Survival of Environment-Derived Opportunistic Bacterial Pathogens to Martian Conditions: Is There a Concern for Human Missions to Mars?

The health of astronauts during space travel to new celestial bodies in the Solar System is a critical factor in the planning of a mission. Despite cleaning and decontamination protocols, microorganisms from the Earth have been and will be identified on spacecraft. This raises concerns for human safety and planetary protection, especially if these microorganisms can evolve and adapt to the new environment. In this study, we examined the tolerance of clinically relevant nonfastidious bacterial species that originate from environmental sources (Burkholderia cepacia, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Serratia marcescens) to simulated martian conditions. Our research showed changes in growth and survival of these species in the presence of perchlorates, under desiccating conditions, exposure to ultraviolet radiation, and exposure to martian atmospheric composition and pressure. In addition, our results demonstrate that growth was enhanced by the addition of a martian regolith simulant to the growth media. Additional future research is warranted to examine potential changes in the infectivity, pathogenicity, and virulence of these species with exposure to martian conditions.

MARSBOx: Fungal and Bacterial Endurance From a Balloon-Flown Analog Mission in the Stratosphere.

Whether terrestrial life can withstand the martian environment is of paramount interest for planetary protection measures and space exploration. To understand microbial survival potential in Mars-like conditions, several fungal and bacterial samples were launched in September 2019 on a large NASA scientific balloon flight to the middle stratosphere (∼38 km altitude) where radiation levels resembled values at the equatorial Mars surface. Fungal spores of Aspergillus niger and bacterial cells of Salinisphaera shabanensis, Staphylococcus capitis subsp. capitis, and Buttiauxella sp. MASE-IM-9 were launched inside the MARSBOx (Microbes in Atmosphere for Radiation, Survival, and Biological Outcomes Experiment) payload filled with an artificial martian atmosphere and pressure throughout the mission profile. The dried microorganisms were either exposed to full UV-VIS radiation (UV dose = 1148 kJ m-2) or were shielded from radiation. After the 5-h stratospheric exposure, samples were assayed for survival and metabolic changes. Spores from the fungus A. niger and cells from the Gram-(-) bacterium S. shabanensis were the most resistant with a 2- and 4-log reduction, respectively. Exposed Buttiauxella sp. MASE-IM-9 was completely inactivated (both with and without UV exposure) and S. capitis subsp. capitis only survived the UV shielded experimental condition (3-log reduction). Our results underscore a wide variation in survival phenotypes of spacecraft associated microorganisms and support the hypothesis that pigmented fungi may be resistant to the martian surface if inadvertently delivered by spacecraft missions.

Taxonomic and functional analyses of intact microbial communities thriving in extreme, astrobiology-relevant, anoxic sites.

BACKGROUND: Extreme terrestrial, analogue environments are widely used models to study the limits of life and to infer habitability of extraterrestrial settings. In contrast to Earth's ecosystems, potential extraterrestrial biotopes are usually characterized by a lack of oxygen. METHODS: In the MASE project (Mars Analogues for Space Exploration), we selected representative anoxic analogue environments (permafrost, salt-mine, acidic lake and river, sulfur springs) for the comprehensive analysis of their microbial communities. We assessed the microbiome profile of intact cells by propidium monoazide-based amplicon and shotgun metagenome sequencing, supplemented with an extensive cultivation effort. RESULTS: The information retrieved from microbiome analyses on the intact microbial community thriving in the MASE sites, together with the isolation of 31 model microorganisms and successful binning of 15 high-quality genomes allowed us to observe principle pathways, which pinpoint specific microbial functions in the MASE sites compared to moderate environments. The microorganisms were characterized by an impressive machinery to withstand physical and chemical pressures. All levels of our analyses revealed the strong and omnipresent dependency of the microbial communities on complex organic matter. Moreover, we identified an extremotolerant cosmopolitan group of 34 poly-extremophiles thriving in all sites. CONCLUSIONS: Our results reveal the presence of a core microbiome and microbial taxonomic similarities between saline and acidic anoxic environments. Our work further emphasizes the importance of the environmental, terrestrial parameters for the functionality of a microbial community, but also reveals a high proportion of living microorganisms in extreme environments with a high adaptation potential within habitability borders. Video abstract.

Questioning the radiation limits of life: Ignicoccus hospitalis between replication and VBNC.

Radiation of ionizing or non-ionizing nature has harmful effects on cellular components like DNA as radiation can compromise its proper integrity. To cope with damages caused by external stimuli including radiation, within living cells, several fast and efficient repair mechanisms have evolved. Previous studies addressing organismic radiation tolerance have shown that radiotolerance is a predominant property among extremophilic microorganisms including (hyper-) thermophilic archaea. The analysis of the ionizing radiation tolerance of the chemolithoautotrophic, obligate anaerobic, hyperthermophilic Crenarchaeon Ignicoccus hospitalis showed a D10-value of 4.7 kGy, fourfold exceeding the doses previously determined for other extremophilic archaea. The genome integrity of I. hospitalis after γ-ray exposure in relation to its survival was visualized by RAPD and qPCR. Furthermore, the discrimination between reproduction, and ongoing metabolic activity was possible for the first time indicating that a potential viable but non-culturable (VBNC) state may also account for I. hospitalis.

Microbial Monitoring in the EDEN ISS Greenhouse, a Mobile Test Facility in Antarctica.

The EDEN ISS greenhouse, integrated in two joined containers, is a confined mobile test facility in Antarctica for the development and optimization of new plant cultivation techniques for future space programs. The EDEN ISS greenhouse was used successfully from February to November 2018 for fresh food production for the overwintering crew at the Antarctic Neumayer III station. During the 9 months of operation, samples from the different plants, from the nutrition solution of the aeroponic planting system, and from diverse surfaces within the three different compartments of the container were taken [future exploration greenhouse (FEG), service section (SS), and cold porch (CP)]. Quantity as well as diversity of microorganisms was examined by cultivation. In case of the plant samples, microbial quantities were in a range from 102 to 104 colony forming units per gram plant material. Compared to plants purchased from a German grocery, the produce hosted orders of magnitude more microorganisms than the EDEN ISS plants. The EDEN ISS plant samples contained mainly fungi and a few bacteria. No classical food associated pathogenic microorganism, like Escherichia and Salmonella, could be found. Probably due to the used cultivation approach, Archaea were not found in the samples. The bioburden in the nutrition solutions increased constantly over time but never reached critical values like 102-103 cfu per 100 mL in irrigation water as it is stated, e.g., for commercial European plant productions. The surface samples revealed high differences in the microbial burden between the greenhouse part of the container and the SS and CP part. However, the numbers of organisms (bacteria and fungi) found in the planted greenhouse were still not critical. The microbial loaded surfaces showed strong temporal as well as spatial fluctuations. In samples of the nutrition solution and the surface, the amount of bacteria exceeded the amount of fungi by many times. For identification, 16S rRNA gene sequencing was performed for the isolated prokaryotic organisms. Phylogenetic analyses revealed that the most abundant bacterial phyla were Firmicutes and Actinobacteria. These phyla include plant- and human-associated bacterial species. In general, it could be shown that it is possible to produce edible fresh food in a remote environment and this food is safe for consumption from a microbiological point of view.

Impact of Simulated Martian Conditions on (Facultatively) Anaerobic Bacterial Strains from Different Mars Analogue Sites.

Five bacterial (facultatively) anaerobic strains, namely Buttiauxella sp. MASE-IM-9, Clostridium sp. MASE-IM-4, Halanaerobium sp. MASE-BB-1, Trichococcus sp. MASE-IM-5, and Yersinia intermedia MASE-LG-1 isolated from different extreme natural environments were subjected to Mars relevant environmental stress factors in the laboratory under controlled conditions. These stress factors encompassed low water activity, oxidizing compounds, and ionizing radiation. Stress tests were performed under permanently anoxic conditions. The survival rate after addition of sodium perchlorate (Na-perchlorate) was found to be species-specific. The inter-comparison of the five microorganisms revealed that Clostridium sp. MASE-IM-4 was the most sensitive strain (D10-value (15 min, NaClO4) = 0.6 M). The most tolerant microorganism was Trichococcus sp. MASE-IM-5 with a calculated D10-value (15 min, NaClO4) of 1.9 M. Cultivation in the presence of Na-perchlorate in Martian relevant concentrations up to 1 wt% led to the observation of chains of cells in all strains. Exposure to Na-perchlorate led to a lowering of the survival rate after desiccation. Consecutive exposure to desiccating conditions and ionizing radiation led to additive effects. Moreover, in a desiccated state, an enhanced radiation tolerance could be observed for the strains Clostridium sp. MASE-IM-4 and Trichococcus sp. MASE-IM-5. These data show that anaerobic microorganisms from Mars analogue environments can resist a variety of Martian-simulated stresses either individually or in combination. However, responses were species-specific and some Mars-simulated extremes killed certain organisms. Thus, although Martian stresses would be expected to act differentially on microorganisms, none of the expected extremes tested here and found on Mars prevent the growth of anaerobic microorganisms.

Microbial Markers Profile in Anaerobic Mars Analogue Environments Using the LDChip (Life Detector Chip) Antibody Microarray Core of the SOLID (Signs of Life Detector) Platform.

One of the main objectives for astrobiology is to unravel and explore the habitability of environments beyond Earth, paying special attention to Mars. If the combined environmental stress factors on Mars are compatible with life or if they were less harsh in the past, to investigate the traces of past or present life is critical to understand its potential habitability. Essential for this research is the characterization of Mars analogue environments on Earth through the development of techniques for biomarker detection in them. Biosensing techniques based on fluorescence sandwich microarray immunoassays (FSMI) have shown to be a powerful tool to detect biosignatures and depict the microbial profiles of different environments. In this study, we described the microbial biomarker profile of five anoxic Mars analogues sites using the Life Detector Chip (LDChip), an antibody microarray for multiple microbial marker detection. Furthermore, we contributed to new targets by developing a new 26-polyclonal antibodies microarray using crude extracts from anaerobic sampling sites, halophilic microorganisms, and anaerobic isolates obtained in the framework of the European Mars Analogues for Space Exploration (MASE) project. The new subset of antibodies was characterized and implemented into a microarray platform (MASE-Chip) for microbial marker searching in salty and anaerobic environments.

Beyond Chloride Brines: Variable Metabolomic Responses in the Anaerobic Organism Yersinia intermedia MASE-LG-1 to NaCl and MgSO4 at Identical Water Activity.

Growth in sodium chloride (NaCl) is known to induce stress in non-halophilic microorganisms leading to effects on the microbial metabolism and cell structure. Microorganisms have evolved a number of adaptations, both structural and metabolic, to counteract osmotic stress. These strategies are well-understood for organisms in NaCl-rich brines such as the accumulation of certain organic solutes (known as either compatible solutes or osmolytes). Less well studied are responses to ionic environments such as sulfate-rich brines which are prevalent on Earth but can also be found on Mars. In this paper, we investigated the global metabolic response of the anaerobic bacterium Yersinia intermedia MASE-LG-1 to osmotic salt stress induced by either magnesium sulfate (MgSO4) or NaCl at the same water activity (0.975). Using a non-targeted mass spectrometry approach, the intensity of hundreds of metabolites was measured. The compatible solutes L-asparagine and sucrose were found to be increased in both MgSO4 and NaCl compared to the control sample, suggesting a similar osmotic response to different ionic environments. We were able to demonstrate that Yersinia intermedia MASE-LG-1 accumulated a range of other compatible solutes. However, we also found the global metabolic responses, especially with regard to amino acid metabolism and carbohydrate metabolism, to be salt-specific, thus, suggesting ion-specific regulation of specific metabolic pathways.

Lack of correlation of desiccation and radiation tolerance in microorganisms from diverse extreme environments tested under anoxic conditions.

Four facultative anaerobic and two obligate anaerobic bacteria were isolated from extreme environments (deep subsurface halite mine, sulfidic anoxic spring, mineral-rich river) in the frame MASE (Mars Analogues for Space Exploration) project. The isolates were investigated under anoxic conditions for their survivability after desiccation up to 6 months and their tolerance to ionizing radiation up to 3000 Gy. The results indicated that tolerances to both stresses are strain-specific features. Yersinia intermedia MASE-LG-1 showed a high desiccation tolerance but its radiation tolerance was very low. The most radiation-tolerant strains were Buttiauxella sp. MASE-IM-9 and Halanaerobium sp. MASE-BB-1. In both cases, cultivable cells were detectable after an exposure to 3 kGy of ionizing radiation, but cells only survived desiccation for 90 and 30 days, respectively. Although a correlation between desiccation and ionizing radiation resistance has been hypothesized for some aerobic microorganisms, our data showed that there was no correlation between tolerance to desiccation and ionizing radiation, suggesting that the physiological basis of both forms of tolerances is not necessarily linked. In addition, these results indicated that facultative and obligate anaerobic organisms living in extreme environments possess varied species-specific tolerances to extremes.

The responses of an anaerobic microorganism, Yersinia intermedia MASE-LG-1 to individual and combined simulated Martian stresses.

The limits of life of aerobic microorganisms are well understood, but the responses of anaerobic microorganisms to individual and combined extreme stressors are less well known. Motivated by an interest in understanding the survivability of anaerobic microorganisms under Martian conditions, we investigated the responses of a new isolate, Yersinia intermedia MASE-LG-1 to individual and combined stresses associated with the Martian surface. This organism belongs to an adaptable and persistent genus of anaerobic microorganisms found in many environments worldwide. The effects of desiccation, low pressure, ionizing radiation, varying temperature, osmotic pressure, and oxidizing chemical compounds were investigated. The strain showed a high tolerance to desiccation, with a decline of survivability by four orders of magnitude during a storage time of 85 days. Exposure to X-rays resulted in dose-dependent inactivation for exposure up to 600 Gy while applied doses above 750 Gy led to complete inactivation. The effects of the combination of desiccation and irradiation were additive and the survivability was influenced by the order in which they were imposed. Ionizing irradiation and subsequent desiccation was more deleterious than vice versa. By contrast, the presence of perchlorates was not found to significantly affect the survival of the Yersinia strain after ionizing radiation. These data show that the organism has the capacity to survive and grow in physical and chemical stresses, imposed individually or in combination that are associated with Martian environment. Eventually it lost its viability showing that many of the most adaptable anaerobic organisms on Earth would be killed on Mars today.

High Tolerance of Hydrogenothermus marinus to Sodium Perchlorate.

On Mars, significant amounts (0.4-0.6%) of perchlorate ions were detected in dry soil by the Phoenix Wet Chemistry Laboratory and later confirmed with the Mars Science Laboratory. Therefore, the ability of Hydrogenothermus marinus, a desiccation tolerant bacterium, to survive and grow in the presence of perchlorates was determined. Results indicated that H. marinus was able to tolerate concentrations of sodium perchlorate up to 200 mM ( 1.6%) during cultivation without any changes in its growth pattern. After the addition of up to 440 mM ( 3.7%) sodium perchlorate, H. marinus showed significant changes in cell morphology; from single motile short rods to long cell chains up to 80 cells. Furthermore, it was shown that the known desiccation tolerance of H. marinus is highly influenced by a pre-treatment with different perchlorates; additive effects of desiccation and perchlorate treatments are visible in a reduced survival rate. These data demonstrate that thermophiles, especially H. marinus, have so far, unknown high tolerances against cell damaging treatments and may serve as model organisms for future space experiments.

Influence of osmotic stress on desiccation and irradiation tolerance of (hyper)-thermophilic microorganisms.

This study examined the influence of prior salt adaptation on the survival rate of (hyper)-thermophilic bacteria and archaea after desiccation and UV or ionizing irradiation treatment. Survival rates after desiccation of Hydrogenothermus marinus and Archaeoglobus fulgidus increased considerably when the cells were cultivated at higher salt concentrations before drying. By doubling the concentration of NaCl, a 30 times higher survival rate of H. marinus after desiccation was observed. Under salt stress, the compatible solute diglycerol phosphate in A. fulgidus and glucosylglycerate in H. marinus accumulated in the cytoplasm. Several different compatible solutes were added as protectants to A. fulgidus and H. marinus before desiccation treatment. Some of these had similar effects as intracellularly produced compatible solutes. The survival rates of H. marinus and A. fulgidus after exposure to UV-C (254 nm) or ionizing X-ray/gamma radiation were irrespective of the salt-induced synthesis or the addition of compatible solutes.