The Combined Effect of Hydrogen Peroxide and Ultraviolet Irradiation on Bacterial Spores

Ultra-violet (u.v.) light irradiation of spores of Bacillus subtilis in the presence of hydrogen peroxide produced a rapid kill which was up to 2000-fold greater than that produced by irradiation alone. A kill of 99–99% was produced by 30s u.v. irradiation of spores of 6 strains of Bacillus and Clostridium in the presence of hydrogen peroxide 1.0 g/100 ml but with the more resistant spores of 9 further strains, irradiation in the presence of hydrogen peroxide 2–5 g/100 ml followed by mild heating was required.     

The kinetics of Bacillus subtilis spore inactivation on filter paper by u.v. light and u.v. light in combination with hydrogen peroxide

Inactivation of spores of Bacillus subtilis (ATCC 6633) on two different grades of cellulose filter paper (Whatman Grades 2 and 6), by ultraviolet light (u.v.), at an intensity of approximately 4·5 Wm⁻² and at fluences of up to 2 × 10³ Jm⁻², and u.v. in the presence of hydrogen peroxide, is described in terms of multi-target and single hit–single target kinetic expressions. Wet spores were inactivated at rates ranging from 6·7 to 10·6 higher than that of dry spores on both grades of filter paper. In addition, spore inactivation was up to 5·6 times more rapid on Grade 2 filter paper. Synergistic inactivation was seen to occur when spores were irradiated in the presence of 1% (w/v) hydrogen peroxide with rates up to 5·3 times higher than with treatment solely by u.v. The results obtained are discussed in general terms with particular reference to surface characteristics which might provide shielding to micro-organisms from incident u.v. light.     

The Effect of Hydrogen Peroxide and Ultraviolet Irradiation on Non-sporing Bacteria

A kill of 99.99% was obtained in cell suspensions of Escherichia coli and Streptococcus faecalis by incubation with hydrogen peroxide 1.0% (w/v) for 75 and 180 min respectively. The same kill was produced by 30 s irradiation with ultraviolet (u.v.) light in the presence of hydrogen peroxide 1.0% (w/v). This simultaneous treatment with u.v. and hydrogen peroxide produced a synergistic kill at least 30-fold greater than that produced by irradiation of cell suspensions of Esch. coli with or without subsequent incubation with hydrogen peroxide.     

Resistance of Serratia marcescens to Hydrogen Peroxide

Irradiation with ultraviolet (u.v.) light (71 J/m²) reduced the viable count of suspenrsions of Serratia marcescens , grown in a glycerol-salts defined medium, to five in 10⁴ cells. Subsequent incubation of irradiated cells in hydrogen peroxide failed to decrease the survivors, but u.v. irradiation in the presence of hydrogen peroxide reduced the viable count to fewer than two in 10⁶ cells. Cells grown in defined medium with added iron had more measurable catalase activity and were more resistant to hydrogen peroxide alone and to simultaneous treatment with u.v. irradiation and hydrogen peroxide. Cells grown in a non-defined medium contained little iron and measurable catalase activity but were more resistant to hydrogen peroxide. Treatment with toluene, heat killing or sonication increased the catalase activity detected in all cell suspensions and showed that resistance to hydrogen peroxide and to u.v. irradiation in hydrogen peroxide was related to the total catalase activity within cells.     

Inactivation of Escherichia coli by ultraviolet light and hydrogen peroxide in a thin film contactor

Apparatus for irradiating enclosed thin liquid films with ultraviolet (u.v.) light (Λ= 253.7 nm) in combination with hydrogen peroxide was used to inactivate Escherichia coli in water. Hydrogen peroxide concentrations of 2.5, 5.0 and 10.0 g/I were used and in each case synergistic inactivation was observed. At the highest concentration, a fractional survival of 1.3 times 10^-3 was obtained after 20 min; this was decreased to 3.1 times 10^-6 by simultaneous u.v. irradiation.     

Sensitized NADH formation of single-stranded breaks in plasmid DNA upon the action of near UV-radiation

It has been shown that NADH photosensitize in vitro single-strand breaks formation in double-strand plasmid DNA pBR 322 upon near-UV (320-400 nm) irradiation. The number of single-strand breaks depends both on UV light dose and sensitizer concentration. Addition of catalase and sodium benzoate strongly decreases the single-strand breaks formation. The results show an important role of hydrogen peroxide (H2O2) and hydroxyl radical (.OH) in inducing single-strand breaks in plasmid DNA irradiated by near-UV radiation in the presence of NADH.     

Carton sterilization by u.v.-C excimer laser light: recovery of Bacillus subtilis spores on vegetable extracts and food simulation matrices

AIMS: To determine the recovery of Bacillus subtilis spores loaded onto preformed cartons and irradiated with u.v.-excimer laser (248 nm) light. METHODS AND RESULTS: Bacillus subtilis spores irradiated with u.v.-excimer laser light retained phase brightness, but were blocked at various stages of germination. In the presence of germinant, the majority of spores began to lose phase brightness but only after an extended lag period (ca 90 min). After 6 h ca 9% of the spores had elongated but failed to form new cells, approx. 12% had undergone partial phase darkening (grey spores), 15% remained phase bright whilst the remainder had turned fully phase dark but failed to elongate. No enhanced recovery of u.v.-treated spores (with intact or permeabilized coats) occurred in media containing hen egg white lysozyme or vegetable extracts (celery, carrot, swede or turnip). However, recovery did occur when irradiated spores were incubated for 26 d, semiaerobically, within cartons containing nutrient broth or milk. CONCLUSIONS: The germination ability of B. subtilis spores is altered following u.v.-excimer laser treatment. Recovery of treated spores was found in liquid systems but not on agar plates supplemented with vegetable extracts or lysozyme. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential recovery of u.v.-excimer laser-treated spores in a range of carton-packed food systems requires further investigation.     

Tailing of Survival Curves of Bacillus licheniformis Spores Treated with Hydrogen Peroxide

An attempt has been made to rule out possible causes of artefacts in establishing survival curves of Bacillus licheniformis spores heated (30–80 °C) in 4.4 mol/l hydrogen peroxide (pH 2.0). A tailing phenomenon apparent as that of a suspension of spores produced by routine subculture was obtained with those grown-up from a single spore selected by micromanipulation. No spore fraction differing in size or density could be separated from the whole population. The tail was not due to decomposition of hydrogen peroxide, protective effect by other spores, release of protective factors, or temperature heterogeneity during treatment. Changing from an open vessel to a closed tube did not influence the tailing. The only apparent artefact was therefore the formation of clumps under the conditions of the treatment. Since the spore catalase was demonstrated to be highly resistant, it was concluded that a spore could be protected against hydrogen peroxide by the catalase of the other spores in the clump. Conditions resembling those arising in spore suspensions could occur under industrial conditions, for example in sterilizing surfaces contaminated with aggregates of Bacillus spores.     

Restoration of u.v.-induced excision repair in Xeroderma D cells transfected with the denV gene of bacteriophage T4.

The heritable DNA repair defect in human Xeroderma D cells, which results in failure to incise at u.v. light-induced pyrimidine dimers, has been partially but stably corrected by transfection of immortalised cells with the denV pyrimidine dimer glycosylase gene of bacteriophage T4. Transfectants selected either for a dominant marker on the mammalian vector carrying the prokaryotic gene or for the dominant marker plus resistance to killing by u.v. light, have been shown to express the denV gene to varying degrees. denV expression results in significant phenotypic change in the initially repair-deficient, u.v.-hypersensitive cells. Increased resistance to u.v. light and more rapid recovery of replicative DNA synthesis following u.v. irradiation have been correlated both with improved repair DNA synthesis and with a novel dimer incision capability present in denV transfected Xeroderma cells but not as evident in transfected normal cells. Most of the transfectants contain a single integrated copy of the denV gene; increase in denV copy number does not result in either increased gene expression or enhanced survival to u.v. light. These results show that expression of a heterologous prokaryotic repair gene can partially compensate for the genetic defect in a human Xeroderma D cell.     

Relationship between physiochemical properties, aggregation and u.v. inactivation of isolated indigenous spores in water

AIMS: The objective of the study was to compare ultraviolet (u.v.) inactivation kinetics of indigenous aerobic spores in surface water with their laboratory-cultured spore isolates and to investigate the relationship between physicochemical characteristics and u.v. inactivation kinetics of spore isolates. METHODS AND RESULTS: Lake water samples were analysed for the presence of indigenous aerobic spores. Different bacterial isolates from the heterogeneous indigenous population were genetically characterized, resporulated and examined for hydrophobicity, surface charge, particle size distribution and survival at different u.v. 254 nm fluence levels. Cultured isolated spores exhibited a three-stage inactivation curve consisting of shoulder, first order and tailing regions whereas indigenous spores exhibited only one stage of linear kinetics. Hydrophobicity of the Bacillus spore isolates was inversely related to the extent of u.v. inactivation before tailing occurred. CONCLUSIONS: Tailing in the u.v. inactivation curves results from aggregation of a portion of the spore population because of hydrophobic interactions, supporting the link between aggregation of spores, hydrophobicity and u.v. inactivation. SIGNIFICANCE AND IMPACT OF THE STUDY: Evidence of the link between spore physicochemical parameters and u.v. disinfection performance furthers the understanding of factors that affect inactivation of microbes in natural waters supplied to drinking water treatment plants.     

The effect of hydrogen peroxide on spores of Clostridium bifermentans.

The effect of hydrogen peroxide on the germination, colony formation and structure of spores of Clostridium bifermentans was examined. Treatment with 0.35 M-hydrogen peroxide increased the germination rate at 25 degrees C but increasing the temperature or concentration of hydrogen peroxide decreased both the germination rate and colony formation. The presence of Cu2+ increased the lethal effect of hydrogen peroxide on colony formation as much as 3000-fold. Pre-incubation of spores with Cu2+ before treatment with hydrogen peroxide produced a similar increase, but this could be eliminated by washing the spores with dilute spores--apparently from the coat--and treatment with dithiothreitol, which also removes spore-coat protein, increased the lethal effect of hydrogen peroxide 500-fold, suggesting that spore-coat protein has a protective effect against hydrogen peroxide.     

Alkyl hydroperoxide reductase, catalase, MrgA, and superoxide dismutase are not involved in resistance of Bacillus subtilis spores to heat or oxidizing agents.

Only a single superoxide dismutase (SodA) was detected in Bacillus subtilis, and growing cells of a sodA mutant exhibited paraquat sensitivity as well as a growth defect and reduced survival at an elevated temperature. However, the sodA mutation had no effect on the heat or hydrogen peroxide resistance of wild-type spores or spores lacking the two major DNA protective alpha/beta-type small, acid-soluble, spore proteins (termed alpha(-)beta(-) spores). Spores also had only a single catalase (KatX), as the two catalases found in growing cells (KatA and KatB) were absent. While a katA mutation greatly decreased the hydrogen peroxide resistance of growing cells, as found previously, katA, katB, and katX mutations had no effect on the heat or hydrogen peroxide resistance of wild-type or alpha(-)beta(-) spores. Inactivation of the mrgA gene, which codes for a DNA-binding protein that can protect growing cells against hydrogen peroxide, also had no effect on spore hydrogen peroxide resistance. Inactivation of genes coding for alkyl hydroperoxide reductase, which has been shown to decrease growing cell resistance to alkyl hydroperoxides, had no effect on spore resistance to such compounds or on spore resistance to heat and hydrogen peroxide. However, Western blot analysis showed that at least one alkyl hydroperoxide reductase subunit was present in spores. Together these results indicate that proteins that play a role in the resistance of growing cells to oxidizing agents play no role in spore resistance. A likely reason for this lack of a protective role for spore enzymes is the inactivity of enzymes within the dormant spore.     

Inactivation of Bacillus subtilis spores on packaging surfaces by u. v. excimer laser irradiation

Ultraviolet (u.v.) laser irradiation has been used to inactivate Bacillus subtilis spores deposited on to planar aluminium- and polyethylene-coated packaging surfaces. Kill kinetics were found to be diphasic, with an initial rapid inactivation phase followed by tailing. Although no definitive evidence was obtained, it is thought that spores located within packaging crevices/pores were primarily responsible for the observed tailing. Surviving spores were also found on the unexposed underside of cards and, to a lesser extent, within clumps. The log count reduction in B. subtilis was dependent on spore loading and total u.v. dose. In comparison, packaging surface composition, fluence (2-18 Jm-2) and frequency (40-150 Hz) had only a negligible effect. By irradiating boards carrying 106 spores, with a dose of 11.5 J cm-2, a log count reduction >5 was obtained. The mode of spore inactivation was primarily through DNA disruption. This was confirmed by the high sensitivity of spores lacking protective, small, acid-soluble proteins, in addition to the high frequency of auxotrophic and asporogenous mutations found amongst survivors.     

The mechanism of bilirubin-photosensitized DNA strand breakage in human cells exposed to phototherapy light

Exposure of normal human fibroblasts to visible light (420–490 nm) in the presence of exogenously added 1–100 μg/ml bilirubin enhanced the level of DNA strand breakage compared with cells irradiated in the absence of added bilirubin. Treatment of cells in the dark with an irradiated bilirubin solution also induced DNA strand breaks. However, strand breakage was not detected in cells treated with an irradiated bilirubin solution that had been incubated with catalase (H₂O₂: H₂O₂ oxidoreductase EC 1.11.1.6). Examination of irradiated bilirubin solutions demonstrated the presence of hydrogen peroxide although, apparently, not at concentrations sufficient to account for the level of DNA strand breakage detected. Hence, irradiation of bilirubin results in the generation of hydrogen peroxide and possibly other peroxides that can cause DNA damage.     

Involvement of calcium and dipicolinic acid in the resistance of Bacillus cereus BIS-59 spores to u.v. and gamma radiations

The role of dipicolinic acid (DPA) in determining the resistance of Bacillus cereus spores to u.v. and gamma radiation was investigated. B. cereus BIS-59 spores containing varying amounts of DPA were prepared by appropriate compositional adjustments in the secondary media. Compared with spores containing 6 per cent DPA (dry weight) those containing 0.8 per cent DPA were far more sensitive to u.v. radiation. Similar u.v. radiation sensitivity was also found in respect of a DPA-less mutant of B. cereus T 6A 1. Pre-treatment of DPA deficient spores (of wild type or mutant B. cereus) with DPA or the presence of DPA during irradiation resulted in increased resistance of these spores to u.v. radiation. In the range 0.2 to 1 per cent DPA content of spores of B. cereus BIS-59, a striking inverse relationship could be discerned between the DPA content and the number of spore photo-products (5-thymidyl, 5,6-dihydrothymine) formed in DNA and spore viability. The resistance of B. cereus spores to gamma radiation did not seem to be influenced by their DPA content.     

A relation between G-, C-, and N-band patterns as revealed by progressive oxidation of chromosomes and a note on the nature of N-bands

Near-ultraviolet irradiation of chromosome preparations mounted in a hydrogen peroxide solution resulted in an oxidative disintegration of the structure of fixed metaphase chromosomes with concomitant production of various band patterns appearing after staining with Giemsa. Neither irradiation nor hydrogen peroxide alone could produce banding. After irradiation in the presence of hydrogen peroxide the gradually increasing effect of oxidation on the chromosomes along the gradient of light intensities from the periphery of the slide towards the radiation focus in the centre of the slide became visible as G-, C-, and N-banding, respectively. Close to the centre only contours of chromosomes were left after this treatment. Although G-banding and differential DNA-extraction often went together, extraction of DNA was not an absolute requirement to obtain a G-band pattern. N-bands appeared to be the chromosomal regions that were most resistant to destruction. Staining methods specific for DNA failed to demonstrate these bands, although with Giemsa an intense staining reaction occurred. On the analogy of the staining behaviour of model protein preparations with Giemsa a phosphoprotein nature is suggested for the N-band material in the chromosomes.     

Production of Conidia by Botrytis fabae grown in vitro

Conidiation in Botrytis fabae was stimulated by irradiating 1 to 3 day old, but not 4 to 5 day old mycelium. Three cycles of 12 h irradiation + 12 h darkness stimulated the production of about twice as many spores compared with only 12 h irradiation. At 18°C all the spores had been produced within 3 days but not within 2 days from the start of irradiation. Near-u.v. irradiation at wavelengths of 375–400 nm induced most sporulation. Red light at 600–650 nm also stimulated conidiation but irradiation at other wavelengths from 300 to 700 nm was ineffective. Fewer conidia were produced when the fungus was kept in darkness at 4°C between periods of irradiaton at 18°C compared with continuous 18°C. The optimum osmotic potential of the culture, medium for conidiation was about-27 bar although more mycelium grew at even lower osmotic potentials. Abundant spore production occurred when the fungus was grown in media with a wide range of pH values.     

Photochemical cross-linking of protein and DNA in chromatin. Synthesis and application of a photosensitive cleavable derivative of 9-aminoacridine with two photoprobes connected through a disulphide-containing linker.

A novel cleavable photo-cross-linking reagent, N-(2-methoxy-6-azidoacridin-9-yl)-N'-(4-azidobenzoyl)cystamine, for analysis of protein-nucleic acid interactions, has been synthesized. The reagent contains two photosensitive groups that can be activated sequentially. The azidoacridinyl moiety is sensitive to u.v. and visible light (lambda less than or equal to 450 nm), whereas the azidobenzoyl part needs higher-energy light (lambda less than or equal to 350 nm). Furthermore, the disulphide bridge connecting the two photoactive groups can be cleaved by reduction with mercaptans. The reagent is shown to induce cleavable cross-links between all five major histones and DNA in chromatin from Ehrlich ascites cells on activation with long-wavelength u.v. light (lambda greater than 300 nm) at an efficiency of approximately 3% of the added reagent.     

Photocatalyzed oxidation of NAD dimers

A mixture of dimers of nicotinamide adenine dinucleotide, largely 4,4?-linked, obtained by electrochemical reduction of NAD⁺, can be photooxidized back to NAD⁺ in the presence of oxygen. Oxygen is consumed during the photooxidation process with the production of hydrogen peroxide. The oxidation is almost pH independent and is stimulated by light whose wavelength exceeds 300 nm. Lactate dehydrogenase and alcohol dehydrogenase added to the solutions under irradiation increased the oxygen uptake by the NAD dimers in a concentration-dependent way. These observations suggest that light induces the homolytic cleavage of NAD dimers to NAD radicals which in turn are oxidized to NAD⁺ by oxygen.     

UV‐irradiation provokes generation of superoxide on cell wall polygalacturonic acid

We examined the redox effects of UV irradiation on cell wall isolates from Pisum sativum leaves, and polygalacturonic and galacturonic acid, in the presence of hydrogen peroxide. For this purpose, electron paramagnetic resonance spectroscopy and two spin‐traps (DEPMPO and BMPO), capable of differentiating between various free radicals, were applied. Systems were exposed to UV‐B (maximum emission at 312 nm) and UV‐A (352 nm) for 10 min (6 J m^–2 s^–1). Cell wall isolates exposed to UV in the presence of hydrogen peroxide, produced hydroxyl radical, carbon dioxide radical and superoxide. The production of superoxide was observed for cell wall isolates, polygalacturonic acid (in the presence and in the absence of calcium) and galacturonic acid, and it was diminished upon superoxide dismutase supplementation. The production is at least partially based on the reaction of hydroxyl radicals with (poly)galacturonic acid having carbon dioxide radicals as a products. Acting as a strong reducing agent, carbon dioxide radical reacts with molecular oxygen to produce superoxide. The results presented here shed a new light on: (1) the redox‐modulating role of cell wall; (2) the production of superoxide in the extracellular compartment; (3) the mechanisms involved in translating UV stress into molecular signaling and (4) some other UV‐related phenomena in plants, such as CO₂ emission.