ALBERT

Description: The most common approach for assessing the abundance of viable bacterial endospores is the culture-based plating method. However, culture-based approaches are heavily biased and oftentimes incompatible with upstream sample processing strategies, which make viable cells/spores uncultivable. This shortcoming highlights the need for rapid molecular diagnostic tools to assess more accurately the abundance of viable spacecraft-associated microbiota, perhaps most importantly bacterial endospores. Propidium monoazide (PMA) has received a great deal of attention due to its ability to differentiate live, viable bacterial cells from dead ones. PMA gains access to the DNA of dead cells through compromised membranes. Once inside the cell, it intercalates and eventually covalently bonds with the double-helix structures upon photoactivation with visible light. The covalently bound DNA is significantly altered, and unavailable to downstream molecular-based manipulations and analyses. Microbiological samples can be treated with appropriate concentrations of PMA and exposed to visible light prior to undergoing total genomic DNA extraction, resulting in an extract comprised solely of DNA arising from viable cells. This ability to extract DNA selectively from living cells is extremely powerful, and bears great relevance to many microbiological arenas.

Description: Contaminant terrestrial microbiota could profoundly impact the scientific integrity of extraterrestrial life-detection experiments. It is therefore important to know what organisms persist on spacecraft surfaces so that their presence can be eliminated or discriminated from authentic extraterrestrial biosignatures. Although there is a growing understanding of the biodiversity associated with spacecraft and cleanroom surfaces, it remains challenging to assess the risk of these microbes confounding life-detection or sample-return experiments. A key challenge is to provide a comprehensive inventory of microbes present on spacecraft surfaces. To assess the phylogenetic breadth of microorganisms on spacecraft and associated surfaces, the Genetic Inventory team used three technologies: conventional cloning techniques, PhyloChip DNA microarrays, and 454 tag-encoded pyrosequencing, together with a methodology to systematically collect, process, and archive nucleic acids. These three analysis methods yielded considerably different results: Traditional approaches provided the least comprehensive assessment of microbial diversity, while PhyloChip and pyrosequencing illuminated more diverse microbial populations. The overall results stress the importance of selecting sample collection and processing approaches based on the desired target and required level of detection. The DNA archive generated in this study can be made available to future researchers as genetic-inventory-oriented technologies further mature.

Description: A report presents a phenotypic and genotypic characterization of a bacterial species that has been found to be of the genus Bacillus and has been tentatively named B. odysseensis because it was isolated from surfaces of the Mars Odyssey spacecraft as part of continuing research on techniques for sterilizing spacecraft to prevent contamination of remote planets by terrestrial species. B. odysseensis is a Gram-positive, facultatively anaerobic, rod-shaped bacterium that forms round spores. The exosporium has been conjectured to play a role in the elevated resistance to sterilization. Research on the exosporium is proposed as a path toward improved means of sterilization, medical treatment, and prevention of biofouling.

Description: This innovation is derived from a proprietary amplification scheme that is based upon random fragmentation of the genome into a series of short, overlapping templates. The resulting shorter DNA strands (〈400 bp) constitute a library of DNA fragments with defined 3 and 5 termini. Specific primers to these termini are then used to isothermally amplify this library into potentially unlimited quantities that can be used immediately for multiple downstream applications including gel eletrophoresis, quantitative polymerase chain reaction (QPCR), comparative genomic hybridization microarray, SNP analysis, and sequencing. The standard reaction can be performed with minimal hands-on time, and can produce amplified DNA in as little as three hours. Post-fragmentation whole genome amplification-based technology provides a robust and accurate method of amplifying femtogram levels of starting material into microgram yields with no detectable allele bias. The amplified DNA also facilitates the preservation of samples (spacecraft samples) by amplifying scarce amounts of template DNA into microgram concentrations in just a few hours. Based on further optimization of this technology, this could be a feasible technology to use in sample preservation for potential future sample return missions. The research and technology development described here can be pivotal in dealing with backward/forward biological contamination from planetary missions. Such efforts rely heavily on an increasing understanding of the burden and diversity of microorganisms present on spacecraft surfaces throughout assembly and testing. The development and implementation of these technologies could significantly improve the comprehensiveness and resolving power of spacecraft-associated microbial population censuses, and are important to the continued evolution and advancement of planetary protection capabilities. Current molecular procedures for assaying spacecraft-associated microbial burden and diversity have inherent sample loss issues at practically every step, particularly nucleic acid extraction. In engineering a molecular means of amplifying nucleic acids directly from single cells in their native state within the sample matrix, this innovation has circumvented entirely the need for DNA extraction regimes in the sample processing scheme.

Description: This innovation is a coupled fluorescence-activated cell sorting (FACS) and fluorescent staining technology for purifying (removing cells from sampling matrices), separating (based on size, density, morphology, and live versus dead), and concentrating cells (spores, prokaryotic, eukaryotic) from an environmental sample.

Description: Frequently there is an inability to process and analyze samples of low biomass due to limiting amounts of relevant biomaterial in the sample. Furthermore, molecular biological protocols geared towards increasing the density of recovered cells and biomolecules of interest, by their very nature, also concentrate unwanted inhibitory humic acids and other particulates that have an adversarial effect on downstream analysis. A novel and robust fluorescence-activated cell-sorting (FACS)-based technology has been developed for purifying (removing cells from sampling matrices), separating (based on size, density, morphology), and concentrating cells (spores, prokaryotic, eukaryotic) from a sample low in biomass. The technology capitalizes on fluorescent cell-sorting technologies to purify and concentrate bacterial cells from a low-biomass, high-volume sample. Over the past decade, cell-sorting detection systems have undergone enhancements and increased sensitivity, making bacterial cell sorting a feasible concept. Although there are many unknown limitations with regard to the applicability of this technology to environmental samples (smaller cells, few cells, mixed populations), dogmatic principles support the theoretical effectiveness of this technique upon thorough testing and proper optimization. Furthermore, the pilot study from which this report is based proved effective and demonstrated this technology capable of sorting and concentrating bacterial endospore and bacterial cells of varying size and morphology. Two commercial off-the-shelf bacterial counting kits were used to optimize a bacterial stain/dye FACS protocol. A LIVE/DEAD BacLight Viability and Counting Kit was used to distinguish between the live and dead cells. A Bacterial Counting Kit comprising SYTO BC (mixture of SYTO dyes) was employed as a broad-spectrum bacterial counting agent. Optimization using epifluorescence microscopy was performed with these two dye/stains. This refined protocol was further validated using varying ratios and mixtures of cells to ensure homogenous staining compared to that of individual cells, and were utilized for flow analyzer and FACS labeling. This technology focuses on the purification and concentration of cells from low-biomass spacecraft assembly facility samples. Currently, purification and concentration of low-biomass samples plague planetary protection downstream analyses. Having a capability to use flow cytometry to concentrate cells out of low-biomass, high-volume spacecraft/ facility sample extracts will be of extreme benefit to the fields of planetary protection and astrobiology. Successful research and development of this novel methodology will significantly increase the knowledge base for designing more effective cleaning protocols, and ultimately lead to a more empirical and true account of the microbial diversity present on spacecraft surfaces. Refined cleaning and an enhanced ability to resolve microbial diversity may decrease the overall cost of spacecraft assembly and/or provide a means to begin to assess challenging planetary protection missions.

Description: A method of discriminating between spore-forming and non-spore-forming bacteria is based on a combination of simultaneous sporulation-specific and non-sporulation-specific quantitative polymerase chain reactions (Q-PCRs). The method was invented partly in response to the observation that for the purposes of preventing or reducing biological contamination affecting many human endeavors, ultimately, only the spore-forming portions of bacterial populations are the ones that are problematic (or, at least, more problematic than are the non-spore-forming portions). In some environments, spore-forming bacteria constitute small fractions of the total bacterial populations. The use of sporulation-specific primers in Q-PCR affords the ability to assess the spore-forming fraction of a bacterial population present in an environment of interest. This assessment can provide a more thorough and accurate understanding of the bacterial contamination in the environment, thereby making it possible to focus contamination- testing, contamination-prevention, sterilization, and decontamination resources more economically and efficiently. The method includes the use of sporulation-specific primers in the form of designed, optimized deoxyribonucleic acid (DNA) oligonucleotides specific for the bacterial spoIVA gene (see table). [In "spoIVA," "IV" signifies Roman numeral four and the entire quoted name refers to gene A for the fourth stage of sporulation.] These primers are mixed into a PCR cocktail with a given sample of bacterial cells. A control PCR cocktail into which are mixed universal 16S rRNA primers is also prepared. ["16S rRNA" denotes a ribosomal ribonucleic acid (rRNA) sequence that is common to all organisms.] Following several cycles of heating and cooling according to the PCR protocol to amplify amounts of DNA molecules, the amplification products can be analyzed to determine the types of bacterial cells present within the samples. If the amplification product is strong, relative to the product of a control PCR sequence, then it is concluded that the bacterial population in the sample consists predominantly of spore-forming cells. If the amplification product is weak or nonexistent, then it is concluded that the bacterial population in the sample consists predominantly or entirely of non-spore-forming cells.

Description: Based on previously reported procedures for permeabilizing vegetative bacterial cells, and numerous trial-and-error attempts with bacterial endospores, a protocol was developed for effectively permeabilizing bacterial spores, which facilitated the applicability of fluorescent in situ hybridization (FISH) microscopy. Bacterial endospores were first purified from overgrown, sporulated suspensions of B. pumilus SAFR-032. Purified spores at a concentration of approx equals 10 million spores/mL then underwent proteinase-K treatment, in a solution of 468.5 L of 100 mM Tris-HCl, 30 L of 10% SDS, and 1.5 microL of 20 mg/mL proteinase-K for ten minutes at 35 C. Spores were then harvested by centrifugation (15,000 g for 15 minutes) and washed twice with sterile phosphate-buffered saline (PBS) solution. This washing process consisted of resuspending the spore pellets in 0.5 mL of PBS, vortexing momentarily, and harvesting again by centrifugation. Treated and washed spore pellets were then resuspended in 0.5 mL of decoating solution, which consisted of 4.8 g urea, 3 mL Milli-Q water, 1 mL 0.5M Tris, 1 mL 1M dithiothreitol (DTT), and 2 mL 10% sodium-dodecylsulfate (SDS), and were incubated at 65 C for 15 minutes while being shaken at 165 rpm. Decoated spores were then, once again, washed twice with sterile PBS, and subjected to lysozyme/mutanolysin treatment (7 mg/mL lysozyme and 7U mutanolysin) for 15 minutes at 35 C. Spores were again washed twice with sterile PBS, and spore pellets were resuspended in 1-mL of 2% SDS. This treatment, facilitating inner membrane permeabilization, lasted for ten minutes at room temperature. Permeabilized spores were washed two final times with PBS, and were resuspended in 200 mkcroL of sterile PBS. At this point, the spores were permeable and ready for downstream processing, such as oligonucleotideprobe infiltration, hybridization, and microscopic evaluation. FISH-microscopic imagery confirmed the effective and efficient (50% successful permeabilization and recovery) permeabilization of numerous spore preparations. The novelty of the technology developed here is in its applicability to bacterial endospores. While protocols abound for the effective permeabilization of bacterial, archaeal, and eukaryotic vegetative cells, there are no such reliable methods for decoating and permeabilizing bacterial endospores in a manner that is amenable to downstream FISH microscopic analyses. This innovation enables the direct visualization and enumeration of spores via FISH-based microscopic techniques, circumventing the complications that accompany previously required germination regimes. The synergistic enzymatic weakening of the many spore layers facilitates a structural compromise that is just enough to render the spores permeable without degrading the spore to a level, which precludes it from recognition.

Description: During space exploration, the control of terrestrial microbes associated with robotic space vehicles intended to land on extraterrestrial solar system bodies is necessary to prevent forward contamination and maintain scientific integrity during the search for life. Microorganisms associated with the spacecraft assembly environment can be a source of contamination for the spacecraft. In this study, we have monitored the microbial burden of air samples of the Mars Exploration Rovers' assembly facility at the Kennedy Space Center utilizing complementary diagnostic tools. To estimate the microbial burden and identify potential contaminants in the assembly facility, several microbiological techniques were used including culturing, cloning and sequencing of 16S rRNA genes, DNA microarray analysis, and ATP assays to assess viable microorganisms. Culturing severely underestimated types and amounts of contamination since many of the microbes implicated by molecular analyses were not cultivable. In addition to the cultivation of Agrobacterium, Burkholderia and Bacillus species, the cloning approach retrieved 16s rDNA sequences of oligotrophs, symbionts, and y-proteobacteria members. DNA microarray analysis based on rational probe design and dissociation curves complemented existing molecular techniques and produced a highly parallel, high resolution analysis of contaminating microbial populations. For instance, strong hybridization signals to probes targeting the Bacillus species indicated that members of this species were present in the assembly area samples; however, differences in dissociation curves between perfect-match and air sample sequences showed that these samples harbored nucleotide polymorphisms. Vegetative cells of several isolates were resistant when subjected to treatments of UVC (254 nm) and vapor H202 (4 mg/L). This study further validates the significance of non-cultivable microbes in association with spacecraft assembly facilities, as our analyses have identified several non-cultivable microbes likely to contaminate the surfaces of spacecraft hardware.

Description: Both culture-based and culture-independent, biomarker-targeted microbial enumeration and identification technologies were employed to estimate total microbial and viral burden and diversity within the cabin air of commercial airliners. Samples from each of twenty flights spanning three commercial carriers were collected via air-impingement. When the total viable microbial population was estimated by assaying relative concentrations of the universal energy carrier ATP, values ranged from below detection limits (BDL) to 4.1 x 106 cells/cubic m of air. The total viable microbial population was extremely low in both of Airline A (approximately 10% samples) and C (approximately 18% samples) compared to the samples collected aboard flights on Airline A and B (approximately 70% samples). When samples were collected as a function of time over the course of flights, a gradual accumulation of microbes was observed from the time of passenger boarding through mid-flight, followed by a sharp decline in microbial abundance and viability from the initiation of descent through landing. It is concluded in this study that only 10% of the viable microbes of the cabin air were cultivable and suggested a need to employ state-of-the art molecular assay that measures both cultivable and viable-but-non-cultivable microbes. Among the cultivable bacteria, colonies of Acinetobacter sp. were by far the most profuse in Phase I, and Gram-positive bacteria of the genera Staphylococcus and Bacillus were the most abundant during Phase II. The isolation of the human pathogens Acinetobacter johnsonii, A. calcoaceticus, Janibacter melonis, Microbacterium trichotecenolyticum, Massilia timonae, Staphylococcus saprophyticus, Corynebacterium lipophiloflavum is concerning, as these bacteria can cause meningitis, septicemia, and a handful of sometimes fatal diseases and infections. Molecular microbial community analyses exhibited presence of the alpha-, beta-, gamma-, and delta- proteobacteria, as well as Gram-positive bacteria, Fusobacteria, Cyanobacteria, Deinococci, Bacterioidetes, Spirochetes, and Planctomyces in varying abundance. Neisseria meningitidis rDNA sequences were retrieved in great abundance from Airline A followed by Streptococcus oralis/mitis sequences. Pseudomonas synxantha sequences dominated Airline B clone libraries, followed by those of N. meningitidis and S. oralis/mitis. In Phase II, Airline C, sequences representative of more than 113 species, enveloping 12 classes of bacteria, were retrieved. Proteobacterial sequences were retrieved in greatest frequency (58% of all clone sequences), followed in short order by those stemming from Gram-positives bacteria (31% of all clone sequences). As for overall phylogenetic breadth, Gram-positive and alpha-proteobacteria seem to have a higher affinity for international flights, whereas beta-and gamma-proteobacteria are far more common about domestic cabin air parcels in Airline C samples. Ultimately, the majority of microbial species circulating throughout the cabin airs of commercial airliners are commensal, infrequently pathogenic normal flora of the human nasopharynx and respiratory system. Many of these microbes likely originate from the oral and nasal cavities, and lungs of passengers and flight crew and are disseminated unknowingly via routine conversation, coughing, sneezing, and stochastic passing of fomites. The data documented in this study will be useful to generate a baseline microbial population database and can be utilized to develop biosensor instrumentation for monitoring microbial quality of cabin or urban air.