ENERGETIC BIOCIDAL MATERIALS AND SPORE NEUTRALIZATION

The objective of this dissertation is to explore a more accurate and versatile approach to investigating the neutralization of spores suffered from ultrafast heating and biocide based stresses, and further to explore and understand novel methods to supply ultrafast heating and biocides through nanostructured energetic materials A surface heating method was developed to apply accurate (± 25 ˚C), high heating rate thermal energy (200 - 800 ˚C, ~103 - ~105 ˚C/s). Uniform attachment of bacterial spores was achieved electrophoretically onto fine wires in liquids, which could be quantitatively detached into suspension for spore enumeration. The spore inactivation increased with temperature and heating rate, and fit a sigmoid response. The neutralization mechanisms of peak temperature and heating rate were correlated to the DNA damage at ~104 ˚C/s, and to the coat rupture by ultrafast vapor pressurization inside spores at ~105 ˚C/s. Humidity was found to have a synergistic effect of rapid heating and chlorine gas to neutralization efficiency. The primary neutralization mechanism of Cl2 and rapid heat is proposed to be chlorine reacting with the spore surface. The stress-kill correlation above provides guidance to explore new biocidal thermites, and to probe mechanisms. Results show that nano-Al/K2S2O8 released more gas at a lower temperature and generated a higher maximum pressure than the other nano-Al/oxysalts. Given that this thermite formulation generates the similar amount of SO2 as O2, it can be considered as a potential candidate for use in energetic biocidal applications. The reaction mechanisms of persulfate and other oxysalts containing thermites can be divided into two groups, with the reactive thermites (e.g. Al/K2S2O8) that generate ~10× higher of pressure and ~10× shorter of burn time ignited via a solid-gas Al/O2 reaction, while the less reactive thermites (e.g. Al/K2SO4) following a condensed phase Al/O reaction mechanism. These different ignition mechanisms were further re-evaluated by investigating the roles of free and bound oxygen. A constant critical reaction rate for ignition was found which is independent to ignition temperature, heating rate and free vs. bound oxygen.

Professional exposure to fungal pathogens: an update to exposure conditions and exposure measurement

In many occupational settings an exposure to fungi occurs. Fungal exposure may occur for instance in the form of dermatocytes, yeasts or mold. Associated to the fungi themselves an exposure to cell wall components like ß(1 ? 3)-D-glucans, to mycotoxins or to microbial volatile compounds can occur. Health hazards may differ across species because fungi may produce different allergens and mycotoxins, and some species can infect humans. Occupational settings are often characterized by special exposure conditions with respect to duration, frequency and especially to the level of exposure resulting at least sometimes to high or very high fungal exposure. Because of these special conditions occupational settings are suitable for epidemiologic studies. However, the knowledge about occupational exposure to fungi and associated compounds like mycotoxins is still fragmentary and not well disseminated. An indication for a high fungal exposure is for instance the handling of dry natural products like grain, hay or herbal plants with a high specific surface and the tendency to release dust during handling. The fungal components often form the determinative part of such dusts and might be a vehicle to respiratory airways. The authors will present results of exposure measurements of occupational settings and exposure conditions which are only rarely investigated.

Isolation and recognition Saprolegnia species from fungal infected eggs of the rainbow trout in Mazandaran province farms

Fungal infection in the eggs of freshwater fish is well known as a problematic disease. That isolation and recognition Saprolegnia fungi from fungal infected eggs of the rainbow trout in Mazandaran province was the aim of this research. For this purpose fungal infected eggs were examined from six fish farm in the fall and winter 2005-2006. The eggs with fungi were inoculated on SDA, CMA, GPagar and hemp seed and sesame seed cultures in sterile tap water at room temperature (18-24°C). In this study recognized three genera and six species Saprolegniaceae members, based on morphological characteristics which contain: Saprolegnia, Achlya, Brevilegnia. Four species were identified in the genus Saprolegnia; S.mixta, S.parasitica, S.moniliphera, S.lapponica and one species was identified in the genus Achlya; A.oblongata. S.parasitica was isolated from almost all the farms. In addition, another nine genera and species were identified; Penicillium, Aspergillus, Paeciliomyces, Acremonium, Fusarium oxysporum, F.solani , Alternaria, Helminthosporium, Mucor.

Fungal contamination in one hotel room: does carpet coating in the floor enhance fungal contamination?

Interest in bioaerosol exposure has increased significantly because it is now recognized that exposure to fungal agents is associated with a wide range of adverse health effects with a major impact on public health. Fungi are able to grow on almost all natural and synthetic materials, especially if they are hygroscopic or wet. Aim of the study: Several materials used indoors can contribute to enhance fungal contamination indoors. This study intended to understand the carpet influence on fungal contamination when used in the floor of a hotel room.

Air fungal contamination in ten hospitals’ food units from Lisbon

A descriptive study was developed to monitor air fungal contamination in ten food units from hospitals. Fifty air samples of 250 litres were collected through impaction method. Samples were collected in food storage facilities, kitchen, food plating, canteen and also, outside premises, since this is the place regarded as reference. Simultaneously, environmental parameters were also monitored, including temperature and relative humidity through the equipment Babouc, LSI Sistems and according to the International Standard ISO 7726.

Assessment of fungal contamination in a Portuguese maternity unit

A descriptive study was developed to monitor air fungal contamination in one Portuguese maternity. Sixty air samples were collected through impaction method. Air sampling was performed in food storage facilities, kitchen, food plating, canteen, pharmacy, sterilization areas, genecology wards, intensive care unit, operating rooms, urgency and also, outside premises, since this was the place regarded as reference. Besides air samples, forty three samples were collected by swabbing the surfaces using a 10 by 10 cm square stencil. Simultaneously, temperature, relative humidity and particles counting (PM10) were registered. Twenty three species of fungi were identified in air, being the two most commonly isolated the genera Penicillium (41,5%) and Cladosporium (28,4%). Regarding yeasts, only Rhodotorula sp. (45,2%), Trichosporon mucoides (51,6%) and Cryptococcus neoformans (3,2%) were found. Thirteen species of fungi were identified in surfaces, being the most frequent the Penicillium genus (91,6%). Concerning yeasts found in surfaces, four species were identified being Rhodotorula sp. (29,1%) the most frequent. There was no coincidence between prevailing genera indoors and outside premises. Moreover, some places presented fungal species different from the ones isolated outside. In the inside environment, Aspergillus species were isolated in air and surfaces. There was no significant relationship (p>0,05) between fungal contamination and the studied environmental variables. Keywords: air, surfaces, fungal contamination, environmental variables, maternity.

Comparison of fungal contamination between hospitals and companies food units

A descriptive study was developed to compare air and surfaces fungal contamination in ten hospitals’ food units and two food units from companies. Fifty air samples of 250 litres through impaction method were collected from hospitals’ food units and 41 swab samples from surfaces were also collected, using a 10 by 10 cm square stencil. Regarding the two companies, ten air samples and eight surface samples were collected. Air and surface samples were collected in food storage facilities, kitchen, food plating and canteen. Outdoor air was also collected since this is the place regarded as a reference. Simultaneously, temperature, relative humidity and meal numbers were registered. Concerning air from hospitals’ food units, 32 fungal species were identified, being the two most commonly isolated genera Penicillium sp.

From the farm to the fork: fungal occupational exposure in the swine meat supply chain

Feed production, swine and slaughterhouses were already reported as occupational environments with high fungal contamination. This condition can ultimately lead to the development of several health conditions. This study aimed to characterize the occupational exposure to fungal burden in three different settings: swine feed unit, swine units and slaughterhouse.

New approach to study the real exposure to fungi in cork industry: nasal swabs mycobiota investigation coupled with screening on fungal resistance to azoles

The permanent contact with cork may lead to constant exposure to fungi, raising awareness as a potential occupational hazard in the cork industry.The presence of fungi belonging to the Penicillium glabrum complex has been associated with the development of respiratory diseases such as suberosis, one of the most prevalent diseases among workers from cork industries, besides occupational asthma. Azoles are used as pesticides but also the first line therapy in the treatment of Aspergillus infections; azole-resistance as been described as to have also an environmental source and is considered an emerging public health problem.The aim of this work was to characterize fungal distribution and to evaluate the presence of azole-resistant Aspergillus isolates in nose swab samples from the cork industry workers.

Fungal contamination in one hotel room: Does carpet coating in the floor enhance fungal contamination?

Study developed in order to know the carpet influence when used in the floor of a hotel room. Twelve air samples of 250L (six in a room with carpet and six more in a room with wood floor) were collected through an impaction method with a flow rate of 140 L/min onto malt extract agar (MEA) supplemented with chloramphenicol (0.05%), using the Millipore air Tester (Millipore), during cleaning activities. Outdoor sample was also performed to be used as a reference. Surface samples from floor and desks, taken at the same time, were collected by the swabbing method. to 7 days. Besides fungal contamination, we also assessed particulate matter contamination in both rooms during the same cleaning tasks. In the analyzed sur- faces, isolates belonging to Aspergillus fumigatus complex were the only fungi found in the carpeted room, whereas in the other room we found Penicllium sp. (63.6%) and Aspergillus sp. (13.6%) as the most frequent genera. In the case of particles the room with carpet obtained significant higher values for both metrics (PMC and PNC), showing that carpet may has influence on particles’ contamination of the room.

What influences fungal communities in cotton soils

Soilborne diseases such as Fusarium wilt, Black root rot and Verticillium wilt have significant impact on cotton production. Fungi are an important component of soil biota with capacity to affect pathogen inoculum levels and their disease causing potential. Very little is known about the soil fungal community structure and management effects in Australian cotton soils. We analysed surface soils from ongoing field experiments monitoring cotton performance and disease incidence in three cotton growing regions, collected prior to 2013 planting, for the genetic diversity and abundance as influenced by soil type, environment and management practices and link it with disease incidence and suppression. Results from the 28S LSU rRNA sequencing based analysis indicated a total of 370 fungal genera in all the cotton soils and the top 25 genera in abundance accounted for the major portion of total fungal community. There were significant differences in the composition and genetic diversity of soil fungi between the different field sites from the three cotton growing regions. Results for diversity indices showed significantly greater diversity in the long-term crop rotation experiment at Narrabri (F6E) and experiments at Cowan and Goondiwindi compared to the Biofumigation and D1 field experiments at ACRI, Narrabri. Diversity was lowest in the soils under brassica crop rotation in Biofumigation experiment. Overall, the diversity and abundance of soil fungal community varied significantly in the three cotton growing regions indicating soil type and environmental effects. These results suggest that changes in soil fungal community may play a notable role in soilborne disease incidence in cotton.

The cellular and mechanistic study of nisin inhibition of bacillus anthracis spore outgrowth, nisin alteration of infection, and native producer immunity

A critical step during Bacillus anthracis infection is the outgrowth of germinated spores into vegetative bacilli that proliferate and disseminate rapidly within the host. An important challenge exists for developing chemotherapeutic agents that act upon and kill B. anthracis immediately after germination initiation when antibiotic resistance is lost, but prior to the outgrowth into vegetative bacilli, which is accompanied by toxin production. Chemical agents must also function in a manner refractive to the development of antimicrobial resistance. In this thesis we have identified the lantibiotics as a class of chemotherapeutics that are predicted to satisfy these two criteria. The objective of this thesis was to evaluate the efficacy of nisin, a prototypical lantibiotic, in prevention of outgrowth of germinated B. anthracis spores. Like all lantibiotics, nisin is a ribosomally translated peptide that undergoes post-translational modification to form (methyl)lanthionine rings that are critical for antimicrobial activity. Our studies indicate that nisin rapidly inhibits the in vitro outgrowth of germinated B. anthracis Sterne 7702 spores. Although germination initiation was shown to be essential for nisin-dependent antimicrobial activity, nisin did not inhibit or promote germination initiation. Nisin irreversibly killed germinated spores by blocking the establishment of a membrane potential and oxidative metabolism, while not affecting the dissolution of the outer spore structures. The membrane permeability of the spore was increased by nisin, but germinated spores did not undergo full lysis. Nisin was demonstrated to localize to lipid II, which is the penultimate precursor for cell wall biogenesis. This localization suggests two possible independent mechanisms of action, membrane pore formation and inhibition of peptidoglycan synthesis. Structure-activity studies with a truncated form of nisin lacking the two C-terminal (methyl)lanthionine rings and with non-pore forming mutants indicated that membrane disruption is essential for nisin-dependent inhibition of spore outgrowth to prevent membrane potential establishment. Finally, utilizing an in vitro infection model, it was shown that nisin reduced the viability of B. anthracis spores within an infection resulting in increased survival of immune cells while reducing infection-mediated cytokine expression. Fluorescence microscopy indicated that nisin localizes with spores within phagosomes of peritioneal macrophages in germinating conditions. These data demonstrate the effectiveness of nisin, as a model lantibiotic, for preventing spore outgrowth. It is speculated that nisin targeting of lipid II, resulting in membrane perturbations, may be effective at inhibiting the outgrowth of spores prepared from bacteria across a number of species.

Potential of Trichoderma harzianum for control of banana leaf fungal pathogens when applied with a food source and an organic adjuvant

Trichoderma isolates were obtained from diseased leaves and fruit collected from plantations in the main banana production area in Northern Queensland. Phylogenetic analyses identified the Trichoderma isolates as T. harzianum and T. virens. The Trichoderma spp. were found to be antagonistic against the banana leaf pathogens Mycosphaerella musicola, Cordana musae, and Deight-oniella torulosa in vitro. Several products used by the banana industry to increase production, including molasses, Fishoil and Seasol, were tested as food source for the Trichoderma isolates. The optimal food substrate was found to be molasses at a concentration of 5 %, which when used in combination with a di-1-p-menthene spreader-sticker enhanced the survivability of Trichoderma populations under natural conditions. This formulation suppressed D. torulosa development under glasshouse conditions. Furthermore, high sensitivity was observed towards the protectant fungicide Mancozeb but Biopest oil (R), a paraffinic oil, only marginally suppressed the growth of Trichoderma isolates in vitro. Thus, this protocol represents a potential to manage banana leaf pathogens as a part of an integrated disease approach.