Qualification of Membrane Filtration for Planetary Protection Flight Implementation.

Planetary protection is the practice of preventing forward and backward contamination of solar system bodies. Spacecraft and associated surfaces are sampled to ensure compliance with bioburden requirements. Current planetary protection sampling and processing methodologies consist of extracting microbial cells from wipe or swab samples through a procedure (NASA Standard Assay) that includes sonication, heat shock, and pour-plate steps. The pour-plate steps are laborious and prolonged. Moreover, results can be imprecise because only a fraction of the sample fluid is plated for CFU enumeration (80% for swabs and 25% for wipes). Thus, analysis requires that a pour fraction extrapolation factor be applied to CFU counts to account for bioburden in the remaining sample volume that is not plated. This extrapolation results in large variances for data, decreasing the accuracy of spore bioburden estimation of spacecraft hardware. In this study, we investigated the use of membrane filtration as an alternative method to pour-plate processing. Membrane filtration is an appealing methodology for planetary protection because it can process greater sample volumes and reduces the data variance for bioburden enumeration. A pour fraction extrapolation factor is still applied for both swabs and wipes (92%), however, it is a greater pour fraction than the pour-plate method. Here we present data collected by the Jet Propulsion Laboratory and the Applied Physics Laboratory to experimentally determine the equivalency of membrane filtration to pour-plate methodology for implementation during the NASA Standard Assay. Additionally, we outline the planned procedures for two membrane filtration systems: Pall® Laboratory Manifold system and Milliflex® Plus Vacuum Pump System. Both systems demonstrated equivalence of the membrane filtration method to the pour-plate method.

Clean room microbiome complexity impacts planetary protection bioburden.

BACKGROUND: The Spacecraft Assembly Facility (SAF) at the NASA's Jet Propulsion Laboratory is the primary cleanroom facility used in the construction of some of the planetary protection (PP)-sensitive missions developed by NASA, including the Mars 2020 Perseverance Rover that launched in July 2020. SAF floor samples (n=98) were collected, over a 6-month period in 2016 prior to the construction of the Mars rover subsystems, to better understand the temporal and spatial distribution of bacterial populations (total, viable, cultivable, and spore) in this unique cleanroom. RESULTS: Cleanroom samples were examined for total (living and dead) and viable (living only) microbial populations using molecular approaches and cultured isolates employing the traditional NASA standard spore assay (NSA), which predominantly isolated spores. The 130 NSA isolates were represented by 16 bacterial genera, of which 97% were identified as spore-formers via Sanger sequencing. The most spatially abundant isolate was Bacillus subtilis, and the most temporally abundant spore-former was Virgibacillus panthothenticus. The 16S rRNA gene-targeted amplicon sequencing detected 51 additional genera not found in the NSA method. The amplicon sequencing of the samples treated with propidium monoazide (PMA), which would differentiate between viable and dead organisms, revealed a total of 54 genera: 46 viable non-spore forming genera and 8 viable spore forming genera in these samples. The microbial diversity generated by the amplicon sequencing corresponded to ~86% non-spore-formers and ~14% spore-formers. The most common spatially distributed genera were Sphinigobium, Geobacillus, and Bacillus whereas temporally distributed common genera were Acinetobacter, Geobacilllus, and Bacillus. Single-cell genomics detected 6 genera in the sample analyzed, with the most prominent being Acinetobacter. CONCLUSION: This study clearly established that detecting spores via NSA does not provide a complete assessment for the cleanliness of spacecraft-associated environments since it failed to detect several PP-relevant genera that were only recovered via molecular methods. This highlights the importance of a methodological paradigm shift to appropriately monitor bioburden in cleanrooms for not only the aeronautical industry but also for pharmaceutical, medical industries, etc., and the need to employ molecular sequencing to complement traditional culture-based assays. Video abstract.

Draft Genome Sequences of Heat Shock-Tolerant Microbes Isolated from a Spacecraft Assembly Facility.

Heat shock-tolerant microorganisms belonging to the orders Bacillales and Micrococcales were isolated from the Spacecraft Assembly Facility at the Jet Propulsion Laboratory, and 63 draft genome sequences were assembled and identified. Further analyses of these genomes can provide insight into methods for preventing forward contamination.

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.

Planetary Protection Implementation on the Interior Exploration Using Seismic Investigations, Geodesy and Heat Transport Mission.

The InSight (Interior Exploration using Seismic Investigations, Geodesy and Heat Transport) Mars flight system is made up of a cruise stage, an aeroshell, and a Phoenix heritage-based lander that successfully landed in the Elysium Planitia on November 26, 2018. InSight has primary science objectives that are aimed at understanding more about the interior of Mars and the formation and evolution of terrestrial planets. As with previous Mars missions, InSight was obligated to satisfy bioburden requirements and verify compliance with international treaty obligations and National Aeronautics and Space Administration requirements before launch. InSight implemented a series of policies and procedures to minimize biological risk and comply with all applicable requirements for a Planetary Protection Category IVa mission. At launch, InSight had an estimated total bacterial spore burden of 1.50 × 105 spores on the entire spacecraft, including the cruise stage, satisfying the requirement of 5 × 105 spores. The landed hardware components required <3 × 105 spores and a bioburden density of <300 spores/m2. At launch, the landed hardware of InSight contained an estimated total spore bioburden of 1.35 × 105 spores and an estimated bioburden density of 129 spores/m2.

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.

KatharoSeq Enables High-Throughput Microbiome Analysis from Low-Biomass Samples.

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.