Bacterial Inactivation by Solar Ultraviolet Radiation Compared with Sensitivity to 254 nm Radiation

Our goal was to derive a quantitative factor that would allow us to predict the solar sensitivity of vegetative bacterial cells to natural solar radiation from the wealth of data collected for cells exposed to UVC (254 nm) radiation. We constructed a solar effectiveness spectrum for inactivation of vegetative bacterial cells by combining the available action spectra for vegetative cell killing in the solar range with the natural sunlight spectrum that reaches the ground. We then analyzed previous studies reporting the effects of solar radiation on vegetative bacterial cells and on bacterial spores. Although UVC-sensitive cells were also more sensitive to solar radiation, we found no absolute numerical correlation between the relative solar sensitivity of vegetative cells and their sensitivity to 254 nm radiation. The sensitivity of bacterial spores to solar exposure during both summer and winter correlated closely to their UVC sensitivity. The estimates presented here should make it possible to reasonably predict the time it would take for natural solar UV to kill bacterial spores or with a lesser degree of accuracy, vegetative bacterial cells after dispersion from an infected host or after an accidental or intentional release.     

Overview of the inactivation by 254 nm ultraviolet radiation of bacteria with particular relevance to biodefense

Abstract Our goal was to ultimately predict the sensitivity of untested bacteria (including those of biodefense interest) to ultraviolet (UV) radiation. In this study, we present an overview and analysis of the relevant 254 nm data previously reported and available in the literature. The amount of variability in this data prevented us from determining an "average" response for any bacterium. Therefore, we developed particular selection criteria to include the data in our analysis and suggested future guidelines for reporting UV sensitivity results. We then compiled a table of the sensitivity to 254 nm UV for 38 bacteria and three bacterial spores. The UV sensitivity was quite similar (within 10%) among the spores of Bacillus anthracis (strains Vollum 1B and Sterne), Bacillus subtilis, and Bacillus megaterium. These data indicate that spores of B. subtilis and B. megaterium could be adequate simulants of B. anthracis spores in UVC experiments. Spores of B. anthracis, B. subtilis and B. megaterium were 5-10 times more resistant to UV than were their corresponding vegetative cells. The vegetative cells of B. anthracis showed similar UV sensitivity to those of Burkholderia pseudomallei, Shigella sonnei, and a wild-type strain of Escherichia coli. Yersinia enterocolitica and Vibrio cholerae appeared more sensitive to UV and Salmonella typhi slightly more resistant to UV than E. coli. The sensitivity (at 254 nm) of all vegetative bacteria ranged from 11 to 80 Jm(2) for a 1 Log(10) kill and from 25-200 Jm(2) for 4 Log(10) kill.     

Inactivation of Vaccinia Virus by Natural Sunlight and by Artificial UVB Radiation

This study determined the sensitivity of vaccinia virus, an orthopox virus commonly used as a surrogate for variola virus (etiological agent of smallpox), exposed to UVB radiation emitted by a solar simulator, or to direct natural sunlight. The data obtained indicate that: (1) the virucidal effect of natural sunlight can be mimicked adequately by an artificial light source with similar spectral characteristics in the UVB, (2) viral sensitivity to UVB or to solar radiation can be correlated with experimental data previously obtained with UVC, (3) the correlation factor between virus inactivation by solar radiation (measured at 300 ± 5 nm) and by UVC (254 nm) is between 33 and 60, and (4) the sensitivity of viruses either dry on glass surfaces or in liquid suspension is similar when in the presence of similar amounts of cellular debris and growth media. The findings reported in this study should assist in estimating the threat posed by the persistence of virus during epidemics or after an accidental or intentional release.     

LETHAL EFFECTS OF NATURAL SOLAR ULTRAVIOLET RADIATION IN REPAIR PROFICIENT AND REPAIR DEFICIENT STRAINS OF ESCHERICHIA COLI: ACTIONS AND INTERACTIONS

Abstract— The inactivation of repair proficient ( Escherichia coli K12 AB 1157, E. coli B/r) and repair deficient ( E. coli K12 AB 1886 uvrA , AB 2463 recA and AB 2480 uvrA recA ) strains of bacteria by noon sunlight has been measured. The use of biological dosimetry based on an ultraviolet (UV) sensitive strain of Bacillus subtilis spores has allowed a quantitative comparison of bacterial inactivation by solar, 254 and 302 nm radiations. Our analysis indicates that: (1) uvrA and recA gene products are involved in repair of a substantial portion of the solar DNA damage, (2) 302 nm is a more appropriate wavelength than 254 nm to represent the DNA-damaging action of sunlight and that (3) repair proficient strains are inactivated by sunlight more rapidly than expected from the levels of DNA damage induced. When populations of repair proficient bacteria are exposed to noon sunlight for 20 min, they become sensitive to the lethal action of far-UV (254 nm), MMS (0.1 M ) and to a lesser extent, mild heat (52°C).     

A COMPARISON OF MAMMALIAN CELL SENSITIVITY TO EITHER 254 nm OR ARTIFICIAL "SOLAR" SIMULATED RADIATION

The sensitivity of six mammalian cell strains to either germicidal (254 nm) or artificial "solar" simulated radiation was tested. The solar simulator used had an output similar, in some respects, to natural sunlight. Cellular capacity for Herpes simplex virus production was used as the assay procedure. The tested cells were a strain of African green monkey kidney cells and five human skin fibroblast cell strains. The latter included a "normal" cell strain, and four photosensitive cell strains; three of which were strains of xeroderma pigmentosum cells, and one strain of Bloom's syndrome cells. When comparing the D¹⁰ values, the different cell strains varied by a factor of six in response to germicidal radiation, but only by a factor of two to artificial "solar" simulated radiation. The relative sensitivity of the cells to either type of radiation also varied from 1.7 to 10.9. Large variations in response occurred even among the xeroderma pigmentosum cell strains. These responses suggest that mammalian cell sensitivity to 254 nm radiation may not be a true indicator of a cell's responses to natural sunlight.     

Both solar UVA and UVB radiation impair conidial culturability and delay germination in the entomopathogenic fungus Metarhizium anisopliae.

The entomopathogenic hyphomycete Metarhizium anisopliae has been used in programs of agricultural pest and disease vector control in several countries. Exposure to simulated solar radiation for a few hours can completely inactivate the conidia of the fungus. In the present study we determined the effect of exposures to full-spectrum sunlight and to solar ultraviolet A radiation at 320-400 nm (UVA) on the conidial culturability and germination of three M. anisopliae strains. The exposures were performed in July and August 2000 in Logan, UT. The strains showed wide variation in tolerance when exposed to full-spectrum sunlight as well as to UVA sunlight. Four-hour exposures to full-spectrum sunlight reduced the relative culturability by approximately 30% for strain ARSEF 324 and by 100% for strains ARSEF 23 and 2575. The relative UV sensitivity of the two more sensitive strains was different under solar UV from that under ultraviolet B radiation at 280-320 nm (UVB) in the laboratory. Four-hour exposures to solar UVA reduced the relative culturability by 10% for strain ARSEF 324, 40% for strain ARSEF 23 and 60% for strain ARSEF 2575. Exposures to both full-spectrum sunlight and UVA sunlight delayed the germination of the surviving conidia of all three strains. These results, in addition to confirming the deleterious effects of UVB, clearly demonstrate the negative effects of UVA sunlight on the survival and germination of M. anisopliae conidia under natural conditions. The negative effects of UVA in sunlight also emphasize that the biological spectral weighting functions for this fungus must not neglect the UVA wavelengths.     

Diel Cycles of DNA Damage and Repair in Eggs and Larvae of Northern Anchovy, Engraulis mordax, Exposed to Solar Ultraviolet Radiation

Abstract— Damage from UVB radiation (280–320 nm) in the form of cyclobutane pyrimidine dimers (CPD) in DNA and the capacity for their repair were measured in newly spawned eggs and yolk-sac larvae of northern anchovy, Engraulis mordax, exposed to natural diel cycles of sunlight. The CPD were measured by a newly developed chemiluminescent immunoblot assay capable of measuring CPD in samples as small as 50 ng DNA. Eggs and yolk-sac larvae exposed to full irradiance levels died. At lower dose rates, equivalent to deeper more natural locations in the water column, there was a diel cycle of dimer concentration that tracked solar intensity. This diel cycle was due to the interaction of damage and repair processes. Repair of CPD in anchovy eggs and larvae could be attributed to true photodependent repair that could be stopped by moving samples into the dark. The CPD present at sunset remained until the following morning. The diel cycles of damage and repair were maintained over at least 4 days without a long-term upward or downward trend in dimer concentration. This indicates that at the UVB doses used for these experiments, there was no long-term accumulation of CPD nor an induction of increased repair capacity. Unhatched embryos spawned in the dark also exhibited a strong photorepair response, suggesting that photolyase expression was innate and not dependent on previous light exposure. The diel cycle observed here indicates that, at least for northern anchovy, the CPD concentration at the time of sampling is a good indicator of dose rate but a poor indicator of cumulative dose (i.e. late afternoon samples have the highest cumulative dose but relatively low CPD concentrations). The CPD immunoassay described here has the required sensitivity for measuring DNA damage in wild populations of ichthyoplankton exposed to natural sunlight. These results will guide the collection and interpretation of field data on natural levels of CPD in wild larvae collected at different depths and times of the day.     

Inactivation of Bacteriophage Infecting Bacteroides Strain GB124 Using UV‐B Radiation

Ultraviolet‐B radiation (280–320 nm) has long been associated with the inactivation of microorganisms in the natural environment. Determination of the environmental inactivation kinetics of specific indicator organisms [used as tools in the field of microbial source tracking (MST)] is fundamental to their successful deployment, particularly in geographic regions subject to high levels of solar radiation. Phage infecting Bacteroides fragilis host strain GB124 (B124 phage) have been demonstrated to be highly specific indicators of human fecal contamination, but to date, little is known about their susceptibility to UV‐B radiation. Therefore, B124 phage (n = 7) isolated from municipal wastewater effluent, were irradiated in a controlled laboratory environment using UV‐B collimated beam experiments. All B124 phage suspensions possessed highly similar first order log‐linear inactivation profiles and the mean fluence required to inactivate phage by 4 ? log¹⁰ was 320 mJ cm^?2. These findings suggest that phage infecting GB124 are likely to be inactivated when exposed to the levels of UV‐B solar radiation experienced in a variety of environmental settings. As such, this may limit the utility of such methods for determining more remote inputs of fecal contamination in areas subject to high levels of solar radiation.     

PHOTODYNAMIC AND SUNLIGHT INACTIVATION OF ESCHERICHIA COLI STRAINS DIFFERING IN NEAR-UV SENSITIVITY AND RECOMBINATION PROFICIENCY

Abstract— Stationary cells of four Escherichia coli strains exhibiting all four possible combinations of genes controlling near-UV sensitivity ( nur vs nur ⁺) and recombination proficiency (far-UV sensitivity; recA1 us recA ⁺) have been inactivated by visible light in the presence of acridine orange (AO, 10µg/m l ) and sunlight. The results demonstrate that strains sensitive to near-UV inactivation are also sensitive to inactivation by visible light in the presence of AO and sunlight irrespective of the recA allele carried by the strain. These results may be interpreted to mean that major mechanisms of inactivation of stationary E. coli cells by near-UV, visible light in the presence of AO and sunlight are similar and not closely related to the mechanism of inactivation by far-UV.     

THE RATE OF REMOVAL OF SUNLIGHT-INDUCED PYRIMIDINE DIMERS FROM THE DNA OF CULTURED HUMAN DIPLOID FIBROBLASTS

Abstract The rate of excision of sunlight-induced pyrimidine dimers in DNA of exposed human cells was determined. Two normal excision repair-proficient human diploid fibroblast strains (WS-1 and KD) and a repair-deficient strain (XP12BE, group A) maintained in a nondividing state were exposed to summer noon-time sunlight for times (5 and 20 min) that induced numbers of dimers equivalent to far UV (254 nm) exposures of 1 and 4 J/m². Pyrimidine dimers were quantified in extracted DNA using a U V-endonuclease-alkaline sedimentation assay. The excision rates of these dimers were similar to those observed for the excision of UV-induced pyrimidine dimers. No sunlight-induced inhibition or stimulation of DNA repair was observed in either strain at these low exposures.     

Functional genetic analysis reveals a 2-Alkyl-4-quinolone signaling system in the human pathogen Burkholderia pseudomallei and related bacteria

Pseudomonas aeruginosa synthesizes diverse 2-alkyl-4(1H)-quinolones (AHQs), including the signaling molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), via the pqsABCDE locus. By examining the genome databases, homologs of the pqs genes were identified in other bacteria. However, apart from P. aeruginosa, only Burkholderia pseudomallei and B. thailandensis contained a complete pqsA-E operon (termed hhqA-E). By introducing the B. pseudomallei hhqA and hhqE genes into P. aeruginosa pqsA and pqsE mutants, we show that they are functionally conserved and restore virulence factor and PQS production. B. pseudomallei, B. thailandensis, B. cenocepacia, and P. putida each produced 2-heptyl-4(1H)-quinolone (HHQ), but not PQS. Mutation of hhqA in B. pseudomallei resulted in the loss of AHQ production, altered colony morphology, and enhanced elastase production, which was reduced to parental levels by exogenous HHQ. These data reveal a role for AHQs in bacterial cell-to-cell communication beyond that seen in P. aeruginosa.     

MEMBRANE DAMAGE CAN BE A SIGNIFICANT FACTOR IN THE INACTIVATION OF ESCHERICHIA COLI BY NEAR-ULTRAVIOLET RADIATION

Abstract A DNA repair competent strain of Escherichia coli K-12 showed sensitivity to inorganic salts (at concentrations routinely used in minimal media) after irradiation with broad spectrum near–UV radiation, at fluences that caused little inactivation when plated on complex growth medium. This effect was not observed with cells that had been exposed to 254 nm radiation. This sensitivity to minimal medium was increased by increasing the salt concentration of the medium and by increasing the pH of the medium. This sensitivity was greatly increased by adding to the medium a low concentration of commercial glassware cleaning detergent that had no effect on unirradiated cells or far-UV irradiated cells. These findings may explain the large variability often observed in near-UV radiation survival data, and demonstrate that, at least on minimal medium plates, membrane damage contributes significantly towards cell killing. This phenomenon is largely oxygen dependent.     

ACTION SPECTRA FOR KILLING NON-DIVIDING NORMAL HUMAN AND XERODERMA PIGMENTOSUM CELLS

Abstract— Non-dividing human cells degenerate and eventually detach from a culture vessel surface when exposed to UV light. Action spectra for this kind of cell inactivation were determined using eight monochromatic wavelengths from 240 to 313 nm and both a normal DNA excision-repair-proficient strain and a repair-deficient Xeroderma pigmentosum (XP12BE) strain. The action spectra for both strains have similar shapes with a broad peak between 254 and 280 nm followed by a steep decline at wavelengths greater than 280 nm. The relative action spectra are similar to those for inactivation of reproductive capacity and pyrimidine dimer formation in rodent cells suggesting that the critical target and critical damage for inactivation of non-dividing human cells is DNA and damage to DNA, respectively. Normal repair-proficient cells are 5–7 times more resistant at all wavelengths, based on a comparison of D_o values, than repair-deficient XP12BE cells, supporting the conclusion that the inactivating damage at all wavelengths is to DNA.     

THE INTERACTION OF A GENE (nur) CONTROLLING NEAR-UV SENSITIVITY AND THE polA1 GENE IN STRAINS OF E. COLI K12

Abstract— When stationary cell populations of the Escherichia coli W3110 strain and the polA1 mutant (p3478) derived directly from it were compared for their sensitivity to near-UV (NUV, 300–400 nm) inactivation, the polA1 strain proved to be more sensitive. By appropriate matings and transductions, four essentially isogenic strains have been developed which carry all four possible combinations of genes conferring far-UV (FUV, 200-300 nm) sensitivity ( polA1 vs. polA ⁺) and NUV sensitivity ( nur vs. nur ⁺). Stationary cells of strains carrying either the polA1 or polA ⁺ allele in combination with the nur allele are indistinguishable in their sensitivity to NUV inactivation and are equivalent in their NUV sensitivity to the original polA1 mutant strain (p3478). With the two strains carrying the nur ⁺ allele, stationary cell populations of the polA1 strain are clearly more sensitive to NUV inactivation than is the polA ⁺ strain. The NUV sensitizing effect of the polA1 mutation in a nur ⁺ genetic background is about the same as that of the nur mutation at the 0.37 survival level. This may mean that the polA1 and nur mutations sensitize E. coli stationary cell populations to NUV inactivation by a common mechanism.     

INDUCTION OF SISTER-CHROMATID EXCHANGES IN ICR 2A FROG CELLS EXPOSED TO 254 NM AND SOLAR UV WAVELENGTHS

Abstract— Exposure of ICR 2A frog cells to 254 nm UV induced the formation of sister-chromatid exchanges (SCEs) in a fluence-dependent manner. Cells were also exposed to the UV produced by a fluorescent sunlamp that was filtered through 8C Mylar in order to simulate the mid-UV(290–320 nm) portion of sunlight reaching the earth's surface. In this instance, SCEs were induced in a linear fashion at low fluences but reached a plateau at a low level of induced SCEs. In addition, pretreatment of cells with the solar UV followed by exposure to 254 nm UV resulted in a significantly lower level of SCEs than in cells exposed to 254 nm UV alone.     

SENSITIVITY OF STRAINS OF ESCHERICHIA COLI DIFFERING IN REPAIR CAPABILITY TO FAR UV,NEAR UV AND VISIBLE RADIATIONS*

Abstract— In stationary phase, strains of Escherichia coli deficient in excision (B/r Her) or recombination repair (K.12 AB2463) were more sensitive than a repair proficient strain (B/r) to monochromatic near-ultraviolet (365 nm) and visible (460 nm) radiations. The relative increase in sensitivity of mutants deficient in excision or recombination repair, in comparision to the wildtype, was less at 365 nm than at 254 nm. However, a strain deficient in both excision and recombination repair (K12 AB2480) showed a large, almost equal, increase in sensitivity over mutants deficient in either excision or recombination repair at 365 nm and 254 nm. All strains tested were highly resistant to 650 nm radiation. Action spectra for lethality of strains B/r and B/r Her in stationary phase reveal small peaks or shoulders in the 330–340, 400–410 and 490–510 nm wavelength ranges. The presence of 5μg/ml acriflavine (an inhibitor of repair) in the plating medium greatly increased the sensitivity of strain B/r to radiation at 254, 365 and 460 nm, while strains E. coli B/r Her and K12 AB2463 were sensitized by small amounts. At each of the wavelengths tested, acriflavine in the plating medium had at most a small effect on E. coli K.12 AB2480. Acriflavine failed to sensitize any strain tested at 650 nm. Evidence supports the interpretation that lesions induced in DNA by 365 nm and 460 nm radiations play the major role in the inactivation of E. coli by these wavelengths. Single-strand breaks (or alkali-labile bonds), but not pyrimidine dimers are candidates for the lethal DNA lesions in uvrA and repair proficient strains. At high fluences lethality may be enhanced by damage to the excision and recombination repair systems.     

RHYTHMS IN BLUE-LIGHT-INDUCED CONIDIATION OF WILD TYPE AND A MUTANT STRAIN OF Trichoderma harzianum

Abstract— Trichoderma harzianum normally requires light for conidiation. Conidiation of colonies grown in continuous light does not appear to be rhythmic, but sharp banding patterns are formed under light/dark cycles. A single pulse of blue light produces a sharp band of conidia that forms where the growing edge of the mycelia is located at the time when the light is given. A period of about 24 h is required following the light pulse to produce mature conidia. During this time colonies are insensitive to further induction by light. The fluence required to produce 50% saturation varies by a factor of about 3 depending on when the pulse is given. This change of sensitivity is rhythmic with a period length of approximately 27 h when grown on medium containing deoxycholate.
The pattern of conidiation in a mutant strain (B119), which is able to form conidia in the dark, is rhythmic and the period length is dependent on the composition of the medium. Addition of deoxycholate to the medium increased the interval between dark bands from 12 to 24 h. The rhythmic banding is suppressed in constant light and a double banding pattern is produced in light/dark cycles. A pulse of blue light induces a band of conidia in this mutant, as in the wild type, but it also delays the reappearance of the dark banding pattern. The extent of this delay depends on when the pulse is given and, although the period length of the dark conidiation rhythm is affected by deoxycholate, the effect of blue light on its phase is not. Of the various rhythmic responses of Trichoderma studied here. the delay in reappearance of the dark banding pattern in B119 is the most promising for further detailed studies, for example of wavelength and temperature dependence.     

Action spectra for inactivation of normal and xeroderma pigmentosum human skin fibroblasts by ultraviolet radiations

—Action spectra for UV-induced lethality as measured by colony forming ability were determined both for a normal human skin fibroblast strain (lBR) and for an excision deficient xeroderma pigmentosum strain (XP4LO) assigned to complementation group A using 7 monochromatic wavelengths in the range 254-365 nm. The relative sensitivity of the XP strain compared to the normal skin fibroblasts shows a marked decrease at wavelengths longer than 313 nm. changing from a ratio of about 20 at the shorter wavelengths to just greater than 1.0 at the longer wavelengths. The action spectra thus indicate that the influence on cell inactivation of the DNA repair defect associated with XP cells is decreased and almost reaches zero at longer UV wavelengths. This would occur, for example, if the importance of pyrimidine dimers as the lethal lesion decreased with increasing wavelength. In common with other studies both in bacterial and mammalian cells, our results are consistent with pyrimidine dimers induced in DNA being the major lethal lesion in both cell strains over the wavelength range 254-313 nm. However, it is indicated that different mechanisms of inactivation operate at wavelengths longer than 313 nm.     

CORRELATION BETWEEN INACTIVATION OF HUMAN CELLS and NUMBERS OF PYRIMIDINE DIMERS INDUCED BY A SUN LAMP and 254 nm RADIATION

Abstract— Nondividing human fibroblasts are inactivated by radiation from a source (a Westinghouse sun lamp) that simulates the UV spectrum of sunlight. Survival curves determined for a DNA excision repair-proficient and a repair-deficient strain (XP12BE) are related to those determined using germicidal light (254 nm) by constant fluence modification factors. In addition, the same fraction of XP12BE cells are killed per pyrimidine dimer by 254 nm and sun lamp light. These results, when related to other survival and photoreactivation studies, suggest that the mechanism for inactivation of nondividing human cells by sun lamp light is the same as that by 254 nm and that pyrimidine dimers are the major responsible photolesion. Repair reverses some of the lethal effects of this light. We suggest that these conclusions apply to sunlight-irradiated skin cells in vivo.