New perspectives on viable microbial communities in low-biomass cleanroom environments.
ABSTRACT: The advent of phylogenetic DNA microarrays and high-throughput pyrosequencing technologies has dramatically increased the resolution and accuracy of detection of distinct microbial lineages in mixed microbial assemblages. Despite an expanding array of approaches for detecting microbes in a given sample, rapid and robust means of assessing the differential viability of these cells, as a function of phylogenetic lineage, remain elusive. In this study, pre-PCR propidium monoazide (PMA) treatment was coupled with downstream pyrosequencing and PhyloChip DNA microarray analyses to better understand the frequency, diversity and distribution of viable bacteria in spacecraft assembly cleanrooms. Sample fractions not treated with PMA, which were indicative of the presence of both live and dead cells, yielded a great abundance of highly diverse bacterial pyrosequences. In contrast, only 1% to 10% of all of the pyrosequencing reads, arising from a few robust bacterial lineages, originated from sample fractions that had been pre-treated with PMA. The results of PhyloChip analyses of PMA-treated and -untreated sample fractions were in agreement with those of pyrosequencing. The viable bacterial population detected in cleanrooms devoid of spacecraft hardware was far more diverse than that observed in cleanrooms that housed mission-critical spacecraft hardware. The latter was dominated by hardy, robust organisms previously reported to survive in oligotrophic cleanroom environments. Presented here are the findings of the first ever comprehensive effort to assess the viability of cells in low-biomass environmental samples, and correlate differential viability with phylogenetic affiliation.
Project description:Spacecraft hardware and assembly cleanroom surfaces (233 m(2) in total) were sampled, total genomic DNA was extracted, hypervariable regions of the 16S rRNA gene (bacteria and archaea) and ribosomal internal transcribed spacer (ITS) region (fungi) were subjected to 454 tag-encoded pyrosequencing PCR amplification, and 203,852 resulting high-quality sequences were analyzed. Bioinformatic analyses revealed correlations between operational taxonomic unit (OTU) abundance and certain sample characteristics, such as source (cleanroom floor, ground support equipment [GSE], or spacecraft hardware), cleaning regimen applied, and location about the facility or spacecraft. National Aeronautics and Space Administration (NASA) cleanroom floor and GSE surfaces gave rise to a larger number of diverse bacterial communities (619 OTU; 20 m(2)) than colocated spacecraft hardware (187 OTU; 162 m(2)). In contrast to the results of bacterial pyrosequencing, where at least some sequences were generated from each of the 31 sample sets examined, only 13 and 18 of these sample sets gave rise to archaeal and fungal sequences, respectively. As was the case for bacteria, the abundance of fungal OTU in the GSE surface samples dramatically diminished (9× less) once cleaning protocols had been applied. The presence of OTU representative of actinobacteria, deinococci, acidobacteria, firmicutes, and proteobacteria on spacecraft surfaces suggests that certain bacterial lineages persist even following rigorous quality control and cleaning practices. The majority of bacterial OTU observed as being recurrent belonged to actinobacteria and alphaproteobacteria, supporting the hypothesis that the measures of cleanliness exerted in spacecraft assembly cleanrooms (SAC) inadvertently select for the organisms which are the most fit to survive long journeys in space.
Project description:Cleanrooms have been considered microbially-reduced environments and are used to protect human health and industrial product assembly. However, recent analyses have deciphered a rather broad diversity of microbes in cleanrooms, whose origin as well as physiological status has not been fully understood. Here, we examined the input of intact microbial cells from a surrounding built environment into a spacecraft assembly cleanroom by applying a molecular viability assay based on propidium monoazide (PMA). The controlled cleanroom (CCR) was characterized by ~6.2*103 16S rRNA gene copies of intact bacterial cells per m2 floor surface, which only represented 1% of the total community that could be captured via molecular assays without viability marker. This was in contrast to the uncontrolled adjoining facility (UAF) that had 12 times more living bacteria. Regarding diversity measures retrieved from 16S rRNA Illumina-tag analyzes, we observed, however, only a minor drop in the cleanroom facility allowing the conclusion that the number but not the diversity of microbes is strongly affected by cleaning procedures. Network analyses allowed tracking a substantial input of living microbes to the cleanroom and a potential enrichment of survival specialists like bacterial spore formers and archaeal halophiles and mesophiles. Moreover, the cleanroom harbored a unique community including 11 exclusive genera, e.g., Haloferax and Sporosarcina, which are herein suggested as indicators of cleanroom environments. In sum, our findings provide evidence that archaea are alive in cleanrooms and that cleaning efforts and cleanroom maintenance substantially decrease the number but not the diversity of indoor microbiomes.
Project description:Strict planetary protection practices are implemented during spacecraft assembly to prevent inadvertent transfer of earth microorganisms to other planetary bodies. Therefore, spacecraft are assembled in cleanrooms, which undergo strict cleaning and decontamination procedures to reduce total microbial bioburden. We wanted to evaluate if these practices selectively favor survival and growth of hardy microorganisms, such as pathogens. Three geographically distinct cleanrooms were sampled during the assembly of three NASA spacecraft: The Lockheed Martin Aeronautics' Multiple Testing Facility during DAWN, the Kennedy Space Center's Payload Hazardous Servicing Facility (KSC-PHSF) during Phoenix, and the Jet Propulsion Laboratory's Spacecraft Assembly Facility during Mars Science Laboratory. Sample sets were collected from the KSC-PHSF cleanroom at three time points: before arrival of the Phoenix spacecraft, during the assembly and testing of the Phoenix spacecraft, and after removal of the spacecraft from the KSC-PHSF facility. All samples were subjected to metagenomic shotgun sequencing on an Illumina HiSeq 2500 platform. Strict decontamination procedures had a greater impact on microbial communities than sampling location Samples collected during spacecraft assembly were dominated by Acinetobacter spp. We found pathogens and potential virulence factors, which determine pathogenicity in all the samples tested during this study. Though the relative abundance of pathogens was lowest during the Phoenix assembly, potential virulence factors were higher during assembly compared to before and after assembly, indicating a survival advantage. Decreased phylogenetic and pathogenic diversity indicates that decontamination and preventative measures were effective against the majority of microorganisms and well implemented, however, pathogen abundance still increased over time. Four potential pathogens, Acinetobacter baumannii, Acinetobacter lwoffii, Escherichia coli and Legionella pneumophila, and their corresponding virulence factors were present in all cleanroom samples. This is the first functional metagenomics study describing presence of pathogens and their corresponding virulence factors in cleanroom environments. The results of this study should be considered for microbial monitoring of enclosed environments such as schools, homes, hospitals and more isolated habitation such the International Space Station and future manned missions to Mars.
Project description:A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments.
Project description:Characterizing the microbiome of spacecraft assembly cleanrooms is important for planetary protection. We report two bacterial metagenome-assembled genomes (MAGs) reconstructed from metagenomes produced from cleanroom samples from the Kennedy Space Center's Payload Hazardous Servicing Facility (KSC-PHSF) during the handling of the Phoenix spacecraft. Characterization of these MAGs will enable identification of the strategies underpinning their survival.
Project description:The ExoMars 2016 mission, consisting of the Trace Gas Orbiter and the Schiaparelli lander, was launched on March 14 2016 from Baikonur, Kazakhstan and reached its destination in October 2016. The Schiaparelli lander was subject to strict requirements for microbial cleanliness according to the obligatory planetary protection policy. To reach the required cleanliness, the ExoMars 2016 flight hardware was assembled in a newly built, biocontrolled cleanroom complex at Thales Alenia Space in Turin, Italy. In this study, we performed microbiological surveys of the cleanroom facilities and the spacecraft hardware before and during the assembly, integration and testing (AIT) activities.Besides the European Space Agency (ESA) standard bioburden assay, that served as a proxy for the microbiological contamination in general, we performed various alternative cultivation assays and utilised molecular techniques, including quantitative PCR and next generation sequencing, to assess the absolute and relative abundance and broadest diversity of microorganisms and their signatures in the cleanroom and on the spacecraft hardware.Our results show that the bioburden, detected microbial contamination and microbial diversity decreased continuously after the cleanroom was decontaminated with more effective cleaning agents and during the ongoing AIT. The studied cleanrooms and change room were occupied by very distinct microbial communities: Overall, the change room harboured a higher number and diversity of microorganisms, including Propionibacterium, which was found to be significantly increased in the change room. In particular, the so called alternative cultivation assays proved important in detecting a broader cultivable diversity than covered by the standard bioburden assay and thus completed the picture on the cleanroom microbiota.During the whole project, the bioburden stayed at acceptable level and did not raise any concern for the ExoMars 2016 mission. The cleanroom complex at Thales Alenia Space in Turin is an excellent example of how efficient microbiological control is performed.
Project description:Space agencies maintain highly controlled cleanrooms to ensure the demands of planetary protection. To study potential effects of microbiome control, we analyzed microbial communities in two particulate-controlled cleanrooms (ISO 5 and ISO 8) and two vicinal uncontrolled areas (office, changing room) by cultivation and 16S rRNA gene amplicon analysis (cloning, pyrotagsequencing, and PhyloChip G3 analysis). Maintenance procedures affected the microbiome on total abundance and microbial community structure concerning richness, diversity and relative abundance of certain taxa. Cleanroom areas were found to be mainly predominated by potentially human-associated bacteria; archaeal signatures were detected in every area. Results indicate that microorganisms were mainly spread from the changing room (68%) into the cleanrooms, potentially carried along with human activity. The numbers of colony forming units were reduced by up to ~400 fold from the uncontrolled areas towards the ISO 5 cleanroom, accompanied with a reduction of the living portion of microorganisms from 45% (changing area) to 1% of total 16S rRNA gene signatures as revealed via propidium monoazide treatment of the samples. Our results demonstrate the strong effects of cleanroom maintenance on microbial communities in indoor environments and can be used to improve the design and operation of biologically controlled cleanrooms.
Project description:Next-generation sequencing techniques, and PhyloChip, have made simultaneous phylogenetic analyses of hundreds of microbial communities possible. Insight into community structure has been limited by the inability to integrate and visualize such vast datasets. Fast UniFrac overcomes these issues, allowing integration of larger numbers of sequences and samples into a single analysis. Its new array-based implementation offers orders of magnitude improvements over the original version. New 3D visualization of principal coordinates analysis results, with the option to view multiple coordinate axes simultaneously, provides a powerful way to quickly identify patterns that relate vast numbers of microbial communities. We show the potential of Fast UniFrac using examples from three data types: Sanger-sequencing studies of diverse free-living and animal-associated bacterial assemblages and from the gut of obese humans as they diet, pyrosequencing data integrated from studies of the human hand and gut, and PhyloChip data from a study of citrus pathogens. We show that a Fast UniFrac analysis using a reference tree recaptures patterns that could not be detected without considering phylogenetic relationships and that Fast UniFrac, coupled with BLAST-based sequence assignment, can be used to quickly analyze pyrosequencing runs containing hundreds of thousands of sequences, showing patterns relating human and gut samples. Finally, we show that the application of Fast UniFrac to PhyloChip data could identify well-defined subcategories associated with infection. Together, these case studies point the way toward a broad range of applications and show some of the new features of Fast UniFrac.
Project description: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.
Project description:Standardized studies examining environmental microbial exposure in populations at risk for asthma are necessary to improve our understanding of the role this factor plays in disease development. Here we describe studies aimed at developing guidelines for high-resolution culture-independent microbiome profiling, using a phylogenetic microarray (PhyloChip), of house dust samples in a cohort collected as part of the NIH-funded Inner City Asthma Consortium (ICAC). We demonstrate that though extracted DNA concentrations varied across dust samples, the majority produced sufficient 16S rRNA to be profiled by the array. Comparison of array and 454-pyrosequencing performed in parallel on a subset of samples, illustrated that increasingly deeper sequencing efforts validated greater numbers of array-detected taxa. Community composition agreement across samples exhibited a hierarchy in concordance, with the highest level of agreement in replicate array profiles followed by samples collected from adjacent 1×1 m(2) sites in the same room, adjacent sites with different sized sampling quadrants (1×1 and 2×2 m(2)), different sites within homes (living and bedroom) to lowest in living room samples collected from different homes. The guidelines for sample collection and processing in this pilot study extend beyond PhyloChip based studies of house-associated microbiota, and bear relevance for other microbiome profiling approaches such as next-generation sequencing.