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Research Containing: Microbes

Rapid culture-independent microbial analysis aboard the International Space Station (ISS)

by on 9 June 2015in Technology Development & Demonstration No comment

A new culture-independent system for microbial monitoring, called the Lab-On-a-Chip Application Development Portable Test System (LOCAD-PTS), was operated aboard the International Space Station (ISS). LOCAD-PTS was launched to the ISS aboard Space Shuttle STS-116 on December 9, 2006, and has since been used by ISS crews to monitor endotoxin on cabin surfaces. Quantitative analysis was performed within 15 minutes, and sample return to Earth was not required. Endotoxin (a marker of Gram-negative bacteria) was distributed throughout the ISS, despite previous indications that mostbacteria on ISS surfaces were Gram-positive [corrected].Endotoxin was detected at 24 out of 42 surface areas tested and at every surface site where colony-forming units (cfu) were observed, even at levels of 4-120 bacterial cfu per 100 cm(2), which is below NASA in-flight requirements (<10,000 bacterial cfu per 100 cm(2)). Absent to low levels of endotoxin (<0.24 to 1.0 EU per 100 cm(2); defined in endotoxin units, or EU) were found on 31 surface areas, including on most panels in Node 1 and the US Lab. High to moderate levels (1.01 to 14.7 EU per 100 cm(2)) were found on 11 surface areas, including at exercise, hygiene, sleeping, and dining facilities. Endotoxin was absent from airlock surfaces, except the Extravehicular Hatch Handle (>3.78 EU per 100 cm(2)). Based upon data collected from the ISS so far, new culture-independent requirements (defined in EU) are suggested, which are verifiable in flight with LOCAD-PTS yet high enough to avoid false alarms. The suggested requirements are intended to supplement current ISS requirements (defined in cfu) and would serve a dual purpose of safeguarding crew health (internal spacecraft surfaces <20 EU per 100 cm(2)) and monitoring forward contamination during Constellation missions (surfaces periodically exposed to the external environment, including the airlock and space suits, <0.24 EU per 100 cm(2)).

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/19845447

Microflow1, a sheathless fiber-optic flow cytometry biomedical platform: demonstration onboard the international space station

by on 9 June 2015in Technology Development & Demonstration No comment

A fiber-optic based flow cytometry platform was designed to build a portable and robust instrument for space applications. At the core of the Microflow1 is a unique fiber-optic flow cell fitted to a fluidic system and fiber coupled to the source and detection channels. A Microflow1 engineering unit was first tested and benchmarked against a commercial flow cytometer as a reference in a standard laboratory environment. Testing in parabolic flight campaigns was performed to establish Microflow1's performance in weightlessness, before operating the new platform on the International Space Station. Microflow1 had comparable performances to commercial systems, and operated remarkably and robustly in weightlessness (microgravity). Microflow1 supported immunophenotyping as well as microbead-based multiplexed cytokine assays in the space environment and independently of gravity levels. Results presented here provide evidence that this fiber-optic cytometer technology is inherently compatible with the space environment with negligible compromise to analytical performance.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/24339248

New molecular technologies against infectious diseases during space flight

by on 9 June 2015in Technology Development & Demonstration No comment

Latent virus reactivation, reduction in the number of immune cells, decreased cell activation and increased sensitivity of astronauts to infections following their return on Earth demonstrate that the immune system is less efficient during space flight. This dysfunction during long-term missions could result in the appearance of opportunistic infections or a decrease in the immuno-surveillance mechanisms that eradicate cancer cells. On the other hand, monitoring of the microbial environment is essential to prevent infectious diseases in space. Therefore, both aspects will have to be monitored continuously during long-term missions in space, using miniature and semi-automated diagnostic systems. In the short term, such equipment will allow the study of the causes of space-related immunodeficiency, developing countermeasures to maintain an optimal immune function and improving our capacity to prevent infectious diseases during space missions. In order to achieve these objectives, a new diagnostic system has been designed to perform a set of biological and immunological assays on board spacecrafts. Through flow cytometric assays and molecular biology analyses, this diagnostic system will improve medical surveillance of astronauts and will be used to test countermeasures aimed at preventing immune deficiency during space missions.

Related URLs:
http://www.sciencedirect.com/science/article/pii/S0094576508000039

Bacillus anthracis-like bacteria and other B. cereus group members in a microbial community within the International Space Station: a challenge for rapid and easy molecular detection of virulent B. anthracis

by on 9 June 2015in Biology & Biotechnology No comment

For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order for B. anthracis to be virulent and its close relationship to Bacillus cereus and other members of the B. cereus group. This is especially the case in environments where build-up of Bacillus spores can occur and several representatives of the B. cereus group may be present, which increases the chance for false-positives. In this study we show the presence of B. anthracis-like bacteria and other members of the B. cereus group in a microbial community within the human environment of the International Space Station and their preliminary identification by using conventional culturing as well as molecular techniques including 16S rDNA sequencing, PCR and real-time PCR. Our study shows that when monitoring the microbial hygiene in a given human environment, health risk assessment is troublesome in the case of virulent B. anthracis, especially if this should be done with rapid, easy to apply and on-site molecular methods.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/24945323

Microbial detection and monitoring in advanced life support systems like the International Space Station

by on 9 June 2015in Biology & Biotechnology No comment

Potentially pathogenic microbes and so-called technophiles may form a serious threat in advanced life support systems, such as the International Space Station (ISS). They not only pose a threat to the health of the crew, but also to the technical equipment and materials of the space station. The development of fast and easy to use molecular detection and quantification methods for application in manned spacecraft is therefore desirable and may also be valuable for applications on Earth. In this paper we present the preliminary results of the SAMPLE experiment in which we performed molecular microbial analysis on environmental samples of the ISS as part of an ESA-MAP project.

Related URLs:
http://dx.doi.org/10.1007/BF02911866

Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

by on 9 June 2015in Biology & Biotechnology No comment

The development of fast and easy on-site molecular detection and quantification methods for hazardous microbes on solid surfaces is desirable for several applications where specialised laboratory facilities are absent. The quantification of bacterial contamination necessitates the assessment of the efficiency of the used methodology as a whole, including the preceding steps of sampling and sample processing. We used quantitative real-time polymerase chain reaction (qrtPCR) for Escherichia coli and Staphylococcus aureus to measure the recovery of DNA from defined numbers of bacterial cells that were subjected to three different DNA extraction methods: the QIAamp DNA Mini Kit, Reischl et al.'s method and FTA Elute. FTA Elute significantly showed the highest median DNA extraction efficiency of 76.9% for E. coli and 108.9% for S. aureus. The Reischl et al. method and QIAamp DNA Mini Kit inhibited the E. coli qrtPCR assay with a 10-fold decrease of detectable DNA. None of the methods inhibited the S. aureus qrtPCR assay. The FTA Elute applicability was demonstrated with swab samples taken from the International Space Station (ISS) interior. Overall, the FTA Elute method was found to be the most suitable to selected criteria in terms of rapidity, easiness of use, DNA extraction efficiency, toxicity, and transport and storage conditions.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/21311936

Characterization of Volume F trash from the three FY11 STS missions: Trash weights and categorization and microbial characterization

by on 9 June 2015in Biology & Biotechnology No comment

This research project provided microbial characterization support to the Waste Management Systems (WMS) element of NASA's Life Support and Habitation Systems (LSHS) program. Conventional microbiological methods were used to detect and enumerate microorganisms in space-generated solid wastes, i.e., STS Volume F Compartment trash returned from orbit and missions to ISS. Crew generated STS trash was characterized for three shuttle missions: STS 133, STS 134, and STS 135. The waste was catalogued into logical categories, weighed, and the water content determined. Results for FY11 STS missions showed more variability than for the FYlO study of STS 129-132 and indicated some waste was probably not included in what was returned to KSC on the STS. Microbial characterization of wastes from each mission and each category determined the presence of high numbers of microbes in food waste and food packaging, in drink pouches, and in personal hygiene wastes. A number of bacteria and fungi were identified, including known pathogens and some likely opportunistic pathogens that could cause problems if these wastes were exposed to an immune compromised crew.

Related URLs:
http://dx.doi.org/10.2514/6.2012-3565

Microbial Characterization of Space Solid Wastes Treated with a Heat Melt Compactor

by on 9 June 2015in Biology & Biotechnology No comment

The purpose of the project has been to characterize and determine the fate of microorganisms in space-generated solid wastes before and after processing by candidate solid waste processing. For FY11, the Heat Melt Compactor (HMC) was the candidate technology that was assessed. Five HMC product disks were produced at ARC from either simulated space-generated trash or from actual space trash. The actual space trash was the STS 130 Volume F compartment wet waste. Conventional microbiological methods were used to detect and enumerate microorganisms in heat melt compaction (HMC) product disks and in surface swab samples of the HMC hardware before and after operation. In addition, biological indicators were added to the STS trash prior to compaction to determine if these spore-forming bacteria could survive the HMC processing conditions, i.e., high temperature (160°C) over a long duration (3 hrs). The HMC disk surfaces were sanitized with 70% alcohol prior to obtaining the core samples to ensure that surface dwelling microbes did not contaminate the HMC product disk interior. Microbiological assays were run before and after sanitization and found that sanitization greatly reduced, but did not eliminate, the number of identified isolate. To characterize the interior of the disks, ten 1.25cm diameter core samples were aseptically obtained from each disk. These were run through the microbial characterization analyses. Low counts of bacteria, on the order of 5 to 50 viable cells per core, were found, indicating that the HMC operating conditions might not be sufficient for absolute sterilization of the waste. However, the direct counts were 6 to 8 orders of magnitude greater, demonstrating that the vast majority of microbes present in the wastes were results from commercial spore test strops that had been added to the wastes prior to HMC operation. Nearly all could be recovered from the HMC disks post-operation and all were showed negative growth when run through the manufacturer’s protocol, meaning that the 1 x 106 or so spores impregnated into the strips were dead. Control test strips, i.e., not exposed to the HMC conditions, were all strongly positive. One area of concern is that the identities of isolates from the cultivable counts included several human pathogens namely Staphylococcus aureus.

Related URLs:
http://dx.doi.org/10.2514/6.2012-3546

Novel quantitative biosystem for modeling physiological fluid shear stress on cells

by on 9 June 2015in Biology & Biotechnology No comment

The response of microbes to changes in the mechanical force of fluid shear has important implications for pathogens, which experience wide fluctuations in fluid shear in vivo during infection. However, the majority of studies have not cultured microbes under physiological fluid shear conditions within a range commonly encountered by microbes during host-pathogen interactions. Here we describe a convenient batch culture biosystem in which (i) the levels of fluid shear force can be varied within physiologically relevant ranges and quantified via mathematical models and (ii) large numbers of cells can be planktonically grown and harvested to examine the effect of fluid shear levels on microbial genomic and phenotypic responses. A quantitative model based on numerical simulations and in situ imaging analysis was developed to calculate the fluid shear imparted by spherical beads of different sizes on bacterial cell cultures grown in a rotating wall vessel (RWV) bioreactor. To demonstrate the application of this model, we subjected cultures of the bacterial pathogen Salmonella enterica serovar Typhimurium to three physiologically-relevant fluid shear ranges during growth in the RVW and demonstrated a progressive relationship between the applied fluid shear and the bacterial genetic and phenotypic responses. By applying this model to different cell types, including other bacterial pathogens, entire classes of genes and proteins involved in cellular interactions may be discovered that have not previously been identified during growth under conventional culture conditions, leading to new targets for vaccine and therapeutic development.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/17142365

Haploid deletion strains of Saccharomyces cerevisiae that determine survival during space flight

by on 9 June 2015in Biology & Biotechnology No comment

This study identifies genes that determine survival during a space flight, using the model eukaryotic organism, Saccharomyces cerevisiae. Select strains of a haploid yeast deletion series grew during storage in distilled water in space, but not in ground based static or clinorotation controls. The survival advantages in space in distilled water include a 133-fold advantage for the deletion of PEX19, a chaperone and import receptor for newly- synthesized class I peroxisomal membrane proteins, to 77–40 fold for deletion strains lacking elements of aerobic respiration, isocitrate metabolism, and mitochondrial electron transport. Following automated addition of rich growth media, the space flight was associated with a marked survival advantage of strains with deletions in catalytically active genes including hydrolases, oxidoreductases and transferases. When compared to static controls, space flight was associated with a marked survival disadvantage of deletion strains lacking transporter, antioxidant and catalytic activity. This study identifies yeast deletion strains with a survival advantage during storage in distilled water and space flight, and amplifies our understanding of the genes critical for survival in space.

Related URLs:
http://www.sciencedirect.com/science/article/pii/S0094576506003225