A microbial survey of the International Space Station (ISS)
Background: Modern advances in sequencing technology have enabled the census of microbial members of many natural ecosystems. Recently, attention is increasingly being paid to the microbial residents of human-made, built ecosystems, both private (homes) and public (subways, office buildings, and hospitals). Here, we report results of the characterization of the microbial ecology of a singular built environment, the International Space Station (ISS). This ISS sampling involved the collection and microbial analysis (via 16S rDNA PCR) of 15 surfaces sampled by swabs onboard the ISS. This sampling was a component of Project MERCCURI (Microbial Ecology Research Combining Citizen and University Researchers on ISS). Learning more about the microbial inhabitants of the "buildings" in which we travel through space will take on increasing importance, as plans for human exploration and colonization of the solar system come to fruition.
Results: Sterile swabs were used to sample 15 surfaces onboard the ISS. The sites sampled were designed to be analogous to samples collected for 1) the Wildlife of Our Homes project and 2) a study of cell phones and shoes that were concurrently being collected for another component of Project MERCCURI. Sequencing of the 16S rDNA genes amplified from DNA extracted from each swab was used to produce a census of the microbes present on each surface sampled. We compared the microbes found on the ISS swabs to those from both Earth homes and data from the Human Microbiome Project.
Conclusions: While significantly different from homes on Earth and the Human Microbiome Project samples analyzed here, the microbial community composition on the ISS was more similar to home surfaces than to the human microbiome samples. The ISS surfaces are species-rich with 1036-4294 operational taxonomic units (OTUs per sample). There was no discernible biogeography of microbes on the 15 ISS surfaces, although this may be a reflection of the small sample size we were able to obtain.
There is a growing appreciation of the importance of microbial communities found in diverse environments from the oceans, to soil, to the insides and outsides of plants and animals. Recently, there has been an expanding focus on the microbial ecology of the "built environment" - human constructed entities like buildings, cars, and trains - places where humans spend a large fraction of their time. One relatively unexplored type of built environment is that found in space. As humans expand their reach into the solar system, with renewed interest in space travel, and with the possibility of the colonization of other planets and moons, it is of critical importance to understand the microbial ecology of the built environments being utilized for such endeavors.
Interest in the microbial occupants of spacecraft long precedes the launch of the International Space Station (ISS) (Trexler 1964)(Silverman 1971). Early work primarily focused on ensuring that the surfaces of spacecraft were free of microbial contaminants in an effort to avoid inadvertent panspermia (seeding other planets with microbes from Earth) (Pierson 2007). Work on human-occupied spacecraft such as Mir, Space Shuttles, and Skylab focused more on microbes with possible human health effects. With the launch of the ISS, it was understood that this new built environment would be permanently housing microbes as well as humans. Calls were made for a better understanding of microbial ecology and human-microbe interactions during extended stays in space (Pierson 2007) (Roberts 2004) (Ott 2004). Efforts were made to establish a baseline microbial census. For example, Novikova et al (Novikova 2006) obtained more than 500 samples from the air, potable water, and surfaces of the ISS, over the course of 6 years.
These early studies were unavoidably limited by their reliance on culturing to identify microbial species. Culture-independent approaches were eventually implemented, including some small-scale 16S rDNA PCR surveys (Castro 2004),(Moissl 2007) and the Lab-On-a-Chip Application Development Portable Test System (LOCAD-PTS) (Maule ), which allows astronauts to test surfaces for lipopolysaccharide (LPS - a marker for Gram negative bacteria). Originally launched in 2006, the capability of the LOCAD-PTS was expanded in 2009 to include an assay for fungi (beta-glucan, a fungal cell wall component) and Gram positive bacteria (lipoteichoic acid, a component of the cell wall of Gram positive bacteria.) The first large-scale, culture-independent 16S rDNA PCR survey was published only in 2014 using the Roche 454 platform (pyrosequencing), looking at dust on the ISS (Venkateswaran 2014). A more recent study examined several samples collected on the Japanese module of the ISS over a period of four years, also sequenced with pyrosequencing (Ichijo 2016). We report here on a further effort involving 16S rDNA PCR and sequencing, using the Illumina platform, to examine the microbial communities found on 15 surfaces inside the ISS.
The 15 surfaces sampled on the ISS were chosen by the Project MERCCURI team in an effort to make them analogous to 1) the surfaces sampled for the "Wildlife of Our Homes" project (http://homes.yourwildlife.org) (Dunn 2013a) (Barberán 2015), which asked citizen scientists to swab nine surfaces in their homes, and 2) cell phone and shoe swab samples that were also being collected via Project MERCCURI. The motivation for choosing the sites in this way was both to increase public awareness of the microbiology of the built environment, as well as to begin to compare the microbial ecology of homes on Earth with the only current human home in space. We also present a comparison of the ISS swab results with data from 13 human body sites sampled via the Human Microbiome Project. This comparison was done to example the potential human contribution to the microbial life on the ISS.
We have also compiled a collection of papers on space microbiology in an online resource to provide a more comprehensive historical perspective of this kind of work (see http://www.mendeley.com/groups/844031/microbiology-of-the-built-environment/papers/added/0/tag/space/).
Astronauts were asked to swab 15 surfaces on the International Space Station. Below are their verbatim instructions.
Audio Terminal Unit (telephone) hand held push-to-talk microphone located in the forward portion of the US Lab Module
Audio Terminal Unit (telephone) hand held push-to-talk microphone located in the aft portion of the US Lab Module
US Lab Robotic Work Station laptop PC keyboard used to control the robotic arm
US Lab Robotic Work Station hand controller used to control the movement of the robotic arm
US Lab Robotic Work Station foothold, left side
US Lab Robotic Work Station foothold, right side
One of the main laptop keyboards in the US Lab used to control science experiments and the systems of the space station
One of the vertical handrails on the equipment racks inside the US Lab
Air vent in the front portion of the US Lab
Air vent in the aft portion of the US Lab
Air vent located on the right crew sleep compartment
Tab used to open, close, and secure the Nomex privacy panel located on the starboard crew sleep compartment
Air vent located on the port crew sleep compartment
Tab used to open, close, and secure the Nomex privacy panel located on the port crew sleep compartment
Crew Choice Surface: Audio Terminal Unit (telephone) hand held push-to-talk microphone located in the starboard portion of the Harmony module (Node 2).
NOTE: An additional large Ziplock Bag is provided (stowed inside the same bag as the NanoRacks Swab Kits) to use per crew preference to separate the used NanoRacks Swab Kits from the clean (unused) NanoRacks Swab Kits for crew efficiency during sampling.
Swabbing was conducted during Expedition 39 (http://www.nasa.gov/mission_pages/station/expeditions/expedition39/index.html). The crew included NASA astronauts Steve Swanson and Rick Mastracchio and Russian cosmonauts Oleg Artemyev, Alexander Skvortsov, and Mikhail Tyurin. Japan Aerospace Exploration Agency (JAXA) astronaut Koichi Wakata was the commander for this expedition, and is the astronaut who performed the swabbing.
These surfaces were chosen in an attempt to sample surfaces analogous to those sampled in the pilot study for the Wildlife of Our Homes project (Dunn 2013). For this study, involving 40 homes, volunteers swabbed nine surfaces in their homes: kitchen cutting board, kitchen counter, a shelf inside a refrigerator, toilet seat, pillowcase, exterior handle of the main door into the house, television screen, the upper door trim on the outside surface of an exterior door, and the upper door trim on an interior door. We were not granted access to all corresponding surfaces aboard the ISS. The kitchen surfaces aboard the ISS are in the Russian module, which we did not have permission to access, swabbing the toilet seat was deemed inappropriate due to biosafety concerns, and the exterior surfaces are accessible only via an "Extra-vehicular Activity" (space walk), which was not requested for this experiment. We also sought to collect samples that would be analogous to the cell phone and shoe samples that were being obtained from thousands of Citizen Scientists across the country in a different component of Project MERCCURI. A final constraint was the limitation of only 15 swabs that was imposed by NASA, severely limiting the number of replicates we could collect. See Table 1 for a list of the ISS sampling sites and to which Earth samples they were intended to be analogous.
Upon successful completion of the swabbing on May 9, 2014, http://blogs.nasa.gov/stationreport/2014/05/09/iss-daily-summary-report-050914/, all swabs were stored at -80 °C in the Minus Eighty-degree Laboratory Freezer for ISS (MELFI) freezer onboard the ISS, until transfer to the SpaceX Dragon spacecraft. In the Dragon, the swabs were stored at -80 °C in the General Laboratory Active Cryogenic ISS Experiment Refrigerator (GLACIER), that runs off of Dragon's batteries until it is plugged in (either to the ISS or on the ground.) The Dragon re-entered the Earth's atmosphere and splashed down in the Pacific Ocean at 12:05 pm PT on May 18, 2014. Samples were transferred to a cooler with dry ice, and shipped to the Earth Microbiome Project (EMP) lab (http://earthmicrobiome.org)(Gilbert 2011).
All samples were prepared using a modified version of the Mo BIO UltraClean®-htp 96 Well Swab DNA Kit (MO BIO). Samples were purified using the Zymo ZR-96 DNA Cleanup and Concentrator™-5 kit according to Zymo Protocol (Zymo). DNA was then amplified using the EMP barcoded primer set, adapted for the Illumina HiSeq2000 and MiSeq by adding nine extra bases in the adapter region of the forward amplification primer that support paired-end sequencing. The V4 region of the 16S rRNA gene (515F-806R) was amplified with region-specific primers that included the Illumina flowcell adapter sequences and a twelve base barcode sequence. Each 25 ul PCR reaction contained the following: 12 ul of PCR water certified DNA-free (MO BIO), 10 ul of 1x 5 Prime HotMasterMix (5 Prime), 1 ul of Forward Primer (5 uM concentration, 200 pM final), 1 ul of Golay Barcode Tagged Reverse Primer (5 uM concentration, 200 pM final), and 1 ul of template DNA. The conditions for PCR were as follows: 94°C for 3 minutes to denature the DNA, with 35 cycles at 94 °C for 45 s, 50 °C for 60 s, and 72 °C for 90 s, with a final extension of 10 min at 72 °C to ensure complete amplification. Amplicons were quantified using PicoGreen (Invitrogen) and a plate reader. Once quantified, different volumes of each of the products were pooled into a single tube so that each amplicon was represented equally. This pool was then cleaned up using UltraClean® PCR Clean-Up Kit (MO BIO), and then quantified using Qubit (Invitrogen). Sequencing of the prepared library was performed on the Illumina MiSeq platform, using the sequencing primers and procedures described in the supplementary methods of (Caporaso 2012).
Unless otherwise noted, all microbial community analyses were conducted using the QIIME workflow version 1.8 or R (R-project 2014). All python scripts referred to are components of QIIME (Caporaso 2010).
An in-house script was used to assign sequences to samples, using dual-index barcoding. This script is available on github (https://github.com/gjospin/Demul_trim_prep). This script allows for 1 base pair difference per barcode. The paired reads were then aligned and a consensus was computed using FLASH (Magoc 2011) with maximum overlap of 120 and a minimum overlap of 70 (other parameters were left as default). The custom script automatically demultiplexes the data into fastq files, executes FLASH, and parses its results to reformat the sequences with appropriate naming conventions for QIIME v. 1.8.0 in fasta format.
Chimeric sequences were identified using usearch61 as implemented in the identify_chimeric_seqs.py script, resulting in the removal of 8760 sequences. The pick_open_reference_otus.py script was used to cluster sequences at 97% similarity to generate OTUs (Operational Taxonomic Units, a proxy for species). Taxonomy was assigned to each OTU by comparing a representative sequence from each cluster to the gg_13_8_otus reference taxonomy provided by the Greengenes Database Consortium (http://greengenes.secondgenome.com) (McDonald 2011). OTUs that were classified as chloroplasts or mitochondria were removed from further analysis. The number of high-quality sequences remaining per sample ranged from 26831 to 77843 (see Table 1). All subsequent beta diversity analyses (comparisons across samples) were performed with all samples rarefied to 26830 sequences.
The sequences and associated metadata from a 40-home pilot study for the Wildlife of Our Homes Project are available for download from Figshare (Menninger; 2013). We also obtained 100 samples from each of 13 body sites from the HMP Data Portal (http://hmpdacc.org/HM16STR/)(Huttenhower 2012)(Gevers 2012). These two additional datasets were used in a combined analysis with the ISS sequences presented here. Because the sequences from the three projects are not all the same lengths, each dataset was independently analyzed using a closed-reference OTU-picking approach, with a 97% similarity cutoff, and the resultant biom tables were merged with the merge_otu_tables.py script. To account for uneven sampling depth, all samples in the combined analysis were rarefied to Shannon diversity, as well as non-metric multidimensional scaling (NMDS) based on Bray-Curtis (Bray 1957)and Unweighted Unifrac (Lozupone 2005) distances were computed and plotted using Phyloseq (McMurdie 2013) and the ggplot2 (Wilkinson 2011) packages in R (R-project 2014). All samples were rarefied to a depth of 1000 samples for the combined analysis.
We obtained a list of human pathogens, compiled by Kembel et al, 2012 from the author. We then used BLAST (Altschul 1990) to search a representative sequence from each of the ISS OTUs against the NCBI Reference Sequence (RefSeq) database (Pruitt 2004). OTUs with 97% similarity to an organism that was on the list of known pathogens were flagged as "related to a known human pathogen". The phylogenetic diversity (Faith's PD) was calculated using the alpha_diversity.py script, with samples rarefied to 700 sequences.