STEM TODAY November 2016, No.14
Microbial Research
STEM TODAY November 2016 , No.14
CONTENTS Microbial Analysis of International Space Station (ISS) Air, Surfaces and Water Prevention of infectious disease in the crew has been the highest priority, but experience gained from the NASAÂMir program showed that microbial contamination of vehicle and lifeÂsupport systems, such as biofouling of water and food, are of equal importance. The major sources of microbiological risk factors for astronauts include food, drinking water, air, surfaces, payloads, research animals, crew members, and personnel in close contact with the astronauts. National Aeronautics and Space Administration (NASA) implemented comprehensive microbial analyses of the major risk factors. This included the establishment of acceptability requirements for food, water, air, surfaces, and crew members .
Editorial Editor: Mr. Abhishek Kumar Sinha Editor / Technical Advisor: Mr. Martin Cabaniss
STEM Today, November 2016, No.14
Cover Page Cygnus Spacecraft Attached to Space Station’s Unity Module Orbital ATK’s Cygnus cargo craft (left) is seen from the Cupola module windows aboard the International Space Station on Oct. 23, 2016. The main robotic work station for controlling the Canadarm2 robotic arm is located inside the Cupola and was used to capture Cygnus upon its arrival. The Expedition 49 crew will unload approximately 5,000 pounds of science investigations, food and supplies from the newly arrived spacecraft. The cargo aboard the Cygnus will support dozens of new and existing investigations as the space station crews of Expeditions 49 and 50 contribute to about 250 science and research studies. The new experiments include studies on fire in space, the effect of lighting on sleep and daily rhythms, collection of health-related data, and a new way to measure neutrons. Image Credit: NASA
Back Cover November Supermoon a Spectacular Sight This image approximates the look of the Nov. 14, 2016, full moon with data from NASA’s Lunar Reconnaissance Orbiter. The moon is a familiar sight in our sky, brightening dark nights and reminding us of space exploration, past and present. But the upcoming supermoon - on Monday, Nov. 14 - will be especially "super" because it’s the closest full moon to Earth since 1948. We won’t see another supermoon like this until 2034. The moon’s orbit around Earth is slightly elliptical so sometimes it is closer and sometimes it’s farther away. When the moon is full as it makes its closest pass to Earth it is known as a supermoon. At perigee - the point at which the moon is closest to Earth - the moon can be as much as 14 percent closer to Earth than at apogee, when the moon is farthest from our planet. The full moon appears that much larger in diameter and because it is larger shines 30 percent more moonlight onto the Earth. Image Credit: NASA Goddard’s Scientific Visualization Studio
STEM Today , November 2016
Editorial Dear Reader
STEM Today, November 2016, No.14
All young people should be prepared to think deeply and to think well so that they have the chance to become the innovators, educators, researchers, and leaders who can solve the most pressing challenges facing our world, both today and tomorrow. But, right now, not enough of our youth have access to quality STEM learning opportunities and too few students see these disciplines as springboards for their careers. According to Marillyn Hewson, "Our children - the elementary, middle and high school students of today - make up a generation that will change our universe forever. This is the generation that will walk on Mars, explore deep space and unlock mysteries that we can’t yet imagine". "They won’t get there alone. It is our job to prepare, inspire and equip them to build the future - and that’s exactly what Generation Beyond is designed to do." STEM Today will inspire and educate people about Spaceflight and effects of Spaceflight on Astronauts. Editor Mr. Abhishek Kumar Sinha
Editorial Dear Reader The Science, Technology, Engineering and Math (STEM) program is designed to inspire the next generation of innovators, explorers, inventors and pioneers to pursue STEM careers. According to President Barack Obama, "[Science] is more than a school subject, or the periodic table, or the properties of waves. It is an approach to the world, a critical way to understand and explore and engage with the world, and then have the capacity to change that world..." STEM Today addresses the inadequate number of teachers skilled to educate in Human Spaceflight. It will prepare , inspire and educate teachers about Spaceflight. STEM Today will focus on NASA’S Human Research Roadmap. It will research on long duration spaceflight and put together latest research in Human Spaceflight in its monthly newsletter. Editor / Technical Advisor Mr. Martin Cabaniss
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MICROBIAL RESEARCH Microbial Analysis of International Space Station (ISS) Air, Surfaces and Water The major sources of microbiological risk factors for astronauts include food, drinking water, air, surfaces, payloads, research animals, crew members, and personnel in close contact with the astronauts.
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Microbial Analysis of International Space Station (ISS)
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Microbial Analysis of International Space Station (ISS) Air, Surfaces and Water (ISS_Micro_Analysis) NASA Life Sciences Data Archive (http://lsda.jsc.nasa.gov) Investigator Name: Mark Ott Mission (Payload): Expeditions 13, 19 - 43, 45 Experiment Title (ID): Microbial Analysis of International Space Station (ISS) Air, Surfaces and Water (ISS_Micro_Analysis) File name/Inventory ID: ISS_Micro_Analysis_Isolates/ ISS_Micro_Analysis_2010311581 Overview • Bacteria from surface and air samples are isolated on Tryptic Soy Agar for identification • Bacteria are isolated from water samples on ISS using a filtration unit, the Microbial Capture Device (MCD) that contains an R3A medium • Bacteria are also isolated from water samples returned from ISS to the Johnson Space Center in an archive water bag. Samples are processed using an R2A medium • Identification of bacteria was performed using either a VITEK identification system (bioMérieux) or 16S genetic analysis. Sample Locations include: U.S. Modules - Node 1, Node 2, Node 3, U.S. Laboratory, PMM Data Source: NASA Life Sciences Data Archive
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Top 5 microbes found at ISS( U.S. Modules )
Staphylococcus epidermidis Staphylococcus epidermidis is a Gram-positive bacterium, and one of over 40 species belonging to the genus Staphylococcus. It is part of the normal human flora, typically the skin flora, and less commonly the mucosal flora. Although S. epidermidis is not usually pathogenic, patients with compromised immune systems are at risk of developing infection. S. epidermidis also has a beneficial role in balancing the microflora on human epithelial surfaces by controlling outgrowth of harmful bacteria such as in particular S. aureus.
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Staphylococcus epidermidis is found in 84 times at ISS at Node 1, Node 2, Node 3, U.S. Laboratory, PMM.
Staphylococcus hominis Staphylococcus hominis is a coagulase-negative member of the bacterial genus Staphylococcus, consisting of Gram-positive, spherical cells in clusters. It occurs very commonly as a harmless commensal on human and animal skin and is known for producing thioalcohol compounds that contribute to body odour. Like many other coagulase-negative staphylococci, S. hominis may occasionally cause infection in patients whose immune systems are compromised, for example by chemotherapy or predisposing illness. Staphylococcus hominis is found in 51 times at ISS at Node 1, Node 2, Node 3, U.S. Laboratory, PMM.
Ralstonia pickettii Ralstonia pickettii, a non-fermenting Gram-negative bacillus, is regarded as being of minor clinical significance; however, many instances of infections with this organism are reported in the literature. Infections can include bacteraemia/septicaemia caused by contaminated solutions, e.g. distilled water, water for injection and aqueous chlorhexidine solutions. Cases of pseudobacteraemia have been recorded in association with R. pickettii, as have many cases of unusual infections, some of which were very invasive and severe, e.g. meningitis, septic arthritis and osteomyelitis. Six cases of death in four separate instances have also been recorded related to R. pickettii. Ralstonia pickettii is found in 33 times at ISS at US Water System.
Staphylococcus capitis Staphylococcus capitis is a coagulase-negative species (CoNS) of Staphylococcus. It is part of the normal flora of the skin of the human scalp, face, neck, and ears and has been associated with prosthetic valve endocarditis, but is rarely associated with native valve infection. Staphylococcus capitis is found in 26 times at ISS at Node 1, Node 2, Node 3, U.S. Laboratory, PMM.
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Burkholderia multivorans Burkholderia multivorans is a species in Phylum proteobacteria. The cells are rod-shaped. It is known to cause human disease, such as colonisation of the lung in cystic fibrosis. Burkholderia multivorans is found in 18 times at ISS at US Water System.
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In Space Skin Falls Off Your Feet (Literally) Video
After 2 1/2 months in Space, astronauts’ calluses start peeling off. NASA astronauts Mike Massimino and Don Pettit explain why and how in this episode of the ISS Science Garage. NOTE:Staphylococcus epidermidis , Staphylococcus hominis and Staphylococcus capitis microbes are part of the normal human flora, typically the skin flora.
Cultivation of Staphylococcus epidermidis in the Human Spaceflight Environment Leads to Alterations in the Frequency and Spectrum of Spontaneous Rifampicin-Resistance Mutations in the rpoB Gene Bacteria of the genus Staphylococcus are persistent inhabitants of human spaceflight habitats and represent potential opportunistic pathogens. The effect of the human spaceflight environment on the growth and the frequency of mutations to antibiotic resistance in the model organism Staphylococcus epidermidis strain ATCC12228 was investigated. Six cultures of the test organism were cultivated in biological research in canisters-Petri dish fixation units for 122 h on orbit in the International Space Station (ISS) as part of the SpaceX-3 resupply mission. Asynchronous ground controls (GCs) consisted of identical sets of cultures cultivated for 122 h in the ISS Environmental Simulator at Kennedy Space Center. S. epidermidis exhibited significantly lower viable counts but significantly higher frequencies of mutation to rifampicin (Rif) resistance in space vs. GC cultures. The spectrum of mutations in the rpoB gene leading to RifR was altered in S. epidermidis isolates cultivated in the ISS compared to GCs. The results suggest that the human spaceflight environment induces unique physiologic stresses on growing bacterial cells leading to changes in mutagenic potential.
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BRIC canisters hold six 60-mm diameter Petri dish bottom halves in small subcompartments called Petri dish fixation units (PDFUs). Each PDFU allows for injection of medium, referred to as actuation, to initiate bacterial growth. For flight (FL) experiments, one BRIC canister was used containing six PDFUs. Immediately, adjacent to the FL canister was deployed a HOBOr temperature data logger (Onset Computer Co., Bourne, MA, USA). Post-flight asynchronous GC experiments were conducted using the same hardware and configuration as in the FL experiment. Each PDFU was loaded with a Petri dish containing air-dried cells, and 13 mL of sterile TSY medium was loaded into a separate reservoir. To prevent contamination, all reagents and equipment used were sterilized prior to use and PDFUs were assembled using aseptic technique within a biological containment hood.
Project MERCCURI Project MERCCURI , aimed at raising public awareness of microbiology and research on board the International Space Station (ISS). Project MERCCURI (Microbial Ecology Research Combining Citizen and University Researchers on the ISS) was a collaborative effort involving the "microbiology of the Built Environment network" (microBEnet) group, Science Cheerleader, NanoRacks, Space Florida, and SciStarter. Of the 48 strains sent to the ISS, 45 of them showed similar growth in space and on Earth using a relative growth measurement adapted for microgravity. The vast majority of species tested in this experiment have also been found in culture-independent surveys of the ISS. Only one bacterial strain showed significantly different growth in space. Bacillus safensis JPL-MERTA-8-2 grew 60% better in space than on Earth.
In order to confirm that the observed results were not due to contamination of the wells, each of the 12 replicates (six space, six ground) for the three bacteria showing statistically different growth between the ISS and Earth were cultured after the experiment. Bacteria were struck from the wells onto LB-agar plates, then single colonies were grown into overnight cultures. DNA was extracted using a Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA) from each of the 36 cultures (3 bacteria X 12 replicates) and the identity was confirmed by BLAST of the Sanger sequenced PCR product using the 27F and 1391R primers as described above.
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Special Edition on Microbial Research Only three bacteria showed a significant difference in the two conditions; Bacillus safensis, Bacillus methylotrophicus, and Microbacterium oleivorans. This Bacillus safensis strain was collected at the Jet Propulsion Laboratory (JPL-NASA) on a Mars Exploration Rover before launch in 2004. As part of standard Planetary Protection protocols, all surface-bound spacecraft are sampled during the assembly process and those strains are then saved for further analysis. Authors obtained this strain as part of a collection of JPL-NASA strains to send to the ISS (Table 1).
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In this experiment, Bacillus safensis grew to a final density of âˆź60% higher in space than on the ground, with very little variation between replicates (Fig. 1). The genome sequence of this strain, Bacillus safensis JPLMERTA-8-2 has just been published and may contain clues as to why this strain behaved so differently in space.
Bacterial monitoring in the ISS - "Kibo"
In this research, authors collected samples from interior surfaces in Kibo every ca. 500 days after Kibo be-
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Special Edition on Microbial Research gan operation. Bacterial abundance and phylogenetic affiliation were determined by fluorescent staining, 16S ribosomal RNA (rRNA) gene-targeted quantitative PCR and pyrosequencing. This is the first report on continuous monitoring of bacterial abundance and phylogenetic affiliation in a space habitat determined by cultureindependent molecular microbiological methods. Bacterial abundance Bacterial abundance on the interior surfaces in Kibo is shown in Table 1. Authors determined bacterial abundance with different approaches, fluorescent microscopy and quantitative PCR targeting the bacterial 16S rRNA gene, to confirm the reliability of the results. Quantitative PCR outputs the copy number of the 16S rRNA gene in a sample. To convert the copy number to a cell number, authors need to know the copy number of the target gene in a single cell. However, bacterial cells carry 1-15 copies of the 16S rRNA gene in their genomes. Authors therefore calculated the average copy number of 16S rRNA genes in the bacterial community in Kibo as 5 copies/cell, based on the community structure determined by pyrosequencing as described below and the ribosomal RNA operon copy number database rrnDB.
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As shown in Table 1, ca. 103 cells/cm2 of bacteria were detected on the interior surfaces in Kibo in Microbe-I (Table 2). In Microbe-II and Microbe-III (Table 2), bacterial abundance was ca. 102 cells/cm2 , or less than the quantification limit. Overall, bacterial abundance did not exceed 104 cells/cm2 . Bacterial community structure Bacterial community structure on the interior surfaces in Kibo was analyzed by 16S rRNA gene-targeted pyrosequencing. Among 99,967 raw reads, 71,190 were high-quality reads after processing with the Quantitative Insights into Microbial Ecology (QIIME) pipeline. These reads were distributed in each sample (13 samples) with an average of 5,472 reads. At first, we estimated sufficient read number for bacterial community analysis using chao1 index (Supplementary Table S1) and a rarefaction curve (Supplementary Figure S1). As shown in Supplementary Table S1, authors found that observed operational taxonomic units (OTUs) revealed 73-100% of bacterial family-level OTUs (equivalent to OTU at 90% similarity) that were present in our sample. Authors therefore confirmed the number of high-quality reads obtained was sufficient to reveal the bacterial community structure in the Kibo sample at the family level. Figure 1a shows the bacterial community structure at the phylum level on the interior surfaces in Kibo. Bacteria of the phyla Proteobacteria (beta- and gamma-subclasses), Firmicutes,and Actinobacteria were frequently detected. The families Enterobacteriaceae and Staphylococcaceae were dominant in each sample (Figure 1b).
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Skin fungal microbiota of astronauts during a half-year stay at the International Space Station This study analyzed the temporal changes in the skin fungal microbiota of 10 astronauts using pyrosequencing and quantitative PCR assay before, during, and after their stay in the ISS. Skin samples from the cheek and chest of the astronauts were obtained using a 6- X 7-cm OpsiteT M transparent dressing (Smith & Nephew Medical Ltd., UK) according to the method of Sugita et al. Samples were collected once prior to the astronautsâ&#x20AC;&#x2122; trip to the ISS, twice during their stay at the ISS (at 2 and 4 months during), and once after their return to Earth. The skin fungal microbiota was characterized by pyrosequencing of the D2 LSU rRNA gene. The data set included 1,092,254 high-quality LSU sequences, with an average of 13,653 sequence reads per sample. Taxonomic assignment of each sequence read identified a total of 230 taxa from the 80 samples (from both cheeks and the chest at the 4 sampling times) of the 10 astronauts. The taxonomic assignment of skin fungal community members is shown in Supplementary Table S1. Of the 230 taxa, 178 were filamentous fungi and 52 were yeasts. Nine Malassezia species were detected from the skin samples. The relative abundances of the taxa are shown in Table 1 for M. restricta, M. globosa, M. sympodialis, Cyberlindnera jadinii, and Cladosporium phylotype 4. Malassezia accounted for >90% of the total of most samples regardless of when the skin sample was collected, the sampled body site, or the astronaut. The samples in which Malassezia accounted for <90% of the total contained other microorganisms with relatively
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high abundances. For example, the samples I, C, E, G, and H contained Trichosporon asahii (22.4%), Aspergillus phylotype 1 (30.6%), Acaromyces (20.4%), Trichosporon asahii (52.4%), and M. slooffiae (65.3%), respectively (Table S1).
Colonization by Malassezia increased in the cheek and chest samples during the crew’s stay at the ISS and decreased upon their return to Earth (Fig. 2). With the level of colonization in the pre-flight sample set at 1, fold changes of 5.2 ± 6.5, 7.6 ± 7.5, and 2.4 ± 2.6 (mean ± standard deviation) were determined in the cheek samples from the in-flight 1, in-flight 2, and post-flight samples, respectively. In the chest samples, the corresponding fold changes were 9.5 ± 24.2, 9.3 ± 17.2, and 1.4 ± 1.0. Because the ascomycetous yeast Cyberlindnera jadinii was highly abundant in the skin samples of 5 of the 10 astronauts, authors recalculated the abundance of Malassezia determined in the pyrosequencing on a species level. M. restricta, M. globosa, and M. sympodialis were the most abundant, although 9 Malassezia species (M. dermatis, M.furfur, M. globosa, M. japonica, M. nana, M. obtusa, M.restricta, M. slooffiae, and M. sympodialis) were identified in the skin samples of the 10 astronauts. Figure 3 shows the temporal changes in the proportion (%) of M. restricta and eight other Malassezia species. The results showed that despite individual variation, there were no major changes in the proportion (%) of M. restricta in the cheek samples collected pre-flight, in-flight, and post-flight (mean ± standard deviation: 83.4% ± 28.0%, 89.6% ± 18.1%, 84.2% ± 28.0%, and 83.8% ± 26.6%, respectively). A similar analysis of the chest samples showed a higher proportion of M. restricta in the two in-flight samples and a decrease in the post-flight sample. The largest contributor to this decrease was M. sympodialis, which accounted for 23.4% ± 25.3%, 10.9% ± 15.7%, 7.6% ± 11.8%, and 30.1% ± 25.9% of the Malassezia species in the pre-flight, in-flight, and post-flight samples, respectively. The pre-flight sample from the chest of astronaut H contained M. sloffiae at an abundance of 65.3%, whereas the abundances of the other Malassezia species (M. globosa, M. restricta, and M. sympodialis) were 32.1%. The ascomycetous yeast Cyberlindnera jadinii was abundantly (>1.0%) detected in the skin samples of 5 (A, B, C, D, and E) of the 10 astronauts. In three of the five astronauts, the yeast was already detected in the preflight samples from the cheek and/or chest at abundances of 1.6% in astronaut B, 4.2% and 41.3% in astronaut C, and 45.2% in astronaut D. In the samples from astronauts A and E, C. jadinii was detected only in the in-flight samples.
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The microorganism was particularly abundant in the skin (cheek and/or chest) samples obtained during the two in-flight samples from the same three astronauts as above: 40.6% (cheek) and 64.9% (chest) of astronaut B, 23.1% and 35.5% (cheek) and 28.8% and 41.3% (chest) in astronaut C, and 23.7% and 45.2% (chest) in astronaut D. However, C. jadiniii was not detected in any of the postflight samples of these five astronauts. These astronauts participated in Expeditions 1, 2 and 3. For one astronaut on Expedition 4, the microorganism was present at very low levels (0.1% and 0.4%) in both in-flight samples. C. jadinii was not detected in any of the skin samples of astronauts H, I, or J.
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The human body is covered by a wide variety of bacteria and fungi. Among the fungi, members of the genus Malassezia are found in abundance at all anatomic sites. Because this yeast requires sebum for its growth, it occurs exclusively in mammals. The fatty acids and glycerin decomposed by the lipase produced by Malassezia are utilized not only by the yeast itself but also by other skin microorganisms. In pathogens such as Salmonella Typhimurium, Pseudomonas aeruginosa, and Candida species, microgravity can affect the production of virulence factors, biofilm formation, and resistance to antimicrobial agents. By contrast, expression of the Malassezia lipase gene is not sensitive to microgravity. Nonetheless, because the host skin serves as a culture medium for skin microorganisms, changes in its chemical composition, sebum production, the synthesis of antimicrobial peptides, and the skin environment (dry vs. oily skin) will impact the interactions of the skin microbiota. In study of members of an Antarctica geological investigation, who during their 3-month stay were only able to bathe once using a wet tissue but were unable to wash their hair, Malassezia colonization increased from 3.0- ± 1.9-fold to 5.3- ± 7.5-fold in cheek samples, from 8.9- ± 10.6-fold to 22.2- ± 40.0-fold in chest samples, and from 96.7- ± 113.8-fold to 916.9- ± 1251.5-fold in scalp samples. The fold changes in Malassezia colonization were similar in samples from the cheek and chest collected from the ISS crew and Antarctica investigation team members. Fungal diversity was reduced during space flight but recovered post-flight, as determined in both cheek and chest samples. The in-flight reduction in microbial diversity was perhaps due to the lack of external microorganisms under the closed environmental conditions of the ISS. Malassezia and C. jadinii together accounted for >90% of the fungi in the skin samples obtained during the crew’s stay at the ISS: 99.1% ± 1.3% and 97.6% ± 5.2% in cheek samples, and 98.7% ± 1.7% and 91.3% ± 18.4% in chest samples (Fig. S1).
Whereas the increased percentage of Malassezia during spaceflight was expected, the higher abundance of C. jadinii in the skin samples of the astronauts was not. C. jadinii is an environmental fungus, not a skin fungus. C. jadinii may have incidentally adhered to the skin during the pre-flight period and persisted on the astronauts’ skin thereafter. Because C. jadinii was detected in the in-flight samples from Expedition 1, it likely arrived there during an earlier mission. Cyberlindnera jadinii was present in large amounts in the preflight samples of ISS Expedition 2 but was virtually nonexistent in the samples collected after ISS Expedition 4.
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Microbial inventories of ISS filter debris During a period spanning portions of ISS Expeditions 30 and 31 (December 2011-July 2012), crewmember reports cited differences in the cabin environment compared to earlier experience as well as allergic responses to the cabin environment. One of the noted observations was a high level of visible dust in the Node 3 cabin of the ISS, to the extent it was sticking to the walls. Flight surgeons indicated that this had been reported not just in Node 3, but also throughout the U.S. On-Orbit Segment, and expressed a concern for crew health. Dust on ISS is expected, with humans being major contributors (via skin shedding, eating, exercising, etc.) and other sources include on-orbit maintenance activities that can release dust from sources such as payloads and systems, clothing, and visiting vehicles.
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As a precautionary measure, in the middle of 2012, an investigation was launched to define and mitigate dust sources, and to determine if exposure to dust might elicit an adverse effect on crew health. As a result of these crewmember reports, particulate and fiber debris samples were collected during ISS Expedition 31 in a vacuum cleaner bag and returned to Earth aboard Soyuz flight 29S in early July 2012 for further analyses. A detailed NASA in-house report (not publicly available) was generated based on culture-based microbiological analyses and concluded that there were no obvious microbiological agents that could be directly correlated with allergic reactions to humans. However, there was no molecular study conducted to comprehensively elucidate the presence of viable but yet to be cultured microorganisms in these samples that might pose problems to crew health.
The items returned aboard Soyuz flight 29S for ground-based analysis included a vacuum cleaner bag containing mixed debris and the vacuum cleaner HEPA filter element with Kaptonr tape over the inlet face as shown by Fig. S2A-B. Blue-grey lint was the predominant material in the vacuum bag. The bulk material is best described as blue-grey fibrous debris matrix with human hair, food, paper, plastic, and miscellaneous granular debris mixed within it. The vacuum bag containing the debris weighed 169.2 g as received. The empty vacuum cleaner bag weighed 93.2 g. Based on the bagâ&#x20AC;&#x2122;s weight difference when full and empty, the total debris weight was approximately 76 g. After establishing the total debris weight, standard test sieves and forceps were used to separate the debris into different size and type fractions and each fraction was weighed. Table S1a summarizes the observed weights for each size and type fraction to <500 Âľm Figure S2C-F shows material retained on the No. 35 test sieve. Fig. S2(A-B). Debris samples and fabric material returned aboard 29S. Views of (A) the mixed debris in the vacuum cleaner bag and (B) the vacuum cleaner HEPA filter. Fig. S2(C-F). Granular debris retained on the No. 35 test sieve. Views at 10X magnification showing (C) debris with dried skin and nail clippings, (D) blue fibers possibly from Velcror closures, (E) mechanical pencil lead, blue fibers, and nail clippings, and (F) metal turnings. Fig. S2(G). Debris that passed through the No.35 test sieve. The debris at 10X magnification shows mixed granular material and human hair.
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The material that passed through the No. 35 test sieve consisted of a uniform tan-colored, powdery mixture of granular material and short human hair fragments (Fig. S2G). The granular material was similar in color to the dried skin fragments and nail clipping found in the larger sized granular material fractions. This material was subjected to further size classification using a test sieve series with mechanical agitation initially for 5 min with successive 1-min agitation periods for a total of 10min. The sieve series consisted of a No. 60 test sieve (250 µm aperture), No. 100 test sieve, 150 µm aperture, No. 140 test sieve, 106 µm aperture, No. 200 test sieve, 75 µm aperture, No. 270 test sieve, 53 µm aperture, and No. 500 test sieve, 25 µm aperture.
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Table S2 summaries the size classification results for the debris <500 µm in size which passed through the No. 35 test sieve. The total material weight after removing it from a storage bag and completing the sieving operation was 3.984 g compared to the earlier 4.0 g that was retained on the No. 35 test sieve. The mass accountability was 99.6 % for this sieving operation (Table S1b). The particles associated with debris were more of solid materials when compared to the fine particles (lint) scraped from the HEPA filter of the vacuum bag. From the visual examination, it was presumed that the materials associated with ISS-debris contained more of solid matter (such as peanuts, paper) compared to the fibrous materials associated with ISS-lint (Fig. S1). Hence, when weighed for microbiological assays, bulk of the weight of debris might be of non-biological origin. The density of the particles collected in the vacuum cleaner bag would be directly related to the efficiency, age, overall usage of the instrument employed, as well as cleanliness and maintenance of the closed environmental surfaces. The "Prime" vacuum cleaner system was used only once in the ISS, whereas the SAF and 103 vacuum cleaner instruments were used for 2 and 6 months, respectively. Even though the vacuum cleaner system used in ISS was different from SAF and 103 clean rooms, all three vacuum cleaners were fitted with HEPA filter bags.
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Total and viable microbiological burden of ISS and other Earth control vacuum cleaner bags, as estimated by conventional and rapid molecular methods, are given in Table 1. The duration in collecting particles (single time to 6 months) varied among all three vacuum cleaners and hence no definitive trend in microbial population was noticed (Table 1). Since all three assays performed during this study independently assess different kinds of populations, cross comparison of various methods and associated microbial population was not attempted.
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The bacteria capable of showing growth at 35 ◦ C in nutrient rich media were in the range of 106 CFU/g in ISS-debris sample where as the SAF and 103 debris samples contain at least 2 logs less than the ISS-debris sample. The cultured bacteria were in the range of 105 CFU/g for all lint samples. When compared to the ISSdebris sample, the cultured bacterial population was ∼50% less in ISS-lint samples. In contrast, the SAF and 103 samples exhibited 1 log more cultured bacteria in lint than their debris samples. The cultured fungal population was 9.3 X 104 CFU/g in ISS-debris and 7.3 X 103 CFU/g in ISS-lint samples, which shows that the readily cultured fungal population was only ∼7 % in desiccated ISS lint samples when compared to the ISS-debris. The fungal burden in ISS and SAF samples were similar, but despite collecting for 6 months, the 103 debris samples exhibited 1-log less fungal burden compared to others.
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The qPCR-based estimate for total bacterial population (dead and alive; PMA-untreated) in the ISS-debris sample (9.5 X 106 16S rRNA copies/g) was 2 logs less than measured in the ISS-lint sample (8.9 X 108 16S rRNA copies/g). The 16S rRNA copy number was 1-log decreased for the viable bacterial population (PMA-treated) in these samples. As depicted in Table 1, ∼12 % of the total bacteria were viable in ISS-debris, whereas only 2.6 % of bacterial population was viable in the ISS-lint sample. The percentage of viable population was higher in SAF (42 % for SAF-debris and 75 % for SAF-lint) compared to the ISS counterparts. When the samples were subjected to ATP assay, the total microbial population (dead and alive) was 5-fold more in the ISS-debris sample (5.8 X 107 RLU/g) than measured in the ISS-lint sample (1.2 X 107 RLU/g). Unlike qPCR measurements, the ATP content was less in the ISS-lint sample when compared to the ISS-debris sample, which was also observed in the cultured assay. Approximately 85 % of the total microorganisms as measured by ATP were viable in the ISS-debris, whereas 34 % of microorganisms were viable in the ISS-lint sample. The SAF-lint and 103-lint samples possessed 1-log more total microbial load than their debris samples where as the viable microbial burden was almost similar in both SAF and 103 samples. Bacterial strains subjected to 16S rRNA-based identification revealed the presence of either spore-forming bacteria or human commensals (Table 2). Bacillus and Staphylococcus species were common in both ISS-debris and ISS-lint samples, whereas the ISS-lint sample contained additional sporeforming bacterial species, Paenibacillus and Brevibacillus. Among the fungal strains tested via morphological and microscopic techniques, members belonging to the fungal phyla, ascomycota (Aspergillus, Penicillium) and basidiomycota (Rhodotorula), were isolated. In total, the next-generation sequencing procedures carried out in this study yielded 65,826 bacterial 16S rRNA gene sequences >350-bp in length from the four sample sets examined. When these sequences were processed with the bioinformatics software MOTHUR, ∼21 % of the sequences were omitted from consideration due to the aforementioned quality control criteria. The remaining 79 % of sequences (51,570; Table 3) whose minimum length was 250 bp were aligned and subjected to cluster analyses to reveal their phylogenetic affiliations. A breakdown of the number of pyrosequences and OTU observed in the various samples examined over the course of this study is provided in Table 3. Even though equivalent materials (weight/volume) were analyzed from the ISS debris and ISS-lint samples, the ISS-debris contained many more pyrosequence reads than the ISS-lint sample. The ISS debris sample gave rise to 86 % of total pyrosequence reads (44,479 sequences), whereas the ISSlint sample had only 7,271 sequences. The PMA-untreated ISS-debris (27,072 sequences) possessed 5.9-fold more bacterial pyrosequences than the ISS-lint samples (4,595 sequences), whereas the OTU numbers were roughly equivalent in both samples (118 in ISS-debris vs. 94 in ISS-lint). Both the samples examined harbored OTU affiliated with physiologically resistant bacteria (Actinobacteria and Firmicutes) and, to some extent, proteobacterial OTU were also present. A closer examination of the pyrosequence reads resulting from the ISSdebris and ISSlint samples indicated a predominance of members of 36 genera (Table 4). It was particularly apparent that members of a few bacterial genera (Corynebacterium, Propionibacterium,Staphylococcus, Streptococcus) were present in great abundance in these samples. Collectively, sequences arising from the members of these abundant genera constituted >90 % of the total in samples that were not treated with PMA. Regardless of sample type, PMA-treated sample (viable) fractions consistently yielded considerably fewer pyrosequences than their untreated counterparts. Overall, the effect of PMA treatment was significantly higher in both ISS debris (61% sequence reduction) and ISS-lint (63% sequence reduction) samples, which indicated the presence of a large number of dead cells or extraneous DNA. Similar to the sequence abundance, the reduction in bacterial OTU numbers was even more in PMA-treated samples for ISS-debris (reduced from 118 to 23 OTU) and ISS-lint samples (reduced from 94 to 12 OTU). Many of the sequences of physiologically recalcitrant bacteria observed in the PMA-untreated samples were absent or scarce in the PMA-treated fractions of
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the same samples. Interestingly, the pyrosequences of the viable members of the three abundant genera were >97 % in both of the PMA-treated samples. The viable bacterial species of Corynebacterium (seven species) and Propionibacterium (three species) required special growth conditions; hence the conventional culture approach employed during this study did not reveal the presence of these bacterial species.
However, the members of the staphylococcal species isolated on the TSA growth medium (Table 5) were also observed in the next-generation sequencing approach (Staphylococcus hominis and S. epidermidis). The presence of viable Propionibacterium acnes pyrosequences was greater in PMA-treated samples, when compared to untreated matrices of both ISS-debris (7-fold increase) and ISS-lint (1.4-fold increase) samples. This might be due to the availability of more beads when the competing DNA template was lower in pyrosequencing. Similarly, more pyrosequences of viable S. epidermidis (1.9-fold), S. pettenkoferi (3.7-fold), and an unidentified Staphylococcus species (2.5-fold) were observed in the ISS debris sample; these sequences were not noticed in high numbers in the ISS-lint sample (Table S2). Some bacterial species (Clostridium, Sphingomonas, Delftia, and Pseudomonas) were observed only in PMA-
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treated samples and none of these sequences, or very few sequences (<1 %), were retrieved from the PMAuntreated samples (Table S2). Incidence of viable bacterial species elucidated by traditional culture and nextgeneration molecular methods is depicted in Table 5. In total, sequences of 36 viable bacterial OTU were recovered, of which 23 and 12O TU were observed in ISS-debris and ISS-lint samples, respectively. Among these 36 OTU, C. kroppenstedtii, Corynebacterium sp., P. acnes,Rothia mucilaginosa, S. pettenkoferi, and Staphylococcus sp. sequences were retrieved from both ISS-debris and ISS-lint samples. Even though high numbers of Streptococcus sequences (9 OTU, Table S2) were noticed in these samples, which were reported to be common human commensals, none of them were viable.
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Unlike members of the phylum actinobacteria, viable members of the other phyla encountered during this study did not overlap between ISS-debris and ISS-lint samples. Presence of anaerobic members (Clostridium species) in the ISS-debris sample might be due to the microaerophilic conditions maintained in the clumps of the ISSdebris, when compared to the ISS-lint sample. Pyrosequences belonging to spore-forming aerobic bacteria were absent; however, these Bacillus and related spore-forming bacterial species were successfully isolated by the traditional culture method. Even though sporeforming bacteria were readily isolated, the molecular method did not retrieve pyrosequences from these samples and warrants further study.
When the incidence of cultured members of the bacterial species was compared, bacterial species other than S. hominis and S. epidermidis were not common. The only bacterial species that was either cultured or had its sequence retrieved by pyrosequencing method was S. epidermidis. When the samples were subjected to the archaeal characterization, both qPCR and next-generation sequencing methods did not generate amplification of archaeal products (data not shown). It is presumed that the presence of archaea in these samples was either at a very low concentration (<100 gene copies per PCR reaction) where the employed method was not able to detect , or materials associated with dust samples collected might have inhibited the archaeal DNA amplification. The latter was confirmed as not true since spiking purified archaeal DNA in these samples before archaeal PCR amplification did exhibit appropriate band (data not shown). Resulting fungal pyrosequence abundance and OTU designations associated with the various samples examined are given in Table 6. In total, 18,635 high-quality fungal pyrosequences comprising 30 distinct fungal OTU were generated in this investigation. Unlike bacterial characterization, where more than 100 OTU pyrosequences were retrieved in both samples, the fungal OTU were less predominant. Among the samples tested, fungal sequences (â&#x2C6;ź8.5-fold increase) and OTU were abundant in ISS-lint (28 OTU), whereas only 1,092 fungal pyrosequences and eight OTU were present in ISS-debris samples. This was surprising since bacterial incidence was higher in ISS debris samples than in ISS-lint samples (Table 4). In addition to the 30 taxa listed in Table 6, an additional 11 OTU were present that were closest to sequences from uncultured fungi (most were fromother metagenomic studies). Four of them had no matches above the 97% identity cut-off value. The others were closest to sequences from species: Aureobasidium, Cladosporium, Cryptococcus, Malassezia, Phaeococcomyces, Pichia, and Rhodotorula. Of the total sequences in the data set, approximately 9 % were closest to sequences from plant pathogens, almost 15 % were closest to sequences from saprobes, 17 % were closest to those from fungi that produce allergens, and more than 32% were closest to sequences from fungi that are either human pathogens or are opportunistic human pathogens.
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Space Flight Induced Reactivation of Latent Epstein-Barr Virus (Epstein-Barr)
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Latent virus reactivation in astronauts during international space station Latent virus reactivation and diurnal salivary cortisol and dehydroepiandrosterone (DHEA) were measured prospectively in 23 astronauts (18 male and 5 female) before, during, and after long duration (60-180 days) space missions on the International Space Station (ISS). Blood, urine, and saliva samples were collected during each of three phases (before, during, and after) spaceflight. Twenty age and sex matched healthy round based subjects were included in the study as a control group. Viral DNA was measured for Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and cytomegalovirus (CMV). One astronaut did not shed targeted viruses in any samples collected during the three mission phases. Simultaneous shedding of all three viruses (EBV, VZV, and CMV) was observed in 8 astronauts. The viruses reactivated independently of each other. Reactivation of EBV, VZV, and CMV increased in frequency, duration, and amplitude (viral copy numbers) when compared to short duration (10-16 days) space shuttle missions. Herpes simplex (HSV 1 and 2) and human herpes virus 6 did not reactivate. Mean salivary cortisol increased significantly during flight as compared to before flight (p = 0.010). There was no difference between the after flight and before-flight (p = .048) levels of cortisol. DHEA concentrations did not change significantly for ISS crew members.
Space Flight Induced Reactivation of Latent Epstein-Barr Virus (Epstein-Barr) The Space Flight-Induced Reactivation of Latent Epstein-Barr Virus (Epstein-Barr) experiment performs tests to study changes in the human immune function using blood and urine samples collected before and after space flight. The study will provide insight for possible countermeasures to prevent the potential development of infectious illness in crewmembers during flight.
Nearly everyone worldwide (up to 95%) has been infected at some point during life with the Epstein-Barr virus (EBV) - a member of the herpes virus family. EBV has been linked to a variety of diseases such as infectious mononucleosis, malignant lymphomas, and nasopharyngeal carcinoma. After primary infection, the virus lies dormant lifelong in the host to avoid detection by the immune system but can be "reactivated" by a number of stressors including those associated with space flight such as preflight stress and immune system changes during flight. A goal of the EBV study was to determine whether changes in EBV gene expression (reactivation) occurred after short- and long-duration space flight. All astronaut and control subjects (24) tested positive for antibodies indicating past EB infection. Peripheral blood samples were collected from 12 astronauts 10 days before launch and on landing/recovery day. The mean mission length for Shuttle astronauts was 11 days, while the mean mission length for ISS astronauts was 180 days. In Shuttle astronauts, a pattern of immediate-early and early viral gene transcription (the first step in gene production) was observed indicative of EBV reactivation. Altogether, there was a significant increase in the number of immediate early and early gene replications in Shuttle astronaut samples as compared to healthy control samples. Although EBV reactivation did occur in Shuttle astronauts, productive EBV replication in peripheral blood B-lymphocytes did not. In contrast, samples from three ISS astronauts after flight show strong evidence that complete productive EBV replication is occurring in the peripheral blood B-cells of these astronauts, and data clearly show activation of the full cascade of replicative EBV gene expression in the B-cells of ISS astronauts. For these crewmembers who flew longer (6 month) in space, latent gene expression and late lytic (involves the destruction of the host cell) gene transcripts were both more frequent and diverse. Overall, there was a significant increase in the number of immediate early and early gene transcripts in ISS astronaut samples as compared to healthy control samples and Shuttle astronauts at landing. The authors acknowledged there were limitations to this study. First, the sample size was limited (the results, however, were quite striking). Second, data from other potential stressors (e.g., sleep deprivation, changes in circadian rhythms, etc.), known to affect immune function, were not available. Additionally, increased radiation exposure on long-duration missions may also affect cellular immunity. Another consideration is that only postflight samples were analyzed and do not necessarily reflect EBV gene expression during flight.
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