Exposure to ultraviolet radiation delays photosynthetic recovery in Arctic kelp zoospores

Seasonal reproduction in some Arctic Laminariales coincides with increased UV-B radiation due to stratospheric ozone depletion and relatively high water temperatures during polar spring. To find out the capacity to cope with different spectral irradiance, the kinetics of photosynthetic recovery was investigated in zoospores of four Arctic species of the order Laminariales, the kelps Saccorhiza dermatodea, Alaria esculenta, Laminaria digitata, and Laminaria saccharina. The physiology of light harvesting, changes in photosynthetic efficiency and kinetics of photosynthetic recovery were measured by in vivo fluorescence changes of Photosystem II (PSII). Saturation irradiance of freshly released spores showed minimal I _k values (photon fluence rate where initial slope intersects horizontal asymptote of the curve) values ranging from 13 to 18 μmol photons m⁻² s⁻¹ among species collected at different depths, confirming that spores are low-light adapted. Exposure to different radiation spectra consisting of photosynthetically active radiation (PAR; 400–700 nm), PAR+UV-A radiation (UV-A; 320–400 nm), and PAR+ UV-A+UV-B radiation (UV-B; 280–320 nm) showed that the cumulative effects of increasing PAR fluence and the additional effect of UV-A and UV-B radiations on photoinhibition of photosynthesis are species specific. After long exposures, Laminaria saccharina was more sensitive to the different light treatments than the other three species investigated. Kinetics of recovery in zoospores showed a fast phase in S. dermatodea, which indicates a reduction of the photoprotective process while a slow phase in L. saccharina indicates recovery from severe photodamage. This first attempt to study photoinhibition and kinetics of recovery in zoospores showed that zoospores are the stage in the life history of seaweeds most susceptible to light stress and that ultraviolet radiation (UVR) effectively delays photosynthetic recovery. The viability of spores is important on the recruitment of the gametophytic and sporophytic life stages. The impact of UVR on the zoospores is related to the vertical depth distribution of the large sporophytes in the field.     

Inactivation of bacteria and spores by pulse electric field and high pressure CO2 at low temperature

The common methods for inactivation of bacteria involve heating or exposure to toxic chemicals. These methods are not suitable for heat-sensitive materials, food, and pharmaceutical products. Recently, a complete inactivation of many microorganisms was achieved with high-pressure carbon dioxide at ambient temperature and in the absence of organic solvent and irradiation. The inactivation of spores with CO(2) required long residence time and high temperatures, such as 60 degrees C. In this study the synergistic effect of pulsed electric field (PEF) in combination with high-pressure CO(2) for inactivation was investigated. The bacteria Escherichia coli, Staphylococcus aureus, and Bacillus cereus were suspended in glycerol solution and treated in the first step with PEF (up to 25 KV/cm) and then with high-pressure CO(2) not higher than 40 degrees C and 200 bar. The inactivation efficiency was determined by counting the colony formation units of control and sample. Samples of the cells subjected to PEF treatment alone and in combination with CO(2) treatment were examined by scanning electron microscopy to determine the effect of the processes on the cell wall. Experimental results indicate that the viability decreased with increasing electrical field strength and number of pulses. A further batch treatment with supercritical CO(2) lead to complete inactivation of bacterial species and decreased the count of the spores by at least three orders of magnitude, the inactivation being enhanced by an increase of contact time between CO(2) and the sample. A synergistic effect between the pulsed electric field and the high-pressure CO(2) was evident in all the species treated. The new low temperature process is an alternative for pasteurization of thermally labile compounds such as protein and plasma and minimizes denaturation of important nutrient compounds in the liquid media.     

Properties of Bacillus anthracis spores prepared under various environmental conditions

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45°C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45°C by wet heat, 2 M HCl, 2 M NaOH and 2 M H₂O₂ was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45°C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45°C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45°C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.     

Photocatalytic disinfection of Giardia intestinalis and Acanthamoeba castellani cysts in water

In this study, disinfection of water containing Giardia intestinalis and Acanthamoeba castellani cysts with TiO2 and modified catalyst silver loaded TiO2 (Ag-TiO2) was investigated. Destruction of the parasites was evaluated after UV illumination of the suspension consisting 5 x 10(8)-13.5 x 10(8)cysts/mL in the presence of 2g/L neat or modified TiO2 at neutral pH. In the initial stage, the solid photocatalyst particles penetrated the cyst wall and then oxidant species produced by TiO2/UV destroyed both cell wall and intracellular structure. In the case of G. intestinalis inactivation (disinfection) performance of TiO2/UV system reached 52.5% only after 25 min illumination and total parasite disinfection was achieved after 30 min illumination. However, silver loaded TiO2 seemed to be more effective as this loading provided better catalytic action as well as additional antimicrobial properties. Cell viability tests showed that parasite cysts, their walls in particular, were irreversibly damaged and cysts did not re-grow. Nevertheless the studied system seemed to be ineffective for the inactivation of A. castellani. Inactivation percentages of TiO2/UV and Ag-TiO2/UV systems were far lower than that of UV alone, being 50.1% and 46.1%, respectively.     

Inactivation and mechanisms of chlorine dioxide on Nosema bombycis

Biological tests demonstrated that the inactivation of Nosema bombycis (N. bombycis) spores by chlorine dioxide (ClO₂) occurs very fast and is highly sensitive. The lowest effective inactivation dosage and time was 15 mg/mL for 30 min. The inactivation of spores was additionally verified by using double color fluorescence stain and spore germination testing. A series of biological changes, including a large number of substrates that were leaked out from the spores included proteins, DNA, polysaccharide, K⁺, and Ca²⁺, occurred a short time after N. bombycis spores were treated with ClO₂. In addition, the lipid of spores was disrupted and ATPase activity was inhibited, which resulted in the destruction of the inner structure of the spores.     

Defensive and simultaneous actions of glycoconjugates during spore decontamination

An estimated $1 billion was lost in decontaminating areas exposed to anthrax in the 2001 attacks. To counter the threat of biological attacks, an effective 'green' decontaminant is vital to minimize the consequences of such attacks. The objective of our research was to study the ability of glycoconjugate ligands to decontaminate Bacillus cereus spores on hard surfaces. Polyvalent glycoconjugates (also known as neoglycoconjugates) were tested during decontamination of B. cereus spores. Resulting colony forming units (CFU) of viable spores were a direct evidence of glycoconjugate decontamination efficacy. Our results indicate a substantial, decreasing CFU count due to defensive and simultaneous actions of glycoconjugates compared to spores only used as controls. Decontamination of B. cereus spores was most efficiently and consistently achieved using Galalpha1-->3GalNAcbeta-PAA-flu glycoconjugate under both defensive and simultaneous conditions. Atomic force microscopy (AFM) allowed us to visualize decontamination at the nanoscale level using glycoconjugates. AFM reveals the size of glycoconjugate agglomerates (clusters) and a noticeably different morphology of glycoconjugate-treated spores during decontamination. Morphological features of untreated spores disappear under a thin layer of glycoconjugate solution. This thin layer is formed due to the defensive action of glycoconjugates. Simultaneous action has shown agglomeration of glycoconjugates in solution with B. cereus spores in glycoconjugate suspensions. Glycoconjugates might be useful for the development of an environment-friendly decontaminant of bacterial spores.     

Effects of moderately high pressure plus heat on the germination and inactivation of Bacillus cereus spores lacking proteins involved in germination

Aims:  To determine the germination and inactivation of Bacillus cereus spores lacking various germination proteins using moderately high pressure (MHP) and heat.
Methods:  The inactivation and germination of wild-type B. cereus spores in buffer by MHP (150 MPa) at various temperatures, as well as the MHP inactivation and germination of B. cereus spores lacking individual germinant receptors and monovalent cation antiporters, was determined.
Results:  Loss of individual germinant receptors had no large effects on spore inactivation or germination, although germination of receptor-deficient spores was generally slightly decreased. Loss of the GerN in particular the GerN and GerT antiporters also decreased spore germination by MHP, especially at 40 and 50°C.
Conclusions:  Both inactivation and germination of B. cereus spores by MHP increased with rise of temperature; however, mutant strains lacking individual germinant receptor had similar levels of germination as compared to wild-type spores. To evaluate the role of germinant receptors in MHP, a strain lacking a large number of germinant receptors is needed.
Significance and Impact of the Study:  The results of this work may lead to a better understanding of how MHP causes germination of spores of B. cereus .     

Inactivation of Geobacillus stearothermophilus spores by high-pressure carbon dioxide treatment

High-pressure CO2 treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO2 treatment at 30 MPa and 35 degrees C, to high-hydrostatic-pressure treatment at 200 MPa and 65 degrees C, or to heat treatment at 0.1 MPa and 85 degrees C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO2 treatment. We also investigated the influence of temperature on CO2 inactivation of G. stearothermophilus. Treatment with CO2 and 30 MPa of pressure at 95 degrees C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95 degrees C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95 degrees C for 120 min had little effect. The activation energy required for CO2 treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO2 treatment of G. stearothermophilus spores, CO2 treatment at 95 degrees C was more effective than treatment at 95 degrees C alone.     

Monoclonal antibodies for use in detection of Bacillus and Clostridium spores.

Five monoclonal antibodies against bacterial spores of Bacillus cereus T and Clostridium sporogenes PA3679 were developed. Two antibodies (B48 and B183) were selected for their reactivity with B. cereus T spores, two (C33 and C225) were selected for their reactivity with C. sporogenes spores, and one (D89) was selected for its reactivity with both B. cereus and C sporogenes spores. The isotypes of the antibodies were determined to be immunoglobulin G2a (IgG2a) (B48), IgG1 (B183), and IgM (C33, C225, and D89). The antibodies reacted with spores of B. cereus T, Bacillus subtilis subsp. globigii, Bacillus megaterium, Bacillus stearothermophilus, C. sporogenes, Clostridium perfringens, and Desulfotomaculum nigrificans. Antibody D89 also reacted with vegetative cells of B. cereus and C. sporogenes. Analysis of B. cereus spore extracts showed that two of the antigens with which the anti-Bacillus antibodies reacted had molecular masses of 76 kDa and approximately 250 kDa. Immunocytochemical localization indicated that antigens with which B48, B183, and D89 react are on the exosporium of the B. cereus T spore. Antibody D89 reacted with the exosporium and outer cortex of C. sporogenes spores in immunocytochemical localization studies but did not react with extracts of C. sporogenes or B. cereus spores in Western blotting. Some C. sporogenes antigens were not stable during long-term storage at -20 degrees C. Antibodies B48, B183, and D89 should prove to be useful tools for developing immunological methods for the detection of bacterial spores.     

Photocatalytic antimicrobial activity of thin surface films of TiO(2), CuO and TiO (2)/CuO dual layers on Escherichia coli and bacteriophage T4

TiO₂-coated surfaces are increasingly studied for their ability to inactivate microorganisms. The activity of glass coated with thin films of TiO₂, CuO and hybrid CuO/TiO₂ prepared by atmospheric Chemical Vapour Deposition (Ap-CVD) and TiO₂ prepared by a sol–gel process was investigated using the inactivation of bacteriophage T4 as a model for inactivation of viruses. The chemical oxidising activity was also determined by measuring stearic acid oxidation. The results showed that the rate of inactivation of bacteriophage T4 increased with increasing chemical oxidising activity with the maximum rate obtained on highly active sol–gel preparations. However, these were delicate and easily damaged unlike the Ap-CVD coatings. Inactivation rates were highest on CuO and CuO/TiO₂ which had the lowest chemical oxidising activities. The inactivation of T4 was higher than that of Escherichia coli on low activity surfaces. The combination of photocatalysis and toxicity of copper acted synergistically to inactivate bacteriophage T4 and retained some self-cleaning activity. The presence of phosphate ions slowed inactivation but NaCl had no effect. The results show that TiO₂/CuO coated surfaces are highly antiviral and may have applications in the food and healthcare industries.     

Polymeric glycoconjugates protect and activate macrophages to promote killing of <Emphasis Type="Italic">Bacillus cereus</Emphasis> spores during phagocytosis

Diseases caused by Bacillus spores might be attenuated if macrophages were able to kill the spores on exposure. Glycoconjugate-bearing polymers, which have been shown to bind to Bacillus spores, were tested for modulation of phagocytosis of B. cereus spores. Without glycoconjugate activation, murine macrophages were ineffective at killing Bacillus spores during phagocytosis. In the presence of glycoconjugates, however, the macrophages efficiently killed the organisms. The glycoconjugates were shown to have a protective influence, sparing macrophages from spore-induced cell death. Very low concentrations of the glycoconjugates prevented macrophage cell death, as shown by lactate dehydrogenase (LDH) release and trypan blue assays. Increased levels of inducible nitric oxide (NO) production by the macrophages in the presence of glycoconjugates suggested that the glycoconjugates provide an activation signal to the macrophages. These results suggest that glycoconjugates promote the killing of Bacillus spores by blocking spore-induced macrophage cell death, while increasing their activation level. Polymeric glycoconjugates may suggest novel approaches to improve existing vaccines as well as prevent and treat infections incurred through either B. cereus or B. anthracis spores.     

Bacterial endospores and their significance in stress resistance

In terms of resistance to extreme environmental stresses, the bacterial spore represents a pinnacle of evolution. Spores are highly resistant to a wide variety of physical stresses such as: wet and dry heat, UV and gamma radiation, oxidizing agents, chemicals, and extremes of both vacuum and ultrahigh hydrostatic pressure. Some of the molecular mechanisms underlying spore resistance properties have been elucidated in the laboratory, and involve both: (i) protection of vital spore macromolecules during dormancy, and (ii) repair of damaged macromolecules during germination. Our group has recently become interested in testing if the laboratory model of spore UV resistance is relevant to spore persistence in the environment. We have constructed a number of Bacillus subtilis strains which are defective in various DNA repair systems and spore structural components. Using spores of these strains, we have been exploring: (i) the types of damage induced in DNA by the UV-B and UV-A components of sunlight; (ii) the relative contribution of the major spore DNA repair systems to spore solar radiation resistance; and (iii) the role of spore structural components such as the spore coats and dipicolinic acid (DPA) in attenuation of the lethal and mutagenic effects of solar UV. The current data are reviewed with the ultimate goal of obtaining a complete model describing spore persistence and longevity in the terrestrial solar UV radiation environment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Virulent spores of Bacillus anthracis and other Bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminants

AIMS: To compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp. deposited on different solid surfaces to inactivation by liquid chemical disinfecting agents. METHODS AND RESULTS: We prepared under similar conditions spores from five different virulent and three attenuated strains of B. anthracis, as well as spores of Bacillus subtilis, Bacillus atrophaeus (previously known as Bacillus globigii), Bacillus cereus, Bacillus thuringiensis and Bacillus megaterium. As spore-surface interactions may bias inactivation experiments, we evaluated the relative binding of different spores to carrier materials. The survival of spores deposited on glass, metallic or polymeric surfaces were quantitatively measured by ASTM standard method E-2414-05 which recovers spores from surfaces by increasing stringency. The number of spores inactivated by each decontaminant was similar and generally within 1 log among the 12 different Bacillus strains tested. This similarity among Bacillus strains and species was observed through a range of sporicidal efficacy on spores deposited on painted metal, polymeric rubber or glass. CONCLUSIONS: The data obtained indicate that the sensitivity of common simulants (B. atrophaeus and B. subtilis), as well as spores of B. cereus, B. thuringiensis, and B. megaterium, to inactivation by products that contain either: peroxide, chlorine or oxidants is similar to that shown by spores from all eight B. anthracis strains studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The comparative results of the present study suggest that decontamination and sterilization data obtained with simulants can be safely extrapolated to virulent spores of B. anthracis. Thus, valid conclusions on sporicidal efficacy could be drawn from safer and less costly experiments employing non-pathogenic spore simulants.     

Dynamics of heat destruction of spores: A new view

Summary Discrepancies between actual, viable spore populations and those predicted by a classical model during heat sterilization of food and pharmaceutical products have long concerned food engineers and scientists as they pursue new sterilization techniques, including ultra-high temperature processes. Among potential causes of those discrepancies, activation of dormant spores is significant, and models addressing that factor were developed recently. This paper reviews historic and current views on the biology and models of microbial spore populations during heat sterilization. Activation and inactivation of viable spores are emphasized, with each viewed as a first-order reaction. Rate constants of those reactions may differ significantly, inactivation rates of dormant and activated spores may differ, and variations of all rate constants with temperature appear to be well described by Arrhenius equations. Model-based analyses show how categories of survivor response curves observed during isothermal heat treatments can arise from simultaneous activation and inactivation of spores in an overall population. Effects of different distributions of initial subpopulations, different distributions of rate constants, and heat shock for homogenizing an indicator population are shown. The complexity of new, multiple process models has not increased greatly, but the potential for accurate, dynamic prediction of product safety after prescribed sterilization has. The relevant biology is understood and accounted for more thoroughly, and it is anticipated that the new models will aid design and evaluation of new and improved sterilization processes for food and pharmaceuticals.Mention of brand or firm names does not constitute an endorsement by the US Department of Agriculture over others of a similar nature not mentioned.     

Photocatalytic inactivation of influenza virus by titanium dioxide thin film

Titanium dioxide (TiO(2)) under ultraviolet (UV) light produces a strong oxidative effect and may therefore be used as a photocatalytic disinfectant. Although many studies on the photocatalytic inactivation of bacteria have been reported, few studies have addressed virus inactivation. In the present study, we demonstrated the inactivation of influenza virus through TiO(2) photocatalysis using TiO(2) nanoparticles immobilized on a glass plate. The influences of the UV intensity, UV irradiation time and bovine serum albumin (BSA) concentration in the viral suspensions on the inactivation kinetics were investigated. Additionally, we also determined whether the International Organization for Standardization (ISO) methodology for the evaluation of antibacterial activity of TiO(2) photocatalysis could be applied to the evaluation of antiviral activity. The viral titers were dramatically reduced by the photocatalytic reaction. Even with a low intensity of UV-A (0.01 mW cm(-2)), a viral reduction of approximately 4-log(10) was observed within a short irradiation time. The viral inactivation kinetics were associated with the exposure time, the UV intensity and the BSA concentration in virus suspensions. These results show that TiO(2) photocatalysis could be used to inactivate the influenza virus. Furthermore, a minor modification of the ISO test method for anti-bacterial effects of TiO(2) photocatalysis could be useful for the evaluation of antiviral activity.     

Airborne fungal spores trend over a highly polluted area of south-west Spain using Cour's trap

Summary This investigation was aimed at studying the mycoflora present in Huelva (South West Spain), a highly polluted region. The study was carried out using the Cour trap and it is worth pointing out that the chemical treatment performed following the Cour methodology might destroy a certain percentage of spores. We analyzed the presence and biannual changes (10 April 1989 to 8 April 1991) of fungal spores of three genera, seventeen form genera, and four groups lacking taxonomic category, looking at their morphology: ascospores, basidiospores, and unidentified unicellular and multicellular spores. Special attention was given to agriculturally interesting groups. The results obtained were correlated with meteorological parameters (rainfall, minimum temperature and humidity).     

5-ALA表面修饰TiO2诱导的光动力疗法体外灭活HL60细胞实验研究

利用表面修饰的方法制备了5-ALA表面修饰的TiO2（5-ALA／TiO2）,并利用傅里叶红外光谱,拉曼光谱以及紫外-可见光吸收光谱（uV—Vis）对样品进行了表征。利用CellCountingKit-8（CCK-8）法检测研究5-ALA／TiO2对HL60细胞的灭活效应。结果显示,5-ALA／TiO2能够显著地抑制HL60细胞的生长,5-ALA／TiO2对HL60细胞的灭活效率要明显高于5-ALA以及TiO2,实验中5-ALA／TiO2的灭活效率达到77．9％,相衬显微镜细胞形态学无损观察表明,细胞凋亡开始于细胞膜的破裂。同时,激光显微拉曼光谱检测显示,TiO2纳米粒子能够进入细胞内部,与细胞发生相互作用。     

Photosynthesis and photoinhibition after night chilling in seedlings of two tropical tree species grown under three irradiances

We investigated the physiological effect of night chilling (CN) on potted seedlings of two tropical tree species, Calophyllum polyanthum and Linociera insignis, in Xishuangbanna, southwest China. Seedlings grown under 8, 25, and 50 % daylight for five months were moved to a 4–6 °C cold storage house for three consecutive nights, and returned to the original shaded sites during the day. CN resulted in strong suppression of photosynthesis and stomatal conductance for L. insignis, and reduced photorespiration rates, carboxylation efficiency, and maximum photochemical efficiency of photosystem 2 (PS2) at dawn and midday for both species. CN increased dawn and midday rates of non-photochemical quenching, and the contents of malondialdehyde and H₂O₂ for both species. CN also induced inactivation or destruction of PS2 reaction centres. The impacts of CN on tropical seedlings increased with the number of CN. Shading could significantly mitigate the adverse effects of CN for both species. After 3-d-recovery, gas exchange and fluorescence parameters for both species returned to pre-treatment levels in most cases. Thus CN induced mainly stomatal limitation of photosynthesis for L. insignis, and non-stomatal limitation for C. polyanthum. C. polyanthum was more susceptible to CN than L. insignis. Fog, which often occurs in Xishuangbanna, could be beneficial to chilling sensitive tropical seedlings in this area through alleviating photoinhibition or photodamage by reducing sunlight.     

A simple method to preserve algal spores of <Emphasis Type="Italic">Ulva</Emphasis> spp. in cold storage with ampicillin

Preservation of algal spores of the green seaweed Ulva fasciata and U. pertusa was enhanced by the addition of ampicillin in f/2 medium at 4°C. The viability of preserved spores was determined by a spore germination assay at various time intervals. The germination rate of U. fasciata remained at 5% to 38% for the first five days, dropping to 1% to 6% on the 10th day of storage with various preservation treatments without ampicillin at 4°C during parameter-selecting experiments. In f/2 medium, 53% of U. fasciata spores were still viable on day 5 and 23% on day 10 at 4°C. By adding 100 μg mL⁻¹ ampicillin to f/2 medium, 90% of the spores were viable at day 40 and 61% after 100 days of storage at 4°C. Spores of U. pertusa had lower preservation rates, with viabilities of 70% at day 40 and 32% at day 100. Algal spore preservation was heavily dependent on the bacterial contamination and subsequent degradation in stock solutions. Handling editor: L. Naselli-Flores