A viability-linked metagenomic analysis of cleanroom environments: eukarya, prokaryotes, and viruses.

BACKGROUND: Recent studies posit a reciprocal dependency between the microbiomes associated with humans and indoor environments. However, none of these metagenome surveys has considered the viability of constituent microorganisms when inferring impact on human health. RESULTS: Reported here are the results of a viability-linked metagenomics assay, which (1) unveil a remarkably complex community profile for bacteria, fungi, and viruses and (2) bolster the detection of underrepresented taxa by eliminating biases resulting from extraneous DNA. This approach enabled, for the first time ever, the elucidation of viral genomes from a cleanroom environment. Upon comparing the viable biomes and distribution of phylotypes within a cleanroom and adjoining (uncontrolled) gowning enclosure, the rigorous cleaning and stringent control countermeasures of the former were observed to select for a greater presence of anaerobes and spore-forming microflora. Sequence abundance and correlation analyses suggest that the viable indoor microbiome is influenced by both the human microbiome and the surrounding ecosystem(s). CONCLUSIONS: The findings of this investigation constitute the literature's first ever account of the indoor metagenome derived from DNA originating solely from the potential viable microbial population. Results presented in this study should prove valuable to the conceptualization and experimental design of future studies on indoor microbiomes aimed at inferring impact on human health.

Microbial existence in controlled habitats and their resistance to space conditions.

The National Research Council (NRC) has recently recognized the International Space Station (ISS) as uniquely suitable for furthering the study of microbial species in closed habitats. Answering the NRC's call for the study, in particular, of uncommon microbial species in the ISS, and/or of those that have significantly increased or decreased in number, space microbiologists have begun capitalizing on the maturity, speed, and cost-effectiveness of molecular/genomic microbiological technologies to elucidate changes in microbial populations in the ISS and other closed habitats. Since investigators can only collect samples infrequently from the ISS itself due to logistical reasons, Earth analogs, such as spacecraft-assembly clean rooms, are used and extensively characterized for the presence of microbes. Microbiologists identify the predominant, problematic, and extremophilic microbial species in these closed habitats and use the ISS as a testbed to study their resistance to extreme extraterrestrial environmental conditions. Investigators monitor the microbes exposed to the real space conditions in order to track their genomic changes in response to the selective pressures present in outer space (external to the ISS) and the spaceflight (in the interior of the ISS). In this review, we discussed the presence of microbes in space research-related closed habitats and the resistance of some microbial species to the extreme environmental conditions of space.

A genetic inventory of spacecraft and associated surfaces.

Terrestrial organisms or other contaminants that are transported to Mars could interfere with efforts to study the potential for indigenous martian life. Similarly, contaminants that make the round-trip to Mars and back to Earth could compromise the ability to discriminate an authentic martian biosignature from a terrestrial organism. For this reason, it is important to develop a comprehensive inventory of microbes that are present on spacecraft to avoid interpreting their traces as authentic extraterrestrial biosignatures. Culture-based methods are currently used by NASA to assess spacecraft cleanliness but deliberately detect only a very small subset of total organisms present. The National Research Council has recommended that molecular (DNA)-based identification techniques should be developed as one aspect of managing the risk that terrestrial contamination could interfere with detection of life on (or returned from) Mars. The current understanding of the microbial diversity associated with spacecraft and clean room surfaces is expanding, but the capability to generate a comprehensive inventory of the microbial populations present on spacecraft outbound from Earth would address multiple considerations in planetary protection, relevant to both robotic and human missions. To this end, a 6-year genetic inventory study was undertaken by a NASA/JPL team. It was completed in 2012 and included delivery of a publicly available comprehensive final report. The genetic inventory study team evaluated the utility of three analytical technologies (conventional cloning techniques, PhyloChip DNA microarrays, and 454 tag-pyrosequencing) and combined them with a systematic methodology to collect, process, and archive nucleic acids as the first steps in assessing the phylogenetic breadth of microorganisms on spacecraft and associated surfaces.

Implementing planetary protection measures on the Mars Science Laboratory.

The Mars Science Laboratory (MSL), comprising a cruise stage; an aeroshell; an entry, descent, and landing system; and the radioisotope thermoelectric generator-powered Curiosity rover, made history with its unprecedented sky crane landing on Mars on August 6, 2012. The mission's primary science objective has been to explore the area surrounding Gale Crater and assess its habitability for past life. Because microbial contamination could profoundly impact the integrity of the mission and compliance with international treaty was required, planetary protection measures were implemented on MSL hardware to verify that bioburden levels complied with NASA regulations. By applying the proper antimicrobial countermeasures throughout all phases of assembly, the total bacterial endospore burden of MSL at the time of launch was kept to 2.78×105 spores, well within the required specification of less than 5.0×105 spores. The total spore burden of the exposed surfaces of the landed MSL hardware was 5.64×104, well below the allowed limit of 3.0×105 spores. At the time of launch, the MSL spacecraft was burdened with an average of 22 spores/m², which included both planned landed and planned impacted hardware. Here, we report the results of a campaign to implement and verify planetary protection measures on the MSL flight system.

Implementing planetary protection on the Atlas V fairing and ground systems used to launch the Mars Science Laboratory.

On November 26, 2011, the Mars Science Laboratory (MSL) launched from Florida's Cape Canaveral Air Force Station aboard an Atlas V 541 rocket, taking its first step toward exploring the past habitability of Mars' Gale Crater. Because microbial contamination could profoundly impact the integrity of the mission, and compliance with international treaty was a necessity, planetary protection measures were implemented on all MSL hardware to verify that bioburden levels complied with NASA regulations. The cleanliness of the Atlas V payload fairing (PLF) and associated ground support systems used to launch MSL were also evaluated. By applying proper recontamination countermeasures early and often in the encapsulation process, the PLF was kept extremely clean and was shown to pose little threat of recontaminating the enclosed MSL flight system upon launch. Contrary to prelaunch estimates that assumed that the interior PLF spore burden ranged from 500 to 1000 spores/m², the interior surfaces of the Atlas V PLF were extremely clean, housing a mere 4.65 spores/m². Reported here are the practices and results of the campaign to implement and verify planetary protection measures on the Atlas V launch vehicle and associated ground support systems used to launch MSL. All these facilities and systems were very well kept and exceeded the levels of cleanliness and rigor required in launching the MSL payload.

Detecting the dormant: a review of recent advances in molecular techniques for assessing the viability of bacterial endospores.

Due to their contribution to gastrointestinal and pulmonary disease, their ability to produce various deadly exotoxins, and their resistance to extreme temperature, pressure, radiation, and common chemical disinfecting agents, bacterial endospores of the Firmicutes phylum are a major concern for public and environmental health. In addition, the hardy and dormant nature of endospores renders them a particularly significant threat to the integrity of robotic extraterrestrial life-detection investigations. To prevent the contamination of critical surfaces with seemingly ubiquitous bacterial endospores, clean rooms maintained at exceedingly stringent cleanliness levels (i.e., fewer than 100,000 airborne particles per ft(3)) are used for surgical procedures, pharmaceutical processing and packaging, and fabrication and assembly of medical devices and spacecraft components. However, numerous spore-forming bacterial species have been reported to withstand typical clean room bioreduction strategies (e.g., UV lights, maintained humidity, paucity of available nutrients), which highlights the need for rapid and reliable molecular methods for detecting, enumerating, and monitoring the incidence of viable endospores. Robust means of evaluating and tracking spore burden not only provide much needed information pertaining to endospore ecophysiology in different environmental niches but also empower decontamination and bioreduction strategies aimed at sustaining the reliability and integrity of clean room environments. An overview of recent molecular advances in detecting and enumerating viable endospores, as well as the expanding phylogenetic diversity of pathogenic and clean room-associated spore-forming bacteria, ensues.

New perspectives on viable microbial communities in low-biomass cleanroom environments.

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.

Pyrosequencing-derived bacterial, archaeal, and fungal diversity of spacecraft hardware destined for Mars.

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.

Rapid detection of viable Bacillus pumilus SAFR-032 encapsulated spores using novel propidium monoazide-linked fluorescence in situ hybridization.

The survival of Bacillus pumilus SAFR-032 spores to standard industrial clean room sterilization practices necessitates the development of rapid molecular diagnostic tool(s) for detection and enumeration of viable bacterial spores in industrial clean room environments. This is of importance to maintaining the sterility of clean room processing products. This paper describes the effect of propidium monoazide (PMA) on fluorescence in situ hybridization (FISH) for detecting and enumerating B. pumilus SAFR-032 viable spores having been artificially encapsulated within poly(methylmethacrylate) (Lucite, Plexiglas) and released via an organic solvent (PolyGone-500). The results of the PMA-FISH experiments discussed herein indicate that PMA was able to permeate only the compromised coat layers of non-viable spores, identifying PMA treatment of bacterial spores prior to FISH analysis as a novel method for selecting out the fraction of the spore population that is non-viable from fluorescence detection. The ability of novel PMA-FISH to selectively distinguish and enumerate only the living spores present in a sample is of potential significance for development of improved strategies to minimize spore-specific microbial burden in a given environment.

Evaluation of fluorescence in situ hybridization to detect encapsulated Bacillus pumilus SAFR-032 spores released from poly(methylmethacrylate).

Bacillus pumilus SAFR-032 spores originally isolated from the Jet Propulsion Laboratory spacecraft assembly facility clean room are extremely resistant to UV radiation, H(2)O(2), desiccation, chemical disinfection and starvation compared to spores of other Bacillus species. The resistance of B. pumilus SAFR-032 spores to standard industrial clean room sterilization practices is not only a major concern for medical, pharmaceutical and food industries, but also a threat to the extraterrestrial environment during search for life via spacecraft. The objective of the present study was to investigate the potential of Alexa-FISH (fluorescence in situ hybridization with Alexa Fluor® 488 labeled oligonucleotide) method as a molecular diagnostic tool for enumeration of multiple sterilant-resistant B. pumilus SAFR-032 spores artificially encapsulated in, and released via organic solvent from, a model polymeric material: poly(methylmethacrylate) (Lucite, Plexiglas). Plexiglas is used extensively in various aerospace applications and in medical, pharmaceutical and food industries. Alexa-FISH signals were not detected from spores via standard methods for vegetative bacterial cells. Optimization of a spore permeabilization protocol capitalizing on the synergistic action of proteinase-K, lysozyme, mutanolysin and Triton X-100 facilitated efficient spore detection by Alexa-FISH microscopy. Neither of the Alexa-probes tested gave rise to considerable levels of Lucite- or solvent-associated background autofluorescence, demonstrating the immense potential of Alexa-FISH for rapid quantification of encapsulated B. pumilus SAFR-032 spores released from poly(methylmethacrylate).

Comparison of innovative molecular approaches and standard spore assays for assessment of surface cleanliness.

A bacterial spore assay and a molecular DNA microarray method were compared for their ability to assess relative cleanliness in the context of bacterial abundance and diversity on spacecraft surfaces. Colony counts derived from the NASA standard spore assay were extremely low for spacecraft surfaces. However, the PhyloChip generation 3 (G3) DNA microarray resolved the genetic signatures of a highly diverse suite of microorganisms in the very same sample set. Samples completely devoid of cultivable spores were shown to harbor the DNA of more than 100 distinct microbial phylotypes. Furthermore, samples with higher numbers of cultivable spores did not necessarily give rise to a greater microbial diversity upon analysis with the DNA microarray. The findings of this study clearly demonstrated that there is not a statistically significant correlation between the cultivable spore counts obtained from a sample and the degree of bacterial diversity present. Based on these results, it can be stated that validated state-of-the-art molecular techniques, such as DNA microarrays, can be utilized in parallel with classical culture-based methods to further describe the cleanliness of spacecraft surfaces.

Comprehensive census of bacteria in clean rooms by using DNA microarray and cloning methods.

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.

Description of Rummeliibacillus stabekisii gen. nov., sp. nov. and reclassification of Bacillus pycnus Nakamura et al. 2002 as Rummeliibacillus pycnus comb. nov.

Strains of aerobic, Gram-positive, rod-shaped, round-spore-forming bacteria were isolated from different geographical locations and a subsequent polyphasic study was undertaken to clarify the taxonomic position of the round-spore-forming isolates strain KSC-SF6g(T), strain M32 and strain NBRC 12622. 16S rRNA gene sequence similarities demonstrated that these strains were most closely affiliated with Bacillus pycnus NRRL NRS-1691(T) (98 %), with species of Kurthia (96 %) and Viridibacillus (94-96 %) as the next nearest relatives. However, while DNA-DNA hybridization studies showed approx. 70 % reassociation among strains KSC-SF6g(T), M32 and NBRC 12622, DNA-DNA hybridization values between these strains and B. pycnus NRRL NRS-1691(T) never exceeded 13 %. Differences in the molecular structure of the cell-wall peptidoglycan could not differentiate these strains sufficiently from other closely related genera (Viridibacillus and Kurthia). However, Lys-Asp was present in strains KSC-SF6g(T), M32 and NBRC 12622, whereas l-Lys-d-Glu was reported in B. pycnus NRRL NRS-1691(T). The menaquinone MK-7 was dominant in strains KSC-SF6g(T), M32 and NBRC 12622 and members of the genus Kurthia, whereas MK-8 was abundant in Viridibacillus species. Strains KSC-SF6g(T), M32 and NBRC 12622 exhibited fatty acid profiles consisting of major amounts of anteiso-C(15 : 0) ( approximately 50 %) and iso-C(15 : 0) ( approximately 25 %) and moderate amounts of anteiso-C(17 : 0) ( approximately 7 %), which discriminated them from closely related B. pycnus NRRL NRS-1691(T) and species of Viridibacillus (iso-C(15 : 0); 46-74 %). The authors propose that strains KSC-SF6g(T), M32 and NBRC 12622 and B. pycnus NRRL NRS-1691(T) be reclassified into a separate genus based on clear-cut differences in discriminative taxonomic markers and the distant placement of B. pycnus and the novel strains described herein from other species of this clade according to current 16S rRNA gene sequence-based relatedness ( approximately 4 % difference in sequence). We propose the placement of these isolates into the novel genus Rummeliibacillus gen. nov. For the new taxon comprising strains KSC-SF6g(T), M32 and NBRC 12622, we propose the name Rummeliibacillus stabekisii gen. nov., sp. nov. (the type species of Rummeliibacillus), represented by the type strain KSC-SF6g(T) (=NRRL B-51320(T) =NBRC 104870(T)). In addition, Bacillus pycnus, which bears traits distinct from other round-spore-forming species [i.e. absence of growth at high NaCl (7 %), positive reaction for gelatin liquefaction], is reclassified as Rummeliibacillus pycnus comb. nov. (type strain JCM 11075(T) =NRRL NRS-1691(T)) based on phylogenetic affiliations and phenotypic characterization.

Microbial burden and diversity of commercial airline cabin air during short and long durations of travel.

Total microbial burden and diversity associated with commercial airliner cabin air was assessed by molecular methods in 125 air samples from the business-class sections of 16 domestic and international flights. Viable microbial burden within these cabin air parcels constituted only 1-10% of the total microbial population and ranged from below detection limits to 1.2 x 10(4) cells m(-3) as determined with a validated ATP-based technology. Cultivable bacterial diversity was almost entirely limited to Gram-positive bacteria such as Staphylococcus and Bacillus. In contrast, cloning and sequencing 16S rRNA gene directly from the samples without cultivation indicated a significantly broader diversity, as sequences representing more than 100 species, and encompassing 12 classes of bacteria, were retrieved in varying abundance. Sequences of proteobacterial and Gram-positive lineage were retrieved most frequently (58% and 31% of all clone sequences, respectively), with Gram-positive and alpha-proteobacterial sequences dominating international flight samples and beta- and gamma-proteobacterial sequences comprising the largest portion of those retrieved from domestic flights. Significant differences in bacterial load and diversity were noted between samples obtained on domestic and international flights. The disparities observed in microbial abundance and diversity further underscore the immense value of state-of-the art molecular assays in augmenting traditional culture-based techniques.

Effect of shadowing on survival of bacteria under conditions simulating the Martian atmosphere and UV radiation.

Spacecraft-associated spores and four non-spore-forming bacterial isolates were prepared in Atacama Desert soil suspensions and tested both in solution and in a desiccated state to elucidate the shadowing effect of soil particulates on bacterial survival under simulated Martian atmospheric and UV irradiation conditions. All non-spore-forming cells that were prepared in nutrient-depleted, 0.2-microm-filtered desert soil (DSE) microcosms and desiccated for 75 days on aluminum died, whereas cells prepared similarly in 60-microm-filtered desert soil (DS) microcosms survived such conditions. Among the bacterial cells tested, Microbacterium schleiferi and Arthrobacter sp. exhibited elevated resistance to 254-nm UV irradiation (low-pressure Hg lamp), and their survival indices were comparable to those of DS- and DSE-associated Bacillus pumilus spores. Desiccated DSE-associated spores survived exposure to full Martian UV irradiation (200 to 400 nm) for 5 min and were only slightly affected by Martian atmospheric conditions in the absence of UV irradiation. Although prolonged UV irradiation (5 min to 12 h) killed substantial portions of the spores in DSE microcosms (approximately 5- to 6-log reduction with Martian UV irradiation), dramatic survival of spores was apparent in DS-spore microcosms. The survival of soil-associated wild-type spores under Martian conditions could have repercussions for forward contamination of extraterrestrial environments, especially Mars.

Molecular bacterial diversity and bioburden of commercial airliner cabin air.

Culture-independent, biomarker-targeted bacterial enumeration and identification strategies were employed to estimate total bacterial burden and diversity within the cabin air of commercial airliners. Samples from each of 4 flights on 2 commercial carriers were collected via air-impingement. The total viable microbial population ranged from below detection limits to 4.1 x 10(6) cells/m(3) of air, as assessed by the ATP assay. A gradual accumulation of microbes was observed from the time of passenger boarding through mid-flight, followed by a sharp decline in bacterial abundance and viability from the initiation of descent through landing. Representatives of the alpha-, beta-, and gamma-Proteobacteria, as well as Gram-positive bacteria, were isolated in varying abundance. Neisseria meningitidis rRNA gene sequences were retrieved in great abundance from Airline A followed by Streptococcus oralis/mitis sequences. Pseudomonas synxantha sequences dominated Airline B clone libraries, followed by those of N. meningitidis and S. oralis/mitis. The cabin air samples examined herein housed low bacterial diversity and were often dominated by a particular subset of bacteria: opportunistic pathogenic inhabitants of the human respiratory tract and oral cavity.

Molecular bacterial community analysis of clean rooms where spacecraft are assembled.

Molecular bacterial community composition was characterized from three geographically distinct spacecraft-associated clean rooms to determine whether such populations are influenced by the surrounding environment or the maintenance of the clean rooms. Samples were collected from facilities at the Jet Propulsion Laboratory (JPL), Kennedy Space Flight Center (KSC), and Johnson Space Center (JSC). Nine clone libraries representing different surfaces within the spacecraft facilities and three libraries from the surrounding air were created. Despite the highly desiccated, nutrient-bare conditions within these clean rooms, a broad diversity of bacteria was detected, covering all the main bacterial phyla. Furthermore, the bacterial communities were significantly different from each other, revealing only a small subset of microorganisms common to all locations (e.g. Sphingomonas, Staphylococcus). Samples from JSC assembly room surfaces showed the greatest diversity of bacteria, particularly within the Alpha- and Gammaproteobacteria and Actinobacteria. The bacterial community structure of KSC assembly surfaces revealed a high presence of proteobacterial groups, whereas the surface samples collected from the JPL assembly facility showed a predominance of Firmicutes. Our study presents the first extended molecular survey and comparison of NASA spacecraft assembly facilities, and provides new insights into the bacterial diversity of clean room environments .

Microbial diversity of Indian Ocean hydrothermal vent plumes: microbes tolerant of desiccation, peroxide exposure, and ultraviolet and gamma-irradiation.

The microbial diversity of Kali chimney plumes, part of a hydrothermal vent field in the Rodriguez Triple Junction, Indian Ocean (depth approximately 2,240 m), was examined in an attempt to discover "extremotolerant" microorganisms that have evolved unique resistance capabilities to this harsh environment. Water and sediment samples were collected from the vent and from sediments located at various distances (2-20 m) away from and surrounding the chimney. Samples were screened for hypertolerant microbes that are able to withstand multiple stresses. A total of 46 isolates were selected for exposure to a number of perturbations, such as heat shock, desiccation, H(2)O(2), and ultraviolet (UV) and gamma-irradiation. The survival of Psychrobacter sp. L0S3S-03b following exposure to >1,000 J/m(2) UV(254) radiation was particularly intriguing amid a background of varying levels of resistance. Vegetative cells of this non-spore-forming microbe not only survived all of the treatments, but also exhibited a 90% lethal dose of 30 s when exposed to simulated martian UV radiation and a 100% lethal dose of 2 min when exposed to full spectrum UV, which is comparable to findings for bacterial endospores.

Isolation and characterization of bacteria capable of tolerating the extreme conditions of clean room environments.

In assessing the bacterial populations present in spacecraft assembly, spacecraft test, and launch preparation facilities, extremophilic bacteria (requiring severe conditions for growth) and extremotolerant bacteria (tolerant to extreme conditions) were isolated. Several cultivation approaches were employed to select for and identify bacteria that not only survive the nutrient-limiting conditions of clean room environments but can also withstand even more inhospitable environmental stresses. Due to their proximity to spacefaring objects, these bacteria pose a considerable risk for forward contamination of extraterrestrial sites. Samples collected from four geographically distinct National Aeronautics and Space Administration clean rooms were challenged with UV-C irradiation, 5% hydrogen peroxide, heat shock, pH extremes (pH 3.0 and 11.0), temperature extremes (4 degrees C to 65 degrees C), and hypersalinity (25% NaCl) prior to and/or during cultivation as a means of selecting for extremotolerant bacteria. Culture-independent approaches were employed to measure viable microbial (ATP-based) and total bacterial (quantitative PCR-based) burdens. Intracellular ATP concentrations suggested a viable microbial presence ranging from below detection limits to 10(6) cells/m(2). However, only 0.1 to 55% of these viable cells were able to grow on defined culture medium. Isolated members of the Bacillaceae family were more physiologically diverse than those reported in previous studies, including thermophiles (Geobacillus), obligate anaerobes (Paenibacillus), and halotolerant, alkalophilic species (Oceanobacillus and Exiguobacterium). Non-spore-forming microbes (alpha- and beta-proteobacteria and actinobacteria) exhibiting tolerance to the selected stresses were also encountered. The multiassay cultivation approach employed herein enhances the current understanding of the physiological diversity of bacteria housed in these clean rooms and leads us to ponder the origin and means of translocation of thermophiles, anaerobes, and halotolerant alkalophiles into these environments.

Bacillus safensis sp. nov., isolated from spacecraft and assembly-facility surfaces.

Thirteen strains of a novel spore-forming, Gram-positive, mesophilic heterotrophic bacterium were isolated from spacecraft surfaces (Mars Odyssey Orbiter) and assembly-facility surfaces at the Jet Propulsion Laboratory in California and the Kennedy Space Center in Florida. Phylogenetic analysis of 16S rRNA gene sequences has placed these novel isolates within the genus Bacillus, the greatest sequence similarity (99.9 %) being found with Bacillus pumilus. However, these isolates share a mere 91.2 % gyrB sequence similarity with Bacillus pumilus, rendering their 16S rRNA gene-derived relatedness suspect. Furthermore, DNA-DNA hybridization showed only 54-66 % DNA relatedness between the novel isolates and strains of B. pumilus. rep-PCR fingerprinting and previously reported matrix-assisted laser desorption/ionization time-of-flight mass spectrometry protein profiling clearly distinguished these isolates from B. pumilus. Phenotypic analyses also showed some differentiation between the two genotypic groups, although the fatty acid compositions were almost identical. The polyphasic taxonomic studies revealed distinct clustering of the tested strains into two distinct species. On the basis of phenotypic characteristics and the results of phylogenetic analyses of 16S rRNA and gyrB gene sequences, repetitive element primer-PCR fingerprinting and DNA-DNA hybridization, the 13 isolates represent a novel species of the genus Bacillus, for which the name Bacillus safensis sp. nov. is proposed. The type strain is FO-36b(T) (=ATCC BAA-1126(T)=NBRC 100820(T)).