From remote to urbanized: Dispersal of antibiotic-resistant bacteria under the aspect of anthropogenic influence.

Antibiotic resistance is a growing global concern, but our understanding of the spread of resistant bacteria in remote regions remains limited. While some level of intrinsic resistance likely contributes to reduced susceptibility to antimicrobials in the environment, it is evident that human actions, particularly the (mis)use of antibiotics, play a significant role in shaping the environmental resistome, even in seemingly distant habitats like glacier ice sheets. Our research aims to bridge this knowledge gap by investigating the direct influence of human activities on the presence of antibiotic-resistant bacteria in various habitats. To achieve a comprehensive assessment of anthropogenic impact across diverse and seemingly isolated sampling sites, we developed an innovative approach utilizing Corine Land Cover data and heatmaps generated from sports activity trackers. This method allowed us to make meaningful comparisons across relatively pristine environments. Our findings indicate a noteworthy increase in culturable antibiotic-resistant bacteria with heightened human influence, as evidenced by our analysis of glacier, snow, and lake water samples. Notably, the most significant concentrations of antibiotic-resistant and multidrug-resistant microorganisms were discovered in two highly impacted sampling locations, namely the Tux Glacier and Gas Station Ellmau.

A comparative approach to confirm antibiotic-resistant microbes in the cryosphere.

Antibiotic-resistant microbes pose one of the biggest challenges of the current century. While areas with proximity to human impact are closely studied, a lot is yet to learn about antimicrobial resistance in remote regions like the cryosphere. Nowadays, antibiotic (AB) resistance is considered a pollution that has reached the Earth's most pristine areas. However, monitoring of resistant environmental bacteria therein faces several challenges that inhibit scientific progress in this field. Due to many cultivation-based antibiotic susceptibility tests being optimized for mesophilic pathogenic microorganisms, many researchers opt for expensive molecular biological approaches to detect antibiotic resistance in the cryosphere. However, some disadvantages of these methods prohibit effective comprehensive monitoring of resistant bacteria in pristine areas, hence we suggest established cultivation-based approaches when looking for antimicrobial resistance in the cryosphere. In this study, we compared two common antibiotic susceptibility tests and optimized them to meet the needs of psychrophilic microorganisms. The resulting cultures thereof originated from cryospheric habitats with differing anthropogenic impacts. The results show that these methods are applicable to detect antibiotic resistance in cryospheric habitats and could potentially increase the comparability between studies.

The AMADEE-18 Mars Analog Expedition in the Dhofar Region of Oman.

From February 1 to 28, 2018, the Austrian Space Forum, in cooperation with the Oman Astronomical Society and research teams from 25 nations, conducted the AMADEE-18 mission, a human-robotic Mars expedition simulation in the Dhofar region in the Sultanate of Oman. A carefully selected field crew, supported by a Mission Support Center in Innsbruck, Austria, conducted 19 experiments relevant to astrobiology, engineering disciplines, geoscience, operations research, and human factors. This expedition was the 12th in a series of analog missions that emulate selected aspects of the science expected for a human Mars mission, including the characterization of the (paleo)geological environment, human factors studies, and the search for biomarkers. In particular, an Exploration Cascade was deployed as a suggested workflow for coordinating the timing and location of the respective instruments and experiments. In validation of this workflow, the decision-making interaction between the field and the Mission Support Center was studied. This article introduces the AMADEE-18 mission and provides the mission-specific context for the other contributions of this special issue.

Comparison of Bacterial and Fungal Composition and Their Chemical Interaction in Free Tropospheric Air and Snow Over an Entire Winter Season at Mount Sonnblick, Austria.

We investigated the interactions of air and snow over one entire winter accumulation period as well as the importance of chemical markers in a pristine free-tropospheric environment to explain variation in a microbiological dataset. To overcome the limitations of short term bioaerosol sampling, we sampled the atmosphere continuously onto quartzfiber air filters using a DIGITEL high volume PM10 sampler. The bacterial and fungal communities, sequenced using Illumina MiSeq, as well as the chemical components of the atmosphere were compared to those of a late season snow profile. Results reveal strong dynamics in the composition of bacterial and fungal communities in air and snow. In fall the two compartments were similar, suggesting a strong interaction between them. The overlap diminished as the season progressed due to an evolution within the snowpack throughout winter and spring. Certain bacterial and fungal genera were only detected in air samples, which implies that a distinct air microbiome might exist. These organisms are likely not incorporated in clouds and thus not precipitated or scavenged in snow. Although snow appears to be seeded by the atmosphere, both air and snow showed differing bacterial and fungal communities and chemical composition. Season and alpha diversity were major drivers for microbial variability in snow and air, and only a few chemical markers were identified as important in explaining microbial diversity. Air microbial community variation was more related to chemical markers than snow microbial composition. For air microbial communities Cl-, TC/OC, SO4 2-, Mg2+, and Fe/Al, all compounds related to dust or anthropogenic activities, were identified as related to bacterial variability while dust related Ca2+ was significant in snow. The only common driver for snow and air was SO4 2-, a tracer for anthropogenic sources. The occurrence of chemical compounds was coupled with boundary layer injections in the free troposphere (FT). Boundary layer injections also caused the observed variations in community composition and chemistry between the two compartments. Long-term monitoring is required for a more valid insight in post-depositional selection in snow.

Cryoconite Hole Location in East-Antarctic Untersee Oasis Shapes Physical and Biological Diversity.

Antarctic cryoconite holes (CHs) are mostly perennially ice-lidded and sediment-filled depressions that constitute important features on glaciers and ice sheets. Once being hydrologically connected, these microbially dominated mini-ecosystems provide nutrients and biota for downstream environments. For example, the East Antarctic Anuchin Glacier gradually melts into the adjacent perennially ice-covered Lake Untersee, and CH biota from this glacier contribute up to one third of the community composition in benthic microbial mats within the lake. However, biogeochemical features of these CHs and associated spatial patterns across the glacier are still unknown. Here we hypothesized about the CH minerogenic composition between the different sources such as the medial moraine and other zones. Further, we intended to investigate if the depth of the CH mirrors the CH community composition, organic matter (OM) content and would support productivity. In this study we show that both microbial communities and biogeochemical parameters in CHs were significantly different between the zones medial moraine and the glacier terminus. Variations in microbial community composition are the result of factors such as depth, diameter, organic matter, total carbon, particle size, and mineral diversity. More than 90% of all ribosomal sequence variants (RSV) reads were classified as Proteobacteria, Cyanobacteria, Bacteroidetes, Actinobacteria, and Acidobacteria. Archaea were detected in 85% of all samples and exclusively belonged to the classes Halobacteria, Methanomicrobia, and Thermoplasmata. The most abundant genus was Halorubrum (Halobacteria) and was identified in nine RSVs. The core microbiome for bacteria comprised 30 RSVs that were affiliated with Cyanobacteria, Bacteroidetes, Actinobacteria, and Proteobacteria. The archaeal fraction of the core microbiome consisted of three RSVs belonging to unknown genera of Methanomicrobiales and Thermoplasmatales and the genus Rice_Cluster_I (Methanocellales). Further, mean bacterial carbon production in cryoconite was exceptionally low and similar rates have not been reported elsewhere. However, bacterial carbon production insignificantly trended toward higher rates in shallow CHs and did not seem to be supported by accumulation of OM and nutrients, respectively, in deeper holes. OM fractions were significantly different between shallower CHs along the medial moraine and deeper CHs at the glacier terminus. Overall, our findings suggest that wind-blown material originating south and southeast of the Anuchin Glacier and deposits from a nunatak are assumed to be local inoculation sources. High sequence similarities between samples from the Untersee Oasis and other Antarctic sites further indicate long-range atmospheric transport mechanisms that complement local inoculation sources.

Laser-Induced Fluorescence Emission (L.I.F.E.) as Novel Non-Invasive Tool for In-Situ Measurements of Biomarkers in Cryospheric Habitats.

Global warming affects microbial communities in a variety of ecosystems, especially cryospheric habitats. However, little is known about microbial-mediated carbon fluxes in extreme environments. Hence, the methodology of sample acquisition described in the very few studies available implies two major problems: A) high resolution data require a large number of samples, which is difficult to obtain in remote areas; B) unavoidable sample manipulation such as cutting, sawing, and melting of ice cores that leads to a misunderstanding of in situ conditions. In this study, a prototype device that requires neither sample preparation nor sample destruction is presented. The device can be used for in situ measurements with a high spectral and spatial resolution in terrestrial and ice ecosystems and is based on the Laser-Induced Fluorescence Emission (L.I.F.E.) technique. Photoautotrophic supraglacial communities can be identified by the detection of L.I.F.E. signatures in photopigments. The L.I.F.E. instrument calibration for the porphyrin derivates chlorophylla (chla) (405 nm laser excitation) and B-phycoerythrin (B-PE) (532 nm laser excitation) is demonstrated. For the validation of this methodology, L.I.F.E. data were ratified by a conventional method for chla quantification that involved pigment extraction and subsequent absorption spectroscopy. The prototype applicability in the field was proven in extreme polar environments. Further testing on terrestrial habitats took place during Mars analog simulations in the Moroccan dessert and on an Austrian rock glacier. The L.I.F.E. instrument enables high resolution scans of large areas with acceptable operation logistics and contributes to a better understanding of the ecological potential of supraglacial communities in the context of global change.

Assessment of Artificial and Natural Transport Mechanisms of Ice Nucleating Particles in an Alpine Ski Resort in Obergurgl, Austria.

Artificial snow production is a crucial part of modern skiing resorts in Austria and globally, and will develop even more so with changing precipitation patterns and a warming climate trend. Producing artificial snow requires major investments in energy, water, infrastructure and manpower for skiing resorts. In addition to appropriate meteorological conditions, the efficiency of artificial snow production depends on heterogeneous ice-nucleation, which can occur at temperatures as high as -2°C when induced by specific bacterial ice nucleating particles (INPs). We aimed to investigate the presence, source and ice nucleating properties of these particles in the water cycle of an alpine ski resort in Obergurgl, Tyrol, Austria. We sampled artificial snow, river water, water pumped from a storage pond and compared it to samples collected from fresh natural snow and aged piste snow from the area. Particles from each sampled system were characterized in order to determine their transport mechanisms at a ski resort. We applied a physical droplet freezing assay [DRoplet Ice Nuclei Counter Zurich (DRINCZ)] to heated and unheated samples to characterize the biological and non-biological component of IN-activity. Bacterial abundance and community structure of the samples was obtained using quantitative PCR and Illumina Mi-Seq Amplicon Sequencing, and their chemical properties were determined by liquid ion-chromatography, energy dispersive X-ray spectroscopy (EDX) and scanning electron microscopy (SEM). The results show the flow of biological and inorganic material from the river to the slopes, an uptake of new microorganisms through the air and the piping, and possible proliferation or introduction of ice nucleation active biological particles in aged piste snow. Natural snow, as the first stage in this system, had the lowest amount of ice nucleation active particles and the least amount of biological and mineral particles in general, yet shares some microbial characteristics with fresh artificial snow.

Source Environments of the Microbiome in Perennially Ice-Covered Lake Untersee, Antarctica.

Ultra-oligotrophic Lake Untersee is among the largest and deepest surface lakes of Central Queen Maud Land in East Antarctica. It is dammed at its north end by the Anuchin Glacier and the ice-cover dynamics are controlled by sublimation - not melt - as the dominating ablation process and therefore surface melt during austral summer does not provide significant amounts of water for recharge compared to subsurface melt of the Anuchin Glacier. Several studies have already described the structure and function of the microbial communities within the water column and benthic environments of Lake Untersee, however, thus far there have been no studies that examine the linkages between the lake ecosystem with that of the surrounding soils or the Anuchin Glacier. The glacier may also play an important role as a major contributor of nutrients and biota into the lake ecosystem. Based on microbial 16S rRNA amplicon sequencing, we showed that the dominant bacterial signatures in Lake Untersee, the Anuchin Glacier and its surrounding soils were affiliated with Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and Proteobacteria. Aerosol and local soil depositions on the glacier surface resulted in distinct microbial communities developing in glacier ice and cryoconite holes. Based on a source tracking algorithm, we found that cryoconite microbial assemblages were a potential source of organisms, explaining up to 36% of benthic microbial mat communities in the lake. However, the major biotic sources for the lake ecosystem are still unknown, illustrating the possible importance of englacial and subglacial zones. The Anuchin Glacier may be considered as a vector in a biological sense for the bacterial colonization of the perennially ice-covered Lake Untersee. However, despite a thick perennial ice cover, observed "lift-off" microbial mats escaping the lake make a bidirectional transfer of biota plausible. Hence, there is an exchange of biota between Lake Untersee and connective habitats possible despite the apparent sealing by a perennial ice cover and the absence of moat areas during austral summer.

Characterisation of Arctic Bacterial Communities in the Air above Svalbard.

Atmospheric dispersal of bacteria is increasingly acknowledged as an important factor influencing bacterial community biodiversity, biogeography and bacteria-human interactions, including those linked to human health. However, knowledge about patterns in microbial aerobiology is still relatively scarce, and this can be attributed, in part, to a lack of consensus on appropriate sampling and analytical methodology. In this study, three different methods were used to investigate aerial biodiversity over Svalbard: impaction, membrane filtration and drop plates. Sites around Svalbard were selected due to their relatively remote location, low human population, geographical location with respect to air movement and the tradition and history of scientific investigation on the archipelago, ensuring the presence of existing research infrastructure. The aerial bacterial biodiversity found was similar to that described in other aerobiological studies from both polar and non-polar environments, with Proteobacteria, Actinobacteria, and Firmicutes being the predominant groups. Twelve different phyla were detected in the air collected above Svalbard, although the diversity was considerably lower than in urban environments elsewhere. However, only 58 of 196 bacterial genera detected were consistently present, suggesting potentially higher levels of heterogeneity. Viable bacteria were present at all sampling locations, showing that living bacteria are ubiquitous in the air around Svalbard. Sampling location influenced the results obtained, as did sampling method. Specifically, impaction with a Sartorius MD8 produced a significantly higher number of viable colony forming units (CFUs) than drop plates alone.

Legal immigrants: invasion of alien microbial communities during winter occurring desert dust storms.

BACKGROUND: A critical aspect regarding the global dispersion of pathogenic microorganisms is associated with atmospheric movement of soil particles. Especially, desert dust storms can transport alien microorganisms over continental scales and can deposit them in sensitive sink habitats. In winter 2014, the largest ever recorded Saharan dust event in Italy was efficiently deposited on the Dolomite Alps and was sealed between dust-free snow. This provided us the unique opportunity to overcome difficulties in separating dust associated from "domestic" microbes and thus, to determine with high precision microorganisms transported exclusively by desert dust. RESULTS: Our metagenomic analysis revealed that sandstorms can move not only fractions but rather large parts of entire microbial communities far away from their area of origin and that this microbiota contains several of the most stress-resistant organisms on Earth, including highly destructive fungal and bacterial pathogens. In particular, we provide first evidence that winter-occurring dust depositions can favor a rapid microbial contamination of sensitive sink habitats after snowmelt. CONCLUSIONS: Airborne microbial depositions accompanying extreme meteorological events represent a realistic threat for ecosystem and public health. Therefore, monitoring the spread and persistence of storm-travelling alien microbes is a priority while considering future trajectories of climatic anomalies as well as anthropogenically driven changes in land use in the source regions.

Planetary Protection and Mars Special Regions--A Suggestion for Updating the Definition.

We highlight the role of COSPAR and the scientific community in defining and updating the framework of planetary protection. Specifically, we focus on Mars "Special Regions," areas where strict planetary protection measures have to be applied before a spacecraft can explore them, given the existence of environmental conditions that may be conducive to terrestrial microbial growth. We outline the history of the concept of Special Regions and inform on recent developments regarding the COSPAR policy, namely, the MEPAG SR-SAG2 review and the Academies and ESF joint committee report on Mars Special Regions. We present some new issues that necessitate the update of the current policy and provide suggestions for new definitions of Special Regions. We conclude with the current major scientific questions that remain unanswered regarding Mars Special Regions.

Aerobiology Over Antarctica - A New Initiative for Atmospheric Ecology.

The role of aerial dispersal in shaping patterns of biodiversity remains poorly understood, mainly due to a lack of coordinated efforts in gathering data at appropriate temporal and spatial scales. It has been long known that the rate of dispersal to an ecosystem can significantly influence ecosystem dynamics, and that aerial transport has been identified as an important source of biological input to remote locations. With the considerable effort devoted in recent decades to understanding atmospheric circulation in the south-polar region, a unique opportunity has emerged to investigate the atmospheric ecology of Antarctica, from regional to continental scales. This concept note identifies key questions in Antarctic microbial biogeography and the need for standardized sampling and analysis protocols to address such questions. A consortium of polar aerobiologists is established to bring together researchers with a common interest in the airborne dispersion of microbes and other propagules in the Antarctic, with opportunities for comparative studies in the Arctic.

Field trial of a dual-wavelength fluorescent emission (L.I.F.E.) instrument and the Magma White rover during the MARS2013 Mars analog mission.

Abstract We have developed a portable dual-wavelength laser fluorescence spectrometer as part of a multi-instrument optical probe to characterize mineral, organic, and microbial species in extreme environments. Operating at 405 and 532 nm, the instrument was originally designed for use by human explorers to produce a laser-induced fluorescence emission (L.I.F.E.) spectral database of the mineral and organic molecules found in the microbial communities of Earth's cryosphere. Recently, our team had the opportunity to explore the strengths and limitations of the instrument when it was deployed on a remote-controlled Mars analog rover. In February 2013, the instrument was deployed on board the Magma White rover platform during the MARS2013 Mars analog field mission in the Kess Kess formation near Erfoud, Morocco. During these tests, we followed tele-science work flows pertinent to Mars surface missions in a simulated spaceflight environment. We report on the L.I.F.E. instrument setup, data processing, and performance during field trials. A pilot postmission laboratory analysis determined that rock samples acquired during the field mission exhibited a fluorescence signal from the Sun-exposed side characteristic of chlorophyll a following excitation at 405 nm. A weak fluorescence response to excitation at 532 nm may have originated from another microbial photosynthetic pigment, phycoerythrin, but final assignment awaits development of a comprehensive database of mineral and organic fluorescence spectra. No chlorophyll fluorescence signal was detected from the shaded underside of the samples.

Coupled cryoconite ecosystem structure-function relationships are revealed by comparing bacterial communities in alpine and Arctic glaciers.

Cryoconite holes are known as foci of microbial diversity and activity on polar glacier surfaces, but are virtually unexplored microbial habitats in alpine regions. In addition, whether cryoconite community structure reflects ecosystem functionality is poorly understood. Terminal restriction fragment length polymorphism and Fourier transform infrared metabolite fingerprinting of cryoconite from glaciers in Austria, Greenland and Svalbard demonstrated cryoconite bacterial communities are closely correlated with cognate metabolite fingerprints. The influence of bacterial-associated fatty acids and polysaccharides was inferred, underlining the importance of bacterial community structure in the properties of cryoconite. Thus, combined application of T-RFLP and FT-IR metabolite fingerprinting promises high throughput, and hence, rapid assessment of community structure-function relationships. Pyrosequencing revealed Proteobacteria were particularly abundant, with Cyanobacteria likely acting as ecosystem engineers in both alpine and Arctic cryoconite communities. However, despite these generalities, significant differences in bacterial community structures, compositions and metabolomes are found between alpine and Arctic cryoconite habitats, reflecting the impact of local and regional conditions on the challenges of thriving in glacial ecosystems.

The dynamic bacterial communities of a melting High Arctic glacier snowpack.

Snow environments can occupy over a third of land surface area, but little is known about the dynamics of snowpack bacteria. The effect of snow melt on bacterial community structure and diversity of surface environments of a Svalbard glacier was examined using analyses of 16S rRNA genes via T-RFLP, qPCR and 454 pyrosequencing. Distinct community structures were found in different habitat types, with changes over 1 week apparent, in particular for the dominant bacterial class present, Betaproteobacteria. The differences observed were consistent with influences from depositional mode (snowfall vs aeolian dusts), contrasting snow with dust-rich snow layers and near-surface ice. Contrary to that, slush as the decompositional product of snow harboured distinct lineages of bacteria, further implying post-depositional changes in community structure. Taxa affiliated to the betaproteobacterial genus Polaromonas were particularly dynamic, and evidence for the presence of betaproteobacterial ammonia-oxidizing bacteria was uncovered, inviting the prospect that the dynamic bacterial communities associated with snowpacks may be active in supraglacial nitrogen cycling and capable of rapid responses to changes induced by snowmelt. Furthermore the potential of supraglacial snowpack ecosystems to respond to transient yet spatially extensive melting episodes such as that observed across most of Greenland's ice sheet in 2012 merits further investigation.

The Aouda.X space suit simulator and its applications to astrobiology.

We have developed the space suit simulator Aouda.X, which is capable of reproducing the physical and sensory limitations a flight-worthy suit would have on Mars. Based upon a Hard-Upper-Torso design, it has an advanced human-machine interface and a sensory network connected to an On-Board Data Handling system to increase the situational awareness in the field. Although the suit simulator is not pressurized, the physical forces that lead to a reduced working envelope and physical performance are reproduced with a calibrated exoskeleton. This allows us to simulate various pressure regimes from 0.3-1 bar. Aouda.X has been tested in several laboratory and field settings, including sterile sampling at 2800 m altitude inside a glacial ice cave and a cryochamber at -110°C, and subsurface tests in connection with geophysical instrumentation relevant to astrobiology, including ground-penetrating radar, geoacoustics, and drilling. The communication subsystem allows for a direct interaction with remote science teams via telemetry from a mission control center. Aouda.X as such is a versatile experimental platform for studying Mars exploration activities in a high-fidelity Mars analog environment with a focus on astrobiology and operations research that has been optimized to reduce the amount of biological cross contamination. We report on the performance envelope of the Aouda.X system and its operational limitations.

Possible interactions between bacterial diversity, microbial activity and supraglacial hydrology of cryoconite holes in Svalbard.

The diversity of highly active bacterial communities in cryoconite holes on three Arctic glaciers in Svalbard was investigated using terminal restriction fragment length polymorphism (T-RFLP) of the 16S rRNA locus. Construction and sequencing of clone libraries allowed several members of these communities to be identified, with Proteobacteria being the dominant one, followed by Cyanobacteria and Bacteroidetes. T-RFLP data revealed significantly different communities in holes on the (cold) valley glacier Austre Brøggerbreen relative to two adjacent (polythermal) valley glaciers, Midtre Lovénbreen and Vestre Brøggerbreen. These population compositions correlate with differences in organic matter content, temperature and the metabolic activity of microbial communities concerned. No within-glacier spatial patterns were observed in the communities identified over the 2-year period and with the 1 km-spaced sampling. We infer that surface hydrology is an important factor in the development of cryoconite bacterial communities.

Laser-induced fluorescence emission (L.I.F.E.): in situ nondestructive detection of microbial life in the ice covers of Antarctic lakes.

Laser-induced fluorescence emission (L.I.F.E.) images were obtained in situ following 532 nm excitation of cryoconite assemblages in the ice covers of annual and perennially frozen Antarctic lakes during the 2008 Tawani International Expedition to Schirmacher Oasis and Lake Untersee in Dronning Maud Land, Antarctica. Laser targeting of a single millimeter-scale cryoconite results in multiple neighboring excitation events secondary to ice/air interface reflection and refraction in the bubbles surrounding the primary target. Laser excitation at 532 nm of cyanobacteria-dominated assemblages produced red and infrared autofluorescence activity attributed to the presence of phycoerythrin photosynthetic pigments. The method avoids destruction of individual target organisms and does not require the disruption of either the structure of the microbial community or the surrounding ice matrix. L.I.F.E. survey strategies described may be of interest for orbital monitoring of photosynthetic primary productivity in polar and alpine glaciers, ice sheets, snow, and lake ice of Earth's cryosphere. The findings open up the possibility of searching from either a rover or from orbit for signs of life in the polar regions of Mars and the frozen regions of exoplanets in neighboring star systems.