AimsTo determine how hydrated Bacillus anthracis spores are killed in a high-temperature gas environment (HTGE), and how spores of several Bacillus species including B.anthracis are killed by UV radiation, dry heat, wet heat and desiccation.Methods and ResultsHydrated B.anthracis spores were HTGE treated at c. 220 degrees C for 50ms, and the treated spores were tested for germination, mutagenesis, rupture and loss of dipicolinic acid. Spores of this and other Bacillus species were also examined for mutagenesis by UV, wet and dry heat and desiccation. There was no rupture of HTGE-treated B.anthracis spores killed 90-99 center dot 9%, no mutagenesis, and release of DPA and loss of germination were much slower than spore killing. However, killing of spores of B.anthracis, Bacillus thuringiensis and Bacillus subtilis by UV radiation or dry heat, but not wet heat in water or ethanol, was accompanied by mutagenesis.ConclusionsIt appears likely that HTGE treatment kills B.anthracis spores by damage to spore core proteins. In addition, various killing regimens inactivate spores of a number of Bacillus species by the same mechanisms.Significance and Impact of the StudyThis work indicates how hydrated spores treated in a HTGE such as might be used to destroy biological warfare agent stocks are killed. The work also indicates that mechanisms whereby different agents kill spores are similar with spores of different Bacillus species.

Stable, insoluble Langmuir monolayer films composed of Staphylococcus aureus-specific lytic bacteriophage were formed at an air–water interface and characterized. The phage monolayer was very strong, withstanding a surface pressure of ∼40mN/m at 20°C. The surface pressure–area (Π–A) isotherm possessed a shoulder at ∼7×104nm2/phage particle, attributed to a change in phage orientation at the air–water interface from horizontal to vertical capsid-down/tail-up orientation as surface pressure was increased. The Π–A-dependence was accurately described using the Volmer equation of state, assuming horizontal orientation to an air–water interface at low surface pressures with an excluded area per phage particle of 4.6×104nm2. At high pressures phage particles followed the space-filling densely packed disks model with a specific area of 8.5×103nm2/phage particle. Lytic phage monolayers were transferred onto gold-coated silica substrates from the air–water interface at a constant surface pressure of 18mN/m by Langmuir–Blodgett method, then dried and analyzed by scanning electron microscopy (SEM) and ellipsometry. Phage specific adsorption (Γ) in Langmuir–Blodgett (LB) films measured by SEM was consistent with that calculated independently from Π–A isotherms at the transfer surface pressure of 18mN/m (Γ=23phage particles/μm2). The 50nm-thickness of phage monolayer measured by ellipsometer agreed well with the horizontal phage average size estimated by SEM. Surface properties of phage Langmuir monolayer compare well with other monolayers formed from nano- and micro-particles at the air–water interface and similar to that of classic amphiphiles 1,2-diphytanoyl-sn-glycero-3-phosphocholine (phospholipid) and stearic acid.

One of the major mechanisms of deactivating spores is to expose them to elevated temperatures. Experiments carried out in an exposure tube to study the effects on spores in a high temperature gas environment provide evidence of spore deactivation, although the deactivation mechanism is not clear. Numerical studies have been performed to complement the experimental work and to study the effects of various parameters, such as gas temperature, exposure time, and internal pressure on spore survivability. The laminar and turbulent flow fields of a sudden expansion inside the thermal exposure system were simulated by computational fluid dynamics. A thermo-structural model with wall porosity was developed to take into account the dynamic response of the spore wall structure to the internal pressure exerted by the heated vapor on the spore wall. Finally, the results of the transient heat transfer and the thermo-structural models are discussed in the context of the uncertainties in biological system parameters and spore survivability.

The microstructure and functionality of methicillin-resistant Staphylococcus aureus (MRSA) biosensors prepared from novel Langmuir-Blodgett monolayers of lytic bacteriophage were characterized using scanning imaging ellipsometry (SIE) and scanning electron microscopy (SEM). SEM revealed uniform distribution of phage monolayers immobilized to biosensor substrates. SIE indicated monolayers were 49.8 ± 18.3 nm thick on average. SIE charged couple imaging analysis of biosensors yielded an average intensity of 159±7 and 194±13 for 108 and 109 CFU/ml MRSA concentrations, respectively.

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: This research was a collaborative effort between experimental program at Auburn University and a computational modeling study at Pennsylvania State University. Determinations were made of the inactivation of aerosolized Bacillus Anthracis (Ba) single spores exposed to temperatures from 165 C to 275 C for times between 25ms and 100ms. The data was used to anchor computational fluid dynamics (CFD) flow modeling of heat transfer into the areosolized Ba spores.

Filamentous phage affinity-selected for streptavidin and Salmonella typhimurium from phage display libraries were transformed with chloroform into spherical forms then deposited as phage coat protein monolayers to quartz crystal microbalances (QCM) by Langmuir-Blodgett (LB) to prepare bioselective sensors. Maximum yield of spheroids was achieved by mixing 8.3 x 10-11 to 2.5 x 10-12 filamentous phage virions/ml with 1 ml chloroform for 60 seconds. Spheroid conversion and binding to S. typhimurium was confirmed by transmission electron microscopy. Phage coat monolayers incorporating phospholipid possessed high elasticity and transference to QCM substrates comparable to antibodies. Sensor responses to increasing concentrations of target analytes were characterized by rapid reaction, steady-state equilibrium, high sensitivity, and linear dose-response that followed mass theory for piezoelectric transducers. Scanning electron microscopy confirmed binding of target analytes to biosensors compared to controls. Phage-based sensors may allow detection of bacterial agents such as S. typhimurium in food safety and security applications.

Monolayers of the antifungal antibiotic amphotericin B undergo the liquid expanded/liquid condensed state transition if spread from chloroform/methanol solvent. The transition disappeared after a long spreading time. The presence of the transition may be due to the retention of solvent and/or the presence of metastable aggregates of amphotericin B.

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The antimycotic activity of amphotericin B depends on its ability to make complexes with cell membrane sterols. Surface pressure (pi) as a function of surface area (A) and pi-A hysteresis were measured for monolayers of amphotericin B/cholesterol mixtures on the water/air interface. Specific area per molecule of amphotericin B and free energy of mixing were calculated as a function of concentration of amphotericin B. When chloroform/methanol was used as a spreading solvent, the pi-A isotherms of the mixed monolayers exhibited characteristic transitions from the gas to liquid-expanded, then liquid-condensed, and finally the solid state. The mean molecular area of the mixed monolayers was significantly higher than the calculated sum of the molecular areas of the pure components. This expanding effect was accompanied by a large pi-A hysteresis and a positive excess of free energy of mixing at high pi. In contrast, when 2-propanol/water was used as spreading solvent, the mixed monolayers at 20 degreesC exhibited pi-A isotherms with no visible transitions, low hysteresis, a condensing effect, and a negative free energy of mixing. The most stable monolayers were produced from molecules of amphotericin B and cholesterol with a 2:1 stoichiometry. At this ratio, amphotericin B and cholesterol form ion channels in lipid bilayers with conductance of 4-400 pS. These results provide a better understanding of the biological activity of amphotericin B. Artificial amphotericin B/cholesterol ion channels having large conductance could be useful in nanotechnology.

The specific and selective detection of Salmonella typhymurium based on the use of a polyclonal antibody immobilized by the Langmuir–Blodgett method on the surface of a quartz crystal acoustic wave device was demonstrated in liquid samples. These biosensors were selective to S. typhymurium in the presence of large concentrations of Escherichia coli O157:H7. They were also specific to S. typhymurium since bacteria preincubated with free antibody produced no signal. Dark-field and electron microscopy showed that two different antibodies, polyvalent somatic O and flagellar H7, were immobilized on the sensor surface producing two distinct attachments of bacteria at the liquid–solid interface. The somatic O antibody exhibits a rigid, binding, while the flagellar H7 antibody forms a flexible connection allowing a large degree of freedom. When the attachment of bacteria was rigid and strong, the responses of the acoustic wave sensors correlated with changes in the mass of bacteria present at the liquid–solid interface. In contrast, when attachment was flexible, the sensor signals were inversely proportional to the additional mass of bound bacteria. This difference is probably determined by the interfacial viscoelasticity and by acoustic and electromagnetic coupling. The signals of environmentally aged sensors with either predominately rigid or flexible positioning of bacteria were correlated with changes in mass at the liquid–solid interface. Sensors with O or H type of binding could be used for analytical purposes.