Chlorine Inactivation of Bacterial Bioterrorism Agents

Seven species of bacterial select agents were tested for susceptibility to free available chlorine (FAC). Under test conditions, the FAC routinely maintained in potable water would be sufficient to reduce six species by 2 orders of magnitude within 10 min. Water contaminated with spores of Bacillus anthracis spores would require further treatment.     

Optimizing acidified bleach solutions to improve sporicidal efficacy on building materials

Aims: We evaluated whether lowering pH (with acetic acid) and raising free available chlorine (FAC) levels in bleach solutions would improve efficacy in inactivating Bacillus spores on different materials. We also determined how varying pH and FAC levels affected bleach stability. Methods and Results: Acidified bleach solutions with pH levels of 4·5, 6 and 7·5 and FAC levels between 5000 and 10 000 ppm were evaluated for decontamination efficacy against Bacillus subtilis spores inoculated onto test coupons made from wood, ceramic and galvanized steel. Lowering the pH or increasing the FAC level improved efficacy in some of the tests, but depended on the material, which significantly affected decontamination efficacy. The acidified bleach at pH of 7·5 was significantly less effective than bleach at a pH of 4·5 or 6. The FAC levels in the bleach were the most stable at pH 4·5, and stability at pH 4·5 was not significantly affected by the initial FAC level. Conclusions: It may be advisable to use bleach solutions with lower pH (rather than high FAC levels) in light of both the decontamination efficacy and bleach stability results. For wood materials, use of sporicides other than acidified bleach may be warranted. Significance and Impact of the Study: These results may be useful in preparing acidified bleach solutions for decontamination of materials contaminated with spores such as Bacillus anthracis.     

A preliminary assessment of Bacillus anthracis spore inactivation using an electrochemically activated solution (ECASOL)

AIM: To evaluate the efficacy of electrochemically activated solution (ECASOL) in decontaminating Bacillus anthracis Ames and Vollum 1B spores, with and without changing the source water hardness and final ECASOL pH. METHODS AND RESULTS: Five different ECASOL formulations were generated, in which the source water hardness and final ECASOL pH were varied, resulting in cases where significant changes in free available chlorine (FAC) and oxidative-reduction potential (ORP) were observed. B. anthracis Ames and Vollum 1B spores were suspended in the various ECASOL formulations for 30 min, and decontamination efficacy was determined; calcium hypochlorite [5% high-test hypochlorite (HTH)] was used as a positive control. The five different ECASOL formulations yielded mean FAC levels ranging from 305 to 464 ppm, and mean ORP levels ranging from +826 to +1000 mV. Exposure to all the ECASOL formulations and 5% HTH resulted in >or=7.0 log reductions in both B. anthracis Ames and Vollum 1B spores. CONCLUSIONS: The present testing demonstrated that ECASOL with a minimum of c. 300-ppm FAC levels and +800-mV ORP inactivated the B. anthracis spores in suspension, similar to 5% HTH. Significance and Impact of the Study: These results provide information for decontaminating B. anthracis Ames and Vollum 1B spores in suspension using ECASOL.     

不同物候时期华西雨屏区四个树种新鲜凋落物可提取腐殖质碳动态

凋落物的可提取腐殖质碳可随着植物生长节律及物候时期的变化而变化,并进而影响物质循环的过程,为深入了解以凋落物为载体的生态系统物质循环特征,该研究以华西雨屏区麻栎(Quercus acutissima)、楠木(Phoebe zhennan)、柳杉(Cryptomeria fortune)和喜树(Camptotheca acuminate)为对象,通过定点动态收集萌芽期、展叶期、盛叶期和落叶期的不同类型凋落物,分析其可提取腐殖质碳(extractable humus carbon,HC)、胡敏酸碳(humic acid carbon,HAC)、富里酸碳(fulvic acid carbon,FAC)以及胡敏酸碳/富里酸碳(humic acid carbon/fulvic acid carbon,HAC/FAC)的差异。结果表明:相对于其他凋落器官,在同一物候时期凋落叶中的HC和HAC含量都最高,大致都表现为凋落叶>凋落枝>凋落果,且在落叶树种中更为显著;相对于其他时期,展叶期四个树种凋落叶均表现出较高的FAC含量,但不同物候期凋落枝和凋落果的HAC含量以及FAC含量受物种的影响较大。尽管HAC/FAC在各器官间差异并不显著,但落叶树种相同器官的HAC/FAC低于常绿树种,表明落叶树种凋落物富里酸相对含量较高,形成速度相对较快,难降解程度也相对较大。统计分析表明关键时期、物种类型、器官类型及其相互作用对凋落物中HC、HAC、FAC含量和HAC/FAC均具有不同程度的影响,这为进一步深入认识区域生态系统以凋落物为载体的物质循环过程提供了理论依据及新的思路。     

Two species of Selaginella cones and their spores from the Bohemian Carboniferous continental basins of the Czech Republic

Two species of Selaginella cones from the Bohemian Upper Carboniferous continental basins of the Bolsovian and Westphalian D age are described, together with their in situ spores. Two specimens of Selaginella gutbierii yielded microspores closely comparable with the dispersed species Cirratriradites saturnii and megaspores closely comparable with the dispersed species Triangulatisporites vermiculatus. Microspores closely comparable with the dispersed species Cirratriradites annulatus and megaspores resemble the dispersed species Triangulatisporites tertius were isolated from cones of Selaginella cf. leptostachys. All the spores isolated from one cone are of the same type and would be referred to one dispersed micro- and megaspore species if found as Sporae dispersae. The paper contains a review of all palynologically studied Carboniferous Selaginella and Selaginella-like cones and reviews of all in situ and dispersed Cirratriradites and Triangulatisporites spores.     

Monochloramine inactivation of bacterial select agents

Seven species of bacterial select agents were tested for susceptibility to monochloramine. Under test conditions, the monochloramine routinely maintained in potable water would reduce six of the species by 2 orders of magnitude within 4.2 h. Bacillus anthracis spores would require up to 3.5 days for the same inactivation with monochloramine.     

Comparative study of the fatty acid composition of some groups of purple nonsulfur bacteria

The fatty acid composition (FAC) of 43 strains of purple nonsulfur bacteria belonging to six genera--Rubrivivax, Rhodopseudomonas, Rhodoplanes, Blastochloris, Rhodobium, and Rhodomicrobium--was studied by capillary gas chromatography. The cultures were grown on standard medium under standard conditions. Automatic identification of the fatty acid methyl esters and statistical processing of the results were performed by the computerized Microbial Identification System (M.I.S). Significant differences between the FACs of different genera, species, and, sometimes, strains were revealed. 16S rRNA genes of some of the new isolates, primarily those having a specific FAC, were sequenced. The taxonomic status of a number of the strains in question was determined using the FAC characteristics as one of the criteria. It was shown that the FAC characteristics may be used both for affiliating the isolates to known species and for revealing new taxa.     

Monochloramine Inactivation of Bacterial Select Agents

Seven species of bacterial select agents were tested for susceptibility to monochloramine. Under test conditions, the monochloramine routinely maintained in potable water would reduce six of the species by 2 orders of magnitude within 4.2 h. Bacillus anthracis spores would require up to 3.5 days for the same inactivation with monochloramine.     

The decay of potentially lethal oxygen-dependent damage in fully hydrated Bacillus megaterium spores exposed to pulsed electron irradiation.

Using a stopped-flow mixing and pulsed irradiation apparatus, a study has been made of the decay, to a harmless form, of radiation-induced species which would otherwise be lethal to spores on contact with oxygen. Aqueous suspensions of Bacillus megaterium spores were irradiated with 600 krad of electrons given over approximately 1 s; at various times after irradiation oxygen in solution was added. As the interval between irradiation and introduction of oxygen increased, the fraction of spores surviving increased. For spores irradiated in a deoxygenated condition the decay of the potentially lethal species, reflected by this change in survival, proceeded as if two parallel first-order reactions with half-lives of 9 and 120 s operate. In contrast, for spores equilibrated with oxygen and then irradiated, the decay is described by a single first-order expression with an associated half-life similar to that of the faster of the two reactions operating in anoxia.     

Importance of timing,spore concentrations,and levels of spore carrier in applications of Nosema locustae (Microsporida: Nosematidae) for control of grasshoppers

In a study of microbial control of grasshoppers, Nosema locustae was applied to 10-acre plots in 2 replications of 24 treatment combinations in which the factors were (1) times of applications: 4 applications spaced over 22 days; (2) concentrations of spores: 1, 30, and 900 spores/in²; and (3) levels of the spore carrier: 1 and 4 lb wheat bran/acre. The concentrations of spores had the most effect in reducing the densities of grasshoppers and the incidence of infection among the survivors at the last sampling (coincided with the initiation of oviposition), the average reduction in density attributed to application of 1 spore/in² was 21%, that attributed to 30 spores/in² was 46%, and that attributed to 900 spores/in² was 73%. The first and second applications caused the greatest reductions in density; the second, third, and fourth applications produced the highest incidence of infections. Also, a higher incidence of infection was observed among grasshoppers from plots treated with 30 spores/in² on 1 lb bran/acre than from plots treated with 30 spores/in² on 4 lb of bran/acre. Therefore, a ratio of about 0.63–0.94 billion spores/lb bran applied at a rate of 1–1.5 lb/acre (= 100–150 spores/in²) at the time when the principal early summer species are third-instar nymphs would have resulted in overall reductions in density of 50–60%, with 35–50% of the survivors sufficiently infected so that fecundity would probably be affected. The reductions and incidence of infections would be higher in species that readily accept and use wheat bran, among which are some of the more economically important species of grasshoppers.     

Spore productivity in Cladosporium

The high incidence ofCladosporia in the airspora indicates a prolific production of spores. Six species ofCladosporium were sampled over a period of 9 weeks, using dry and wet (mist-laden) air, and over a period of 4 weeks using humid air. Many more spores were released in wet air than in dry air: numbers released in humid air were generally intermediate between those of wet and dry samples. None of the cultures was exhausted of spores at the end of the sampling periods although samples generally decreased in size from the fifth or sixth week onwards. Removal of spores would seem to be conducive to further sporulation provided the substrate is not exhausted. Maximum productivities recorded for the six species (all in mist-laden air) ranged from 730 to 26 100 spores per mg dry weight of mycelium. Differences in the levels of spore production in culture by the six species do not correlate with their individual frequencies in the airspora, indicating that the latter are more dependent on the distribution and substrate relationships of each species.     

Systematic implications of flavonoid pigments in the fern genus hemionitis (adiantiaceae)

The closely related fern generaHemionitis L. andGymnopteris Bernhardi are separated primarily on differences in leaf architecture and venation. Studies indicate that these characters are highly variable and unreliably diagnostic. Further, the type species of the two genera readily hybridize with each other. Spore morphology, as exhibited by SEM, does not support the traditional alignment of the species in these two genera: some species ofHemionitis andGymnopteris have the same rugose to papillate spores, while other species from both genera possess crested spores. The flavonoid chemistry of these taxa coincides with spore type, i.e., taxa from both genera which possess crested spores produce kaempferol and quercetin 3-0-glycosides, while species with tuberculate spores produce only quercetin 3,4′-0-glycosides. The spore and chemical data suggest a realignment of these taxa within a single genus, which would avoid the rather tenuous dependence on a single vegetative character for generic distinctions.     

Comparative study of the fatty acid composition of some groups of purple nonsulfur bacteria

The fatty acid composition (FAC) of 43 strains of purple nonsulfur bacteria belonging to six genera—Rubrivivax, Rhodopseudomonas, Rhodoplanes, Blastochloris, Rhodobium, and Rhodomicrobium—was studied by capillary gas chromatography. The cultures were grown on standard medium under standard conditions. Automatic identification of the fatty acid methyl esters and statistical processing of the results were performed by the computerized Microbial Identification System (MIS). Significant differences between the FACs of different genera, species, and, sometimes, strains were revealed. 16S rRNA genes of some of the new isolates, primarily those having a specific FAC, were sequenced. The taxonomic status of a number of the strains in question was determined using the FAC characteristics as one of the criteria. It was shown that the FAC characteristics may be used both for affiliating isolates to known species and for revealing new taxa.     

Larger capsules enhance short‐range spore dispersal in Sphagnum,but what happens further away?

I made a dispersal experiment with six Sphagnum (peat moss) species, representing a wide range of spore and capsule sizes. Spore deposition was recorded at nine distances up to 3.2 m during two four-day periods with sunny conditions in July and August.
Deposition patterns in all species fitted well to the inverse power law (deposition per unit area proportional to distance^b), with rates of decline b ranging from −1.84 to −2.35 among species (R²>0.99). However, even when these curves are extended to infinity for the four species with b<−2, they fail to explain all spores being dispersed (e.g. only 11% in S. squarrosum ). Difference in rates of decline b between the periods, despite similar horizontal wind speeds and directions, indicates the importance of thermal updrafts. Spore capsule diameter negatively correlated with the proportions of spores remaining in dehisced capsules (range 5–16%), being deposited within the colony (range 2–14%), and being deposited between the colony edge and the outer sampled perimeter (range 7–22%), probably because a larger capsule shoots the spores higher into the air, in effect meaning an increased initial release height. Spore diameter positively affected deposition outside the colony edge. The deposition curves together with observed long-distance colonisations, omnipresence in mires of many Sphagnum species and genetic evidence, suggest that Sphagnum spores regularly travel far.
Spatial extension of the empirical deposition curves at a regional scale indicate that with increasing isolation of target sites, the higher the proportion of spores would originate from sources farther away than the nearest sources. Given spatial patterns in the source populations, this would result in higher genetic (and species) diversity per coloniser with increasing isolation, thus counteracting differentiation.     

Detection of Clostridium tyrobutyricum spores using polyclonal antibodies and flow cytometry

Aims: The present work investigates the feasibility of using flow cytometry (FCM) combined with fluorescent‐labelled specific polyclonal antibodies for the detection and presumptive identification of Clostridium tyrobutyricum spores in bovine milk. Methods and Results: Two fluorescent molecules (fluorescein isothiocyanate and Alexa Fluor 488) were conjugated to antispores polyclonal antibodies. Side scatter and forward scatter profiles of the Cl. tyrobutyricum spores marked with fluorescent antibodies permitted the detection of spores and differentiated them from other related microbial species. The detection limit of this method was 10³ spores per 100 ml of milk, and results could be achieved in 2 h. Conclusions: FCM combined with fluorochrome‐conjugated antibodies, especially Alexa Fluor, could be an efficacious means to detect and provide presumptive identification of Cl. tyrobutyricum spores, as well as differentiation from other Clostridium species that can also cause late blowing in cheese. Significance and Impact of the Study: This study describes the basis for the development of a method suitable for analysis of milk destined for cheese manufacture that would permit the detection of Cl. tyrobutyricum spores in a short period. This would enable the industry to use contaminated milk for dairy products other than cheese where Cl. tyrobutyricum does not cause a problem.     

ELECTROPHORETIC EVIDENCE FOR THE ORIGIN OF FERN SPECIES BY UNREDUCED SPORES

Allopolyploid speciation is well documented in the ferns, but data from enzyme electrophoresis have only recently shown that certain sexual and agamosporous taxa are autopolyploids. Autopolyploidy may arise through fertilization involving gametophytes from unreduced spores, a mechanism previously proposed to account for the origin of allopolyploid Asplenium plenum. This report assesses the ability of unreduced spores to function in the origin of polyploid fern species by using enzyme electrophoresis to test their hypothesized role in the origin of A. plenum. Six isozymes of the enzymes PGI, PGM, TPI, 6PGD, and LAP are species-specific for the taxa proposed as parental under two competing hypotheses for the origin of this species. Electrophoretic data reject the more conventional hypothesis involving simple hybridization and agree perfectly with expectations under the more complex hypothesized origin via unreduced spores. The mechanism whereby unreduced spores have functioned in this case is no different from that by which they would function in the origin of autopolyploid taxa and may be more common in the origin of fern species than previously suspected.     

Glycolaldehyde Dehydrogenase,Its Involvement in Vitamin B6 Biosynthetic Pathway of Escherichia coli B

The deoxyribonucleic acid (DNA) polymerases were partially purified from spores and vegetative cells of Bacillus subtilis. Some biochemical properties of the enzymes from the spores were studied in comparison with those from the vegetative cells. The spores and vegetative cells had at least three species of DNA polymerases (DNA polymerase I, II and III). These DNA polymerases in spores could not be distinguished from those in vegetative cells, respectively, with regard to the reresponses to ionic strength, the sensitivity to thiol-blocking agents, the template specificity, pH and temperature optima in assay, and the sedimentation behavior. It is inferred that DNA polymerases from spores was essentially identical to those from vegetative cells.

The DNA polymerase activity decreased rapidly in the course of sporulation, and only about 20% is recovered in the spores, suggesting that an extentive inactivation mechanism of the enzymes would be involved during sporulation.     

The high-resolution architecture and structural dynamics of Bacillus spores

The capability to image single microbial cell surfaces at nanometer scale under native conditions would profoundly impact mechanistic and structural studies of pathogenesis, immunobiology, environmental resistance, and biotransformation. Here, using in vitro atomic force microscopy, we have directly visualized high-resolution native structures of bacterial endospores, including the exosporium and spore coats of four Bacillus species in air and water environments. Our results demonstrate that the mechanisms of spore coat self-assembly are similar to those described for inorganic and macromolecular crystallization. The dimensions of individual Bacillus atrophaeus spores decrease reversibly by 12% in response to a change in the environment from fully hydrated to air-dried state, establishing that the dormant spore is a dynamic physical structure. The interspecies distributions of spore length and width were determined for four species of Bacillus spores in water and air environments. The dimensions of individual spores differ significantly depending upon species, growth regimes, and environmental conditions. These findings may be useful in the reconstruction of environmental and physiological conditions during spore formation and for modeling the inhalation and dispersal of spores. This study provides a direct insight into molecular architecture and structural variability of bacterial endospores as a function of spatial and developmental organizational scales.     

Observations on the pathogenicity of Pasteuria penetrans, a parasite of root-knot nematodes

A simple test was used to determine whether or not Pasteuria penetrans spores would attach to 17 species of nematodes. All susceptible individuals had spores attached to their cuticles after 24 h of gentle agitation in suspensions containing 10⁵spores/ml. Spores of P. penetrans from six populations of Meloidogyne only adhered to species of Meloidogyne and they adhered in greatest numbers to the species from which they had been originally isolated. Sonication of spores from infected females increased attachment but the effect was dependent on pH and whether the test was conducted in tap or distilled water. Invasion of tomato roots was reduced by up to 86% when, rather than using healthy juveniles, second-stage juveniles bearing 15 or more spores were added to soil at high densities (1000 or 3000/plant); at low densities (500/plant) invasion was not significantly affected. The rate of development of M. incognita juveniles infected with P. penetrans was slower than that of healthy juveniles. The numbers of second-generation of M. incognita were reduced by 82–93% when juveniles encumbered with 1–15 spores were added to soil instead of those bearing no spores. Pasteuria penetrans populations differed in their aggressiveness and when juveniles encumbered with the same number of spores from two populations were added to soil there were differences in the numbers of females that became infected. The implications of these results for the development of P. penetrans as a biological control agent are discussed.     

木耳和毛木耳的极性研究

从木耳(Auricularia auricula)和毛木耳(A.polytricha)的同一子实体弹射、分离30个单孢子并发育成单核菌丝体,各自分成3组,以10×10方式进行单核体两两配对。取两配对单核体交结处菌丝体块到新的平板上继续发育并插入无菌的盖玻片让其菌丝爬上。后利用双苯并咪唑(Hoechst 33258)染色,在萤光显微镜下逐块检查配对后菌丝体细胞中核的数目。如果出现双核,再加以检查锁状联合以验证,则为配对亲和。不亲和者仍为单核。根据配对行为进行不亲和因子分配决定其交配型。检测结果表明,木耳和毛木耳担孢子的性别是由一对遗传因子A.a所控制。属典型性二极性(bipolar)异宗结合。