS and the CFA ratios for E. coli after exposure to an LP UV lamp (♦), an MP UV lamp (▵), or an MPF UV lamp (○). The data are the results of five independent exposures to each type of lamp.

FIG. 5.

Gel images for ESS assays of E. coli after exposure to fluorescent light after LP, MP, or MPF UV inactivation. (A) Exposure to LP UV. Lane 1, standard marker; lane 2, no UV; lane 3, UV dose of 5.7 mJ · cm⁻²; lanes 4 to 8, UV dose of 5.7 mJ · cm⁻², followed by exposure to fluorescent light for 30, 60, 90, 120, and 180 min, respectively. (B) Exposure to MP UV. Lane 1, no UV; lane 2, UV dose of 6.3 mJ · cm⁻²; lanes 3 to 6, UV dose of 6.3 mJ · cm⁻², followed by exposure to fluorescent light for 60, 90, 120, and 180 min, respectively; lane 7, standard marker. (C) Exposure to MPF UV. Lane 1, standard marker; lane 2, no UV; lane 3, UV dose of 5.4 mJ · cm⁻²; lanes 4 to 6, UV dose of 5.4 mJ · cm⁻², followed by exposure to fluorescent light for 60, 120, and 180 min, respectively.

FIG. 6.

ESS remaining ratios after exposure to fluorescent light after LP UV (♦), MP UV (▵), or MPF UV (○) inactivation. The symbols indicate the means from two or three independent experiments, and the bars indicate the maximum and minimum values.

FIG. 7.

Photorepair of ESS in vivo (▪), in vitro with intact photolyase (□), or in vitro with MP-exposed photolyase (▵) after MP inactivation. Lane 1, standard marker; lane 2, no UV; lane 3, MP UV dose of 6.3 mJ · cm⁻²; lane 4, MP UV dose of 6.3 mJ · cm⁻², followed by photorepair in vivo; lanes 5 and 6, MP UV dose of 6.3 mJ · cm⁻², followed by photorepair in vitro with intact photolyase (lane 5) or with MP-exposed photolyase (lane 6). For photorepair in vivo, MP UV-irradiated E. coli was subsequently exposed to fluorescent light. For photorepair in vitro, DNA of MP UV-irradiated E. coli was exposed to fluorescent light in vitro with intact or MP UV-exposed photolyase. The symbols indicate the means from two or three independent experiments, and the bars indicate the maximum and minimum values.

FIG. 8.

CFA ratios after exposure to fluorescent light after LP UV (♦), MP UV (▵), or MPF UV (○) inactivation. The symbols indicate the means from two or three independent experiments, and the bars indicate the maximum and minimum values.

FIG. 9.

Relationships between the ESS remaining ratios and the CFA ratios after exposure to fluorescent light after LP UV (♦), MP UV (▵), or MPF UV (○) inactivation. The data indicate three independent results for each type of lamp.