Influence of culture media on the radiation resistance of Micrococcus radiodurans.
Culture medium effect on radiation resistance of microorganism Micrococcus radiodurans
Anderson, A. W.↗
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A salt-inhibited cytochrome c reductase obtained from the moderately halophilic bacterium, Micrococcus halodenitrificans.
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Miller, J. E.↗
Deficiency of the DNA of Micrococcus radiodurans methyladenine and methylcytosine
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Schein, A.↗
Microbial Monitoring of New Cleanrooms Used to Curate Astrobiologically Relevant Asteroid Samples from Bennu and Ryugu
Introduction: NASA has constructed two new cleanrooms to house materials from the OSRIS-REx and Hayabusa2 missions to the asteroids Ryugu (162173) and Bennu (101955), respectively. In accordance with standard astromaterials curation practices, these cleanrooms will be monitored for particulate contamination and maintained to ISO 5 equivalent standards1. Since the samples in these collections are expected to contain prebiotic organic compounds that may help explain the origin of life on Earth, these labs will also be monitored for organic and biological contamination2. Samples from Ryugu arrived on Earth in December, 2020. After basic characterization in Japan, NASA received a subset of these samples at the astromaterials curation facility in Houston in December of 2021. OSIRIS-REx is expected to return samples in September, 2023. Here we present preliminary microbial monitoring results from monthly monitoring of these new labs and the connected microtomy and staging areas that support them, as they are being commissioned. We also compare these results to baseline values for other astromaterials curation labs. We will also briefly describe additional cleaning efforts employed to reduce the bioburden in these new cleanrooms. Methods: Microbial samples were collected from surfaces using a dry macrofoam swab (Puritan Brand 2518051PFRNDFD). Swabs were also opened in the lab but not touched to any surfaces to function as negative controls. Samples and controls were processed inside a class II biosafety cabinet to avoid inadvertent cross contamination. The swabs were suspended in 15 ml of PBS (Phosphate Buffered Saline) and vortexed for 20 seconds to remove cells from the swab surface. The PBS was used to inoculate Petri dishes filled with TSA (Tryptic Soy Agar), Blood Agar, or Reasoners 2 agar to check for microbial growth. Each plate was inoculated with 0.1 ml of PBS. The TSA and blood agar plates were incubated at 35˚C and the Reasoners 2 agar plates were incubated at 25˚C for seven days. Petri dishes filled with Potato dextrose agar, Saboraud dextrose agar, or Saboraud dextrose agar with 0.1 mg/ml of chloramphenicol, an antibiotic, were used to check for fungal growth. These plates were inoculated with 0.3 ml of PBS and incubated at 30˚C. The remaining PBS was frozen at -80 ˚C for DNA sequencing. After incubation, isolates were counted and reisolated for identification. Isolates were identified using the VITEK23 system or by sequencing a portion of the 16S rRNA gene for bacteria or the ribosomal internal transcribed spacer (ITS) for fungi. Sequencing was performed with an ABI 3500 Sanger sequencer. Results: During our initial sampling, six of the seven sites sampled (86%) displayed bacterial or fungal growth. Samples collected from the staging areas and microtomy labs are not included in this calculation since those areas are maintained at a lower ISO 7 equivalent cleanliness standard. A month later, only three of the seven sites (43%) displayed bacterial growth. No fungal growth was detected in the second sampling. Since new equipment had been introduced to the Hayabusa2 lab since the first round of sampling, an additional three sampling sites were included in the second round of sampling. None of these sites displayed microbial growth. These sites will be included in all future sampling efforts. Bacterial isolates have been identified from the following genera at multiple time points: Micrococcus, Staphylococcus, and Bacillus. Isolates from the genera: Microbacterium, Nocardioides, Methylocystis, and Microvirga were identified in the initial sampling, but were not present at later time points. Identification of fungal isolates is in progress. Results are summarized in Table 1. Discussion: The recovery rate or percentage of positive samples4 was initially 86%, which is higher than the median recovery rate for comparable ISO 5 equivalent curation labs like Stardust (33%), Hayabusa (33%), and Cosmic Dust (50%). However, after a month of operation, the recovery rate for these same sites decreased to 43%, which is similar to what we observe in comparable curation cleanrooms with no microbial control requirements. Adding in the new sampling sites further decreases the recovery rate to 30%. With the reduction in recovery rate, we also observed a decrease in microbial diversity. At the first time point, we observed at least 10 different bacterial species and at least two different fungi. This is a higher diversity than the median values for comparable ISO 5 equivalent labs (2-4 isolates per sampling event). After the second sampling, we observed at least 4 bacterial species and no fungi, which is more consistent with comparable labs. We expect the recovery rate and diversity in both labs to continue to decrease as routine operation continues. We will use ultrapure hydrogen peroxide to disinfect equipment and work areas prior to opening any sample containers. Most of the bacterial and fungal isolates were detected on samples from the cleanroom floors. This is consistent with baseline results from other curation labs. Organisms from the genera Bacillus, Staphylococcus, and Micrococcus that were repeatedly detected are common in cleanrooms and on human skin5,6. These organisms are generally thought to be introduced when people enter the cleanroom. Microbacterium, Nocardioides, and Microvirga have also previously been identified in astromaterials cleanrooms, but not as frequently as Bacillus, Staphylococcus, and Micrococcus. Methylocystis is a novel genus in the astromaterials cleanrooms, but it was identified with low accuracy (93% match in the sequenced region of the 16S rRNA gene) and further work is needed to confirm this identification. Microbacterium is a diverse genus with isolates identified from terrestrial and aquatic sediments. Some species of Microbacterium are capable of degrading complex organic compounds found in crude oil. The presence of these bacteria in the OSIRIS REx and Hayabusa2 cleanrooms should be closely monitored. Methylocystis is a genus of methanotrophic bacteria capable of oxidizing methane. If this identification proves to be correct and it is detected again, it should be closely monitored as well. Under nominal operating conditions, samples should not ever encounter the cleanroom floor or other high traffic areas. If we observe an increase in the bioburden in sensitive work areas that appears to be influenced by organism transfer from high traffic areas like the floors, we can employ additional hydrogen peroxide treatments to disinfect high traffic areas. Routine microbial monitoring of these labs will ensure that NASA’s astromaterials collections remain pristine and useful for scientific study. Table 1. Sampling Locations and Colony Counts Bacterial CFUa Fungal CFU Bacterial CFU Fungal CFU Lab - Location 11/2/2021 11/2/2021 12/13/2021 12/13/2021 H2b-Floor 4 8 1 0 H2-staging pass through 3 0 0 0 H2-microtomy pass through TNTCc 0 0 0 H2 Microscope 1 NA NA 0 0 H2 Microscope 2 NA NA 0 0 H2-Table NA NA 0 0 OREXd- microtomy pass through 0 0 6 0 OREX – Anteroom pass through 0 0 0 0 OREX – Floor 1 2 0 0 OREX Witness Foil Table 3 0 1 0 Staging-Floor 16 0 15 0 Microtomy-Floor 3 0 2 0 a: CFU = Colony Forming Unit b: H2 = Hayabusa2 Lab c: TNTC = too numerous to count d: OREX = OSIRIS-REx Lab References: 1. ISO 14644-1:2015 - Cleanrooms and associated controlled environments -- Part 1: Classification of air cleanliness by particle concentration. 37 (2015). 2. McCubbin, F. M. et al. Space Sci Rev 215, (2019). 3. Pincus, D. H. Encyclopedia of Rapid Microbiological Methods (2005). 4. The United States Pharmacopeial Convention. USP General Chapter <1116> 17, 784–794 (2013). 5. Sheraba, N. S., Yassin, A. S. & Amin, M. BMC Research Notes 3, 278 (2010). 6. Utescher, C. L. de A., Franzolin, M. R., Trabulsi, L. R. & Gambale, V. Brazilian Journal of Microbiology 38, 710–716 (2007).
A B Regberg↗
The Effect of Temperature on the Survival of Microorganisms in a Deep Space Vacuum
A space molecular sink research facility (Molsink) was used to evaluate the ability of microorganisms to survive the vacuum of outer space. This facility could be programmed to simulate flight spacecraft vacuum environments at pressures in the .1 nanotorr range and thermal gradients (30 to 60 C) closely associated to surface temperatures of inflight spacecraft. Initial populations of Staphylococcus epidermidis and a Micrococcus sp. were reduced approximately 1 log while exposed to -105 and 34 C, and approximately 2 logs while exposed to 59 C for 14 days in the vacuum environment. Spores of Bacillus subtilis var. niger were less affected by the environment. Initial spore populations were reduced 0.2, 0.3, and 0.8 log during the 14-day vacuum exposure at -124, 34, and 59 C, respectively.
Hagen, C. A.↗
Isoelectric focusing analysis of antibody clonotype changes occurring during immune responses using immobilized pH gradients
Serum was collected from rabbits at 2-day intervals following a single injection with tetanus toxoid or at weekly intervals following multiple injections with Micrococcus lysodeikticus cell walls. These sera were analyzed for the presence of individual clonotypes of specific antitetanus or antimicrococcal antibodies by isoelectric focusing in immobilized pH gradients with added carrier ampholytes followed by affinity immunoblotting. The affinity immunoblots obtained clearly defined both the rapid disappearance and late appearance of distinct subsets of antibody clonotypes during the response. These data demonstrate the application of affinity immunoblotting combined with immobilized pH gradients for detecting the subtle changes in specific antibody clonotype patterns which occur during an immune response.
Knisley, Keith A.↗
Survival of epiphytic bacteria from seed stored on the Long Duration Exposure Facility (LDEF)
This study was designed to determine the survival of microorganisms exposed to the relatively harsh conditions found in low Earth orbit (LEO). Seed of corn, sunflower, canteloupe, zucchini, bean, pea, and pumpkin cultivars were packaged in two 18 x 2.5 cm aluminum tubes; wall thickness for each tube was 1.33 mm. One seed tube was attacked to payload M0006, tray C-2; a second tube was stored at room temperature in a lab on Earth. Five lithium fluoride thermoluminescent dosimetry wafers (TLD-100 wafers) were placed in each aluminum tube. The total mean dosages for flight and ground-control TLD wafers were 210.0 and 0.9 rads, respectively. Seeds were washed for 2 hrs in a phosphate buffered saline solution. Bacteria were isolated by plating samples of the seed-washings onto dilute tryptic soy agar. Pure isolates of morphologically distinct bacteria were obtained by standard microbiological procedures. Bacteria were grouped according to colony-type and preliminary identification was completed using a fatty-acid analysis system. Bacillus spp. were the primary microoganisms that survived on seed during the experiment. Bacterial diversity and relative abundance were similar for the ground flight seed. Bacillus subtilus, B. pumilus, B. licheniformis, B. polymyxa, B. megaterium, and B. pabuli were isolated most frequently. Members of the genera Kurthia, Listeria, Micrococcus, and Arthrobacter were also isolated from flight and ground control seed. Results support the hypothesis that terrestrial microorganisms can survive long periods of time in the relatively harsh LEO environment.
Schuerger, Andrew C.↗
Enzymatic synthesis of polymers containing nicotinamide mononucleotide
Nicotinamide mononucleoside 5'-diphosphate in its reduced form is an excellent substrate for polynucleotide phosphorylase from Micrococcus luteus both in de novo polymerization reactions and in primer extension reactions. The oxidized form of the diphosphate is a much less efficient substrate; it can be used to extend primers but does not oligomerize in the absence of a primer. The cyanide adduct of the oxidized substrate, like the reduced substrate, polymerizes efficiently. Loss of cyanide yields high molecular weight polymers of the oxidized form. Terminal transferase from calf thymus accepts nicotinamide mononucleoside 5'-triphosphate as a substrate and efficiently adds one residue to the 3'-end of an oligodeoxynucleotide. T4 polynucleotide kinase accepts oligomers of nicotinamide mononucleotide as substrates. However, RNA polymerases do not incorporate nicotinamide mononucleoside 5'-triphosphate into products on any of the templates that we used.
Liu, Rihe↗
Microbial colonization of a closed growth chamber during hydroponic cultivation of lettuce
The goal of this study was to characterize sessile and planktonic microbiota that developed during two successive hydroponic cultures of lettuce in a closed chamber system. Coupons of polyvinyl chloride (PVC) placed in the nutrient solution lines were removed periodically, as were samples of the nutrient solutions and condensate from the air-handling system. The bacteria and fungi present on the coupons and in fluid samples were enumerated by direct plate counts. Disinfecting the hydroponic system with 0.1% hypochlorite and 0.1 N nitric acid reduced the bacterial densities in biofilm samples from 1 x 10 7 CFU/10 cm 2 to 1 x 10 1 CFU/10 cm 2 and eliminated culturable fungi; Staphylococcus sp., Pseudomonas sp., and Micrococcus sp. survived this procedure. Bacterial and fungal concentrations in all samples returned to predisinfection levels after 2 days of plant growth. Pseudomonas and Acremonium predominated both before and after disinfection. Fungal concentrations never exceeded 7 x 10 2 CFU/10 cm 2 . The coupon microbiota differed from that of the rhizoplane at harvest. Overall, the greatest numbers of species were found on the rhizoplane samples collected during the second crop. The microbial community changed little during individual crops or between successive crops. Diversity indices remained relatively constant for all samples.
Bacteria growth & development↗
Enzymatic Synthesis of Polymers Containing Nicotinamide Mononucleotide
Nicotinamide mononucleoside 5'-diphosphate in its reduced form is an excellent substrate for polynucleotide phosphorylase from Micrococcus luteus both in de novo polymerization reactions and in primer extension reactions. The oxidized form of the diphosphate is a much less efficient substrate; it can be used to extend primers but does not oligomerize in the absence of a primer. The cyanide adduct of the oxidized substrate, like the reduced substrate, polymerizes efficiently. Loss of cyanide yields high molecular weight polymers of the oxidized form. Terminal transferase from calf thymus accepts nicotinamide mononucleoside 5'-triphosphate as a substrate and efficiently adds one residue to the 3'-end of an oligodeoxynucleotide. T4 polynucleotide kinase accepts oligomers of nicotinamide mononucleotide as substrates. However, RNA polymerases do not incorporate nicotinamide mononucleoside 5'-triphosphate into products on any of the templates that we used.
Liu, Rihe↗
Characterization of 15 selected coccal bacteria isolated from Antarctic rock and soil samples from the McMurdo-Dry Valleys (South-Victoria Land)
Approximately 1500 cultures of microorganisms were isolated from rocks and soils of the Ross Desert (McMurdo-Dry Valleys). From these, 15 coccoid strains were chosen for more detailed investigation. They were characterized by morphological, physiological and chemotaxonomical properties. All isolates were Gram-positive, catalase-positive and nonmotile. Six strains showed red pigmentation and could be identified as members of the genera Micrococcus (M. roseus, M. agilis) or Deinococcus. In spite of their coccoid morphology, the remaining nine strains had to be associated with coryneform bacteria (Arthrobacter, Brevibacterium), because of their cell wall composition and G+C ratios. Most of the strains were psychrotrophic, but one strain was even obligately psychrophilic, with a temperature maximum below 20 degrees C. Red cocci had in vitro pH optima above 9.0 although they generally originated from acid samples. Most isolates showed a preference for sugar alcohols and organic acids, compounds which are commonly known to be released by lichens, molds and algae, the other components of the cryptoendolithic ecosystem. These properties indicate that our strains are autochthonous members of the natural Antarctic microbial population.
NASA Program Exobiology↗
Molecular microbial diversity of a spacecraft assembly facility
In ongoing investigations to map and archive the microbial footprints in various components of the spacecraft and its accessories, we have examined the microbial populations of the Jet Propulsion Laboratory's Spacecraft Assembly Facility (JPL-SAF). Witness plates made up of spacecraft materials, some painted with spacecraft qualified paints, were exposed for approximately 7 to 9 months at JPL-SAF and examined the particulate materials collected for the incidence of total cultivable aerobic heterotrophs and heat-tolerant (80 degrees C for 15-min.) spore-formers. The results showed that the witness plates coated with spacecraft qualified paints attracted more dust particles than the non-coated stainless steel witness plates. Among the four paints tested, witness plates coated with NS43G accumulated the highest number of particles, and hence attracted more cultivable microbes. The conventional microbiological examination revealed that the JPL-SAF harbors mainly Gram-positive microbes and mostly spore-forming Bacillus species. Most of the isolated microbes were heat resistant to 80 degrees C and proliferate at 60 degrees C. The phylogenetic relationships among 23 cultivable heat-tolerant microbes were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. By 16S rDNA sequence analysis, the isolates fell into seven clades: Bacillus licheniformis, B. pumilus, B. cereus, B. circulans, Staphylococcus capitis, Planococcus sp. and Micrococcus lylae. In contrast to the cultivable approach, direct DNA isolation, cloning and 16S rDNA sequencing analysis revealed equal representation of both Gram-positive and Gram-negative microorganisms.
Industry↗
Evaluation of portable air samplers for monitoring airborne culturable bacteria
Airborne culturable bacteria were monitored at five locations (three in an office/laboratory building and two in a private residence) in a series of experiments designed to compare the efficiency of four air samplers: the Andersen two-stage, Burkard portable, RCS Plus, and SAS Super 90 samplers. A total of 280 samples was collected. The four samplers were operated simultaneously, each sampling 100 L of air with collection on trypticase soy agar. The data were corrected by applying positive hole conversion factors for the Burkard portable, Andersen two-stage, and SAS Super 90 air samplers, and were expressed as log10 values prior to statistical analysis by analysis of variance. The Burkard portable air sampler retrieved the highest number of airborne culturable bacteria at four of the five sampling sites, followed by the SAS Super 90 and the Andersen two-stage impactor. The number of bacteria retrieved by the RCS Plus was significantly less than those retrieved by the other samplers. Among the predominant bacterial genera retrieved by all samplers were Staphylococcus, Bacillus, Corynebacterium, Micrococcus, and Streptococcus.
NASA Center JSC↗
Population dynamics of transgenic strain Escherichia coli Z905/pPHL7 in freshwater and saline lake water microcosms with differing microbial community structures
Populations of Escherichia coli Z905/pPHL7, a transgenic microorganism, were heterogenic in the expression of plasmid genes when adapting to the conditions of water microcosms of various mineralization levels and structure of microbial community. This TM has formed two subpopulations (ampicillin-resistant and ampicillin-sensitive) in every microcosm. Irrespective of mineralization level of a microcosm, when E. coli Z905/pPHL7 alone was introduced, the ampicillin-resistant subpopulation prevailed, while introduction of the TM together with indigenous bacteria led to the dominance of the ampicillin-sensitive subpopulation. A high level of lux gene expression maintained longer in the freshwater microcosms than in sterile saline lake water microcosms. A horizontal gene transfer has been revealed between the jointly introduced TM and Micrococcus sp. 9/pSH1 in microcosms with the Lake Shira sterile water. c2005 COSPAR. Published by Elsevier Ltd. All rights reserved.
Ecosystem↗
Medical Significance of Microorganisms in Spacecraft Environment
Microorganisms can spoil food supplies, contaminate drinking water, release noxious volatile compounds, initiate allergic responses, contaminate the environment, and cause infectious diseases. International acceptability limits have been established for bacterial and fungal contaminants in air and on surfaces, and environmental monitoring is conducted to ensure compliance. Allowable levels of microorganism in water and food have also been established. Environmental monitoring of the space shuttle, the Mir, and the ISS have allowed for some general conclusions. Generally, the bacteria found in air and on interior surfaces are largely of human origin such as Staphylococcus spp., Micrococcus spp. Common environmental genera such as Bacillus spp. are the most commonly isolated bacteria from all spacecraft. Yeast species associated with humans such as Candida spp. are commonly found. Aspergillus spp., Penicillium spp., and Cladosporium spp. are the most commonly isolated filamentous fungi. Microbial levels in the environment differ significantly depending upon humidity levels, condensate accumulation, and availability of carbon sources. However, human "normal flora" of bacteria and fungi can result in serious, life-threatening diseases if human immunity is compromised. Disease incidence is expected to increase as mission duration increases.
Pierson, Duane L.↗
A Lab with a View: Findings from NASA's Aircraft Bioaerosol Collector
Airborne microorganisms in the upper troposphere and lower stratosphere remain elusive due to a lack of reliable sample collection systems. To address this problem, we designed, installed, and flight-validated a novel Aircraft Bioaerosol Collector (ABC) for NASA's C-20A that can make collections for microbiological research investigations up to altitudes of 13.7 km. Herein we report results from the first set of science flights-four consecutive missions flown over the United States (US) from 30 October to 2 November, 2017. To ascertain how the concentration of airborne bacteria changed across the tropopause, we collected air during aircraft Ascent/Descent (0.3 to 11 km), as well as sustained Cruise altitudes in the lower stratosphere (~12 km). Bioaerosols were captured on DNA-treated gelatinous filters inside a cascade air sampler, then analyzed with molecular and culture-based characterization. Several viable bacterial isolates were recovered from flight altitudes, including Bacillus sp., Micrococcus sp., Arthrobacter sp., and Staphylococcus sp. from Cruise samples and Brachybacterium sp. from Ascent/Descent samples. Using 16S V4 sequencing methods for a culture-independent analysis of bacteria, the average number of total OTUs was 305 for Cruise samples and 276 for Ascent/Descent samples. Surprisingly, our results revealed a homogeneous distribution of bacteria in the atmosphere up to 12 km. The observation could be due to atmospheric conditions producing similar background aerosols across the western US, as suggested by modeled back trajectories and satellite measurements. However, the influence of aircraft-associated bacterial contaminants could not be fully eliminated and that background signal was reported throughout our dataset. Considering the tremendous engineering challenge of collecting biomass at extreme altitudes where contamination from flight hardware remains an ever-present issue, we note the utility of using the stratosphere as a proving ground for planned life detection missions across the solar system.
bioaerosols↗
Nanopore Sequencing-Based Microbial Air Profiling Method for Crewed Spacecraft
Microbial monitoring of the International Space Station (ISS) atmosphere is vital to maintaining the health of the spacecraft and crew. Key to NASA’s microbial risk assessment is the identity of contaminating microorganisms in the environment. Historically, this has been achieved through impaction-based air sampling followed by culture. Identification of the microorganisms present requires sample return to Earth and lab-based analyses. While this culture-based approach has served to provide alerts to anomalies and overall confidence in the controls in place, it is not suitable for exploration missions with no sample return. Recently, significant advancements in molecular-based microbial monitoring via nanopore sequencing have been implemented onboard the ISS. Building on this work, multiple commercially available air samplers, compatible with downstream molecular analysis, were evaluated for use in the spaceflight environment. Through this assessment, the Coriolis Compact (Bertin Technologies), which uses cyclonic technology to collect bioaerosols onto the surface of a sterile cone, was selected for a larger-scale comparison to the current culture-based monitoring method. Using the Coriolis Compact, 1000 L of air was collected from the breakroom of an office building and a fitness center. The buffer used to dissociate the microbial cells from the surface of the cone was split between the NASA Microbiology Laboratory’s standard culture and Sanger sequencing-based method and the culture-independent nanopore sequencing method. The bacteria identified through culture were present in the nanopore data, with Micrococcus, Staphylococcus, and Moraxella being the most common cultured isolates, which is expected based on the media and growth conditions. Not surprisingly, the nanopore data yielded much higher diversity and paralleled that of previous atmospheric microbiome studies of human-occupied built environments. As compared to the culture-based data where the breakroom and fitness center data sets cluster in proximity, the nanopore data depicts the contrast of these atmospheric microbiomes. Moreover, the nanopore data were sufficient to meet NASA’s risk assessment needs and noted the culturable isolates routinely observed. This nanopore-based atmospheric microbial profiling method will enable near real-time environmental monitoring of crewed spacecraft as future missions extend beyond low-Earth orbit.
Brandon Dunbar↗