Joanne Horn

Bioaerosol Mass Spectrometry for Rapid Detection of Individual Airborne Mycobacterium tuberculosis H37Ra Particles

ABSTRACT Single-particle laser desorption/ionization time-of-flight mass spectrometry, in the form of bioaerosol mass spectrometry (BAMS), was evaluated as a rapid detector for individual airborne, micron-sized, Mycobacterium tuberculosis H37Ra particles, comprised of a single cell or a small number of clumped cells. The BAMS mass spectral signatures for aerosolized M. tuberculosis H37Ra particles were found to be distinct from M. smegmatis, Bacillus atrophaeus, and B. cereus particles, using a distinct biomarker. This is the first time a potentially unique biomarker was measured in M. tuberculosis H37Ra on a single-cell level. In addition, M. tuberculosis H37Ra and M. smegmatis were aerosolized into a bioaerosol chamber and were sampled and analyzed using BAMS, an aerodynamic particle sizer, a viable Anderson six-stage sampler, and filter cassette samplers that permitted direct counts of cells. In a background-free environment, BAMS was able to sample and detect M. tuberculosis H37Ra at airborne concentrations of >1 M. tuberculosis H37Ra-containing particles/liter of air in 20 min as determined by direct counts of filter cassette-sampled particles, and concentrations of >40 M. tuberculosis H37Ra CFU/liter of air in 1 min as determined by using viable Andersen six-stage samplers. This is a first step toward the development of a rapid, stand-alone airborne M. tuberculosis particle detector for the direct detection of M. tuberculosis bioaerosols generated by an infectious patient. Additional instrumental development is currently under way to make BAMS useful in realistic environmental and respiratory particle backgrounds expected in tuberculosis diagnostic scenarios.

Bacterial Growth Dynamics, Limiting Factors, and Community Diversity in a Proposed Geological Nuclear Waste Repository Environment

Microbiological growth parameters, including limiting factors, kinetics, and minimal cell densities were assessed for subsurface microbiological communities collected with rock from an area proposed for a nuclear waste repository at Yucca Mountain, Nevada. Phospholipid fatty acid analysis revealed that approximately 104–105 viable cells per gram of dry rock are extant, and water availability was shown to be the primary factor limiting microbial growth in situ. Phosphate and carbon limitation, however, also suppress final cell densities by at least one order of magnitude under saturated conditions. Despite these limiting factors, significant growth of aerobic chemoheterotrophic microorganisms was shown to occur in unconcentrated simulated groundwater with or without addition of a reduced carbon source (7 × 107 and 8 × 106 planktonic cells/mL, respectively), indicating that when water becomes available in the repository environment, microbial growth will ensue. Organisms that were isolated from stationary cultures grown from Yucca Mountain rock in concentrated and unconcentrated simulated groundwaters showed significant 16S rDNA sequence divergence from reference organisms. Different (but related) organisms were isolated from concentrated and unconcentrated groundwater-grown cultures. Generally, as experimental conditions approached those expected to be encountered in situ, the organisms isolated were more divergent from reference organisms. Organisms that were isolated have metabolic properties that could allow them to be active and grow within the repository environment if water availability is sufficient.

Microbial Impacts to the Near-Field Environment Geochemistry: a model for estimating microbial communities in repository drifts at Yucca Mountain

Geochemical and microbiological modeling was performed to evaluate the potential quantities and impact of microorganisms on the geochemistry of the area adjacent to and within nuclear waste packages in the proposed repository drifts at Yucca Mountain, Nevada. The microbial growth results from the introduction of water, ground support, and waste package materials into the deep unsaturated rock. The simulations, which spanned 1 million years, were accomplished using a newly developed computer code, Microbial Impacts to the Near-Field Environment Geochemistry (MING). MING uses environmental thresholds for limiting microbial growth to temperatures below 120 degrees C and above relative humidities of 90% in repository drifts. Once these thresholds are met, MING expands upon a mass balance and thermodynamic approach proposed by McKinley et al. [FEMS Microbiol. Rev. 20 (1997) 545] by using kinetic rates to supply constituents from design materials and constituent fluxes including solubilized rock components into the drift to perform two separate mass balance calculations as a function of time. The first (nutrient limit) assesses the available nutrients (C, N, P and S) and calculates how many microorganisms can be produced based on a microorganism stoichiometry of C(160)(H(280)O(80))N(30)P(2)S. The second (energy limit) calculates the energy available from optimally combined redox couples for the temperature and pH at that time. This optimization maximizes those reactions that produce >15 kJ/mol (limit on useable energy) using an iterative linear optimization technique. The final available energy value is converted to microbial mass at a rate of 1 kg of biomass (dry weight) for every 64 MJ of energy. These two values (nutrient limit and energy limit) are then compared and the smaller value represents the number of microorganisms that can be produced over a specified time. MING can also be adapted to investigate other problems of interest as the model can be used in saturated and unsaturated environments and in laboratory situations to establish microbial growth limitations. Other projected uses include investigations of contaminated locations where monitored natural attenuation or engineered bioremediation could be employed.

Non-DNA Methods for Biological Signatures

Publisher Summary This chapter focuses on the methods that can determine chemical or structural features of biological agent particles that are signatures of particular methods of growth and post-growth processing (often referred to as “weaponization”). The detection of these signatures in a sample of a bio-weapon (BW) agent can aid the attribution by indicating: (1) the level of sophistication of the producer, (2) the access to particular types of agent weaponization information, (3) the likelihood that the material could be or has been produced at a significant scale, (4) and by providing essential sample matching data for ascertaining a putative relationship with other samples obtained in other venues. An example of the use of biologicals in forensic science is DNA, amplied by the Polymerase Chain Reaction (PCR) technique, legally admissible in courtas evidence. DNA evidence is successfully used in the court to convict or clear people of crimes because each persons DNA is unique. High-resolution techniques are being applied to investigations; such as Environmental scanning electron microscopy (ESEM) is used for taking high-resolution images under hydrated conditions; this avoids any artifacts associated with the critical point drying process that is required under normal Scanning Electron Microscopy (SEM) operations. ESEM is also equipped with Energy Dispersive X-ray (EDX) microanalysis and Backscatter capabilities. SEM is a standard “workhorse” technique for characterizing particulate samples, found in many laboratories worldwide. It provides excellent imaging of the surfaces of agent particles and other material in a sample, and is used for identifying likely agent particles for analysis by other instruments. When combined with EDX, the elemental composition of the material in the imaged region can be determined. These techniques continue to signature libraries of correlations between analyses and growth and processing conditions of growth, it will be necessary to develop an information system which combines types of data to determine unique signatures.