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1.  Resistance of Bacterial Endospores to Outer Space for Planetary Protection Purposes—Experiment PROTECT of the EXPOSE-E Mission 
Astrobiology  2012;12(5):445-456.
Abstract
Spore-forming bacteria are of particular concern in the context of planetary protection because their tough endospores may withstand certain sterilization procedures as well as the harsh environments of outer space or planetary surfaces. To test their hardiness on a hypothetical mission to Mars, spores of Bacillus subtilis 168 and Bacillus pumilus SAFR-032 were exposed for 1.5 years to selected parameters of space in the experiment PROTECT during the EXPOSE-E mission on board the International Space Station. Mounted as dry layers on spacecraft-qualified aluminum coupons, the “trip to Mars” spores experienced space vacuum, cosmic and extraterrestrial solar radiation, and temperature fluctuations, whereas the “stay on Mars” spores were subjected to a simulated martian environment that included atmospheric pressure and composition, and UV and cosmic radiation. The survival of spores from both assays was determined after retrieval. It was clearly shown that solar extraterrestrial UV radiation (λ≥110 nm) as well as the martian UV spectrum (λ≥200 nm) was the most deleterious factor applied; in some samples only a few survivors were recovered from spores exposed in monolayers. Spores in multilayers survived better by several orders of magnitude. All other environmental parameters encountered by the “trip to Mars” or “stay on Mars” spores did little harm to the spores, which showed about 50% survival or more. The data demonstrate the high chance of survival of spores on a Mars mission, if protected against solar irradiation. These results will have implications for planetary protection considerations. Key Words: Planetary protection—Bacterial spores—Space experiment—Simulated Mars mission. Astrobiology 12, 445–456.
doi:10.1089/ast.2011.0737
PMCID: PMC3371261  PMID: 22680691
2.  Persistence of Biomarker ATP and ATP-Generating Capability in Bacterial Cells and Spores Contaminating Spacecraft Materials under Earth Conditions and in a Simulated Martian Environment▿  
Applied and Environmental Microbiology  2008;74(16):5159-5167.
Most planetary protection research has concentrated on characterizing viable bioloads on spacecraft surfaces, developing techniques for bioload reduction prior to launch, and studying the effects of simulated martian environments on microbial survival. Little research has examined the persistence of biogenic signature molecules on spacecraft materials under simulated martian surface conditions. This study examined how endogenous adenosine-5′-triphosphate (ATP) would persist on aluminum coupons under simulated martian conditions of 7.1 mbar, full-spectrum simulated martian radiation calibrated to 4 W m−2 of UV-C (200 to 280 nm), −10°C, and a Mars gas mix of CO2 (95.54%), N2 (2.7%), Ar (1.6%), O2 (0.13%), and H2O (0.03%). Cell or spore viabilities of Acinetobacter radioresistens, Bacillus pumilus, and B. subtilis were measured in minutes to hours, while high levels of endogenous ATP were recovered after exposures of up to 21 days. The dominant factor responsible for temporal reductions in viability and loss of ATP was the simulated Mars surface radiation; low pressure, low temperature, and the Mars gas composition exhibited only slight effects. The normal burst of endogenous ATP detected during spore germination in B. pumilus and B. subtilis was reduced by 1 or 2 orders of magnitude following, respectively, 8- or 30-min exposures to simulated martian conditions. The results support the conclusion that endogenous ATP will persist for time periods that are likely to extend beyond the nominal lengths of most surface missions on Mars, and planetary protection protocols prior to launch may require additional rigor to further reduce the presence and abundance of biosignature molecules on spacecraft surfaces.
doi:10.1128/AEM.00891-08
PMCID: PMC2519281  PMID: 18567687
3.  Effects of Simulated Mars Conditions on the Survival and Growth of Escherichia coli and Serratia liquefaciens▿  
Escherichia coli and Serratia liquefaciens, two bacterial spacecraft contaminants known to replicate under low atmospheric pressures of 2.5 kPa, were tested for growth and survival under simulated Mars conditions. Environmental stresses of high salinity, low temperature, and low pressure were screened alone and in combination for effects on bacterial survival and replication, and then cells were tested in Mars analog soils under simulated Mars conditions. Survival and replication of E. coli and S. liquefaciens cells in liquid medium were evaluated for 7 days under low temperatures (5, 10, 20, or 30°C) with increasing concentrations (0, 5, 10, or 20%) of three salts (MgCl2, MgSO4, NaCl) reported to be present on the surface of Mars. Moderate to high growth rates were observed for E. coli and S. liquefaciens at 30 or 20°C and in solutions with 0 or 5% salts. In contrast, cell densities of both species generally did not increase above initial inoculum levels under the highest salt concentrations (10 and 20%) and the four temperatures tested, with the exception that moderately higher cell densities were observed for both species at 10% MgSO4 maintained at 20 or 30°C. Growth rates of E. coli and S. liquefaciens in low salt concentrations were robust under all pressures (2.5, 10, or 101.3 kPa), exhibiting a general increase of up to 2.5 orders of magnitude above the initial inoculum levels of the assays. Vegetative E. coli cells were maintained in a Mars analog soil for 7 days under simulated Mars conditions that included temperatures between 20 and −50°C for a day/night diurnal period, UVC irradiation (200 to 280 nm) at 3.6 W m−2 for daytime operations (8 h), pressures held at a constant 0.71 kPa, and a gas composition that included the top five gases found in the martian atmosphere. Cell densities of E. coli failed to increase under simulated Mars conditions, and survival was reduced 1 to 2 orders of magnitude by the interactive effects of desiccation, UV irradiation, high salinity, and low pressure (in decreasing order of importance). Results suggest that E. coli may be able to survive, but not grow, in surficial soils on Mars.
doi:10.1128/AEM.02147-09
PMCID: PMC2849189  PMID: 20154104
4.  Potassium ferrate [Fe(VI)] does not mediate self-sterilization of a surrogate mars soil 
BMC Microbiology  2003;3:4.
Background
Martian soil is thought to be enriched with strong oxidants such as peroxides and/or iron in high oxidation states that might destroy biological materials. There is also a high flux of ultraviolet radiation at the surface of Mars. Thus, Mars may be inhospitable to life as we know it on Earth. We examined the hypothesis that if the soil of Mars contains ferrates [Fe(VI)], the strongest of the proposed oxidizing species, and also is exposed to high fluxes of UV radiation, it will be self-sterilizing.
Results
Under ambient conditions (25°C, oxygen and water present) K2FeO4 mixed into sand mineralized some reactive organic molecules to CO2, while less reactive compounds were not degraded. Dried endospores of Bacillus subtilis incubated in a Mars surrogate soil comprised of dry silica sand containing 20% by weight K2FeO4 and under conditions similar to those now on Mars (extreme desiccation, cold, and a CO2-dominated atmosphere) were resistant to killing by the ferrate-enriched sand. Similar results were observed with permanganate. Spores in oxidant-enriched sand exposed to high fluxes of UV light were protected from the sporocidal activity of the radiation below about 5 mm depths.
Conclusion
Based on our data and previously published descriptions of ancient but dormant life forms on Earth, we suggest that if entities resembling bacterial endospores were produced at some point by life forms on Mars, they might still be present and viable, given appropriate germination conditions. Endospores delivered to Mars on spacecraft would possibly survive and potentially compromise life detection experiments.
doi:10.1186/1471-2180-3-4
PMCID: PMC153549  PMID: 12694634
5.  Survival of Spacecraft-Associated Microorganisms under Simulated Martian UV Irradiation 
Applied and Environmental Microbiology  2005;71(12):8147-8156.
Spore-forming microbes recovered from spacecraft surfaces and assembly facilities were exposed to simulated Martian UV irradiation. The effects of UVA (315 to 400 nm), UVA+B (280 to 400 nm), and the full UV spectrum (200 to 400 nm) on the survival of microorganisms were studied at UV intensities expected to strike the surfaces of Mars. Microbial species isolated from the surfaces of several spacecraft, including Mars Odyssey, X-2000 (avionics), and the International Space Station, and their assembly facilities were identified using 16S rRNA gene sequencing. Forty-three Bacillus spore lines were screened, and 19 isolates showed resistance to UVC irradiation (200 to 280 nm) after exposure to 1,000 J m−2 of UVC irradiation at 254 nm using a low-pressure mercury lamp. Spores of Bacillus species isolated from spacecraft-associated surfaces were more resistant than a standard dosimetric strain, Bacillus subtilis 168. In addition, the exposure time required for UVA+B irradiation to reduce the viable spore numbers by 90% was 35-fold longer than the exposure time required for the full UV spectrum to do this, confirming that UVC is the primary biocidal bandwidth. Among the Bacillus species tested, spores of a Bacillus pumilus strain showed the greatest resistance to all three UV bandwidths, as well as the total spectrum. The resistance to simulated Mars UV irradiation was strain specific; B. pumilus SAFR-032 exhibited greater resistance than all other strains tested. The isolation of organisms like B. pumilus SAFR-032 and the greater survival of this organism (sixfold) than of the standard dosimetric strains should be considered when the sanitation capabilities of UV irradiation are determined.
doi:10.1128/AEM.71.12.8147-8156.2005
PMCID: PMC1317311  PMID: 16332797
6.  Response of terrestrial microorganisms to a simulated Martian environment. 
Soil samples from Cape Canaveral were subjected to a simulated Martian environment and assayed periodically over 45 days to determine the effect of various environmental parameters on bacterial populations. The simulated environment was based on the most recent available data, prior to the Viking spacecraft, describing Martian conditions and consisted of a pressure of 7 millibars, an atmosphere of 99.9% CO2 and 0.1% O2, a freeze-thaw cycle of -65 degrees C for 16 h and 24 degrees C for 8 h, and variable moisture and nutrients. Reduced pressure had a significant effect, reducing growth under these conditions. Slight variations in gaseous composition of the simulated atmosphere had negligible effect on growth. The freeze-thaw cycle did not inhibit growth but did result in a slower rate of decline after growth had occurred. Dry samples exhibited no change during the 45-day experiment, indicating that the simulated Martian environment was not toxic to bacterial populations. Psychotrophic organisms responded more favorably to this environment than mesophiles, although both types exhibited increases of approximately 3 logs in 7 to 14 days when moisture and nutrients were available.
PMCID: PMC242914  PMID: 646358
7.  Survival of Microorganisms in a Simulated Martian Environment 
Applied Microbiology  1964;12(3):215-218.
Survival of Bacillus subtilis var. globigii in a simulated Martian environment was demonstrated. Previous contact with the simulated Martian soil or atmosphere reduced germination or outgrowth of unheated spores, or both. Inoculation into simulated Martian soil and then flushing with a simulated Martian atmosphere were lethal to both vegetative cells and spores. After one diurnal temperature cycle (26 to -60 C), the majority of of cells present were spores. No further effect of the diurnal cycle on survival was noted in any of the experimental samples.
PMCID: PMC1058102  PMID: 14170958
8.  Bacterial Growth at the High Concentrations of Magnesium Sulfate Found in Martian Soils 
Astrobiology  2012;12(2):98-106.
Abstract
The martian surface environment exhibits extremes of salinity, temperature, desiccation, and radiation that would make it difficult for terrestrial microbes to survive. Recent evidence suggests that martian soils contain high concentrations of MgSO4 minerals. Through warming of the soils, meltwater derived from subterranean ice-rich regolith may exist for an extended period of time and thus allow the propagation of terrestrial microbes and create significant bioburden at the near surface of Mars. The current report demonstrates that halotolerant bacteria from the Great Salt Plains (GSP) of Oklahoma are capable of growing at high concentrations of MgSO4 in the form of 2 M solutions of epsomite. The epsotolerance of isolates in the GSP bacterial collection was determined, with 35% growing at 2 M MgSO4. There was a complex physiological response to mixtures of MgSO4 and NaCl coupled with other environmental stressors. Growth also was measured at 1 M concentrations of other magnesium and sulfate salts. The complex responses may be partially explained by the pattern of chaotropicity observed for high-salt solutions as measured by agar gelation temperature. Select isolates could grow at the high salt concentrations and low temperatures found on Mars. Survival during repetitive freeze-thaw or drying-rewetting cycles was used as other measures of potential success on the martian surface. Our results indicate that terrestrial microbes might survive under the high-salt, low-temperature, anaerobic conditions on Mars and present significant potential for forward contamination. Stringent planetary protection requirements are needed for future life-detection missions to Mars. Key Words: Analogue—Mars—Planetary protection—Salts—Life in extreme environments. Astrobiology 12, 98–106.
doi:10.1089/ast.2011.0720
PMCID: PMC3277918  PMID: 22248384
9.  Dry-heat resistance of selected psychrophiles. 
The dry-heat resistance characteristics of spores of psychrophilic organisms isolated from soil samples from the Viking spacecraft assembly areas at Cape Kennedy Space Flight Center, Cape Canaveral, Fla., were studied. Spore suspensions were produced, and dry-heat D values were determined for the microorganisms that demonstrated growth or survival under a simulated Martian environment. The dry-heat tests were carried out by using the planchet-boat-hot plate system at 110 and 125 degrees C with an ambient relative humidity of 50% at 22 degrees C. The spores evaluated had a relatively low resistance to dry heat. D(110 degrees C) values ranged from 7.5 to 122 min, whereas the D(123 degrees C) values ranged from less than 1.0 to 9.8 min.
PMCID: PMC242613  PMID: 410367
10.  Protective Role of Spore Structural Components in Determining Bacillus subtilis Spore Resistance to Simulated Mars Surface Conditions 
Applied and Environmental Microbiology  2012;78(24):8849-8853.
Spores of wild-type and mutant Bacillus subtilis strains lacking various structural components were exposed to simulated Martian atmospheric and UV irradiation conditions. Spore survival and mutagenesis were strongly dependent on the functionality of all of the structural components, with small acid-soluble spore proteins, coat layers, and dipicolinic acid as key protectants.
doi:10.1128/AEM.02527-12
PMCID: PMC3502913  PMID: 23064347
11.  Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars 
Radiation research  2003;159(6):722-729.
Kminek, G., Bada, J. L., Pogliano, K. and Ward, J. F. Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars. Radiat. Res. 159, 722–729 (2003).
When claims for the long-term survival of viable organisms are made, either within terrestrial minerals or on Mars, considerations should be made of the limitations imposed by the naturally occurring radiation dose to which they have been exposed. We investigated the effect of ionizing radiation on different bacterial spores by measuring the inactivation constants for B. subtilis and S. marismortui spores in solution as well as for dry spores of B. subtilis and B. thuringiensis. S. marismortui is a halophilic spore that is genetically similar to the recently discovered 2-9-3 bacterium from a halite fluid inclusion, claimed to be 250 million years old (Vreeland et al., Nature 407, 897–900, 2000). B. thuringiensis is a soil bacterium that is genetically similar to the human pathogens B. anthracis and B. cereus (Helgason et al., Appl. Environ. Microbiol. 66, 2627–2630, 2000). To relate the inactivation constant to some realistic environments, we calculated the radiation regimen in a halite fluid inclusion and in the Martian subsurface over time. Our conclusion is that the ionizing dose of radiation in those environments limits the survival of viable bacterial spores over long periods. In the absence of an active repair mechanism in the dormant state, the long-term survival of spores is limited to less than 109 million years in halite fluid inclusions, to 100 to 160 million years in the Martian subsurface below 3 m, and to less than 600,000 years in the uppermost meter of Mars.
PMCID: PMC3919141  PMID: 12751954
12.  The Apparent Involvement of ANMEs in Mineral Dependent Methane Oxidation, as an Analog for Possible Martian Methanotrophy 
Life : Open Access Journal  2011;1(1):19-33.
On Earth, marine anaerobic methane oxidation (AOM) can be driven by the microbial reduction of sulfate, iron, and manganese. Here, we have further characterized marine sediment incubations to determine if the mineral dependent methane oxidation involves similar microorganisms to those found for sulfate-dependent methane oxidation. Through FISH and FISH-SIMS analyses using 13C and 15N labeled substrates, we find that the most active cells during manganese dependent AOM are primarily mixed and mixed-cluster aggregates of archaea and bacteria. Overall, our control experiment using sulfate showed two active bacterial clusters, two active shell aggregates, one active mixed aggregate, and an active archaeal sarcina, the last of which appeared to take up methane in the absence of a closely-associated bacterial partner. A single example of a shell aggregate appeared to be active in the manganese incubation, along with three mixed aggregates and an archaeal sarcina. These results suggest that the microorganisms (e.g., ANME-2) found active in the manganese-dependent incubations are likely capable of sulfate-dependent AOM. Similar metabolic flexibility for Martian methanotrophs would mean that the same microbial groups could inhabit a diverse set of Martian mineralogical crustal environments. The recently discovered seasonal Martian plumes of methane outgassing could be coupled to the reduction of abundant surface sulfates and extensive metal oxides, providing a feasible metabolism for present and past Mars. In an optimistic scenario Martian methanotrophy consumes much of the periodic methane released supporting on the order of 10,000 microbial cells per cm2 of Martian surface. Alternatively, most of the methane released each year could be oxidized through an abiotic process requiring biological methane oxidation to be more limited. If under this scenario, 1% of this methane flux were oxidized by biology in surface soils or in subsurface aquifers (prior to release), a total of about 1020 microbial cells could be supported through methanotrophy with the cells concentrated in regions of methane release.
doi:10.3390/life1010019
PMCID: PMC4187123  PMID: 25382054
Archaea; methane; methanotrophy; Mars; subsurface biosphere
13.  Survival of Bacillus thuringiensis Spores in Soil † 
Bacillus thuringiensis spores and parasporal crystals were incubated in natural soil, both in the laboratory and in nature. During the first 2 weeks, the spore count decreased by approximately 1 log. Thereafter, the number of spore CFU remained constant for at least 8 months. B. thuringiensis did not lose its ability to make the parasporal crystals during its residence in soil. Spore survival was similar for a commercial spore-crystal preparation (the insecticide) and for laboratory-grown spores. In contrast to these results, spores that were produced in situ in soil through multiplication of added vegetative cells survived for only a short time. For spore additions to soil, variations in soil pH had little effect on survival for those spores that survived the first 2 weeks of incubation. Also without effect were various pretreatments of the spores before incubation in soil or nutritional amendment or desiccation of the soil. Remoistening of a desiccated soil, however, caused a decrease in spore numbers. Spores incubated in soil in the field did not show this, but the degree of soil desiccation in nature probably never reached that for the laboratory samples. The good survival of B. thuringiensis spores after the first 2 weeks in soil seemed to be a result of their inability to germinate in soil. We found no evidence for the hypothesis that rapid germination ability for spores in soil conferred a survival advantage.
PMCID: PMC238786  PMID: 16346949
14.  Investigating the Effects of Simulated Martian Ultraviolet Radiation on Halococcus dombrowskii and Other Extremely Halophilic Archaebacteria 
Astrobiology  2009;9(1):104-112.
The isolation of viable extremely halophilic archaea from 250-million-year-old rock salt suggests the possibility of their long-term survival under desiccation. Since halite has been found on Mars and in meteorites, haloarchaeal survival of martian surface conditions is being explored. Halococcus dombrowskii H4 DSM 14522T was exposed to UV doses over a wavelength range of 200–400 nm to simulate martian UV flux. Cells embedded in a thin layer of laboratory-grown halite were found to accumulate preferentially within fluid inclusions. Survival was assessed by staining with the LIVE/DEAD kit dyes, determining colony-forming units, and using growth tests. Halite-embedded cells showed no loss of viability after exposure to about 21 kJ/m2, and they resumed growth in liquid medium with lag phases of 12 days or more after exposure up to 148 kJ/m2. The estimated D37 (dose of 37 % survival) for Hcc. dombrowskii was ≥ 400 kJ/m2. However, exposure of cells to UV flux while in liquid culture reduced D37 by 2 orders of magnitude (to about 1 kJ/m2); similar results were obtained with Halobacterium salinarum NRC-1 and Haloarcula japonica. The absorption of incoming light of shorter wavelength by color centers resulting from defects in the halite crystal structure likely contributed to these results. Under natural conditions, haloarchaeal cells become embedded in salt upon evaporation; therefore, dispersal of potential microscopic life within small crystals, perhaps in dust, on the surface of Mars could resist damage by UV radiation.
doi:10.1089/ast.2007.0234
PMCID: PMC3182532  PMID: 19215203
Halococcus dombrowskii; Simulated martian UV radiation; LIVE/DEAD staining; Halite fluid inclusions; UV transmittance and reflectance; Desiccation
15.  Perchlorate Radiolysis on Mars and the Origin of Martian Soil Reactivity 
Astrobiology  2013;13(6):515-520.
Abstract
Results from the Viking biology experiments indicate the presence of reactive oxidants in martian soils that have previously been attributed to peroxide and superoxide. Instruments on the Mars Phoenix Lander and the Mars Science Laboratory detected perchlorate in martian soil, which is nonreactive under the conditions of the Viking biology experiments. We show that calcium perchlorate exposed to gamma rays decomposes in a CO2 atmosphere to form hypochlorite (ClO−), trapped oxygen (O2), and chlorine dioxide (ClO2). Our results show that the release of trapped O2 (g) from radiation-damaged perchlorate salts and the reaction of ClO− with amino acids that were added to the martian soils can explain the results of the Viking biology experiments. We conclude that neither hydrogen peroxide nor superoxide is required to explain the results of the Viking biology experiments. Key Words: Mars—Radiolysis—Organic degradation—in situ measurement—Planetary habitability and biosignatures. Astrobiology 13, 515–520.
doi:10.1089/ast.2013.0999
PMCID: PMC3691774  PMID: 23746165
16.  Activation of Methanogenesis in Arid Biological Soil Crusts Despite the Presence of Oxygen 
PLoS ONE  2011;6(5):e20453.
Methanogenesis is traditionally thought to occur only in highly reduced, anoxic environments. Wetland and rice field soils are well known sources for atmospheric methane, while aerated soils are considered sinks. Although methanogens have been detected in low numbers in some aerated, and even in desert soils, it remains unclear whether they are active under natural oxic conditions, such as in biological soil crusts (BSCs) of arid regions. To answer this question we carried out a factorial experiment using microcosms under simulated natural conditions. The BSC on top of an arid soil was incubated under moist conditions in all possible combinations of flooding and drainage, light and dark, air and nitrogen headspace. In the light, oxygen was produced by photosynthesis. Methane production was detected in all microcosms, but rates were much lower when oxygen was present. In addition, the δ13C of the methane differed between the oxic/oxygenic and anoxic microcosms. While under anoxic conditions methane was mainly produced from acetate, it was almost entirely produced from H2/CO2 under oxic/oxygenic conditions. Only two genera of methanogens were identified in the BSC-Methanosarcina and Methanocella; their abundance and activity in transcribing the mcrA gene (coding for methyl-CoM reductase) was higher under anoxic than oxic/oxygenic conditions, respectively. Both methanogens also actively transcribed the oxygen detoxifying gene catalase. Since methanotrophs were not detectable in the BSC, all the methane produced was released into the atmosphere. Our findings point to a formerly unknown participation of desert soils in the global methane cycle.
doi:10.1371/journal.pone.0020453
PMCID: PMC3105065  PMID: 21655270
17.  Utilization of Low-Pressure Plasma to Inactivate Bacterial Spores on Stainless Steel Screws 
Astrobiology  2013;13(7):597-606.
Abstract
A special focus area of planetary protection is the monitoring, control, and reduction of microbial contaminations that are detected on spacecraft components and hardware during and after assembly. In this study, wild-type spores of Bacillus pumilus SAFR-032 (a persistent spacecraft assembly facility isolate) and the laboratory model organism B. subtilis 168 were used to study the effects of low-pressure plasma, with hydrogen alone and in combination with oxygen and evaporated hydrogen peroxide as a process gas, on spore survival, which was determined by a colony formation assay. Spores of B. pumilus SAFR-032 and B. subtilis 168 were deposited with an aseptic technique onto the surface of stainless steel screws to simulate a spore-contaminated spacecraft hardware component, and were subsequently exposed to different plasmas and hydrogen peroxide conditions in a very high frequency capacitively coupled plasma reactor (VHF-CCP) to reduce the spore burden. Spores of the spacecraft isolate B. pumilus SAFR-032 were significantly more resistant to plasma treatment than spores of B. subtilis 168. The use of low-pressure plasma with an additional treatment of evaporated hydrogen peroxide also led to an enhanced spore inactivation that surpassed either single treatment when applied alone, which indicates the potential application of this method as a fast and suitable way to reduce spore-contaminated spacecraft hardware components for planetary protection purposes. Key Words: Bacillus spores—Contamination—Spacecraft hardware—Plasma sterilization—Planetary protection. Astrobiology 13, 597–606.
doi:10.1089/ast.2012.0949
PMCID: PMC3713438  PMID: 23768085
18.  Searching for signatures of life on Mars: an Fe-isotope perspective 
Recent spacecraft and lander missions to Mars have reinforced previous interpretations that Mars was a wet and warm planet in the geological past. The role of liquid water in shaping many of the surface features on Mars has long been recognized. Since the presence of liquid water is essential for survival of life, conditions on early Mars might have been more favourable for the emergence and evolution of life. Until a sample return mission to Mars, one of the ways of studying the past environmental conditions on Mars is through chemical and isotopic studies of Martian meteorites. Over 35 individual meteorite samples, believed to have originated on Mars, are now available for lab-based studies. Fe is a key element that is present in both primary and secondary minerals in the Martian meteorites. Fe-isotope ratios can be fractionated by low-temperature processes which includes biological activity. Experimental investigations of Fe reduction and oxidation by bacteria have produced large fractionation in Fe-isotope ratios. Hence, it is considered likely that if there is/were any form of life present on Mars then it might be possible to detect its signature by Fe-isotope studies of Martian meteorites. In the present study, we have analysed a number of Martian meteorites for their bulk-Fe-isotope composition. In addition, a set of terrestrial analogue material has also been analysed to compare the results and draw inferences. So far, our studies have not found any measurable Fe-isotopic fractionation in bulk Martian meteorites that can be ascribed to any low-temperature process operative on Mars.
doi:10.1098/rstb.2006.1899
PMCID: PMC1664681  PMID: 17008212
Mars; Martian meteorites; SNC; terrestrial analogues; iron isotopes; life
19.  Triggering Germination Represents a Novel Strategy to Enhance Killing of Clostridium difficile Spores 
PLoS ONE  2010;5(8):e12285.
Background
Clostridium difficile is an anaerobic, spore-forming bacterium that is the most common cause of healthcare-associated diarrhea in developed countries. Control of C. difficile is challenging because the spores are resistant to killing by alcohol-based hand hygiene products, antimicrobial soaps, and most disinfectants. Although initiation of germination has been shown to increase susceptibility of spores of other bacterial species to radiation and heat, it was not known if triggering of germination could be a useful strategy to increase susceptibility of C. difficile spores to radiation or other stressors.
Principal Findings
Here, we demonstrated that exposure of dormant C. difficile spores to a germination solution containing amino acids, minerals, and taurocholic acid resulted in initiation of germination in room air. Germination of spores in room air resulted in significantly enhanced killing by ultraviolet-C (UV-C) radiation and heat. On surfaces in hospital rooms, application of germination solution resulted in enhanced eradication of spores by UV-C administered by an automated room decontamination device. Initiation of germination under anaerobic, but not aerobic, conditions resulted in increased susceptibility to killing by ethanol, suggesting that exposure to oxygen might prevent spores from progressing fully to outgrowth. Stimulation of germination also resulted in reduced survival of spores on surfaces in room air, possibly due to increased susceptibility to stressors such as oxygen and desiccation.
Conclusions
Taken together, these data demonstrate that stimulation of germination could represent a novel method to enhance killing of spores by UV-C, and suggest the possible application of this strategy as a means to enhance killing by other agents.
doi:10.1371/journal.pone.0012285
PMCID: PMC2924393  PMID: 20808871
20.  Validation of a Clostridium Endospore Viability Assay and Analysis of Greenland Ices and Atacama Desert Soils▿ †  
A microscopy-based endospore viability assay (micro-EVA) capable of enumerating germinable Clostridium endospores (GCEs) in less than 30 min has been validated and employed to determine GCE concentrations in Greenland ices and Atacama Desert soils. Inoculation onto agarose doped with Tb3+ and d-alanine triggers Clostridium spore germination and the concomitant release of ∼108 molecules of dipicolinic acid (DPA) per endospore, which, under pulsed UV excitation, enables enumeration of resultant green Tb3+-DPA luminescent spots as GCEs with time-gated luminescence microscopy. The intensity time courses of the luminescent spots were characteristic of stage I Clostridium spore germination dynamics. Micro-EVA was validated against traditional CFU cultivation from 0 to 1,000 total endospores/ml (i.e., phase-bright bodies/ml), yielding 56.4% ± 1.5% GCEs and 43.0% ± 1.0% CFU. We also show that d-alanine serves as a Clostridium-specific germinant (three species tested) that inhibits Bacillus germination of spores (five species tested) in that endospore concentration regime. Finally, GCE concentrations in Greenland ice cores and Atacama Desert soils were determined with micro-EVA, yielding 1 to 2 GCEs/ml of Greenland ice (versus <1 CFU/ml after 6 months of incubation) and 66 to 157 GCEs/g of Atacama Desert soil (versus 40 CFU/g soil).
doi:10.1128/AEM.01966-10
PMCID: PMC3067429  PMID: 21296951
21.  Combined Effects of High Hydrostatic Pressure and Temperature for Inactivation of Bacillus anthracis Spores 
Spores of Bacillus anthracis are known to be extremely resistant to heat treatment, irradiation, desiccation, and disinfectants. To determine inactivation kinetics of spores by high pressure, B. anthracis spores of a Sterne strain-derived mutant deficient in the production of the toxin components (strain RP42) were exposed to pressures ranging from 280 to 500 MPa for 10 min to 6 h, combined with temperatures ranging from 20 to 75°C. The combination of heat and pressure resulted in complete destruction of B. anthracis spores, with a D value (exposure time for 90% inactivation of the spore population) of approximately 4 min after pressurization at 500 MPa and 75°C, compared to 160 min at 500 MPa and 20°C and 348 min at atmospheric pressure (0.1 MPa) and 75°C. The use of high pressure for spore inactivation represents a considerable improvement over other available methods of spore inactivation and could be of interest for antigenic spore preparation.
doi:10.1128/AEM.70.1.635-637.2004
PMCID: PMC321244  PMID: 14711702
22.  Artificial and Solar UV Radiation Induces Strand Breaks and Cyclobutane Pyrimidine Dimers in Bacillus subtilis Spore DNA 
The loss of stratospheric ozone and the accompanying increase in solar UV flux have led to concerns regarding decreases in global microbial productivity. Central to understanding this process is determining the types and amounts of DNA damage in microbes caused by solar UV irradiation. While UV irradiation of dormant Bacillus subtilis endospores results mainly in formation of the “spore photoproduct” 5-thyminyl-5,6-dihydrothymine, genetic evidence indicates that an additional DNA photoproduct(s) may be formed in spores exposed to solar UV-B and UV-A radiation (Y. Xue and W. L. Nicholson, Appl. Environ. Microbiol. 62:2221–2227, 1996). We examined the occurrence of double-strand breaks, single-strand breaks, cyclobutane pyrimidine dimers, and apurinic-apyrimidinic sites in spore DNA under several UV irradiation conditions by using enzymatic probes and neutral or alkaline agarose gel electrophoresis. DNA from spores irradiated with artificial 254-nm UV-C radiation accumulated single-strand breaks, double-strand breaks, and cyclobutane pyrimidine dimers, while DNA from spores exposed to artificial UV-B radiation (wavelengths, 290 to 310 nm) accumulated only cyclobutane pyrimidine dimers. DNA from spores exposed to full-spectrum sunlight (UV-B and UV-A radiation) accumulated single-strand breaks, double-strand breaks, and cyclobutane pyrimidine dimers, whereas DNA from spores exposed to sunlight from which the UV-B component had been removed with a filter (“UV-A sunlight”) accumulated only single-strand breaks and double-strand breaks. Apurinic-apyrimidinic sites were not detected in spore DNA under any of the irradiation conditions used. Our data indicate that there is a complex spectrum of UV photoproducts in DNA of bacterial spores exposed to solar UV irradiation in the environment.
PMCID: PMC91806  PMID: 10618224
23.  Use of UV-C radiation to disinfect non-critical patient care items: a laboratory assessment of the Nanoclave Cabinet 
BMC Infectious Diseases  2012;12:174.
Background
The near-patient environment is often heavily contaminated, yet the decontamination of near-patient surfaces and equipment is often poor. The Nanoclave Cabinet produces large amounts of ultraviolet-C (UV-C) radiation (53 W/m2) and is designed to rapidly disinfect individual items of clinical equipment. Controlled laboratory studies were conducted to assess its ability to eradicate a range of potential pathogens including Clostridium difficile spores and Adenovirus from different types of surface.
Methods
Each test surface was inoculated with known levels of vegetative bacteria (106 cfu/cm2), C. difficile spores (102-106 cfu/cm2) or Adenovirus (109 viral genomes), placed in the Nanoclave Cabinet and exposed for up to 6 minutes to the UV-C light source. Survival of bacterial contaminants was determined via conventional cultivation techniques. Degradation of viral DNA was determined via PCR. Results were compared to the number of colonies or level of DNA recovered from non-exposed control surfaces. Experiments were repeated to incorporate organic soils and to compare the efficacy of the Nanoclave Cabinet to that of antimicrobial wipes.
Results
After exposing 8 common non-critical patient care items to two 30-second UV-C irradiation cycles, bacterial numbers on 40 of 51 target sites were consistently reduced to below detectable levels (≥ 4.7 log10 reduction). Bacterial load was reduced but still persisted on other sites. Objects that proved difficult to disinfect using the Nanoclave Cabinet (e.g. blood pressure cuff) were also difficult to disinfect using antimicrobial wipes. The efficacy of the Nanoclave Cabinet was not affected by the presence of organic soils. Clostridium difficile spores were more resistant to UV-C irradiation than vegetative bacteria. However, two 60-second irradiation cycles were sufficient to reduce the number of surface-associated spores from 103 cfu/cm2 to below detectable levels. A 3 log10 reduction in detectable Adenovirus DNA was achieved within 3 minutes; after 6 minutes, viral DNA was undetectable.
Conclusion
The results of this study suggest that the Nanoclave Cabinet can provide rapid and effective disinfection of some patient-related equipment. However, laboratory studies do not necessarily replicate ‘in-use’ conditions and further tests are required to assess the usability, acceptability and relative performance of the Nanoclave Cabinet when used in situ.
doi:10.1186/1471-2334-12-174
PMCID: PMC3449183  PMID: 22856652
Ultraviolet radiation; Surface disinfection; Nosocomial pathogens; Adenovirus
24.  Colonization patterns of soil microbial communities in the Atacama Desert 
Microbiome  2013;1:28.
Background
The Atacama Desert is one of the driest deserts in the world and its soil, with extremely low moisture, organic carbon content, and oxidizing conditions, is considered to be at the dry limit for life.
Results
Analyses of high throughput DNA sequence data revealed that bacterial communities from six geographic locations in the hyper-arid core and along a North-South moisture gradient were structurally and phylogenetically distinct (ANOVA test for observed operating taxonomic units at 97% similarity (OTU0.03), P <0.001) and that communities from locations in the hyper-arid zone displayed the lowest levels of diversity. We found bacterial taxa similar to those found in other arid soil communities with an abundance of Rubrobacterales, Actinomycetales, Acidimicrobiales, and a number of families from the Thermoleophilia. The extremely low abundance of Firmicutes indicated that most bacteria in the soil were in the form of vegetative cells. Integrating molecular data with climate and soil geochemistry, we found that air relative humidity (RH) and soil conductivity significantly correlated with microbial communities’ diversity metrics (least squares linear regression for observed OTU0.03 and air RH and soil conductivity, P <0.001; UniFrac PCoA Spearman’s correlation for air RH and soil conductivity, P <0.0001), indicating that water availability and salt content are key factors in shaping the Atacama soil microbiome. Mineralization studies showed communities actively metabolizing in all soil samples, with increased rates in soils from the southern locations.
Conclusions
Our results suggest that microorganisms in the driest soils of the Atacama Desert are in a state of stasis for most of the time, but can potentially metabolize if presented with liquid water for a sufficient duration. Over geological time, rare rain events and physicochemical factors potentially played a major role in selecting micro-organisms that are most adapted to extreme desiccating conditions.
doi:10.1186/2049-2618-1-28
PMCID: PMC3971613  PMID: 24451153
Soil microbial communities; Extreme environment; Arid soil; Atacama Desert; Desertification; High-throughput 16S rRNA sequencing
25.  Germination and Amplification of Anthrax Spores by Soil-Dwelling Amoebas 
Applied and Environmental Microbiology  2012;78(22):8075-8081.
While anthrax is typically associated with bioterrorism, in many parts of the world the anthrax bacillus (Bacillus anthracis) is endemic in soils, where it causes sporadic disease in livestock. These soils are typically rich in organic matter and calcium that promote survival of resilient B. anthracis spores. Outbreaks of anthrax tend to occur in warm weather following rains that are believed to concentrate spores in low-lying areas where runoff collects. It has been concluded that elevated spore concentrations are not the result of vegetative growth as B. anthracis competes poorly against indigenous bacteria. Here, we test an alternative hypothesis in which amoebas, common in moist soils and pools of standing water, serve as amplifiers of B. anthracis spores by enabling germination and intracellular multiplication. Under simulated environmental conditions, we show that B. anthracis germinates and multiplies within Acanthamoeba castellanii. The growth kinetics of a fully virulent B. anthracis Ames strain (containing both the pX01 and pX02 virulence plasmids) and vaccine strain Sterne (containing only pX01) inoculated as spores in coculture with A. castellanii showed a nearly 50-fold increase in spore numbers after 72 h. In contrast, the plasmidless strain 9131 showed little growth, demonstrating that plasmid pX01 is essential for growth within A. castellanii. Electron and time-lapse fluorescence microscopy revealed that spores germinate within amoebal phagosomes, vegetative bacilli undergo multiplication, and, following demise of the amoebas, bacilli sporulate in the extracellular milieu. This analysis supports our hypothesis that amoebas contribute to the persistence and amplification of B. anthracis in natural environments.
doi:10.1128/AEM.02034-12
PMCID: PMC3485947  PMID: 22983962

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