The Surface, Water, and Air Biocharacterization (SWAB) Flight Experiment was designed with the general aim of developing advanced collection and analysis techniques to better understand the microbial ecology and potential health risks in spacecraft, specifically focusing on the application of molecular identification techniques. The specific aims of this experiment were:
Surface samples were taken at a variety of locations, including air supply surfaces, food locker surfaces, and water dispenser surfaces. A surface sampling tube containing a Dacron swab and phosphate buffer (pH 7.0) was removed from the SWAB kit. The swab was used to swab a 25 cm squared surface area. The swab was then returned to the vial and marked with the date and location where the sample was collected using a Sharpie marker. Upon return to Earth, the swab and buffer were sonicated to facilitate microbial detachment into the liquid.
Potable water samples were collected directly into 1 liter Teflon bags currently approved for in-flight use. ISS water sampling sites included the US Potable Water Dispenser (PWD) that distributes water for potable use. Surface samples were collected using a modified damp swab and liquid vial produced by Charm Sciences. Air samples were collected using gelatin filters incorporated into the Sartorius MD 8 Air Port System.
Denaturing Gradient Gel Electrophoresis (DGGE):
DNA samples were analyzed for relative concentration and identity of ribosomal sequences using a combination of PCR, Denaturing Gradient Gel Electrophoresis (DGGE), and 16S ribosomal analysis for identification. 16S-ribosomal DNA was extracted from the consortium and amplified using a universal primer set. This amplified mixture of various ribosomal sequences was separated using DGGE to determine the number of bacterial species present in a sample. The isolated DNA was eluted from the gels and sequenced for identification. Identification was determined by comparison of the sequences with the National Center for Biotechnology Information database.
Real Time PCR:
All samples except potable water were analyzed for specific fungal species using PCR primers for medically significant or indicative fungi, including Aspergillus fumigates and Stachybotrys chartarum. A relative moldiness index (RMI) multiplex technique developed by the Environmental Protection Agency was also utilized to provide a thorough fungal characterization. Samples were also analyzed for the presence of Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), and Varicella Zoster Virus (VZV).
Whole Genome Analysis:
ISS water samples were analyzed by whole genome sequencing performed at the University of Houston Institute for Molecular Design using technology developed by Illumina, Inc.
Preflight sampling was completed for air, surface, and water samples. In-flight sample collection was completed for a total of eight air, surface, and water sample collection sessions. In-flight hardware performed well, and the surface sampling device received positive feedback from the crew for its ease of use.
Denaturing Gradient Gel Electrophoresis (DGGE)
The DGGE technique was found to be less sensitive than culture-based methods. One million cells need to be present to detect a PCR band on an agarose gel. In addition, DGGE has a greater incidence of Gram-negative bacteria on surface samples when compared to culture methods. Subsequent experiments suggested that primer bias or contamination was not the cause.
Real Time PCR
To determine the utility of QPCR for monitoring and the potential presence of two toxigenic fungi, preflight and in flight samples were analyzed using QPCR analysis developed for the detection of Aspergillus fumigates and Stachybotrys chartarum at the University of Nevada Las Vegas. These fungi were not detected. The use of QPCR was further investigated in a collaborative effort with colleagues from the Environmental Protection Agency (EPA). In this study, dust from four ISS vacuum bags and two HEPA filters used from 2007 to 2009 were returned to the ground for QPCR-based molecular analysis. An environmental relative moldiness index (ERMI) multiplex technique developed by the EPA was also utilized to provide a thorough fungal characterization. The most common species measured was P. chrysogenum followed by Aureobasidium pullulans. Of the 36 species in the standard ERMI analysis, 17 were detected at some concentration. Results were comparable to what would be found in a home.
Finally, QPCR technology was tested by analyzing 336 environmental samples (both pre- and in flight) from spacecraft surfaces (266), water (5) and air (70) for the presence of Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), and Varicella Zoster Virus (VZV). These latent viruses may be carried asymptomatically by the crew and have the potential to be “reactivated” into live, infectious viruses. This reactivation has been associated with stress and suppressed immune function. Analyses revealed no CMV in any samples. VZV was detected in 6 air samples and 5 surface samples. EBV was detected in 2 air samples and 10 surface samples.
|Mission||Launch/Start Date||Landing/End Date||Duration|
|Expedition 13||03/22/2006||09/24/2006||186 days|
|Expedition 14||09/18/2006||04/21/2007||215 days|
|Expedition 15||04/07/2007||10/21/2007||197 days|
|Expedition 16||10/10/2007||04/19/2008||192 days|
|Expedition 20||05/27/2009||10/11/2009||137 days|
|Expedition 21||10/11/2009||12/01/2009||51 days|
|Expedition 22||11/30/2009||03/18/2010||109 days|
|Expedition 23||03/18/2010||06/01/2010||75 days|
|Expedition 24||06/01/2010||09/25/2010||117 days|