Application of the ATP assay to rapidly assess cleanliness of spacecraft surfaces: a path to set a standard for future missions

Benardini, James N.; Venkateswaran, Kasthuri

doi:10.1186/s13568-016-0286-9

Cited by 10 publications

(4 citation statements)

References 12 publications

(20 reference statements)

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“… a NG, no growth. b The ATP standard curve was generated by the method of Benardini and Venkateswaran ( 89 ) with 50 RLU = 1.169 × 10 −11 mmol of ATP. …”

Section: Resultsmentioning

confidence: 99%

Microbiome and Metagenome Analyses of a Closed Habitat during Human Occupation

et al. 2020

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Microbial contamination during long-term confinements of space exploration presents potential risks for both crew members and spacecraft life support systems. A novel swab kit was used to sample various surfaces from a submerged, closed, analog habitat to characterize the microbial populations. Samples were collected from various locations across the habitat which were constructed from various surface materials (linoleum, dry wall, particle board, glass, and metal), and microbial populations were examined by culture, quantitative PCR (qPCR), microbiome 16S rRNA gene sequencing, and shotgun metagenomics. Propidium monoazide (PMA)-treated samples identified the viable/intact microbial population of the habitat. The cultivable microbial population ranged from below the detection limit to 106 CFU/sample, and their identity was characterized using Sanger sequencing. Both 16S rRNA amplicon and shotgun sequencing were used to characterize the microbial dynamics, community profiles, and functional attributes (metabolism, virulence, and antimicrobial resistance). The 16S rRNA amplicon sequencing revealed abundance of viable (after PMA treatment) Actinobacteria (Brevibacterium, Nesternkonia, Mycobacterium, Pseudonocardia, and Corynebacterium), Firmicutes (Virgibacillus, Staphylococcus, and Oceanobacillus), and Proteobacteria (especially Acinetobacter) on linoleum, dry wall, and particle board (LDP) surfaces, while members of Firmicutes (Leuconostocaceae) and Proteobacteria (Enterobacteriaceae) were high on the glass/metal surfaces. Nonmetric multidimensional scaling determined from both 16S rRNA and metagenomic analyses revealed differential microbial species on LDP surfaces and glass/metal surfaces. The shotgun metagenomic sequencing of samples after PMA treatment showed bacterial predominance of viable Brevibacterium (53.6%), Brachybacterium (7.8%), Pseudonocardia (9.9%), Mycobacterium (3.7%), and Staphylococcus (2.1%), while fungal analyses revealed Aspergillus and Penicillium dominance. IMPORTANCE This study provides the first assessment of monitoring cultivable and viable microorganisms on surfaces within a submerged, closed, analog habitat. The results of the analyses presented herein suggest that the surface material plays a role in microbial community structure, as the microbial populations differed between LDP and metal/glass surfaces. The metal/glass surfaces had less-complex community, lower bioburden, and more closely resembled the controls. These results indicated that material choice is crucial when building closed habitats, even if they are simply analogs. Finally, while a few species were associated with previously cultivated isolates from the International Space Station and MIR spacecraft, the majority of the microbial ecology of the submerged analog habitat differs greatly from that of previously studied analog habitats.

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“… a NG, no growth. b The ATP standard curve was generated by the method of Benardini and Venkateswaran ( 89 ) with 50 RLU = 1.169 × 10 −11 mmol of ATP. …”

Section: Resultsmentioning

confidence: 99%

Microbiome and Metagenome Analyses of a Closed Habitat during Human Occupation

et al. 2020

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“…Environmental samples were obtained from JPL facilities (non-cleanroom facilities were used due to ease of access) using a cotton-tipped swab (806-WC, Puritan, Guilford, ME) as follows: swabs were moistened with sterile DI water before sampling a 25 cm 2 surface (National Aeronautics and Space Administration, 2010; Probst et al, 2010;Benardini and Venkateswaran, 2016). Following sampling, the swab head was aseptically broken off from the shaft and transferred into a test tube containing 10 ml sterile DI water.…”

Section: Environmental Samples For Enumerationmentioning

confidence: 99%

Dehydrated thin film media to rapidly estimate bioburden for planetary protection flight implementation

Dean

Stott

Schubert

et al. 2023

International Journal of Astrobiology

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Planetary Protection (PP) is the practice of safeguarding solar system bodies from terrestrial biological contamination and screening the Earth against potentially harmful extraterrestrial biological contamination. On Earth, cleanrooms and spacecraft surfaces are assayed using swabs and wipes that are then heat shocked for 15 min at 80°C to select for spores. The samples are further processed using the pour-plate method and Petri plates (TSA plates), with trypticase soy agar (TSA) serving as the growth medium. This sampling and processing procedure, called the NASA Standard Assay (NSA), is used by PP engineers around the world. Recent years have seen an increase in the incorporation of state-of-the-art technology, such as membrane filtration, into the NSA, with a push for implementing environmentally friendly technology into day-to-day activities. Dehydrated thin film media, such as Petrifilm Rapid Aerobic Count (RAC) plates, suit these goals as an alternative method to TSA plates. RAC plates show bacterial growth (and distinguish colonies from foreign particles such as bubbles) faster than TSA plates due to the incorporation of chromogenic colour indicators in the media. RAC plates also possess a much smaller environmental footprint than TSA plates, and are designed to evaluate even some of the challenging-to-detect environmental organisms, including spreaders that fill over 25% of the plate area in only a few hours. With these benefits in mind the PP Group at the NASA Jet Propulsion Laboratory took on the task of comparing RAC plates directly to TSA plates within the context of the NSA. Not only were the RAC plates able to detect surface environmental samples and in vitro spiked samples equivalent to NSA-processed TSA plates, but spreader organisms were countable on RAC plates at culture densities 10- to 100-fold greater than on TSA plates. In addition, RAC plates showed a robust, linear detection capability when challenged with membrane filter incorporation and organisms were easily acquired from RAC plates for archiving or post-processing experiments including MALDI-TOF bacterial identification. With their ease of use, small footprint, and both rapid and accurate bioburden measurements, RAC plates have the potential to overcome limitations posed by current PP culturing protocols.

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“…In addition to the NSA, NASA has certified other bioburden assessments, including two rapid assays approved in 2005 as alternative measures of microbial contamination ( Table 1 ). The adenosine triphosphate (ATP) assay and the Limulus Amoebocyte Lysate (LAL) assay (Benardini and Venkateswaran, 2016 ) were implemented principally to not only provide rapid analysis to circumvent the 3-day incubation period of the NSA, but also offer a means to screen spacecraft hardware for microbial contamination (Kern et al , 2005 ; Morris et al , 2010 ).…”

Section: Current Technologies For Bioburden Assessmentmentioning

confidence: 99%

Metagenomic Methods for Addressing NASA's Planetary Protection Policy Requirements on Future Missions: A Workshop Report

et al. 2023

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Molecular biology methods and technologies have advanced substantially over the past decade. These new molecular methods should be incorporated among the standard tools of planetary protection (PP) and could be validated for incorporation by 2026. To address the feasibility of applying modern molecular techniques to such an application, NASA conducted a technology workshop with private industry partners, academics, and government agency stakeholders, along with NASA staff and contractors. The technical discussions and presentations of the Multi-Mission Metagenomics Technology Development Workshop focused on modernizing and supplementing the current PP assays. The goals of the workshop were to assess the state of metagenomics and other advanced molecular techniques in the context of providing a validated framework to supplement the bacterial endospore-based NASA Standard Assay and to identify knowledge and technology gaps. In particular, workshop participants were tasked with discussing metagenomics as a stand-alone technology to provide rapid and comprehensive analysis of total nucleic acids and viable microorganisms on spacecraft surfaces, thereby allowing for the development of tailored and cost-effective microbial reduction plans for each hardware item on a spacecraft. Workshop participants recommended metagenomics approaches as the only data source that can adequately feed into quantitative microbial risk assessment models for evaluating the risk of forward (exploring extraterrestrial planet) and back (Earth harmful biological) contamination. Participants were unanimous that a metagenomics workflow, in tandem with rapid targeted quantitative (digital) PCR, represents a revolutionary advance over existing methods for the assessment of microbial bioburden on spacecraft surfaces. The workshop highlighted low biomass sampling, reagent contamination, and inconsistent bioinformatics data analysis as key areas for technology development. Finally, it was concluded that implementing metagenomics as an additional workflow for addressing concerns of NASA's robotic mission will represent a dramatic improvement in technology advancement for PP and will benefit future missions where mission success is affected by backward and forward contamination.

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Application of the ATP assay to rapidly assess cleanliness of spacecraft surfaces: a path to set a standard for future missions

Cited by 10 publications

References 12 publications

Microbiome and Metagenome Analyses of a Closed Habitat during Human Occupation

Microbiome and Metagenome Analyses of a Closed Habitat during Human Occupation

Dehydrated thin film media to rapidly estimate bioburden for planetary protection flight implementation

Metagenomic Methods for Addressing NASA's Planetary Protection Policy Requirements on Future Missions: A Workshop Report

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