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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 wasBacillus subtilis, and the most temporally abundant spore-former wasVirgibacillus 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 wereSphinigobium,Geobacillus, andBacilluswhereas temporally distributed common genera wereAcinetobacter,Geobacilllus, andBacillus. Single-cell genomics detected 6 genera in the sample analyzed, with the most prominent beingAcinetobacter. 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.
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This content will become publicly available on August 11, 2026
Tersicoccus phoenicis (Actinobacteria), a spacecraft clean room isolate, exhibits dormancy
ABSTRACT Space missions or spacecraft equipment destined for sensitive environments, such as Mars, Europa, or Enceladus, are required to be designed to avoid forward contamination. Spacecraft are assembled in clean rooms (SACs) employing treatments to eliminate microbial contamination. However, some organisms can survive the cleaning procedures. Characterization of these populations, through both culture-based and sequencing methods, reveals that the majority consists of spore-forming bacteria. However, a smaller group of non-spore-forming organisms, primarily classified within the orderMicrococcalesof the phylumActinobacteria(Actinomycetota), exists in some SACs. Despite their repeated occurrence and isolation, actinobacterial strains associated with SACs have not been studied for their dormancy potential. Here, we show for the first time that a non-spore-forming SAC isolate,Tersicoccus phoenicis(Micrococcales), enters dormancy under nutrient starvation. Dormancy inMicrococcus luteusinvolves a universal stress protein and a resuscitation-promoting factor (Rpf). Genes for these proteins are widely found in actinobacteria, includingT. phoenicis. We show that dormantT. phoenicis(Micrococcales) can be revived through the addition of the Rpf to the media. Dormancy, as observed in the SAC actinobacterial isolateT. phoenicis, could well be a common trait adopted by other actinobacterial strains under the stressful conditions of spacecraft clean rooms or the ISS (International Space Station). This has implications for the persistence, identification, and recovery of such microbes from cleanroom facilities.IMPORTANCENASA’s long-range goal of a human mission to the Mars surface raises issues relating to planetary protection. The concerns of forward contamination require that spacecraft assembly clean rooms (SACs) be maintained to inhibit microbial survival. However, despite these efforts, distinct microbial communities persist. Here, we show that a SAC isolate,T. phoenicis, exhibits dormancy, a state in which the cells are viable but not cultivable. Dormancy may help non-spore-forming organisms survive in the clean room environments utilized for space missions. This has implications for improving cleaning procedures.
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- Award ID(s):
- 2227347
- PAR ID:
- 10627508
- Editor(s):
- Tischler, Dirk
- Publisher / Repository:
- American Society for Microbiology
- Date Published:
- Journal Name:
- Microbiology Spectrum
- ISSN:
- 2165-0497
- Page Range / eLocation ID:
- e01692-25.
- Subject(s) / Keyword(s):
- bacterial dormancy, resuscitation, spacecraft assembly facilities, clean rooms, human built environments, planetary protection, astrobiology
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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