Clean room microbiome complexity impacts planetary protection bioburden

Ryan Hendrickson,Rob Knight,Camilla Urbaniak,Heidi S Aronson,Ramunas Stepanauskas,Kasthuri Venkateswaran,Cameron Martino,Jeremiah J Minich

doi:10.1186/s40168-021-01159-x

Abstract

BackgroundThe 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.ResultsCleanroom 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.ConclusionThis 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.-vsg9JJhywtr3shQUX5nF9Video abstract

Highlights

Planetary protection (PP) is a scientific discipline aimed at preventing microbial contamination on spacecraft outbound to a celestial body and on spacecraft hardware and samples returning to Earth to avoid harmful contamination to a native environment or compromising scientific data
Sanger sequencing of the full-length 16S rRNA gene on the NASA standard assay (NSA) isolates from the 98 samples resulted in 130 isolates, belonging to 16 genera and 49 species (Fig. 2). 97% of these isolates (126/130) belonged to species of Bacillus, Brevibacillus, Cohnella, Gracilibacillus, Oceanobacillus, Paenibacillus, Psychrobacillus, Rummeliibacillus, Sporosarcina, Streptomyces, Terribacillus, and Virgibacillus, which are commonly considered spore-formers
The remaining ~3% of the isolates cultured from the NSA (4/130) were non-spore forming bacteria consisting of Brevibacterium luteolum, Massilia consociata, Micrococcus yunnanensis, and Staphylococcus haemolyticus

Summary

Introduction

Planetary protection (PP) is a scientific discipline aimed at preventing microbial contamination on spacecraft outbound to a celestial body (forward planetary protection) and on spacecraft hardware and samples returning to Earth (backward planetary protection) to avoid harmful contamination to a native environment or compromising scientific data. The presence of other spore-formers (i.e., non-NSA spore-formers) and the remaining viable microbial population that are relevant to PP are underestimated [9, 17, 20] These underestimated microbial populations can be determined via culture-independent molecular methods [21] or approximated using a conversion ratio between the NSA sporeformers and the viable microbial population. A recent publication of the SAF cleanroom environment revealed that the ratio of NSA spores to viable microbial cells was in the range of 150 to 12,000 depending on the methodology used [23] This earlier SAF report quantitatively measured the microbial burden in SAF, additional research was required to gain a more thorough understanding of the microbial community composition of the SAF. 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

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