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The ability to form endospores allows certain Gram-positive bacteria (e.g. Bacillus subtilis) to challenge the limits of microbial resistance and survival. Thus, B. subtilis is able to tolerate many environmental extremes by transitioning into a dormant state as spores, allowing survival under otherwise unfavorable conditions. Despite thorough study of spore resistance to external stresses, precisely how long B. subtilis spores can lie dormant while remaining viable, a period that potentially far exceeds the human lifespan; is not known although convincing examples of long term spore survival have been recorded. In this study, we report the first data from a 500-year microbial experiment, which started in 2014 and will finish in 2514. A set of vials containing a defined concentration of desiccated B. subtilis spores is opened and tested for viability every two years for the first 24 years and then every 25 years until experiment completion. Desiccated baseline spore samples were also exposed to environmental stresses, including X-rays, 254 nm UV-C, 10% H2O2, dry heat (120°C) and wet heat (100°C) to investigate how desiccated spores respond to harsh environmental conditions after long periods of storage. Data from the first 2 years of storage show no significant decrease in spore viability. Additionally, spores of B. subtilis were subjected to various short-term storage experiments, revealing that space-like vacuum and high NaCl concentration negatively affected spore viability. [ABSTRACT FROM AUTHOR]

The immune system is one of the most affected systems of the human body during space flight. The cells of the immune system are exceptionally sensitive to microgravity. Thus, serious concerns arise, whether space flight associated weakening of the immune system ultimately precludes the expansion of human presence beyond the Earth's orbit. For human space flight, it is an urgent need to understand the cellular and molecular mechanisms by which altered gravity influences and changes the functions of immune cells. The CELLBOX-PRIME (= CellBox-Primary Human Macrophages in Microgravity Environment) experiment investigated for the first time microgravity-associated long-term alterations in primary human macrophages, one of the most important effector cells of the immune system. The experiment was conducted in the U.S. National Laboratory on board of the International Space Station ISS using the NanoRacks laboratory and Biorack type I standard CELLBOX EUE type IV containers. Upload and download were performed with the SpaceX CRS-3 and the Dragon spaceship on April 18th, 2014 / May 18th, 2014. Surprisingly, primary human macrophages exhibited neither quantitative nor structural changes of the actin and vimentin cytoskeleton after 11 days in microgravity when compared to 1g controls. Neither CD18 or CD14 surface expression were altered in microgravity, however ICAM-1 expression was reduced. The analysis of 74 metabolites in the cell culture supernatant by GC–TOF–MS, revealed eight metabolites with significantly different quantities when compared to 1g controls. In particular, the significant increase of free fucose in the cell culture supernatant was associated with a significant decrease of cell surface–bound fucose. The reduced ICAM-1 expression and the loss of cell surface–bound fucose may contribute to functional impairments, e.g. the activation of T cells, migration and activation of the innate immune response. We assume that the surprisingly small and non-significant cytoskeletal alterations represent a stable “steady state” after adaptive processes are initiated in the new microgravity environment. Due to the utmost importance of the human macrophage system for the elimination of pathogens and the clearance of apoptotic cells, its apparent robustness to a low gravity environment is crucial for human health and performance during long-term space missions. [ABSTRACT FROM AUTHOR]

Living organisms in space are constantly exposed to radiation, toxic chemicals or reactive oxygen species generated due to increased levels of environmental and psychological stresses. Understanding the impact of spaceflight factors, microgravity in particular, on cellular responses to DNA damage is essential for assessing the radiation risk for astronauts and the mutation rate in microorganisms. In a study conducted on the International Space Station, confluent human fibroblasts in culture were treated with bleomycin for three hours in the true microgravity environment. The degree of DNA damage was quantified by immunofluorescence staining for γ-H2AX, which is manifested in three types of staining patterns. Although similar percentages of these types of patterns were found between flight and ground cells, there was a slight shift in the distribution of foci counts in the flown cells with countable numbers of γ-H2AX foci. Comparison of the cells in confluent and in exponential growth conditions indicated that the proliferation rate between flight and the ground may be responsible for such a shift. We also performed a microarray analysis of gene expressions in response to bleomycin treatment. A qualitative comparison of the responsive pathways between the flown and ground cells showed similar responses with the p53 network being the top upstream regulator. The microarray data was confirmed with a PCR array analysis containing a set of genes involved in DNA damage signaling; with BBC3, CDKN1A, PCNA and PPM1D being significantly upregulated in both flight and ground cells after bleomycin treatment. Our results suggest that whether microgravity affects DNA damage response in space can be dependent on the cell type and cell growth condition. [ABSTRACT FROM AUTHOR]

Restoring all facility operations after the 2001 Amerithrax attacks took years to complete, highlighting the need to reduce remediation time. Some of the most time intensive tasks were environmental sampling and sample analyses. Composite sampling allows disparate samples to be combined, with only a single analysis needed, making it a promising method to reduce response times. We developed a statistical experimental design to test three different composite sampling methods: 1) single medium single pass composite (SM-SPC): a single cellulose sponge samples multiple coupons with a single pass across each coupon; 2) single medium multi-pass composite: a single cellulose sponge samples multiple coupons with multiple passes across each coupon (SM-MPC); and 3) multi-medium post-sample composite (MM-MPC): a single cellulose sponge samples a single surface, and then multiple sponges are combined during sample extraction. Five spore concentrations of Bacillus atrophaeus Nakamura spores were tested; concentrations ranged from 5 to 100 CFU/coupon (0.00775 to 0.155 CFU/cm2). Study variables included four clean surface materials (stainless steel, vinyl tile, ceramic tile, and painted dry wallboard) and three grime coated/dirty materials (stainless steel, vinyl tile, and ceramic tile). Analysis of variance for the clean study showed two significant factors: composite method (p< 0.0001) and coupon material (p = 0.0006). Recovery efficiency (RE) was higher overall using the MM-MPC method compared to the SM-SPC and SM-MPC methods. RE with the MM-MPC method for concentrations tested (10 to 100 CFU/coupon) was similar for ceramic tile, dry wall, and stainless steel for clean materials. RE was lowest for vinyl tile with both composite methods. Statistical tests for the dirty study showed RE was significantly higher for vinyl and stainless steel materials, but lower for ceramic tile. These results suggest post-sample compositing can be used to reduce sample analysis time when responding to a Bacillus anthracis contamination event of clean or dirty surfaces. [ABSTRACT FROM AUTHOR]

This study investigates the quantitative bacterial recovery of Methicillin-resistant Staphylococcus aureus (MRSA) in nasal screenings by utilizing dry or moistened swabs within an in vivo and an in vitro experimental setting. 135 nasal MRSA carriers were each swabbed in one nostril with a dry and in the other one with a moistened rayon swab. Quantitative bacterial recovery was measured by standard viable count techniques. Furthermore, an anatomically correct artificial nose model was inoculated with a numerically defined suspension of MRSA and swabbed with dry and moistened rayon, polyurethane-foam and nylon-flocked swabs to test these different settings and swab-materials under identical laboratory conditions. In vivo, quantities of MRSA per nostril in carriers varied between <101 and >107 colony forming units, with a median of 2.15x104 CFU. However, no statistically significant differences could be detected for the recovery of MRSA quantities when swabbing nasal carriers with moist or dry rayon swabs. In vitro testing confirmed the in vivo data for swabs with rayon, polyurethane and nylon-flocked tips, since pre-moistening of swabs did not significantly affect the quantities of retrieved bacteria. Therefore, pre-moistening of swabs prior to nasal MRSA sampling provides no advantage in terms of recovering greater bacterial quantities and therefore can be omitted. In addition, this situation can be mimicked in an in vitro model, thereby providing a useful basis for future in vitro testings of new swab types or target organisms for screening approaches. [ABSTRACT FROM AUTHOR]

Walruses, Odobenus rosmarus, play a key role in the Arctic ecosystem, including northern Indigenous communities, which are reliant upon walruses for aspects of their diet and culture. However, walruses face varied environmental threats including rising sea-water temperatures and decreasing ice cover. An underappreciated threat may be the large amount of solar ultraviolet radiation (UV) that continues to reach the Arctic as a result of ozone loss. UV has been shown to negatively affect whales. Like whales, walrus skin is unprotected by fur, but in contrast, walruses spend long periods of time hauled-out on land. In this study, we combined the results of histological analyses of skin sections from five Atlantic walruses, Odobenus rosmarus rosmarus, collected in Nunavik (Northern Quebec, Canada) with qualitative data obtained through the interviews of 33 local walrus hunters and Inuit Elders. Histological analyses allowed us to explore UV-induced cellular lesions and interviews with experienced walrus hunters and Elders helped us to study the incidences and temporal changes of UV-induced gross lesions in walruses. At the microscopic scale, we detected a range of skin abnormalities consistent with UV damage. However, currently such UV effects do not seem to be widely observed at the whole-animal level (i.e., absence of skin blistering, erythema, eye cataract) by individuals interviewed. Although walruses may experience skin damage under normal everyday UV exposure, the long-term data from local walrus hunters and Inuit Elders did not report a relation between the increased sun radiation secondary to ozone loss and walrus health. [ABSTRACT FROM AUTHOR]

Astronaut intestinal health may be impacted by microgravity, radiation, and diet. The aim of this study was to characterize how high and low linear energy transfer (LET) radiation, microgravity, and elevated dietary iron affect colon microbiota (determined by 16S rDNA pyrosequencing) and colon function. Three independent experiments were conducted to achieve these goals: 1) fractionated low LET γ radiation (137Cs, 3 Gy, RAD), high Fe diet (IRON) (650 mg/kg diet), and a combination of low LET γ radiation and high Fe diet (IRON+RAD) in male Sprague-Dawley rats; 2) high LET 38Si particle exposure (0.050 Gy), 1/6 G partial weight bearing (PWB), and a combination of high LET38Si particle exposure and PWB in female BalbC/ByJ mice; and 3) 13 d spaceflight in female C57BL/6 mice. Low LET radiation, IRON and spaceflight increased Bacteroidetes and decreased Firmicutes. RAD and IRON+RAD increased Lactobacillales and lowered Clostridiales compared to the control (CON) and IRON treatments. Low LET radiation, IRON, and spaceflight did not significantly affect diversity or richness, or elevate pathogenic genera. Spaceflight increased Clostridiales and decreased Lactobacillales, and similar trends were observed in the experiment using a ground-based model of microgravity, suggesting altered gravity may affect colonic microbiota. Although we noted no differences in colon epithelial injury or inflammation, spaceflight elevated TGFβ gene expression. Microbiota and mucosal characterization in these models is a first step in understanding the impact of the space environment on intestinal health. [ABSTRACT FROM AUTHOR]

After a potential biological incident the sampling strategy and sample analysis are crucial for the outcome of the investigation and identification. In this study, we have developed a simple sandwich ELISA based on commercial components to quantify BSA (used as a surrogate for ricin) with a detection range of 1.32–80 ng/mL. We used the ELISA to evaluate different protein swabbing procedures (swabbing techniques and after-swabbing treatments) for two swab types: a cotton gauze swab and a flocked nylon swab. The optimal swabbing procedure for each swab type was used to obtain recovery efficiencies from different surface materials. The surface recoveries using the optimal swabbing procedure ranged from 0–60% and were significantly higher from nonporous surfaces compared to porous surfaces. In conclusion, this study presents a swabbing procedure evaluation and a simple BSA ELISA based on commercial components, which are easy to perform in a laboratory with basic facilities. The data indicate that different swabbing procedures were optimal for each of the tested swab types, and the particular swab preference depends on the surface material to be swabbed. [ABSTRACT FROM AUTHOR]

Objectives: Swab-based nasal screening is commonly used to identify asymptomatic carriage of Staphylococcus aureus in patients. Bacterial detection depends on the uptake and release capacities of the swabs and on the swabbing technique itself. This study investigates the performance of different swab-types in nasal MRSA-screening by utilizing a unique artificial nose model to provide realistic and standardized screening conditions. Methods: An anatomically correct artificial nose model was inoculated with a numerically defined mixture of MRSA and Staphylococcus epidermidis bacteria at quantities of 4×102 and 8×102 colony forming units (CFU), respectively. Five swab-types were tested following a strict protocol. Bacterial recovery was measured for direct plating and after elution into Amies medium by standard viable count techniques. Results: Mean recovered bacteria quantities varied between 209 and 0 CFU for MRSA, and 365 and 0 CFU for S. epidermidis, resulting swab-type-dependent MRSA-screening-sensitivities ranged between 0 and 100%. Swabs with nylon flocked tips or cellular foam tips performed significantly better compared to conventional rayon swabs referring to the recovered bacterial yield (p<0.001). Best results were obtained by using a flocked swab in combination with Amies preservation medium. Within the range of the utilized bacterial concentrations, recovery ratios for the particular swab-types were independent of the bacterial species. Conclusions: This study combines a realistic model of a human nose with standardized laboratory conditions to analyze swab-performance in MRSA-screening situations. Therefore, influences by inter-individual anatomical differences as well as diverse colonization densities in patients could be excluded. Recovery rates vary significantly between different swab-types. The choice of the swab has a great impact on the laboratory result. In fact, the swab-type contributes significantly to true positive or false negative detection of nasal MRSA carriage. These findings should be considered when screening a patient. [ABSTRACT FROM AUTHOR]

Objectives: Swabs are widely used to collect samples for microbiological analyses from various clinical settings. They vary by material, size, and structure of the tip. This study investigates the uptake and release capacities for liquid and bacteria. Methods: Five swabs were analyzed for their uptake and release capacities of Staphylococcus aureus and Staphylococcus epidermidis suspensions. Two approaches were investigated providing volume-restricted and unrestricted amounts of bacterial suspensions to mimic various clinical situations. Volume and bacterial uptake and release were measured in milligrams and by counting colony forming units (CFU), respectively. Results: Volume uptake and release in the unrestricted setting varied highly significant between 239.6 mg and 88.7 mg (p<0.001) and between 65.2 mg and 2.2 mg (p<0.001), respectively. In the volume-restricted setting the complete volume was absorbed by all swabs, volume release could only be detected for flocked swabs (2.7 mg; p<0.001). Highest amount of CFU release was detected for the MWE Dryswab in the unrestricted setting for both S. aureus and S. epidermidis with 1544 CFU and 553 CFU, respectively, lowest release for the Sarstedt neutral swab with 32 CFU and 17 CFU, respectively (p<0.001). In the volume-restricted setting MWE Σ-Swab released the highest bacterial amount with 135 CFU S. aureus and 55 CFU S. epidermidis, respectively, the lowest amount was released by Mast Mastaswab with 2 CFU S. aureus and 1 CFU S. epidermidis, respectively (p<0.001). Within the range of the utilized bacterial concentrations, uptake/release ratios were identical for the particular swab types and independent of the bacterial species. Conclusions: The influence of the swab type on subsequent diagnostic results is often underestimated. Uptake and release of the investigated bacteria vary significantly between different swab types and sampling conditions. For best diagnostic outcome swabs should be chosen according to the examined situation and the swab performance profile. [ABSTRACT FROM AUTHOR]

Bioburden encapsulated in spacecraft polymers (such as adhesives and coatings) poses a potential risk to jeopardize scientific exploration of other celestial bodies. This is particularly critical for spacecraft components intended for hard landing. So far, it remained unclear if polymers are indeed a source of microbial contamination. In addition, data with respect to survival of microbes during the embedding/polymerization process are sparse. In this study we developed testing strategies to quantitatively examine encapsulated bioburden in five different polymers used frequently and in large quantities on spaceflight hardware. As quantitative extraction of the bioburden from polymerized (solid) materials did not prove feasible, contaminants were extracted from uncured precursors. Cultivation-based analyses revealed <0.1–2.5 colony forming units (cfu) per cm3 polymer, whereas quantitative PCR-based detection of contaminants indicated considerably higher values, despite low DNA extraction efficiency. Results obtained from this approach reflect the most conservative proxy for encapsulated bioburden, as they give the maximum bioburden of the polymers irrespective of any additional physical and chemical stress occurring during polymerization. To address the latter issue, we deployed an embedding model to elucidate and monitor the physiological status of embedded Bacillus safensis spores in a cured polymer. Staining approaches using AlexaFluor succinimidyl ester 488 (AF488), propidium monoazide (PMA), CTC (5-cyano-2,3-diotolyl tetrazolium chloride) demonstrated that embedded spores retained integrity, germination and cultivation ability even after polymerization of the adhesive Scotch-Weld 2216 B/A. Using the methods presented here, we were able to estimate the worst case contribution of encapsulated bioburden in different polymers to the bioburden of spacecraft. We demonstrated that spores were not affected by polymerization processes. Besides Planetary Protection considerations, our results could prove useful for the manufacturing of food packaging, pharmacy industry and implant technology. [ABSTRACT FROM AUTHOR]

Opportunistic fungal pathogens may cause an array of superficial infections or serious invasive infections, especially in immunocompromised patients. Cryptococcus neoformans is a pathogen causing cryptococcosis in HIV/AIDS patients, but treatment is limited due to the relative lack of potent antifungal agents. Photodynamic inactivation (PDI) uses the combination of non-toxic dyes called photosensitizers and harmless visible light, which produces singlet oxygen and other reactive oxygen species that produce cell inactivation and death. We report the use of five structurally unrelated photosensitizers (methylene blue, Rose Bengal, selenium derivative of a Nile blue dye, a cationic fullerene and a conjugate between poly-L-lysine and chlorin(e6)) combined with appropriate wavelengths of light to inactivate C. neoformans. Mutants lacking capsule and laccase, and culture conditions that favoured melanin production were used to probe the mechanisms of PDI and the effect of virulence factors. The presence of cell wall, laccase and melanin tended to protect against PDI, but the choice of the appropriate photosensitizers and dosimetry was able to overcome this resistance. [ABSTRACT FROM AUTHOR]

Background: Ionizing radiation (IR) can be extremely harmful for human cells since an improper DNA-damage response (DDR) to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation, being microRNAs (miRNAs) small noncoding RNA that act as posttranscriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity, a condition of weightlessness experienced by astronauts during space missions, which could have a synergistic action on cells, increasing the risk of radiation exposure. Methodology/Principal Findings: We analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL) incubated for 4 and 24 h in normal gravity (1 g) and in modeled microgravity (MMG) during the repair time after irradiation with 0.2 and 2Gy of γ-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover, let-7iး, miR-7, miR-7-1း, miR-27a, miR-144, miR-200a, miR-598, miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles, carried out on PBL of the same donors, identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of "Response to DNA damage" is enriched when PBL are incubated in 1 g but not in MMG. Moreover, some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses. Conclusions/Significance: On the whole, by integrating the transcriptome and microRNome, we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL. [ABSTRACT FROM AUTHOR]