Development and optimization of a quantitative cell culture infectivity assay for the microsporidium Encephalitozoon intestinalis and application to ultraviolet light inactivation

Microsporidia are unique parasites recognized as a major cause of intestinal illness among immunocompromised patients and occasionally in otherwise healthy hosts. These organisms have been detected in water and are likely transmitted by the fecal-oral route. The most common human pathogenic microsporidia for which cell culture methods have been established is Encephalitozoon intestinalis. This study describes the development of a quantitative cell culture infectivity assay for E. intestinalis and its application to assess inactivation by ultraviolet (UV) light irradiation. The method described here employs calcofluor white, a fluorescent brightener that targets the chitin spore wall, to visualize groups of developing spores in order to confirm infectivity. Serial dilutions of the spore suspension were seeded into tissue culture well slides containing RK-13 cells. Slides were then rinsed, fixed in methanol and stained with calcofluor white and examined microscopically. Large masses of developing spores were easily visible on infected cell monolayers. Positive and negative wells at each dilution step were used to quantify the number of infectious spores in the original suspension using a most-probable-number (MPN) statistical analysis. This assay was used to evaluate the disinfecting potential of ultraviolet light on E. intestinalis spores in water. The ultraviolet dose required for a 3-log(10) or 99.9% reduction in the number of infective spores was determined to be 8.43 mW s/cm(2).     

Susceptibility of spores of the microsporidian Nosema algerae to sunlight and germicidal ultraviolet radiation

Spores of the microsporidian Nosema algerae exposed to 1, 2, and 4 hr of sunlight and 121 μW/cm² of germicidal ultraviolet light were fed to first-instar Anopheles albimanus. Twenty days after feeding, the incidence and intensity of infection (spores/mosquito) were recorded from adult mosquitoes. While sunlight-treated spores showed no significant decline in incidence of infection after 1-, 2-, and 4-hr exposures, intensity of infection decreased significantly after the 2- and 4-hr exposures. Incidence of infection of mosquitoes fed bactericidal ultraviolet light-treated spores declined 48.2, 76.2, and 99.9% after 1-, 2-, and 8-min exposures, respectively. Measured by intensity of infection, activity of bactericidal light-treated spores decreased 87.2% after 1 min, 91.7% after 2 min, and 99.9% after 8 min. Levels of radiation required to inactivate spores of N. algerae fell within the range reported for other Microsporidia.     

Increased numbers of heat-resistnat spores produced by two strains of Clostridium perfringens bearing temperate phage s9.

Sporulation kinetics and spore heat resistance data were compared for a lysogenic strain of Clostridium perfringens, s9, before and after curing with ultraviolet irradiation. The cured strain showed the same growth rate in broth media as the lysogenic strain but took 6 h longer to form refractile spores. For lysogenized and cured strains the percentages of refractile spores produced that were heat-resistant (80 degrees C for 15 min) were 50 and 0.2, respectively. When reinfected with the temperature phage, the cured strain produced spores in 2 to 3 h, like the original lysogenic culture, and 10% of the spores produced were heat-resistnat.     

Method to sensitize bacterial spores to subsequent killing by dry heat or ultraviolet irradiation

Hydrogen peroxide and ultraviolet irradiation are known to interact synergistically for killing of bacterial spores. Synergy could be demonstrated with spores of Bacillus megaterium ATCC19213 adsorbed to filter paper strips or glass coverslips treated first with the peroxide and then dried for as long as 48 h prior to UV irradiation. This delayed action was considered to be due to absorption of the peroxide by the spores in an active but not readily vaporized form, which could become sporicidal also if the spores were heated to 50 degrees C. B. megaterium spores mixed with 0.1% (32.6 mM) H(2)O(2) solution appeared to absorb as much as 15 micromol/mg dry weight or about 0.5 mg/mg, but only a third to half of the peroxide could be recovered by water washing. A part of the unrecovered peroxide was degraded in reactions resulting in measurable production of oxygen. Degradation was not reduced by heating the spores to 65 degrees C or by azide and so appeared to be non-enzymatic. Spores of the anaerobe Clostridium sporogenes were also sensitized to ultraviolet killing by H(2)O(2) treatment followed by drying. They appear to absorb less peroxide, only about 2 micromol/mg, but had lower capacities to degrade H(2)O(2) so that nearly all of the peroxide could be recovered by washing with water. The findings presented should be helpful in the design of new methods for synergistic killing of spores by H(2)O(2) and UV irradiation or dry heat, especially involving, for example, packaging materials.     

The composition and structure of walls of dark fungus spores

Summary Dark pigmented fungi predominate in desert soils. The dark pigment of the spores is a melanin. These dark spores resist ultraviolet radiation of 2537 Å. The degree of opacity depends on the amount and location of the melanin.In the presence of hexachloracetone, about half of the spores produced in culture are light colored, and easily killed by u.v. light. Electronmicrographs are presented showing spore wall structure of several representative fungi at high resolution.     

Relationship of Dipicolinic Acid Content in Spores of Bacillus cereus T to Ultraviolet and Gamma Radiation Resistance

Spores of Bacillus cereus T lacking dipicolinic acid showed a statistically significant reduction in resistance to ultraviolet and gamma radiation as compared with spores with high dipicolinic acid content.     

THE EFFECTS OF ULTRAVIOLET RADIATION ON SPORES OF THE FUNGUS ASPERGILLUS NIGER

The survival ratio of Aspergillus spores exposed to ultraviolet radiation has been measured as a function of total incident energy for wave lengths of 2537 Å, 3022 Å, 3129 Å, and 3650 Å. The effect of humidity on killing of Aspergillus spores by ultraviolet radiation has been found to be negligible. A delay in germination as a result of irradiation has been found. The Bunsen-Roscoe reciprocity law has been found to hold within the limits of the radiation intensities studied. Certain morphological changes have been observed.     

-aminobutyric acid as a required germinant for mutant spores of Bacillus megaterium

Germinated spores of Bacillus megaterium QM B1551 were irradiated with ultraviolet light, and spore-forming survivors were screened for germination requirements. Spore strains which failed to germinate in a variety of defined solutions germinative for spores of the parent strain were obtained. Mutant spores germinated readily in solutions containing yeast extract or one of numerous complex preparations. gamma-Aminobutyric acid, obtained from yeast extract by column chromatography, was shown to be required for germination by the mutant spores. gamma-Aminobutyric acid and l-alanine at final concentrations of 1 mm each, in solutions of KI (40 mm), equaled the potency of yeast extract (1 mg/ml) in the germination of the mutant spores. One of several other amino acids could be substituted, though less effectively, for l-alanine. alpha-Aminobutyric acid, beta-aminobutyric acid, beta-alanine, and 5-aminovaleric acid were ineffective substitutes for gamma-aminobutyric acid in mutant spore germination.     

Localization of low-molecular-weight basic proteins in Bacillus megaterium spores by cross-linking with ultraviolet light.

Two low-molecular-weight basic proteins, termed A and B proteins, comprise about 15% of the protein of dormant spores of Bacillus megaterium. Irradiation of intact dormant spores with ultraviolet light results in covalent cross-linking of the A and B proteins to other spore macromolecules. The cross-linked A and B proteins are precipitated by ethanol and can be solubilized by treatment with deoxyribonuclease (75%) or ribonuclease (25%). Irradiation of complexes formed in vitro between deoxyribonucleic acid (DNA) or ribonucleic acid and a mixture of the low-molecular-weight basic proteins from spores also resulted in cross-linking of A and B proteins to nucleic acids. The dose-response curves for formation of covalent cross-links were similar for irradiation of both a protein-DNA complex in vitro and intact spores. However, if irradiation was carried out in vitro under conditions where DNA-protein complexes were disrupted, no covalent cross-links were formed. These data suggest that significant amounts of the low-molecular-weight basic proteins unique to bacterial spores are associated with spore DNA in vivo.     

Production of Aseptic Spores of Vesicular-Arbuscular Endophytes and Their Viability After Chemical and Physical Stress

The survival of germinating spores of vesicular-arbuscular endophytes after treatments with oxidizing agents, antibiotics, moist heat, ultrasonic radiation, and ultraviolet radiation was compared with that of their contaminating microbes. Spores of three species were rapidly decontaminated by treatment with 0.42% (wt/vol) chlorine available from 5.0% (wt/vol) chloramine-T at 30°C for 20 to 40 min depending on the species and the soil from which they were extracted. This treatment did not change spore viability. The survival of spores was reduced by exposure for 20 min to 1.11% chlorine at 30°C for Glomus caledonius or at 35°C for Acaulospora laevis. Growth of any bacteria surviving treatment with oxidizing agents was inhibited by 100 μg of chloramphenicol per ml in agar; however, spore germination and germ tube growth were reduced only by concentrations greater than 200 μg/ml in agar. Spore germination was decreased by concentration of pimaracin, which controlled fungal growth. The spores survived moist heat at 40°C for 80 min, 55°C for 10 min, and 60°C for less than 1 min. The viability of spores was unaffected by ultrasonic irradiation for up to 4 min. Spores of G. caledonius and A. laevis were extremely resistant to ultraviolet radiation. Their viability was unaffected by exposure to 5 × 10⁸ ergs cm⁻² from an ultraviolet source of 253.7nm. The spores had very thick, pigmented walls, and the possibility that these provided some protection against the physical and chemical treatments is discussed. The degree of physiological damage to the spores caused by the treatments demonstrated some adverse effects of basic laboratory procedures. This information, together with that on the comparative sensitivity of contaminating microbes to the treatments, was used in the development of protocol for producing large numbers of uncontaminated spores.     

Temperature-Induced Changes in the Sporicidal Activity and Chemical Properties of Glutaraldehyde

Freshly prepared 2% acid and alkaline glutaraldehyde solutions were stored at 4, 20, and 37 C. At intervals, samples were removed and changes in pH, ultraviolet spectrum, and sporicidal activity (against Bacillus pumilus spores) were recorded. Alkaline solutions stored at 4 C showed little changes in these properties, whereas such solutions stored at 37 C became turbid and showed a decrease in pH, marked changes in ultraviolet spectrum, and an almost complete loss of sporicidal activity. Intermediate results were obtained with alkaline solutions stored at 20 C. In contrast, acid 2% glutaraldehyde solutions (initial pH 3.5) showed comparatively few changes in their properties. Treatment of spores with freshly prepared glutaraldehyde solutions (0.5%) at temperature above 40 C reduced the effect of pH on sporicidal activity.     

Resistance and recovery studies on ultraviolet-irradiated spores of Bacillus pumilus.

A spore suspension model and a procedure for recovering ultraviolet (UV)-irradiated spores of Bacillus pumilus were investigated. A most-probable-number tube dilution method using double-strength Trypticase soy broth was found to be superior to the agar plate method for recovering optimal numbers of spores irradiated with sublethal doses of UV energy. Aqueous suspensions of B. pumilus survived UV doses up to 108,000 ergs/mm2 as determined by a most-probable-number recovery and estimation procedure. Resistance and stability data were consistent and reproducible, indicating the dependability of this method for recovering UV-damaged spores. The procedures used to collect information concerning resistance characteristics for two strains of B. pumilus are discussed.     

Resistivity of Spores to Ultraviolet and γ Radiation while Exposed to Ultrahigh Vacuum or at Atmospheric Pressure

Viability studies were conducted on microbial spores subjected to ultrahigh vacuum (UHV) in the 10(-9) to 10(-10) torr range. After 5 to 7 days in vacuum, they were exposed to ultraviolet (UV) or to gamma radiation either while still under vacuum or in the presence of dried air. Among the four test organisms subjected to UHV and ultraviolet radiation, Aspergillus niger was the most resistant; Bacillus megaterium, B. subtilis var. niger, and B. stearothermophilus were about equally less resistant. All four spores were more sensitive to ultraviolet radiation when UHV-dried than when desiccant-dried. Of the four test organisms subjected to UHV and gamma radiation, B. megaterium proved to be the most resistant; A. niger was the least resistant; and the remaining two organisms were of intermediate resistivity. All four organisms were less radiation resistant when UHV-dried than when irradiated in their normally hydrated state, and all showed an increased radiosensitivity after vacuum drying when oxygen was present. In addition, spores of B. subtilis var. niger and A. niger were less radiosensitive when UHV-dried and irradiated in vacuum than when "wet" and irradiated in air, whereas the reverse relationship was observed for the remaining two organisms. Based on the fact that microbial contaminants can be readily shielded from UV light by soils, metal particles, etc., and considering that the levels of ionizing radiations reported to be present in interstellar space are generally lower than those used in these experiments, the decrease in radioresistivity imparted by UHV drying is not of a sufficient magnitude to sterilize dependably portions of a spacecraft while on a mission.     

Fate of bacterial indicators,viruses and protozoan parasites in a wastewater multi-component treatment system

The extent of reduction in selected microrganisms was tested at a multi-component wastewater treatment plant that treats sewage for a potential re-use in agriculture. The aim of the investigation was to evaluate possible reciprocal correlation among the different microrganisms and to compare the removal of two encysted pathogenic protozoa with that of microbial indicators, Clostridium perfringens spores, enteroviruses and bacteriophages. Samples collected included the raw wastewater, the chlorinated effluent and the effluent after an ultraviolet light treatment. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested but the bacteriophage B40-8. The data obtained confirm the removal efficiency of the entire process for indicator bacteria but also show the low and variable removal efficiency for the other microbial parameters, such as Giardia and Cryptosporidium, enteroviruses and Clostridium perfringens spores. Reciprocal correlation between Cryptosporidium and Giardia (oo)cysts and the other microbial groups was not demonstrated. The results confirm the resistance of Clostridium perfringens spores, enteroviruses and protozoa to chlorination and demonstrate the relative persistence of these organisms in the effluents even during the ultraviolet light treatment. The yields also emphasise the influence of the analytical method for the determination of protozoan parasites.     

Increasing Activity of Germinating Bacillus subtilis Spores to Incorporate Thymidine Triphosphate into Deoxyribonucleic Acid After Detergent Treatment

The incorporation of (3)H-labeled thymidine triphosphate ((3)H-dTTP) into deoxyribonucleic acid (DNA) of germinated and then Brij 58-treated Bacillus subtilis spores was measured to study DNA replication activity of cells. The dTTP incorporation rate was very low in dormant spores, gradually increased as germination proceeded, and reached a level of the vegetative cell activity approximately 4 hr after the start of germination. This is in contrast to the DNA polymerase activity in the cell extract which remained at the same level throughout the germination period. The increase of the dTTP incorporation activity was inhibited by chloramphenicol or phenethyl alcohol. When these inhibitors were added after germination had proceeded, the elevated dTTP incorporation activity gradually decreased. Permeability to dTTP of spores germinated in the presence of chloramphenicol and then treated with Brij 58 was confirmed by (i) (3)H-dTTP incorporation into the treated spores following either electron or ultraviolet irradiation and (ii) release of radioactivity from the treated spores containing radioactively labeled DNA after deoxyribonuclease I treatment.     

Ultraviolet Sensitivity of Bacillus subtilis Spores upon Germination and Outgrowth

A strain of Bacillus subtilis, UVSSP-42-1, which produces ultraviolet (UV)-sensitive spores and vegetative cells, was found to possess germinated spores 25 times more UV resistant than the resting spores. This relative resistance achieved upon germination was associated with the transition of the heat-resistant refractile spores to the heat-sensitive phase-dark forms. Several generations of outgrowth were required before the cells attained the level of UV sensitivity characteristic of the vegetative cell. The UV sensitivity of germinated spores was compared with other strains with various combinations of mutations affecting deoxyribonucleic acid repair capabilities. The presence of hcr and ssp mutations which are known to abolish the removal of photoproducts from deoxyribonucleic acid did not alter significantly the sensitivity of the germinated forms. However, the addition of the recA mutation and, to some extent, the pol mutation increased the UV sensitivity of the germinated spores. These results indicate that deoxyribonucleic acid repair mechanisms dependent on the recA gene are active in the germinated spores. The chemical nature of the damage repaired by the recA gene product is not known. This study indicates that the life cycle of sporulating bacilli consists of at least three photobiologically distinct forms: spore, germinated spore, and vegetative cell.     

Mutants produced by X-irradiation of spores of Chaetomium globosum and a comparison with those produced by ultraviolet irradiation

1. Mutants produced by x-irradiation of fungal spores of Chaetomium globosum have been compared with those produced by ultraviolet irradiation. 2. The most striking difference between the mutants produced by x-irradiation and ultraviolet irradiation is the absence in x-ray experiments of the K mutant which is produced in large numbers at short ultraviolet wave lengths. 3. A comparison is made of the relation between x-ray dose and numbers of lethal mutants, and the relation between the short ultraviolet wave length 2804 dose and numbers of lethal mutants. Both are compared with theoretical curves for 1, 2, 5, and 8 quantum hits. 4. The production of lethal mutants by x-rays is shown to be consistent with the theoretical curve for five quantum hits on the sensitive spot of the spore, whereas the production of lethal mutants by the ultraviolet wave length 2804 A.u. is consistent with two quantum hits.     

The effect of hypochlorite on spores of Bacillus subtilis lacking small acid-soluble proteins

M.Z.H. SABLI, P. SETLOW AND W.M. WAITES. 1996. α/β-Type small acid-soluble proteins (SASP) bind to spore DNA and protect it against ultraviolet light, heat, hydrogen peroxide and freeze drying, making the spores much more resistant than vegetative cells to these agents. Spores of a mutant of Bacillus subtilis lacking the two major α/β-type SASP were almost 30 000-fold less resistant to hypochlorite than were wild-type spores. After treatment with hypochlorite, surviving spores of the mutant, but not those of the wild type, showed higher levels of mutation, suggesting that SASP contribute to hypochlorite resistance by protecting spore DNA.     

Transitory germinative excision repair in Bacillus subtilis.

Bacillus subtilis strains UVSSP-42-1 (hcr42 ssp1) and UVSSP-1-1 (hcr1 ssp1) are ultraviolet (UV) radiation sensitive both as dormant spores and as vegetative cells. These strains are unable to excise cyclobutane-type dimers from the deoxyribonucleic acid (DNA) of irradiated vegetative cells and fail to remove spore photoproduct from the DNA of irradiated spores either by excision (controlled by gene hcr) or by spore repair (controlled by gene ssp1). When irradiated soon after spore germination, these strains excise dimers, but not spore photoproduct, from their DNA. This process, termed germinative excision repair, functions only transiently in the germination phase and is responsible for the high UV resistance of germinated spores and for their temporary capacity to host cell reactivate irradiated phages infecting them. The recA1 mutation confers higher UV sensitivity to the germinated spores, but does not interfere with dimer removal by germinative excision repair.     

The effect of sunlight on allergen release from spores of the fungus Alternaria

Airborne fungal spores are known carriers of allergen. Correlations between spore counts and allergen concentrations are poor. It is known that germination increases allergen release, implicating spore viability as a determinant of allergen release. During aerial dispersal, spores can be exposed to prolonged periods of ultraviolet (UV) light which can reduce viability of spores. We examined the relation between spore viability and allergen release in two experiments: firstly spores from culture were treated with a UV wavelength of 254?nm (not present in sunlight reaching the earth's surface) or autoclaved, and secondly, spores were exposed to simulated sunlight over three days. In both studies viability was measured (by germination on agar and by metabolic activity with nitro-blue tetrazolium vital stain) and allergen release by the Halogen immunoassay. The UV light reduced the proportion of spores able to germinate but did not affect metabolic activity or allergen release. Autoclaving reduced the proportion of spores releasing allergen by half (p<0.0001). Three days' exposure to simulated sunlight correlated negatively with spore germination and metabolic activity (p<0.0001), but did not affect allergen release (p=0.799). In conclusion, simulated sunlight reduced the metabolic activity and germinability of spores however the proportion releasing allergen remained unaffected. These findings suggest that spore counts may reflect allergen concentrations in the air if spores are dead or dormant. The contribution of viable spores to concentrations of airborne allergen, as well as the role of germination in allergen delivery to the respiratory tract, remains to be resolved.