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. 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. 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.

The International Space Station (ISS) is a unique built environment due to the effects of microgravity, space radiation, elevated carbon dioxide levels, and especially continuous human habitation. Understanding the composition of the ISS microbial community will facilitate further development of safety and maintenance practices. The primary goal of this study was to characterize the viable microbiome of the ISS-built environment. A second objective was to determine if the built environments of Earth-based cleanrooms associated with space exploration are an appropriate model of the ISS environment. Samples collected from the ISS and two cleanrooms at the Jet Propulsion Laboratory (JPL, Pasadena, CA) were analyzed by traditional cultivation, adenosine triphosphate (ATP), and propidium monoazide–quantitative polymerase chain reaction (PMA-qPCR) assays to estimate viable microbial populations. The 16S rRNA gene Illumina iTag sequencing was used to elucidate microbial diversity and explore differences between ISS and cleanroom microbiomes. Statistical analyses showed that members of the phyla Actinobacteria, Firmicutes, and Proteobacteria were dominant in the samples examined but varied in abundance. Actinobacteria were predominant in the ISS samples whereas Proteobacteria, least abundant in the ISS, dominated in the cleanroom samples. The viable bacterial populations seen by PMA treatment were greatly decreased. However, the treatment did not appear to have an effect on the bacterial composition (diversity) associated with each sampling site. The results of this study provide strong evidence that specific human skin-associated microorganisms make a substantial contribution to the ISS microbiome, which is not the case in Earth-based cleanrooms. For example, Corynebacterium and Propionibacterium (Actinobacteria) but not Staphylococcus (Firmicutes) species are dominant on the ISS in terms of viable and total bacterial community composition. The results obtained will facilitate future studies to determine how stable the ISS environment is over time. The present results also demonstrate the value of measuring viable cell diversity and population size at any sampling site. This information can be used to identify sites that can be targeted for more stringent cleaning. Finally, the results will allow comparisons with other built sites and facilitate future improvements on the ISS that will ensure astronaut health.

In assessing the bacterial populations present in spacecraft assembly, spacecraft test, and launch preparation facilities, extremophilic bacteria (requiring severe conditions for growth) and extremotolerant bacteria (tolerant to extreme conditions) were isolated. Several cultivation approaches were employed to select for and identify bacteria that not only survive the nutrient-limiting conditions of clean room environments but can also withstand even more inhospitable environmental stresses. Due to their proximity to spacefaring objects, these bacteria pose a considerable risk for forward contamination of extraterrestrial sites. Samples collected from four geographically distinct National Aeronautics and Space Administration clean rooms were challenged with UV-C irradiation, 5% hydrogen peroxide, heat shock, pH extremes (pH 3.0 and 11.0), temperature extremes (4°C to 65°C), and hypersalinity (25% NaCl) prior to and/or during cultivation as a means of selecting for extremotolerant bacteria. Culture-independent approaches were employed to measure viable microbial (ATP-based) and total bacterial (quantitative PCR-based) burdens. Intracellular ATP concentrations suggested a viable microbial presence ranging from below detection limits to 10⁶cells/m². However, only 0.1 to 55% of these viable cells were able to grow on defined culture medium. Isolated members of theBacillaceaefamily were more physiologically diverse than those reported in previous studies, including thermophiles (Geobacillus), obligate anaerobes (Paenibacillus), and halotolerant, alkalophilic species (OceanobacillusandExiguobacterium). Non-spore-forming microbes (α- and β-proteobacteria and actinobacteria) exhibiting tolerance to the selected stresses were also encountered. The multiassay cultivation approach employed herein enhances the current understanding of the physiological diversity of bacteria housed in these clean rooms and leads us to ponder the origin and means of translocation of thermophiles, anaerobes, and halotolerant alkalophiles into these environments.

Microbiome analyses of low-biomass samples are challenging because of contamination and inefficiencies, leading many investigators to employ low-throughput methods with minimal controls. We developed a new automated protocol, KatharoSeq (from the Greek katharos [clean]), that outperforms single-tube extractions while processing at least five times as fast. KatharoSeq incorporates positive and negative controls to reveal the whole bacterial community from inputs of as few as 50 cells and correctly identifies 90.6% (standard error, 0.013%) of the reads from 500 cells. To demonstrate the broad utility of KatharoSeq, we performed 16S rRNA amplicon and shotgun metagenome analyses of the Jet Propulsion Laboratory spacecraft assembly facility (SAF; n = 192, 96), 52 rooms of a neonatal intensive care unit (NICU; n = 388, 337), and an endangered-abalone-rearing facility (n = 192, 123), obtaining spatially resolved, unique microbiomes reproducible across hundreds of samples. The SAF, our primary focus, contains 32 sOTUs (sub-OTUs, defined as exact sequence matches) and their inferred variants identified by the deblur algorithm, with four (Acinetobacter lwoffii, Paracoccus marcusii, Mycobacterium sp., and Novosphingobium) being present in >75% of the samples. According to microbial spatial topography, the most abundant cleanroom contaminant, A. lwoffii, is related to human foot traffic exposure. In the NICU, we have been able to discriminate environmental exposure related to patient infectious disease, and in the abalone facility, we show that microbial communities reflect the marine environment rather than human input. Consequently, we demonstrate the feasibility and utility of large-scale, low-biomass metagenomic analyses using the KatharoSeq protocol. IMPORTANCE Various indoor, outdoor, and host-associated environments contain small quantities of microbial biomass and represent a niche that is often understudied because of technical constraints. Many studies that attempt to evaluate these low-biomass microbiome samples are riddled with erroneous results that are typically false positive signals obtained during the sampling process. We have investigated various low-biomass kits and methods to determine the limit of detection of these pipelines. Here we present KatharoSeq, a high-throughput protocol combining laboratory and bioinformatic methods that can differentiate a true positive signal in samples with as few as 50 to 500 cells. We demonstrate the application of this method in three unique low-biomass environments, including a SAF, a hospital NICU, and an abalone-rearing facility.

The International Space Station (ISS) is an ideal test bed for studying the effects of microbial persistence and succession on a closed system during long space flight. Culture-based analyses, targeted gene-based amplicon sequencing (bacteriome, mycobiome, and resistome), and shotgun metagenomics approaches have previously been performed on ISS environmental sample sets using whole genome amplification (WGA). However, this is the first study reporting on the metagenomes sampled from ISS environmental surfaces without the use of WGA. Metagenome sequences generated from eight defined ISS environmental locations in three consecutive flights were analyzed to assess the succession and persistence of microbial communities, their antimicrobial resistance (AMR) profiles, and virulence properties. Metagenomic sequences were produced from the samples treated with propidium monoazide (PMA) to measure intact microorganisms. The intact microbial communities detected in Flight 1 and Flight 2 samples were significantly more similar to each other than to Flight 3 samples. Among 318 microbial species detected, 46 species constituting 18 genera were common in all flight samples. Risk group or biosafety level 2 microorganisms that persisted among all three flights were Acinetobacter baumannii, Haemophilus influenzae, Klebsiella pneumoniae, Salmonella enterica, Shigella sonnei, Staphylococcus aureus, Yersinia frederiksenii, and Aspergillus lentulus. Even though Rhodotorula and Pantoea dominated the ISS microbiome, Pantoea exhibited succession and persistence. K. pneumoniae persisted in one location (US Node 1) of all three flights and might have spread to six out of the eight locations sampled on Flight 3. The AMR signatures associated with β-lactam, cationic antimicrobial peptide, and vancomycin were detected. Prominent virulence factors were cobalt-zinc-cadmium resistance and multidrug-resistance efflux pumps. There was an increase in AMR and virulence gene factors detected over the period sampled, and metagenome sequences of human pathogens persisted over time. Comparative analysis of the microbial compositions of ISS with Earth analogs revealed that the ISS environmental surfaces were different in microbial composition. Metagenomics coupled with PMA treatment would help future space missions to estimate problematic risk group microbial pathogens. Cataloging AMR/virulence characteristics, succession, accumulation, and persistence of microorganisms would facilitate the development of suitable countermeasures to reduce their presence in the closed built environment.