Potential repair of Escherichia coli DNA following exposure to UV radiation from both medium- and low-pressure UV sources used in drinking water treatment

The increased use of UV radiation as a drinking water treatment technology has instigated studies of the repair potential of microorganisms following treatment. This study challenged the repair potential of an optimally grown nonpathogenic laboratory strain of Escherichia coli after UV radiation from low- and medium-pressure lamps. Samples were irradiated with doses of 5, 8, and 10 mJ/cm(2) from a low-pressure lamp and 3, 5, 8, and 10 mJ/cm(2) from a medium-pressure UV lamp housed in a bench-scale collimated beam apparatus. Following irradiation, samples were incubated at 37 degrees C under photoreactivating light or in the dark. Sample aliquots were analyzed for up to 4 h following incubation using a standard plate count. Results of this study showed that E. coli underwent photorepair following exposure to the low-pressure UV source, but no repair was detectable following exposure to the medium-pressure UV source at the initial doses examined. Minimal repair was eventually observed upon medium-pressure UV lamp exposure when doses were lowered to 3 mJ/cm(2). This study clearly indicates differences in repair potential under laboratory conditions between irradiation from low-pressure and medium-pressure UV sources of the type used in water treatment.     

Low-Pressure UV Inactivation and DNA Repair Potential of Cryptosporidium parvum Oocysts

Because Cryptosporidium parvum oocysts are very resistant to conventional water treatment processes, including chemical disinfection, we determined the kinetics and extent of their inactivation by monochromatic, low-pressure (LP), mercury vapor lamp UV radiation and their subsequent potential for DNA repair of UV damage. A UV collimated-beam apparatus was used to expose suspensions of purified C. parvum oocysts in phosphate-buffered saline, pH 7.3, at 25°C to various doses of monochromatic LP UV. C. parvum infectivity reductions were rapid, approximately first order, and at a dose of 3 mJ/cm² (=30 J/m²), the reduction reached the cell culture assay detection limit of ~3 log₁₀. At UV doses of 1.2 and 3 mJ/cm², the log₁₀ reductions of C. parvum oocyst infectivity were not significantly different for control oocysts and those exposed to dark or light repair conditions for UV-induced DNA damage. These results indicate that C. parvum oocysts are very sensitive to inactivation by low doses of monochromatic LP UV radiation and that there is no phenotypic evidence of either light or dark repair of UV-induced DNA damage.     

Endotoxin Inactivation in Water by Using Medium-Pressure UV Lamps

Deionized water was spiked with various concentrations of endotoxin and exposed to UV irradiation from medium-pressure UV lamps to assess endotoxin inactivation. It was found that endotoxin inactivation was proportional to the UV dose under the conditions examined. The inactivation rate was determined to be ~0.55 endotoxin unit/ml per mJ/cm² of irradiation delivered.     

Efficacy of UV Irradiation in Inactivating Cryptosporidiumparvum Oocysts

To evaluate the effectiveness of UV irradiation in inactivating Cryptosporidium parvum oocysts, the animal infectivities and excystation abilities of oocysts that had been exposed to various UV doses were determined. Infectivity decreased exponentially as the UV dose increased, and the required dose for a 2-log₁₀ reduction in infectivity (99% inactivation) was approximately 1.0 mWs/cm² at 20°C. However, C. parvum oocysts exhibited high resistance to UV irradiation, requiring an extremely high dose of 230 mWs/cm² for a 2-log₁₀ reduction in excystation, which was used to assess viability. Moreover, the excystation ability exhibited only slight decreases at UV doses below 100 mWs/cm². Thus, UV treatment resulted in oocysts that were able to excyst but not infect. The effects of temperature and UV intensity on the UV dose requirement were also studied. The results showed that for every 10°C reduction in water temperature, the increase in the UV irradiation dose required for a 2-log₁₀ reduction in infectivity was only 7%, and for every 10-fold increase in intensity, the dose increase was only 8%. In addition, the potential of oocysts to recover infectivity and to repair UV-induced injury (pyrimidine dimers) in DNA by photoreactivation and dark repair was investigated. There was no recovery in infectivity following treatment by fluorescent-light irradiation or storage in darkness. In contrast, UV-induced pyrimidine dimers in the DNA were apparently repaired by both photoreactivation and dark repair, as determined by endonuclease-sensitive site assay. However, the recovery rate was different in each process. Given these results, the effects of UV irradiation on C. parvum oocysts as determined by animal infectivity can conclusively be considered irreversible.     

Enhanced UV Inactivation of Adenoviruses under Polychromatic UV Lamps

Adenovirus is recognized as the most UV-resistant waterborne pathogen of concern to public health microbiologists. The U.S. EPA has stipulated that a UV fluence (dose) of 186 mJ cm⁻² is required for 4-log inactivation credit in water treatment. However, all adenovirus inactivation data to date published in the peer-reviewed literature have been based on UV disinfection experiments using UV irradiation at 253.7 nm produced from a conventional low-pressure UV source. The work reported here presents inactivation data for adenovirus based on polychromatic UV sources and details the significant enhancement in inactivation achieved using these polychromatic sources. When full-spectrum, medium-pressure UV lamps were used, 4-log inactivation of adenovirus type 40 is achieved at a UV fluence of less than 60 mJ cm⁻² and a surface discharge pulsed UV source required a UV fluence of less than 40 mJ cm⁻². The action spectrum for adenovirus type 2 was also developed and partially explains the improved inactivation based on enhancements at wavelengths below 230 nm. Implications for water treatment, public health, and the future of UV regulations for virus disinfection are discussed.     

Effects of UV Radiation on Photolyase and Implications with Regards to Photoreactivation following Low- and Medium-Pressure UV Disinfection

Photolyase activity following exposure to low-pressure (LP) and medium-pressure (MP) UV lamps was evaluated. MP UV irradiation resulted in a greater reduction in photolyase activity than LP UV radiation. The results suggest that oxidation of the flavin adenine dinucleotide in photolyase may have caused the decrease in activity.     

Inactivation of Feline Calicivirus and Adenovirus Type 40 by UV Radiation

Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm², respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm² in treated groundwater. A dose of 103 mJ/cm² was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.     

Low-pressure UV inactivation and DNA repair potential of Cryptosporidium parvum oocysts.

Because Cryptosporidium parvum oocysts are very resistant to conventional water treatment processes, including chemical disinfection, we determined the kinetics and extent of their inactivation by monochromatic, low-pressure (LP), mercury vapor lamp UV radiation and their subsequent potential for DNA repair of UV damage. A UV collimated-beam apparatus was used to expose suspensions of purified C. parvum oocysts in phosphate-buffered saline, pH 7.3, at 25 degrees C to various doses of monochromatic LP UV. C. parvum infectivity reductions were rapid, approximately first order, and at a dose of 3 mJ/cm(2) (=30 J/m(2)), the reduction reached the cell culture assay detection limit of approximately 3 log(10). At UV doses of 1.2 and 3 mJ/cm(2), the log(10) reductions of C. parvum oocyst infectivity were not significantly different for control oocysts and those exposed to dark or light repair conditions for UV-induced DNA damage. These results indicate that C. parvum oocysts are very sensitive to inactivation by low doses of monochromatic LP UV radiation and that there is no phenotypic evidence of either light or dark repair of UV-induced DNA damage.     

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

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.     

Assessment of the Effectiveness of Low-Pressure UV Light for Inactivation of Helicobacter pylori

Three strains of Helicobacter pylori were exposed to UV light from a low-pressure source to determine log inactivation versus applied fluence. Results indicate that H. pylori is readily inactivated at UV fluences typically used in water treatment regimens. Greater than 4-log₁₀ inactivation was demonstrated on all three strains at fluences of less than 8 mJ cm⁻².     

Using UVC Light-Emitting Diodes at Wavelengths of 266 to 279 Nanometers To Inactivate Foodborne Pathogens and Pasteurize Sliced Cheese

UVC light is a widely used sterilization technology. However, UV lamps have several limitations, including low activity at refrigeration temperatures, a long warm-up time, and risk of mercury exposure. UV-type lamps only emit light at 254 nm, so as an alternative, UV light-emitting diodes (UV-LEDs) which can produce the desired wavelengths have been developed. In this study, we validated the inactivation efficacy of UV-LEDs by wavelength and compared the results to those of conventional UV lamps. Selective media inoculated with Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes were irradiated using UV-LEDs at 266, 270, 275, and 279 nm in the UVC spectrum at 0.1, 0.2, 0.5, and 0.7 mJ/cm², respectively. The radiation intensity of the UV-LEDs was about 4 μW/cm², and UV lamps were covered with polypropylene films to adjust the light intensity similar to those of UV-LEDs. In addition, we applied UV-LED to sliced cheese at doses of 1, 2, and 3 mJ/cm². Our results showed that inactivation rates after UV-LED treatment were significantly different (P < 0.05) from those of UV lamps at a similar intensity. On microbiological media, UV-LED treatments at 266 and 270 nm showed significantly different (P < 0.05) inactivation effects than other wavelength modules. For sliced cheeses, 4- to 5-log reductions occurred after treatment at 3 mJ/cm² for all three pathogens, with negligible generation of injured cells.     

Physical Inactivation of Toxoplasma gondii Oocysts in Water

Inactivation of Toxoplasma gondii oocysts occurred with exposure to pulsed and continuous UV radiation, as evidenced by mouse bioassay. Even at doses of ≥500 mJ/cm², some oocysts retained their viability.     

Inactivation of murine norovirus, feline calicivirus and echovirus 12 as surrogates for human norovirus (NoV) and coliphage (F+) MS2 by ultraviolet light (254 nm) and the effect of cell association on UV inactivation

Aims: To determine inactivation profiles of three human norovirus (NoV) surrogate viruses and coliphage MS2 by ultraviolet (UV) irradiation and the protective effect of cell association on UV inactivation. Methods and Results: The inactivation rate for cell‐free virus or intracellular echovirus 12 was determined by exposure to 254‐nm UV light at fluence up to 100 mJ cm^?2. The infectivity of murine norovirus (MNV), feline calicivirus (FCV) and echovirus 12 was determined by cell culture infectivity in susceptible host cell lines, and MS2 infectivity was plaque assayed on Escherichia coli host cells. The UV fluencies to achieve 4‐log₁₀ inactivation were 25, 29, 30 and 70 (mJ cm^?2) for cell‐free FCV, MNV, echovirus 12 and MS2, respectively. However, a UV fluence of 85 mJ cm^?2 was needed to inactivate intracellular echovirus 12 by 4 log₁₀. Conclusions: Murine norovirus and echoviruses 12 are more conservative surrogates than FCV to predict the UV inactivation response of human NoV. Intracellular echovirus 12 was 2·8‐fold more resistant to UV irradiation than cell‐free one. Significance and Impact of the Study: Variation in UV susceptibilities among NoV surrogate viruses and a likely protective effect of cell association on virus susceptibility to UV irradiation should be considered for effective control of human NoV in water.     

Distinct Melanogenic Response of Human Melanocytes in Mono‐culture,in Co‐Culture with Keratinocytes and in Reconstructed Epidermis,to UV Exposure

Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co‐cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm²), an increase in melanin synthesis whereas in melanocyte mono‐cultures, UVB doses up to 50 mJ/cm² had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono‐ and co‐cultures. Removing certain keratinocyte growth factors from the co‐culture medium abolished the melanogenic response to UVB, but not to UVA exposure. When integrated into the basal layer of a reconstructed human epidermis, human melanocytes similarly reacted to UVA and UVB irradiation as in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. The presence of a dense stratum corneum, known to scatter and absorb UV light, is responsible for higher minimal UVB and UVA doses required to trigger a melanogenic response in the reconstructed epidermis compared to keratinocyte–melanocyte co‐cultures. Furthermore, an immediate tanning response was observed in the pigmented epidermis following UVA irradiation. From these results we conclude that: (i) keratinocytes play an important role in mediating UVB‐induced pigmentation, (ii) UVA‐induced pigmentation is the result of a rather direct effect on melanocytes and (iii) reconstructed pigmented epidermis is the most appropriate model to study UV‐induced pigmentation in vitro.     

UV-induced repair inEscherichia coli B/r Hcr− thy trp cells: Its dependence on UV damage

Irradiation ofEscherichia coli B/r Hcr^? thy trp cells with a low UV-dose permits a post-replication repair of DNA and decreases the breakdown of DNA after a successive irradiation of cells with high UV doses. The usefulness of a repair function of the protein synthesized after a low irradiation dose increases with the increasing damage of DNA.     

Combination of high‐frequency ultrasound and UV radiation of excilamp for surface disinfection

The combination of high‐frequency ultrasound (HFUS) and UV represents a new approach to disinfecting surfaces. This study aimed to examine the inactivation efficiency of HFUS (1.7 MHz) and monochromatic UV radiation of KrCl excilamp (222 nm) in a single and a sequential mode against Bacillus cereus cells and spores added to glass surfaces. When treated by UV only, cells at populations of 10³, 10⁴, and 10⁵ colony‐forming units (CFU)/cm² showed 100% disinfection at high doses up to 1760 mJ/cm². Spores at 10⁴ CFU/cm² were completely inactivated at a dose of 1170 mJ/cm². Treatment with aqueous aerosol (produced by HFUS) reduced cell counts by 100% within a 40‐min exposure, whereas it was ineffective in inactivating spores under these conditions. In a sequential mode, the contaminated surface was pretreated with the sonicated aqueous aerosol and subsequently irradiated with the excilamp. It was found that HFUS exposure times and UV doses for complete inactivation decreased by a factor of 2 and 6–7, respectively, compared to sole HFUS or UV. A portable apparatus for surface disinfection was designed. The combined HFUS/UV method may be a promising technique for rapid disinfection of microbially contaminated surfaces.     

Ultraviolet disinfection of Schistosoma mansoni cercariae in water

BackgroundSchistosomiasis is a parasitic disease that is transmitted by skin contact with waterborne schistosome cercariae. Mass drug administration with praziquantel is an effective control method, but it cannot prevent reinfection if contact with cercariae infested water continues. Providing safe water for contact activities such as laundry and bathing can help to reduce transmission. In this study we examine the direct effect of UV light on Schistosoma mansoni cercariae using ultraviolet light-emitting diodes (UV LEDs) and a low-pressure (LP) mercury arc discharge lamp.MethodologyS. mansoni cercariae were exposed to UV light at four peak wavelengths: 255 nm, 265 nm, 285 nm (UV LEDs), and 253.7 nm (LP lamp) using bench scale collimated beam apparatus. The UV fluence ranged from 0–300 mJ/cm² at each wavelength. Cercariae were studied under a stereo-microscope at 0, 60, and 180 minutes post-exposure and the viability of cercariae was determined by assessing their motility and morphology.ConclusionVery high UV fluences were required to kill S. mansoni cercariae, when compared to most other waterborne pathogens. At 265 nm a fluence of 247 mJ/cm² (95% confidence interval (CI): 234–261 mJ/cm²) was required to achieve a 1-log₁₀ reduction at 0 minutes post-exposure. Cercariae were visibly damaged at lower fluences, and the log reduction increased with time post-exposure at all wavelengths. Fluences of 127 mJ/cm² (95% CI: 111–146 mJ/cm²) and 99 mJ/cm² (95% CI: 85–113 mJ/cm²) were required to achieve a 1-log₁₀ reduction at 60 and 180 minutes post-exposure at 265 nm. At 0 minutes post-exposure 285 nm was slightly less effective, but there was no statistical difference between 265 nm and 285 nm after 60 minutes. The least effective wavelengths were 255 nm and 253.7 nm. Due to the high fluences required, UV disinfection is unlikely to be an energy- or cost-efficient water treatment method against schistosome cercariae when compared to other methods such as chlorination, unless it can be demonstrated that UV-damaged cercariae are non-infective using alternative assay methods or there are improvements in UV LED technology.     

Studies on the resistance/reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts exposed to medium-pressure ultraviolet radiation

The ex vivo and in vivo reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts after exposure to different doses of ultraviolet (UV) radiation was determined using animal infectivity. The infectivity of UV-treated parasites stored for 1-4 days (G. muris) or 1-17 days (C. parvum) at room temperature in the dark was similar to that of organisms administered immediately after UV treatment, indicating that the parasites did not reactivate ex vivo. In contrast, we observed in vivo reactivation of G. muris in three of seven independent animal infectivity experiments, when parasites were treated with relatively low doses of medium-pressure UV (<25 mJ/cm(2)). Our observations indicate that G. muris cysts and C. parvum oocysts exposed to medium-pressure UV doses of 60 mJ/cm(2) or higher did not exhibit resistance to and/or reactivation following treatment. This suggests that when appropriate doses of UV are used, significant and permanent inactivation of these parasites may be achieved.     

Genetic Inactivation of European Sea Bass (Dicentrarchus labrax L.) Eggs Using UV-Irradiation: Observations and Perspectives

Androgenesis is a form of uniparental reproduction leading to progenies inheriting only the paternal set of chromosomes. It has been achieved with variable success in a number of freshwater species and can be attained by artificial fertilization of genetically inactivated eggs following exposure to gamma (γ), X-ray or UV irradiation (haploid androgenesis) and by restoration of diploidy by suppression of mitosis using a pressure or thermal shock. The conditions for the genetic inactivation of the maternal genome in the European sea bass (Dicentrarchus labrax L.) were explored using different combinations of UV irradiation levels and durations. UV treatments significantly affected embryo survival and generated a wide range of developmental abnormalities. Despite the wide range of UV doses tested (from 7.2 to 720 mJ.cm⁻²), only one dose (60 mJ.cm⁻².min⁻¹ with 1 min irradiation) resulted in a small percentage (14%) of haploid larvae at hatching in the initial trials as verified by flow cytometry. Microsatellite marker analyses of three further batches of larvae produced by using this UV treatment showed a majority of larvae with variable levels of paternal and maternal contributions and only one larva displaying pure paternal inheritance. The results are discussed also in the context of an assessment of the UV-absorbance characteristics of egg extracts in this species that revealed the presence of gadusol, a compound structurally related to mycosporine-like amino acids (MAAs) with known UV-screening properties.     

Survival of Spacecraft-Associated Microorganisms under Simulated Martian UV Irradiation

Spore-forming microbes recovered from spacecraft surfaces and assembly facilities were exposed to simulated Martian UV irradiation. The effects of UVA (315 to 400 nm), UVA+B (280 to 400 nm), and the full UV spectrum (200 to 400 nm) on the survival of microorganisms were studied at UV intensities expected to strike the surfaces of Mars. Microbial species isolated from the surfaces of several spacecraft, including Mars Odyssey, X-2000 (avionics), and the International Space Station, and their assembly facilities were identified using 16S rRNA gene sequencing. Forty-three Bacillus spore lines were screened, and 19 isolates showed resistance to UVC irradiation (200 to 280 nm) after exposure to 1,000 J m⁻² of UVC irradiation at 254 nm using a low-pressure mercury lamp. Spores of Bacillus species isolated from spacecraft-associated surfaces were more resistant than a standard dosimetric strain, Bacillus subtilis 168. In addition, the exposure time required for UVA+B irradiation to reduce the viable spore numbers by 90% was 35-fold longer than the exposure time required for the full UV spectrum to do this, confirming that UVC is the primary biocidal bandwidth. Among the Bacillus species tested, spores of a Bacillus pumilus strain showed the greatest resistance to all three UV bandwidths, as well as the total spectrum. The resistance to simulated Mars UV irradiation was strain specific; B. pumilus SAFR-032 exhibited greater resistance than all other strains tested. The isolation of organisms like B. pumilus SAFR-032 and the greater survival of this organism (sixfold) than of the standard dosimetric strains should be considered when the sanitation capabilities of UV irradiation are determined.