Modeling of recovery efficiency of sampling devices used in planetary protection bioburden estimation.

IMPORTANCE: Planetary protection at the National Aeronautics and Space Administration (NASA) requires bioburden on certain spacecraft to be estimated via sampling in order to comply with biological cleanliness requirements. To achieve this, the recovery efficiency of devices used to sample the spacecraft pre-launch must be understood and their uncertainty quantified in order to produce the most reasonable estimates of bioburden. This study brings together experiments performed by NASA and the European Space Agency with approved swab and wipe sampling devices, inoculating steel coupons with laboratory strains of Bacillus spp. spores commonly recovered from spacecraft assembly clean rooms (B. atrophaeus, B. megaterium, B. safensis and B. thuringiensis), with a mathematical model of the assay process to assess recovery efficiency. The statistical treatment developed in this study allows comparison of bioburden estimates made from different devices processed by different methods. This study also gives stakeholders and practitioners a statistically rigorous approach to predict bioburden that can be folded into future modeling efforts.

Multi-faceted metagenomic analysis of spacecraft associated surfaces reveal planetary protection relevant microbial composition.

The National Aeronautics and Space Administration (NASA) has been monitoring the microbial burden of spacecraft since the 1970's Viking missions. Originally culture-based and then focused 16S sequencing techniques were used, but we have now applied whole metagenomic sequencing to a variety of cleanroom samples at the Jet Propulsion Lab (JPL), including the Spacecraft Assembly Facility (SAF) with the goals of taxonomic identification and for functional assignment. Our samples included facility pre-filters, cleanroom vacuum debris, and surface wipes. The taxonomic composition was carried out by three different analysis tools to contrast marker, k-mer, and true alignment approaches. Hierarchical clustering analysis of the data separated vacuum particles from other SAF DNA samples. Vacuum particle samples were the most diverse while DNA samples from the ISO (International Standards Organization) compliant facilities and the SAF were the least diverse; all three were dominated by Proteobacteria. Wipe samples had higher diversity and were predominated by Actinobacteria, including human commensals Cutibacterium acnes and Corynebacterium spp. Taxa identified by the three methods were not identical, supporting the use of multiple methods for metagenome characterization. Likewise, functional annotation was performed using multiple methods. Vacuum particles and SAF samples contained strong signals of the tricarboxylic acid cycle and of amino acid biosynthesis, suggesting that many of the identified microorganisms have the ability to grow in nutrient-limited environments. In total, 18 samples generated high quality metagenome assembled genomes (MAG), which were dominated by Moraxella osloensis or Malassezia restricta. One M. osloensis MAG was assembled into a single circular scaffold and gene annotated. This study includes a rigorous quantitative determination of microbial loads and a qualitative dissection of microbial composition. Assembly of multiple specimens led to greater confidence for the identification of particular species and their predicted functional roles.

Draft Genome Sequences of Spacecraft-Associated Microbes Isolated from Six NASA Missions.

Whole-genome sequencing can be used to better understand and assess the functional abilities of microorganisms isolated from spacecraft hardware and associated surfaces for planetary protection (PP) purposes. We sequenced 191 isolates from 6 spaceflight missions with PP requirements and identified them using Illumina-based sequencing methods and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.

Draft Genome Sequences of Fungi Isolated from Mars 2020 Spacecraft Assembly Facilities.

During the Mars 2020 mission, several fungal strains were isolated from surfaces where spacecraft components were assembled. Draft genome sequencing and characterization will help identify the genes responsible for radiation resistance, supporting the development of countermeasures to prevent fungal contamination of extraterrestrial environments.

Performance of Multiple Metagenomics Pipelines in Understanding Microbial Diversity of a Low-Biomass Spacecraft Assembly Facility.

NASA planetary protection (PP) requires an assessment of the biological contamination of the potential microbial burden on spacecraft destined to explore planetary bodies that may harbor signs of life, like Mars and Europa. To help meet these goals, the performance of multiple metagenomic pipelines were compared and assessed for their ability to detect microbial diversity of a low-biomass clean room environment used to build spacecraft destined to these planetary bodies. Four vendors were chosen to implement their own metagenomic analysis pipeline on the shotgun sequences retrieved from environmental surfaces in the relevant environments at NASA's Jet Propulsion Laboratory. None of the vendors showed the same microbial profile patterns when analyzing same raw dataset since each vendor used different pipelines, which begs the question of the validity of a single pipeline to be recommended for future NASA missions. All four vendors detected species of interest, including spore-forming and extremotolerant bacteria, that have the potential to hitch-hike on spacecraft and contaminate the planetary bodies explored. Some vendors demonstrated through functional analysis of the metagenomes that the molecular mechanisms for spore-formation and extremotolerance were represented in the data. However, relative abundances of these microorganisms varied drastically between vendor analyses, questioning the ability of these pipelines to quantify the number of PP-relevant microorganisms on a spacecraft surface. Metagenomics offers tantalizing access to the genetic and functional potential of a microbial community that may offer NASA a viable method for microbial burden assays for planetary protection purposes. However, future development of technologies such as streamlining the processing of shotgun metagenome sequence data, long read sequencing, and all-inclusive larger curated and annotated microbial genome databases will be required to validate and translate relative abundances into an actionable assessment of PP-related microbes of interest. Additionally, the future development of machine learning and artificial intelligence techniques could help enhance the quality of these metagenomic analyses by providing more accurate identification of the genetic and functional potential of a microbial community.

Application of a Bayesian Statistical Framework for Planetary Protection as a Means of Verifying Low-Biomass, Zero-Inflated Test Data from Spacecraft.

Planetary Protection is applicable for missions to biologically sensitive targets of interest in the solar system. For robotic missions landing on the Martian surface, Earth-based biological contamination must be reduced, controlled, and monitored to adhere to forward planetary protection requirements. To address the overall biological load limit and microbial density requirements per spacecraft each component is tracked based on its manufacturing pedigree and/or directly assessed using a direct sampling technique with either a swab or wipe. The tracking and reporting of requirements compliance has varied from mission to mission and reporting of numbers has consistently leaned towards the conservative worst-case scenario. With an increase in the number of missions and mission complexities, the need to establish a technically sound, statistical, and biological solution that provides a single point solution which addresses the distribution of spacecraft contamination becomes critical. Select components of the InSight mission, launched in 2018, have been used as a test case to evaluate the efficacy of applying Bayesian statistics to planetary protection data sets. Eight representative components covering the various bounding cases of high and low surface area, biological count, and sampling devices were analyzed as well as an assembly level case to evaluate the rollup of directly sampled and manufacturing pedigree components. A Bayesian approach was developed leveraging different priors from the zero-inflated data sets and compared to the heritage and existing NASA bioburden assessment approaches. In addition, several non-informative priors were evaluated for use in performing bioburden calculations. The results have demonstrated a viable framework to enable a Bayesian statistical approach to be further developed and utilized for planetary protection requirements assessment.

A comprehensive metagenomics framework to characterize organisms relevant for planetary protection.

BACKGROUND: Clean rooms of the Space Assembly Facility (SAF) at the Jet Propulsion Laboratory (JPL) at NASA are the final step of spacecraft cleaning and assembly before launching into space. Clean rooms have stringent methods of air-filtration and cleaning to minimize microbial contamination for exoplanetary research and minimize the risk of human pathogens, but they are not sterile. Clean rooms make a selective environment for microorganisms that tolerate such cleaning methods. Previous studies have attempted to characterize the microbial cargo through sequencing and culture-dependent protocols. However, there is not a standardized metagenomic workflow nor analysis pipeline for spaceflight hardware cleanroom samples to identify microbial contamination. Additionally, current identification methods fail to characterize and profile the risk of low-abundance microorganisms. RESULTS: A comprehensive metagenomic framework to characterize microorganisms relevant for planetary protection in multiple cleanroom classifications (from ISO-5 to ISO-8.5) and sample types (surface, filters, and debris collected via vacuum devices) was developed. Fifty-one metagenomic samples from SAF clean rooms were sequenced and analyzed to identify microbes that could potentially survive spaceflight based on their microbial features and whether the microbes expressed any metabolic activity or growth. Additionally, an auxiliary testing was performed to determine the repeatability of our techniques and validate our analyses. We find evidence that JPL clean rooms carry microbes with attributes that may be problematic in space missions for their documented ability to withstand extreme conditions, such as psychrophilia and ability to form biofilms, spore-forming capacity, radiation resistance, and desiccation resistance. Samples from ISO-5 standard had lower microbial diversity than those conforming to ISO-6 or higher filters but still carried a measurable microbial load. CONCLUSIONS: Although the extensive cleaning processes limit the number of microbes capable of withstanding clean room condition, it is important to quantify thresholds and detect organisms that can inform ongoing Planetary Protection goals, provide a biological baseline for assembly facilities, and guide future mission planning. Video Abstract.

End-to-End Protocol for the Detection of SARS-CoV-2 from Built Environments.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is a respiratory virus primarily transmitted person to person through inhalation of droplets or aerosols, laden with viral particles. However, as recent studies have shown, virions can remain infectious for up to 72 h on surfaces, which can lead to transmission through contact. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end (E2E) study showed that the effective combination for monitoring SARS-CoV-2 on surfaces includes using an Isohelix swab collection tool, DNA/RNA Shield as a preservative, an automated system for RNA extraction, and reverse transcriptase quantitative PCR (RT-qPCR) as the detection assay. Using this E2E approach, this study showed that, in some cases, noninfectious viral fragments of SARS-CoV-2 persisted on surfaces for as long as 8 days even after bleach treatment. Additionally, debris associated with specific built environment surfaces appeared to inhibit and negatively impact the recovery of RNA; Amerstat demonstrated the highest inhibition (>90%) when challenged with an inactivated viral control. Overall, it was determined that this E2E protocol required a minimum of 1,000 viral particles per 25 cm2 to successfully detect virus from test surfaces. Despite our findings of viral fragment longevity on surfaces, when this method was employed to evaluate 368 samples collected from various built environmental surfaces, all samples tested negative, indicating that the surfaces were either void of virus or below the detection limit of the assay.IMPORTANCE The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the virus responsible for coronavirus disease 2019 [COVID-19]) pandemic has led to a global slowdown with far-reaching financial and social impacts. The SARS-CoV-2 respiratory virus is primarily transmitted from person to person through inhalation of infected droplets or aerosols. However, some studies have shown that virions can remain infectious on surfaces for days and can lead to human infection from contact with infected surfaces. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end study showed that the effective combination for monitoring SARS-CoV-2 on surfaces required a minimum of 1,000 viral particles per 25 cm2 to successfully detect virus from surfaces. This comprehensive study can provide valuable information regarding surface monitoring of various materials as well as the capacity to retain viral RNA and allow for effective disinfection.

Planetary Protection Implementation of the InSight Mission Launch Vehicle and Associated Ground Support Hardware.

The InSight (Interior Exploration using Seismic Investigations, Geodesy and Heat Transport) Mars mission launched from Vandenberg Air Force Base on an Atlas V 401 rocket on May 5, 2018. Prior to launch, the InSight spacecraft, associated launch vehicle hardware, and ground support equipment were required to satisfy Planetary Protection requirements to comply with international treaty obligations and demonstrate compliance with National Aeronautics and Space Administration (NASA) levied bioburden requirements. InSight was the first bioburden-controlled mission to launch from Vandenberg Air Force Base and required mission-unique policies and procedures to ensure Planetary Protection requirements were satisfied. All the launch vehicle hardware and associated ground support equipment with direct contact or line of sight to flight hardware were required to demonstrate a bioburden density of less than 1,000 spores/m2. Additionally, the environmental control system air ducts were required to demonstrate more stringent bioburden limits on internal duct surfaces (<100 spore/m2) and on air passing through the ducts (88 colony-forming units/m3). Although conservative approaches were used with the data analysis and launch recontamination analysis, InSight, the launch vehicle hardware, and ground support equipment were able to demonstrate compliance with the Planetary Protection requirements needed for launch approval. Here we detail the biological practices implemented on the launch vehicle hardware and ground support equipment that resulted in biologically clean hardware and the satisfaction of Planetary Protection.

Draft Genome Sequences of Bacillus glennii V44-8, Bacillus saganii V47-23a, Bacillus sp. Strain V59.32b, Bacillus sp. Strain MER_TA_151, and Paenibacillus sp. Strain MER_111, Isolated from Cleanrooms Where the Viking and Mars Exploration Rover Spacecraft Were Assembled.

We report the draft genome sequences of Bacillus glennii V44-8, Bacillus saganii V47-23a, and Bacillus sp. strain V59.32b, isolated from the Viking spacecraft assembly cleanroom, and Bacillus sp. strain MER_TA_151 and Paenibacillus sp. strain MER_111, isolated from the Mars Exploration Rover (MER) assembly cleanroom.

Draft Genome Sequences of Six Strains Isolated from the InSight Spacecraft and Associated Surfaces Using Oxford Nanopore- and Illumina-Based Sequencing.

Whole-genome sequencing and annotation have allowed planetary protection engineers to assess the functional capabilities of microorganisms isolated from spacecraft hardware and associated surfaces. Here, we report draft genomes of six strains isolated from the InSight mission, determined using Oxford Nanopore- and Illumina-based sequencing.

Bacillus glennii sp. nov. and Bacillus saganii sp. nov., isolated from the vehicle assembly building at Kennedy Space Center where the Viking spacecraft were assembled.

Two Gram-stain-positive, motile, endospore-forming, aerobic strains, designated V44-8T and V47-23aT, were isolated from environmental air sampling at the vehicle assembly building at Cape Canaveral, Florida, where the Viking spacecraft were assembled. Growth was observed at pH 7-9 (optimum, pH 9) for strain V44-8T, and pH 5-10 (pH 9) for strain V47-23aT. Both strains displayed growth in 0-5 % NaCl with an optimum at 1 % for strain V44-8T; 0 % for strain V47-23aT. Strains V44-8T and V47-23aT grew optimally at 32 °C, (15-32 °C) and 25 °C (20-45 °C), respectively. The cell wall of both strains contained meso-diaminopimelic acid as the diagnostic diamino acid. Both strains contained phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. The predominant cellular fatty acids were anteiso-C15 : 0, iso-C14 : 0 and iso-C15 : 0. Strain V47.23aT shared its highest 16S rRNA sequence similarity with Bacillus cavernae DSM-105484T at 96.9%, and V44.8T with Bacillus zeae DSM-103964T at 96.6 %. Based on their phenotypic characteristics and phylogenetic position inferred from 16S rRNA gene sequence analyses, the isolates were identified as being a members of the genus Bacillus that forms a separate clade when compared to close relatives. Average nucleotide identity and average amino acid identity values between strains V44-8T and DSM-103964T were 72.1% and 67.5 %; V47-23aT and DSM-105484T were 62.4% and 69.1%, respectively. Based on the phenotypic, genomic and biochemical data, strains V44-8T and V47-23aT represent two novel species in the genus Bacillus for which the names Bacillus glennii sp. nov. [type strain, V44-8T (=ATCC BAA-2860T =DSM 105192T)], and Bacillus saganii sp. nov. [V47-23aT (=ATCC BAA-2861T=DSM 105190T)] are proposed.

Survival of Extremotolerant Bacteria from the Mukundpura Meteorite Impact Crater.

Carbonaceous meteorites provide clues with regard to prebiotic chemistry and the origin of life. Geological Survey of India recorded a carbonaceous chondrite meteorite fall in Mukundpura, India, on June 6, 2017. We conducted a study to investigate the microbial community that survived the meteorite impact. 16S rRNA metagenomic sequencing indicates the presence of Actinobacteria, Proteobacteria, and Acidobacteria in meteorite impact soil. Comparative phylogenetic analysis revealed an intriguing abundance of class Bacilli in the impact soil. Bacillus thermocopriae IR-1, a moderately thermotolerant organism, was isolated from a rock, impacted by the Mukundpura meteorite. We investigated the resilience of B. thermocopriae IR-1 to environmental stresses and impact shock in a Reddy shock tube. Bacillus thermocopriae IR-1 survived (28.82% survival) the effect of shock waves at a peak shock pressure of 300 kPa, temperature 400 K, and Mach number of 1.47. This investigation presents the first report on the effect of impact shock on B. thermocopriae IR-1. The study is also the first report on studying the microbial diversity and isolation of bacteria from impact crater soil immediately after meteorite impact event.

Paenibacillus xerothermodurans sp. nov., an extremely dry heat resistant spore forming bacterium isolated from the soil of Cape Canaveral, Florida.

A Gram-stain-positive, motile, endospore-producing, facultative anaerobic bacterial strain, designated ATCC 27380T, was isolated from heat-stressed soil of Cape Canaveral, Florida, USA. Growth was observed at 20-42 °C (optimum, 37 °C), at pH 6.0-10.0 (optimum pH 7.0) and in the presence of 0.5-3 % NaCl (optimum 0.5 %). The cell wall contained meso-diaminopimelic acid as the diagnostic amino acid and the isoprenoid quinone was MK-7. The polar lipids present were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and one unknown phospholipid. The main fatty acids were iso-C15 : 0 and anteiso-C15 : 0. Phylogenetic analysis based on 16S rRNA gene sequencing affiliated strain ATCC 27380T to the genus Paenibacillus, and showed the highest sequence similarity to Paenibacillus rigui JCM 16352T (97.0 %). The other closely related type strains exhibited 16S rRNA gene sequence similarity values below 95.9 %. The draft genome of ATCC 27380T had a size of 4,361,187 bases, with a G+C content of 51.0 %. The average nucleotide identity and in silico DNA-DNA hybridization values between strain ATCC 27380T and P. rigui JCM 16352T were 72.5% and 18.5 %, respectively, which were below the threshold suggested for species differentiation (96% and 70 %, respectively). The average amino acid identity between strain ATCC 27380T and P. rigui JCM 16352T was 68.72 %, which was above the suggested genus level demarcation of 65 %. Based on phenotypic, genotypic and chemotaxonomic data, strain ATCC 27380T represents a novel species in the genus Paenibacillus, for which the name Paenibacillusxerothermodurans sp. nov. (=DSM 520T=NRRL NRS-1629T=ATCC 27380T) is proposed.

Taxonomic description and draft genome of Pseudomonas sediminis sp. nov., isolated from the rhizospheric sediment of Phragmites karka.

The taxonomic position of a Gram-stain-negative, rod-shaped bacterial strain, designated PI11T, isolated from the rhizospheric sediment of Phragmites karka was characterized using a polyphasic approach. Strain PI11T could grow optimally at 1.0% NaCl concentration with pH 7.0 at 30°C and was positive for oxidase and catalase but negative for hydrolysis of starch, casein, and esculin ferric citrate. Phylogenetic analysis of 16S rRNA gene sequences indicated that the strain PI11T belonged to the genus Pseudomonas sharing the highest sequence similarities with Pseudomonas indoloxydans JCM 14246T (99.72%), followed by, Pseudomonas oleovorans subsp. oleovorans DSM 1045T (99.29%), Pseudomonas toyotomiensis JCM 15604T (99.15%), Pseudomonas chengduensis DSM 26382T (99.08%), Pseudomonas oleovorans subsp. lubricantis DSM 21016T (99.08%), and Pseudomonas alcaliphila JCM 10630T (99.01%). Experimental DNA-DNA relatedness between strain PI11T and P. indoloxydans JCM 14246T was 49.4%. The draft genome of strain PI11T consisted of 4,884,839 bp. Average nucleotide identity between the genome of strain PI11T and other closely related type strains ranged between 77.25-90.74%. The polar lipid pattern comprised of phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylcholine. The major (> 10%) cellular fatty acids were C18:1ω6c/ω7c, C16:1ω6c/ω7c, and C16:0. The DNA G + C content of strain PI11T was 62.4 mol%. Based on the results of polyphasic analysis, strain PI11T was delineated from other closely related type strains. It is proposed that strain PI11T represents represents a novel species of the genus Pseudomonas, for which the name Pseudomonas sediminis sp. nov. is proposed. The type strain is PI11T (= KCTC 42576T = DSMZ 100245T).

Development of a Custom MALDI-TOF MS Database for Species-Level Identification of Bacterial Isolates Collected From Spacecraft and Associated Surfaces.

Since the 1970s, the Planetary Protection Group at the Jet Propulsion Laboratory (JPL) has maintained an archive of spacecraft-associated bacterial isolates. Identification of these isolates was routinely performed by sequencing the 16S rRNA gene. Although this technique is an industry standard, it is time consuming and has poor resolving power for some closely related taxa. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry is widely used in clinical diagnostics and is a promising method to substitute standard 16S rRNA sequencing. However, manufacturer-provided databases lack the bacterial diversity found in spacecraft-assembly cleanrooms. This study reports the development of the first custom database of MALDI-TOF MS profiles of bacterial isolates obtained from spacecraft and associated cleanroom environments. With the use of this in-house database, 454 bacterial isolates were successfully identified in concurrence with their 16S rRNA sequence-based classifications. Additionally, MALDI-TOF MS resolved strain-level variations, identified potential novel species and distinguished between members of taxonomic groups, which is not possible using conventional 16S rRNA sequencing. MALDI-TOF MS has proved to be an accurate, high-throughput approach for real-time identification of bacterial isolates during the spacecraft assembly process.

Draft Genome Sequences of 12 Dry-Heat-Resistant Bacillus Strains Isolated from the Cleanrooms Where the Viking Spacecraft Were Assembled.

Spore-forming microorganisms are of concern for forward contamination because they can survive harsh interplanetary travel. Here, we report the draft genome sequences of 12 spore-forming strains isolated from the Manned Spacecraft Operations Building (MSOB) and the Vehicle Assembly Building (VAB) in Cape Canaveral, FL, where the Viking spacecraft were assembled.

Draft Genome Sequences of Acinetobacter and Bacillus Strains Isolated from Spacecraft-Associated Surfaces.

We report here the draft genome sequences of four strains isolated from spacecraft-associated surfaces exhibiting increased resistance to stressors such as UV radiation and exposure to H2O2 The draft genomes of strains 1P01SCT, FO-92T, 50v1, and 2P01AA had sizes of 5,500,894 bp, 4,699,376 bp, 3,174,402 bp, and 4,328,804 bp, respectively.

Draft Genome Sequence of Solibacillus kalamii, Isolated from an Air Filter Aboard the International Space Station.

We report here the draft genome of Solibacillus kalamii ISSFR-015, isolated from a high-energy particulate arrestance filter aboard the International Space Station. The draft genome sequence of this strain contains 3,809,180 bp with an estimated G+C content of 38.61%.