Characterization of the total and viable bacterial and fungal communities associated with the International Space Station surfaces

Background The International Space Station (ISS) is a closed system inhabited by microorganisms originating from life support systems, cargo, and crew that are exposed to unique selective pressures such as microgravity. To date, mandatory microbial monitoring and observational studies of spacecraft and space stations have been conducted by traditional culture methods, although it is known that many microbes cannot be cultured with standard techniques. To fully appreciate the true number and diversity of microbes that survive in the ISS, molecular and culture-based methods were used to assess microbial communities on ISS surfaces. Samples were taken at eight pre-defined locations during three flight missions spanning 14 months and analyzed upon return to Earth. Results The cultivable bacterial and fungal population ranged from 104 to 109 CFU/m2 depending on location and consisted of various bacterial (Actinobacteria, Firmicutes, and Proteobacteria) and fungal (Ascomycota and Basidiomycota) phyla. Amplicon sequencing detected more bacterial phyla when compared to the culture-based analyses, but both methods identified similar numbers of fungal phyla. Changes in bacterial and fungal load (by culture and qPCR) were observed over time but not across locations. Bacterial community composition changed over time, but not across locations, while fungal community remained the same between samplings and locations. There were no significant differences in community composition and richness after propidium monoazide sample treatment, suggesting that the analyzed DNA was extracted from intact/viable organisms. Moreover, approximately 46% of intact/viable bacteria and 40% of intact/viable fungi could be cultured. Conclusions The results reveal a diverse population of bacteria and fungi on ISS environmental surfaces that changed over time but remained similar between locations. The dominant organisms are associated with the human microbiome and may include opportunistic pathogens. This study provides the first comprehensive catalog of both total and intact/viable bacteria and fungi found on surfaces in closed space systems and can be used to help develop safety measures that meet NASA requirements for deep space human habitation. The results of this study can have significant impact on our understanding of other confined built environments on the Earth such as clean rooms used in the pharmaceutical and medical industries. Electronic supplementary material The online version of this article (10.1186/s40168-019-0666-x) contains supplementary material, which is available to authorized users.

copies or ITS region of PMA (intact/viable) by non-PMA (dead and intact/viable) treated samples are shown in Y-axis. (A) Bar graph showing the total read count for each sample and each control. "CTL" indicates the sampling wipe that was exposed to the ISS environment but did not touch any surfaces and "DNACTL" was water added to the negative control for extraction instead of material obtained from the surface/control wipe. (B) SourceTracker was used to examine if and in what proportion the ISS surface bacterial microbiome was associated with contaminating bacterial DNA from the sampling wipes or contaminated with DNA during processing, extraction and sequencing. This was done by comparing the ASV sequences found in the controls ("source") with that present in the samples ("sink"). Each pie graph represents one sample. The blue represents the ASVs unique to the ISS surface samples. The pink, beige and purple slices represent the proportion of ASVs in the ISS surface samples that were similar to those found in 3 controls from F1 (pink), 3 from F2 (beige) and 4 from F3 (purple). The number after F1, F2, F3 refers to the location sampled and "P" indicates a sample that was treated with PMA before DNA extraction. Read counts were rarified to 1000 reads. Each bar represents a sample collected from particular location and during a specific Flight sampling session. F1, F2, F3 refers to one of the three flight sampling sessions, with the number after the underscore indicating the location it was sampled from (i.e. F1_1= flight 1, location 1)..
The colors in each bar represent a taxon and the height of that colored box represents the relative proportion of that taxon within a sample. The remaining fraction, colored grey, groups taxa that were less than 2% abundance in each sample. The legend is read from bottom to top, with the bottom taxon on the legend corresponding to the bottom colored on the barplot. (A) untreated samples and (B) PMA treated samples.

Fig. S4 Assessment of microbial alpha diversity.
Bacterial diversity within a sample (based on sequences that were summarized down to the Family level) was measured with (A) Shannon's diversity index, which measures organism presence/absence and its relative abundance within a sample and (B) Taxa richness, which reports the number of unique taxa within a particular sample. (A) Each symbol on the graph represents one location sampled (1-8) with the line representing the median for all samples. The higher the index the greater the bacterial diversity found within a sample. Since Shannon's diversity index is a logarithmic number with base 2, a value of "4" is 2x higher than a value of "3". The average index for F2 samples was statistically significantly higher than that of F1 and F3 samples (P <0.05). There were no differences (P >0.05) in the diversity index between PMA and non-PMA treated samples for each flight. (B) Each point on the graph represents a location with the line representing the median for all samples. F2 samples were statistically significantly richer than F3 samples but not F1 samples. There were no differences in richness between PMA and non-PMA treated samples at each Flight. Fungal alpha diversity was also measured using (C) Shannon's diversity index and (D) Taxa richness. There were no statistically significant differences (P > 0.05) in Shannon's diversity index nor taxa richness between F1 and F2 samples and between PMA and non-PMA treated samples at each Flight. The Kruskal-Wallis test with the Benjamini Hochberg FDR multiple test correction was used for both bacterial and fungal statistical analyses. 24 surface wipes, were collected from 8 locations over 3 flight sampling sessions, spanning 14 months. DNA was sent for 16S rRNA gene targeted sequencing and the sequences taxonomically assigned using the SILVA database. The ASV table was summarized to the genus level and read counts were transformed using centered log ratios. With centered log ratio data, zero represents the geometric mean abundance (i.e. the average relative abundance of all organisms within a sample), thus the higher a value is above zero the more abundant that organism is compared to all others in that sample. The highest relative abundance is depicted in red and the lowest in light yellow. Grey represents those taxa that had raw read counts of zero. F1, F2, F3 refers to one of the three flight sampling sessions, with the number after the underscore indicating the location it was sampled from (i.e. F1_1= flight 1, location 1). "P" at the end indicates that the sample was treated with PMA before DNA extraction. Out of 121 taxa identified, 77 could be assigned to the genus level. The genera were classified based on whether published studies have found them to form spores and/or biofilms and whether they are associated with the environment or humans.
The pie chart represents the percent of each classification detected in the dataset SourceTracker was used to examine if and in what proportion the ISS fungal surface microbiome was associated with contaminating fungal DNA from the sampling wipes or contaminated with DNA during processing, DNA extraction and sequencing. This was done by comparing the OTUs sequences found in the controls ("source") with that present in the samples ("sink"). Each pie graph represents one sample. The beige represents the OTUs unique to the ISS samples and the pink represents the proportion of OTUs in the ISS samples that were similar to those found in three controls from F1 and F2. Read counts were rarified to 1000 reads. The number after F1 and F2 refer to the location sampled and "P" indicates a sample that was treated with PMA before DNA extraction. (B) Bar graph showing the total read count for each sample and each control.
"CTL" indicates the sampling wipe that was exposed to the ISS environment but did not touch any surfaces. NB: A DNA control "DNACTL" (water added for extraction instead of material obtained from the surface/control wipe) and a F1 CTL_P sample were processed however they did not produce any amplicons and thus was not sent for sequencing.

Fig. S7 Barplot showing the relative abundance of fungal genera.
Barplot showing the relative abundances of fungi, summarized to the genus level, of 16 wipes collected during Flight 1 and Flight 2, at 8 different locations across the ISS. Each bar represents a sample collected from particular location and during a specific Flight sampling session. The letter "P" after the location number signifies PMA treatment of the sample (i.e F1_1P means sample collected from Flight 1, location 1, PMA treatment whereas F2_5 means sample collected from Flight 2, location 5, not treated with PMA). The colors in each bar represent a genus taxon and the height of that colored box represents the relative proportion of that genus within a sample. The legend is read from bottom to top, with the bottom taxon on the legend corresponding to the bottom colored on the barplot. ALDEx2 did not report any statistically significant differences in the mycobiome between F1 and F2.   This chart summarizes which cultured bacteria were also detected with 16S rRNA sequencing.
The far left column lists all bacteria that were cultured, summarized to the genus level. A black box indicates that the genus was cultured from a sample (i.e. Acinetobacter was cultured from the sample collected from location "1", flight 2 "ISS2: for Acinetobacter). A green box indicates that the genus was detected from a sample treated with PMA (i.e. intact, potentially viable) and a red box indicates that the genus was detected from a sample that was not treated with PMA (i.e. total).   I II III I II III I II III I II III I II III I II III I II III I II I II III I II III I II III I II III I II III I II III I II III I II   F 1 _ 1 F 1 _ 1 P F 1 _ 2 F 1 _ 2 P F 1 _ 3 F 1 _ 3 P F 1 _ 4 F 1 _ 4 P F 1 _ 5 F 1 _ 5 P F 1 _ 6 F 1 _ 6 P F 1 _ 7 F 1 _ 7 P F 1 _ 8 F 1 _ 8 P F 1 _ C T L         F1   F2   ISS1_1P   ISS1_1  ISS1_2P  ISS1_2  ISS1_3P  ISS1_3  ISS1_4P  ISS1_4  ISS1_5P  ISS1_5  ISS1_6P  ISS1_6  ISS1_7P  ISS1_7  ISS1_8P  ISS1_8  ISS2_1P  ISS2_1  ISS2_2P  ISS2_2  ISS2_3P  ISS2_3  ISS2_4P  ISS2_4  ISS2_5P  ISS2_5  ISS2_6P  ISS2_6  ISS2_7P  ISS2_7  ISS2_8P  ISS2_8