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Clostridium difficile-associated diarrhoea (CDAD) is a frequently occurring healthcare-associated infection, which is responsible for significant morbidity and mortality amongst elderly patients in healthcare facilities. Environmental contamination is known to play an important contributory role in the spread of CDAD and it is suspected that contamination might be occurring as a result of aerial dissemination of C. difficile spores. However previous studies have failed to isolate C. difficile from air in hospitals. In an attempt to clarify this issue we undertook a short controlled pilot study in an elderly care ward with the aim of culturing C. difficile from the air. In a survey undertaken during February (two days) 2006 and March (two days) 2007, air samples were collected using a portable cyclone sampler and surface samples collected using contact plates in a UK hospital. Sampling took place in a six bedded elderly care bay (Study) during February 2006 and in March 2007 both the study bay and a four bedded orthopaedic bay (Control). Particulate material from the air was collected in Ringer's solution, alcohol shocked and plated out in triplicate onto Brazier's CCEY agar without egg yolk, but supplemented with 5 mg/L of lysozyme. After incubation, the identity of isolates was confirmed by standard techniques. Ribotyping and REP-PCR fingerprinting were used to further characterise isolates. On both days in February 2006, C. difficile was cultured from the air with 23 samples yielding the bacterium (mean counts 53 ¿ 426 cfu/m3 of air). One representative isolate from each of these was characterized further. Of the 23 isolates, 22 were ribotype 001 and were indistinguishable on REP-PCR typing. C. difficile was not cultured from the air or surfaces of either hospital bay during the two days in March 2007. This pilot study produced clear evidence of sporadic aerial dissemination of spores of a clone of C. difficile, a finding which may help to explain why CDAD is so persistent within hospitals and difficult to eradicate. Although preliminary, the findings reinforce concerns that current C. difficile control measures may be inadequate and suggest that improved ward ventilation may help to reduce the spread of CDAD in healthcare facilities.

Morphologically and anatomically the Russulales (Basidiomycetes) are a homogeneous group of higher fungi, which contains both ballistosporic and statismosporic, agaricoid and gasteroid taxa. Spore symmetry and ability to forcibly discharge spores are therefore fundamental systematic characteristics in the Russulales. Ballistosporic and statismosporic basidiosporogenesis however, has not been critically compared. Early and late basidiosporogenesis, spore-wall tegumentation, and differentiation of the hilar appendix were ultrastructurally characterized in species selected from eight genera of agaricoid and gasteroid Russulales including: Russula, Lactarius, Macowanites, Arcangeliella, Elasmomyces, Gymnomyces, Martellia, and Zelleromyces. Six spore-wall layers are present in developing spores in all genera. Two wall layers are associated with an evanescent pellicle and four wall layers are derived from the sterigma and young spore. The amyloid portion of the spore wall in the Russulales is an electron-translucent wall layer covered by an electron-dense surface layer. Ontogeny of spore-wall ornamentation is similar in all genera, however diversity in the degree of ornamentation and amyloidity results from differentiation and intermixing of the two outermost enduring wall layers. Establishment of early spore asymmetry in both ballistosporic- and statismosporic-heterotropic basidiospores is highly correlated with the presence of a hilar appendix body. Observation of a demarcated cytoplasmic region, reminiscent of the hilar appendix body, in asymmetric sterigmata of orthotropic Russulales suggests that basidiospore morphology and symmetry are variable features in the Russulales. Late spore development subsequent to nuclear migration is similar in orthotropic and heterotropic Russulales. Orthotropic basidiospores appear to be released from sterigmata upon breakdown of the sterigmata. Heterotropic basidiospores are released while sterigmata are intact. Plugging layers develop in both the sterigma and in the hilar appendix. The hilar appendix plugging material appears to be produced by a plug-forming body which originates in the spore, and possibly controls liquid droplet formation during ballistosporic discharge. Ballistosporic discharge appears to be a conservative phenomenon in most Basidiomycetes resulting from a prescribed sequence of biochemical and developmental processes. The use of ballistospory and statismospory in distinguishing families and genera must be re-evaluated in the Russulales. Recognition of the Lactariaceae and Russulaceae is discussed. / Ph. D.

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No description available.

Bacillus anthracis has been classified as one of the most dangerous bioterrorism agents causing high mortality rates in short periods of time. Anthrax spores are extremely resistant to chemical and environmental factors, and have the ability to return into a vegetative (virulent) state during the process of germination. Previous research has suggested that spores can be eradicated with common disinfectants after germination and release of spore coats. During germination, the spore coat is degraded, making the spore susceptible to penetration of chemicals into the spore core. While previous research has focused on a qualitative understanding of germination of spores by obtaining high-resolutions images of spore coats to understand how protein coat layers change during germination, very few studies have evaluated changes in mechanical properties of spores during germination, and how germination affects virulence of macrophages. In this study, we performed a series of in vitro experiments to do an in-depth analysis of germination and virulence of B. anthracis. Atomic force microscopy (AFM) was used to investigate changes in spore surface properties during germination including morphology, roughness, elasticity, and spring constant. AFM results suggested that germination mechanisms depend on germinants used to trigger germination and roughness of Bacillus species increase during germination. In addition, the elasticity and spring cell constant of B. anthracis spores are affected during germination since the elastic moduli and cell spring constant values decreased with time as the spore was germinating, making the cells more susceptible. Spore killing was also tested both in sporulated and vegetative B. anthracis using the antimicrobial peptide chrysophsin-3 and the surfactant dodecylamine (DDA). Both killing agents were capable of eradicating B. anthracis spores, but more killing was observed for spores that were germinating or had become vegetative. The presence of germinant receptors from the Ger operon and its role on germination kinetics of B. anthracis was also investigated. The germination of mutant spores that carried one receptor or lacked all germinant receptors was compared to the germination kinetics of wild-type B. anthracis. Our results suggest that germination of spores is modified by the presence or absence of germinant receptors. Furthermore, the mutant B. anthracis strain lacking all receptors germinated suggesting that other receptor independent pathways may exist in B. anthracis. Finally the ability of B. anthracis to adhere, grow, and invade macrophages was investigated. Invasion of macrophages by B. anthracis was dependent on germinant receptors and the ability of spores to germinate and multiply. Our results suggest that macrophages were not capable of killing infecting spores, and on the contrary, germination of spores inside macrophages caused the lysis of macrophages. An uncontrolled release of cytokines by macrophages was elicited by spores and germinated B. anthracis. Our study helps understand the process of germination of B. anthracis spores at a nanomolecular level. Our investigation may be a valuable tool in the design and development of antisporal compounds.

Anomalous behaviour of water confined in nanoscale gaps influences many biological and technological processes. However, due to the small size of confining structures, it is historically difficult to manipulate and study water’s dimension-dependent transport character. Experimental studies of nanoconfined water are generally limited to artificial test structures, and/or single-file channels, and so transport behavior of biologically nanoconfined water remains elusive. We utilize poroelastic bacterial spores coated onto a nanomechanical sensor to probe photo-thermal evaporative relaxation in a biological setting and report viscous water, 7 orders of magnitude larger than that of bulk liquid, and via thermodynamic investigations reveal an activation energy close to ice. Overall, these experiments characterize transport behaviour of nanoconfined water in vivo, and highlight the dramatic effects of nanoscale confinement on water that could impact myriad natural and synthetic processes. Following from this work, a hypothesis is pursued in which the bacterial lifecycle is intimately connected with transitions in the physical structure of the internal water. We expand an initial idea proposed in Science, 1960 by J.C. Lewis, N.S. Snell and H.K. Burr that the low water content of the spore core is accomplished through compressive contraction during development3. During sporulation, the genetic material is packaged with chelating chemicals within a special water-responsive, layered coating that electrostatically pulls the water out of the core. Together, these agents produce the extremely dehydrated, hydraulically tensioned, and stable spore-phase organism. During germinative re-awakening, an event lacking a complete mechanistic theory of sensation, the core is rehydrated and the organism subsequently reanimated. This work’s findings regarding the spore’s physically restrained but exchangeable water support the idea that the physical state of the water contributes significantly to tensioning the organisms into a ‘charged’ but dormant configuration. This dormant but spring-loaded phase of the bacterial lifecycle is subject to awakening by agents (nutrient or otherwise) which disrupt surface tension including amino acids, salts, surfactants, and hydrostatic pressures. In the least, it must be acknowledged that the slowed water observed herein enforces slowed biochemistry and thus dormancy. Taken together we present a picture where internal spore water, even that which is exchanged with the external environment, is nanoconfined and slowed under tremendous tension (negative pressure). The mechanism governing this slow water appears to be unlike that any previously described, the majority of which are typically based upon crystalline surfaces, the likes of which are not found in the spore. We consider that the spore water structure itself participates, in certain environments, in the signaling chain of the organism through stabilizing a delicately balanced and highly tensioned architecture. Presently we are working toward testing the hypothesis and expanding our understanding with new methods, including additional structural mutants and expanded biophysical techniques.

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No description available.

The survival and germination of Clostridium perfringens spores in different heating media and at different heating rates was studied to determine the fate of C. perfringens spores during abusive cooking and cooling of pork products. The heat resistance (HR) of C. perfringens spores from three strains that were either previously heat shocked (HS) or non-heat shocked (NHS) was determined individually and as a cocktail in phosphate buffer solution (pH 7.4) (PBS), beef gravy (BG), ground pork (GP) and cured ground pork (CGP) at 75ºC. The effect of the heating rate on HR, germination and outgrowth of C. perfringens spores in CGP was determined by increasing the temperature from 20 to 75ºC at a rate of 4, 8, and 12ºC/h prior to heating and holding at 75ºC for 48 h. Heating rates at 4ºC/h in GP and CGP were repeated with additional cooling from 54.4 to 7.2ºC within 20 h (temperature abuse). Linear survival curves were observed on NHS spores in the four heating media, whereas HS spores showed linear curves when heated in PBS and BG, and biphasic curves when heated in GP and CGP. In general, HS spores were more heat sensitive than NHS spores. NHS spores heated in GP had greater HR than spores heated in CGP, BG or PBS. There were no significant differences (P>0.05) on the HR of C. perfringens spores in CGP heated from 20 to 75ºC at 4, 8, or 12ºC/h. Heating rates of 8 and 12ºC/h showed no difference in germination and outgrowth of inoculated spores, whereas at 4ºC/h, growth of C. perfringens occurred between 44 and 56ºC. Temperature abuse during cooling of GP resulted in 2.8 log CFU/g increase of C. perfringens counts. In CGP, C. perfringens counts decreased by 1.1 log CFU/g during cooling from 54.4 to 36.3ºC and then increased by 1 log CFU/g until the product reached 7.2ºC. However, with an initial inoculum in raw CGP of 5 log CFU C. perfringens spores/g, C. perfringens counts did not exceed 3.4 log CFU/g during a 20 h abusive cooling. These results suggest there is no risk associated with C. perfringens in cured pork products under the conditions tested. Results from the present study indicate that different behavior may be expected with different meat products.