Temporal variations of antimicrobial resistance genes in aerosols: A one-year monitoring at the puy de Dôme summit (Central France).

The recent characterization of antibiotic resistance genes (ARGs) in clouds evidenced that the atmosphere actively partakes in the global spreading of antibiotic resistance worldwide. Indeed, the outdoor atmosphere continuously receives large quantities of particles of biological origins, emitted from both anthropogenic or natural sources at the near Earth's surface. Nonetheless, our understanding of the composition of the atmospheric resistome, especially at mid-altitude (i.e. above 1000 m a.s.l.), remains largely limited. The atmosphere is vast and highly dynamic, so that the diversity and abundance of ARGs are expected to fluctuate both spatially and temporally. In this work, the abundance and diversity of ARGs were assessed in atmospheric aerosol samples collected weekly between July 2016 and August 2017 at the mountain site of puy de Dôme (1465 m a.s.l., central France). Our results evidence the presence of 33 different subtypes of ARGs in atmospheric aerosols, out of 34 assessed, whose total concentration fluctuated seasonally from 59 to 1.1 × 105 copies m-3 of air. These were heavily dominated by genes from the quinolone resistance family, notably the qepA gene encoding efflux pump mechanisms, which represented >95 % of total ARGs concentration. Its abundance positively correlated with that of bacteria affiliated with the genera Kineococcus, Neorhizobium, Devosia or Massilia, ubiquitous in soils. This, along with the high abundance of Sphingomonas species, points toward a large contribution of natural sources to the airborne ARGs. Nonetheless, the increased contribution of macrolide resistance (notably the erm35 gene) during winter suggests a sporadic diffusion of ARGs from human activities. Our observations depict the atmosphere as an important vector of ARGs from terrestrial sources. Therefore, monitoring ARGs in airborne microorganisms appears necessary to fully understand the dynamics of antimicrobial resistances in the environment and mitigate the threats they may represent.

The aeromicrobiome: the selective and dynamic outer-layer of the Earth's microbiome.

The atmosphere is an integral component of the Earth's microbiome. Abundance, viability, and diversity of microorganisms circulating in the air are determined by various factors including environmental physical variables and intrinsic and biological properties of microbes, all ranging over large scales. The aeromicrobiome is thus poorly understood and difficult to predict due to the high heterogeneity of the airborne microorganisms and their properties, spatially and temporally. The atmosphere acts as a highly selective dispersion means on large scales for microbial cells, exposing them to a multitude of physical and chemical atmospheric processes. We provide here a brief critical review of the current knowledge and propose future research directions aiming at improving our comprehension of the atmosphere as a biome.

Quantification of antibiotic resistance genes (ARGs) in clouds at a mountain site (puy de Dôme, central France).

Antibiotic resistance in bacteria is becoming a major sanitary concern worldwide. The extensive use of large quantities of antibiotics to sustain human activity has led to the rapid acquisition and maintenance of antibiotic resistant genes (ARGs) in bacteria and to their spread into the environment. Eventually, these can be disseminated over long distances by atmospheric transport. Here, we assessed the presence of ARGs in clouds as an indicator of long-distance travel potential of antibiotic resistance in the atmosphere. We hypothesized that a variety of ARGs can reach the altitude of clouds mainly located within the free troposphere. Once incorporated in the atmosphere, they are efficiently transported and their respective concentrations should differ depending on the sources and the geographical origin of the air masses. We deployed high-flow rate impingers and collected twelve clouds between September 2019 and October 2021 at the meteorological station of the puy de Dôme summit (1465 m a.s.l., France). Total airborne bacteria concentration was assessed by flow cytometry, and ARGs subtypes of the main families of antibiotic resistance (quinolone, sulfonamide, tetracycline; glycopeptide, aminoglycoside, ß-lactamase, macrolide) including one mobile genetic element (transposase) were quantified by qPCR. Our results indicate the presence of 29 different ARGs' subtypes at concentrations ranging from 1.01 × 103 to 1.61 × 104 copies m-3 of air. Clear distinctions could be observed between clouds in air masses transported over marine areas (Atlantic Ocean) and clouds influenced by continental surfaces. Specifically, quinolones (mostly qepA) resistance genes were prevalent in marine clouds (54 % of the total ARGs on average), whereas higher contributions of sulfonamide, tetracycline; glycopeptide, ß-lactamase and macrolide were found in continental clouds. This study constitutes the first evidence for the presence of microbial ARGs in clouds at concentrations comparable to other natural environments. This highlights the atmosphere as routes for the dissemination of ARGs at large scale.

High-quality genome of the basidiomycete yeast Dioszegia hungarica PDD-24b-2 isolated from cloud water.

The genome of the basidiomycete yeast Dioszegia hungarica strain PDD-24b-2 isolated from cloud water at the summit of puy de Dôme (France) was sequenced using a hybrid PacBio and Illumina sequencing strategy. The obtained assembled genome of 20.98 Mb and a GC content of 57% is structured in 16 large-scale contigs ranging from 90 kb to 5.56 Mb, and another 27.2 kb contig representing the complete circular mitochondrial genome. In total, 8,234 proteins were predicted from the genome sequence. The mitochondrial genome shows 16.2% cgu codon usage for arginine but has no canonical cognate tRNA to translate this codon. Detected transposable element (TE)-related sequences account for about 0.63% of the assembled genome. A dataset of 2,068 hand-picked public environmental metagenomes, representing over 20 Tbp of raw reads, was probed for D. hungarica related ITS sequences, and revealed worldwide distribution of this species, particularly in aerial habitats. Growth experiments suggested a psychrophilic phenotype and the ability to disperse by producing ballistospores. The high-quality assembled genome obtained for this D. hungarica strain will help investigate the behavior and ecological functions of this species in the environment.

Complete Genome of Sphingomonas aerolata PDD-32b-11, Isolated from Cloud Water at the Summit of Puy de Dôme, France.

The complete genome of Sphingomonas aerolata PDD-32b-11, a bacterium isolated from cloud water, was sequenced. It features four circular replicons, a chromosome of 3.99 Mbp, and three plasmids. Two putative rhodopsin-encoding genes were detected which might act as proton pumps to harvest light energy.

Antimicrobial Resistance in the Environment: Towards Elucidating the Roles of Bioaerosols in Transmission and Detection of Antibacterial Resistance Genes.

Antimicrobial resistance (AMR) is continuing to grow across the world. Though often thought of as a mostly public health issue, AMR is also a major agricultural and environmental problem. As such, many researchers refer to it as the preeminent One Health issue. Aerial transport of antimicrobial-resistant bacteria via bioaerosols is still poorly understood. Recent work has highlighted the presence of antibiotic resistance genes in bioaerosols. Emissions of AMR bacteria and genes have been detected from various sources, including wastewater treatment plants, hospitals, and agricultural practices; however, their impacts on the broader environment are poorly understood. Contextualizing the roles of bioaerosols in the dissemination of AMR necessitates a multidisciplinary approach. Environmental factors, industrial and medical practices, as well as ecological principles influence the aerial dissemination of resistant bacteria. This article introduces an ongoing project assessing the presence and fate of AMR in bioaerosols across Canada. Its various sub-studies include the assessment of the emissions of antibiotic resistance genes from many agricultural practices, their long-distance transport, new integrative methods of assessment, and the creation of dissemination models over short and long distances. Results from sub-studies are beginning to be published. Consequently, this paper explains the background behind the development of the various sub-studies and highlight their shared aspects.

Microbial ecology of the atmosphere.

The atmosphere connects habitats across multiple spatial scales via airborne dispersal of microbial cells, propagules and biomolecules. Atmospheric microorganisms have been implicated in a variety of biochemical and biophysical transformations. Here, we review ecological aspects of airborne microorganisms with respect to their dispersal, activity and contribution to climatic processes. Latest studies utilizing metagenomic approaches demonstrate that airborne microbial communities exhibit pronounced biogeography, driven by a combination of biotic and abiotic factors. We quantify distributions and fluxes of microbial cells between surface habitats and the atmosphere and place special emphasis on long-range pathogen dispersal. Recent advances have established that these processes may be relevant for macroecological outcomes in terrestrial and marine habitats. We evaluate the potential biological transformation of atmospheric volatile organic compounds and other substrates by airborne microorganisms and discuss clouds as hotspots of microbial metabolic activity in the atmosphere. Furthermore, we emphasize the role of microorganisms as ice nucleating particles and their relevance for the water cycle via formation of clouds and precipitation. Finally, potential impacts of anthropogenic forcing on the natural atmospheric microbiota via emission of particulate matter, greenhouse gases and microorganisms are discussed.

Rainfalls sprinkle cloud bacterial diversity while scavenging biomass.

Bacteria circulate in the atmosphere, through clouds and precipitation to surface ecosystems. Here, we conducted a coordinated study of bacteria assemblages in clouds and precipitation at two sites distant of ∼800 m in elevation in a rural vegetated area around puy de Dôme Mountain, France, and analysed them in regard to meteorological, chemical and air masses' history data. In both clouds and precipitation, bacteria generally associated with vegetation or soil dominated. Elevated ATP-to-cell ratio in clouds compared with precipitation suggested a higher proportion of viable cells and/or specific biological processes. The increase of bacterial cell concentration from clouds to precipitation indicated strong below-cloud scavenging. Using ions as tracers, we derive that 0.2 to 25.5% of the 1.1 × 107 to 6.6 × 108 bacteria cell/m2/h1 deposited with precipitation originated from the source clouds. Yet, the relative species richness decreased with the proportion of inputs from clouds, pointing them as sources of distant microbial diversity. Biodiversity profiles, thus, differed between clouds and precipitation in relation with distant/local influencing sources, and potentially with bacterial phenotypic traits. Notably Undibacterium, Bacillus and Staphylococcus were more represented in clouds, while epiphytic bacteria such as Massilia, Sphingomonas, Rhodococcus and Pseudomonas were enriched in precipitation.

Cyanobacteria and Algae in Clouds and Rain in the Area of puy de Dôme, Central France.

The atmosphere contains diverse living microbes, of which the heterotrophic community has been the best studied. Microbes with other trophic modes, such as photoautotrophy, have received much less attention. In this study, culture-independent and dependent methods were used to examine the presence and diversity of oxygenic photoautotrophic microbes in clouds and rain collected at or around puy de Dôme Mountain, central France. Cloud water was collected from the summit of puy de Dôme (1,465 m above sea level [a.s.l.]) for cultivation and metagenomic analysis. Cyanobacteria, diatoms, green algae, and other oxygenic photoautotrophs were found to be recurrent members of clouds, while green algae affiliated with the Chlorellaceae were successfully cultured from three different clouds. Additionally, rain samples were collected below the mountain from Opme meteorological station (680 m a.s.l.). The abundance of chlorophyll a-containing cells and the diversity of cyanobacteria and green algae in rain were assessed by flow cytometry and amplicon sequencing. The corresponding downward flux of chlorophyll a-containing organisms to the ground, entering surface ecosystems with rain, varied with time and was estimated to be between ∼1 and >300 cells cm-2 day-1 during the sampling period. Besides abundant pollen from Pinales and Rosales, cyanobacteria of the Chroococcidiopsidales and green algae of the Trebouxiales were dominant in rain samples. Certain members of these taxa are known to be ubiquitous and stress tolerant and could use the atmosphere for dispersal. Overall, our results indicate that the atmosphere carries diverse, viable oxygenic photoautotrophic microbes and acts as a dispersal vector for this microbial guild.IMPORTANCE Information regarding the diversity and abundance of oxygenic photoautotrophs in the atmosphere is limited. More information from diverse locations is needed. These airborne organisms could have important impacts upon atmospheric processes and on the ecosystems they enter after deposition. Oxygenic photoautotrophic microbes are integral to ecosystem functioning, and some have the potential to affect human health. A better understanding of the diversity and the movements of these aeolian dispersed organisms is needed to understand their ecology, as well as how they could affect ecosystems and human health.

Seasonal shift in airborne microbial communities.

Microorganisms are ubiquitous in the atmosphere. Global investigations on the geographical and temporal distribution of airborne microbial communities are critical for identifying the sources and the factors shaping airborne communities. At mid-latitude sites, a seasonal shift in both the concentration and diversity of airborne microbial communities has been systematically observed in the planetary boundary layer. While the factors suspected of affecting this seasonal change were hypothesized (e.g., changes in the surface conditions, meteorological parameters and global air circulation), our understanding on how these factors influence the temporal variation of airborne microbial communities, especially at the microbial taxon level, remains limited. Here, we investigated the distribution of both airborne bacterial and fungal communities on a weekly basis over more than one year at the mid-latitude and continental site of puy de Dôme (France; +1465 m altitude above sea level). The seasonal shift in microbial community structure was likely correlated to the seasonal changes in the characteristics of puy de Dôme's landscape (croplands and natural vegetation). The airborne microbial taxa that were the most affected by seasonal changes trended differently throughout the seasons in relation with their trophic mode. In addition, the windy and variable local meteorological conditions found at puy de Dôme were likely responsible for the intraseasonal variability observed in the composition of airborne microbial communities.

Metabolic modulations of Pseudomonas graminis in response to H2O2 in cloud water.

In cloud water, microorganisms are exposed to very strong stresses especially related to the presence of reactive oxygen species including H2O2 and radicals, which are the driving force of cloud chemistry. In order to understand how the bacterium Pseudomonas graminis isolated from cloud water respond to this oxidative stress, it was incubated in microcosms containing a synthetic solution of cloud water in the presence or in the absence of H2O2. P. graminis metabolome was examined by LC-MS and NMR after 50 min and after 24 hours of incubation. After 50 min, the cells were metabolizing H2O2 while this compound was still present in the medium, and it was completely biodegraded after 24 hours. Cells exposed to H2O2 had a distinct metabolome as compared to unexposed cells, revealing modulations of certain metabolic pathways in response to oxidative stress. These data indicated that the regulations observed mainly involved carbohydrate, glutathione, energy, lipid, peptides and amino-acids metabolisms. When cells had detoxified H2O2 from the medium, their metabolome was not distinguishable anymore from unexposed cells, highlighting the capacity of resilience of this bacterium. This work illustrates the interactions existing between the cloud microbial metabolome and cloud chemistry.

Methylotrophs and Methylotroph Populations for Chloromethane Degradation.

Chloromethane is a halogenated volatile organic compound, produced in large quantities by terrestrial vegetation. After its release to the troposphere and transport to the stratosphere, its photolysis contributes to the degradation of stratospheric ozone. A better knowledge of chloromethane sources (production) and sinks (degradation) is a prerequisite to estimate its atmospheric budget in the context of global warming. The degradation of chloromethane by methylotrophic communities in terrestrial environments is a major underestimated chloromethane sink. Methylotrophs isolated from soils, marine environments and more recently from the phyllosphere have been grown under laboratory conditions using chloromethane as the sole carbon source. In addition to anaerobes that degrade chloromethane, the majority of cultivated strains were isolated in aerobiosis for their ability to use chloromethane as sole carbon and energy source. Among those, the Proteobacterium Methylobacterium (recently reclassified as Methylorubrum) harbours the only characterisized 'chloromethane utilization' (cmu) pathway, so far. This pathway is not representative of chloromethane-utilizing populations in the environment as cmu genes are rare in metagenomes. Recently, combined 'omics' biological approaches with chloromethane carbon and hydrogen stable isotope fractionation measurements in microcosms, indicated that microorganisms in soils and the phyllosphere (plant aerial parts) represent major sinks of chloromethane in contrast to more recently recognized microbe-inhabited environments, such as clouds. Cultivated chloromethane-degraders lacking the cmu genes display a singular isotope fractionation signature of chloromethane. Moreover, 13CH3Cl labelling of active methylotrophic communities by stable isotope probing in soils identify taxa that differ from the taxa known for chloromethane degradation. These observations suggest that new biomarkers for detecting active microbial chloromethane-utilizers in the environment are needed to assess the contribution of microorganisms to the global chloromethane cycle.

Methods to Investigate the Global Atmospheric Microbiome.

The interplay between microbes and atmospheric physical and chemical conditions is an open field of research that can only be fully addressed using multidisciplinary approaches. The lack of coordinated efforts to gather data at representative temporal and spatial scales limits aerobiology to help understand large scale patterns of global microbial biodiversity and its causal relationships with the environmental context. This paper presents the sampling strategy and analytical protocols developed in order to integrate different fields of research such as microbiology, -omics biology, atmospheric chemistry, physics and meteorology to characterize atmospheric microbial life. These include control of chemical and microbial contaminations from sampling to analysis and identification of experimental procedures for characterizing airborne microbial biodiversity and its functioning from the atmospheric samples collected at remote sites from low cell density environments. We used high-volume sampling strategy to address both chemical and microbial composition of the atmosphere, because it can help overcome low aerosol and microbial cell concentrations. To account for contaminations, exposed and unexposed control filters were processed along with the samples. We present a method that allows for the extraction of chemical and biological data from the same quartz filters. We tested different sampling times, extraction kits and methods to optimize DNA yield from filters. Based on our results, we recommend supplementary sterilization steps to reduce filter contamination induced by handling and transport. These include manipulation under laminar flow hoods and UV sterilization. In terms of DNA extraction, we recommend a vortex step and a heating step to reduce binding to the quartz fibers of the filters. These steps have led to a 10-fold increase in DNA yield, allowing for downstream omics analysis of air samples. Based on our results, our method can be integrated into pre-existing long-term monitoring field protocols for the atmosphere both in terms of atmospheric chemistry and biology. We recommend using standardized air volumes and to develop standard operating protocols for field users to better control the operational quality.

Metatranscriptomic exploration of microbial functioning in clouds.

Clouds constitute the uppermost layer of the biosphere. They host diverse communities whose functioning remains obscure, although biological activity potentially participates to atmospheric chemical and physical processes. In order to gain information on the metabolic functioning of microbial communities in clouds, we conducted coordinated metagenomics/metatranscriptomics profiling of cloud water microbial communities. Samples were collected from a high altitude atmospheric station in France and examined for biological content after untargeted amplification of nucleic acids. Living microorganisms, essentially bacteria, maintained transcriptional and translational activities and expressed many known complementary physiological responses intended to fight oxidants, osmotic variations and cold. These included activities of oxidant detoxification and regulation, synthesis of osmoprotectants/cryoprotectants, modifications of membranes, iron uptake. Consistently these energy-demanding processes were fueled by central metabolic routes involved in oxidative stress response and redox homeostasis management, such as pentose phosphate and glyoxylate pathways. Elevated binding and transmembrane ion transports demonstrated important interactions between cells and their cloud droplet chemical environments. In addition, polysaccharides, potentially beneficial for survival like exopolysaccharides, biosurfactants and adhesins, were synthesized. Our results support a biological influence on cloud physical and chemical processes, acting notably on the oxidant capacity, iron speciation and availability, amino-acids distribution and carbon and nitrogen fates.

Chloromethane Degradation in Soils: A Combined Microbial and Two-Dimensional Stable Isotope Approach.

Chloromethane (CHCl, methyl chloride) is the most abundant volatile halocarbon in the atmosphere and involved in stratospheric ozone depletion. The global CHCl budget, and especially the CHCl sink from microbial degradation in soil, still involves large uncertainties. These may potentially be resolved by a combination of stable isotope analysis and bacterial diversity studies. We determined the stable isotope fractionation of CHCl hydrogen and carbon and investigated bacterial diversity during CHCl degradation in three soils with different properties (forest, grassland, and agricultural soils) and at different temperatures and headspace mixing ratios of CHCl. The extent of chloromethane degradation decreased in the order forest > grassland > agricultural soil. Rates ranged from 0.7 to 2.5 µg g dry wt. d for forest soil, from 0.1 to 0.9 µg g dry wt. d for grassland soil, and from 0.1 to 0.4 µg g dry wt. d for agricultural soil and increased with increasing temperature and CHCl supplementation. The measured mean stable hydrogen enrichment factor of CHCl of -50 ± 13‰ was unaffected by temperature, mixing ratio, or soil type. In contrast, the stable carbon enrichment factor depended on CHCl degradation rates and ranged from -38 to -11‰. Bacterial community composition correlated with soil properties was independent from CHCl degradation or isotope enrichment. Nevertheless, increased abundance after CHCl incubation was observed in 21 bacterial operational taxonomical units (OTUs at the 97% 16S RNA sequence identity level). This suggests that some of these bacterial taxa, although not previously associated with CHCl degradation, may play a role in the microbial CHCl sink in soil.

Chloromethane formation and degradation in the fern phyllosphere.

Chloromethane (CH3Cl) is the most abundant halogenated trace gas in the atmosphere. It plays an important role in natural stratospheric ozone destruction. Current estimates of the global CH3Cl budget are approximate. The strength of the CH3Cl global sink by microbial degradation in soils and plants is under discussion. Some plants, particularly ferns, have been identified as substantial emitters of CH3Cl. Their ability to degrade CH3Cl remains uncertain. In this study, we investigated the potential of leaves from 3 abundant ferns (Osmunda regalis, Cyathea cooperi, Dryopteris filix-mas) to produce and degrade CH3Cl by measuring their production and consumption rates and their stable carbon and hydrogen isotope signatures. Investigated ferns are able to degrade CH3Cl at rates from 2.1 to 17 and 0.3 to 0.9µggdw-1day-1 for C. cooperi and D. filix-mas respectively, depending on CH3Cl supplementation and temperature. The stable carbon isotope enrichment factor of remaining CH3Cl was -39±13‰, whereas negligible isotope fractionation was observed for hydrogen (-8±19‰). In contrast, O. regalis did not consume CH3Cl, but produced it at rates ranging from 0.6 to 128µggdw-1day-1, with stable isotope values of -97±8‰ for carbon and -202±10‰ for hydrogen, respectively. Even though the 3 ferns showed clearly different formation and consumption patterns, their leaf-associated bacterial diversity was not notably different. Moreover, we did not detect genes associated with the only known chloromethane utilization pathway "cmu" in the microbial phyllosphere of the investigated ferns. Our study suggests that still unknown CH3Cl biodegradation processes on plants play an important role in global cycling of atmospheric CH3Cl.

High-Flow-Rate Impinger for the Study of Concentration, Viability, Metabolic Activity, and Ice-Nucleation Activity of Airborne Bacteria.

The study of airborne bacteria relies on a sampling strategy that preserves their integrity and in situ physiological state, e.g. viability, cultivability, metabolic activity, and ice-nucleation activity. Because ambient air harbors low concentrations of bacteria, an effective bioaerosol sampler should have a high sampling efficiency and a high airflow. We characterize a high-flow-rate impinger with respect to particle collection and retention efficiencies in the range 0.5-3.0 µm, and we investigated its ability to preserve the physiological state of selected bacterial species and seawater bacterial community in comparison with four commercial bioaerosol samplers. The collection efficiency increased with particle size and the cutoff diameter was between 0.5 and 1 µm. During sampling periods of 120-300 min, the impinger retained the cultivability, metabolic activity, viability, and ice-nucleation activity of investigated bacteria. Field studies in semiurban, high-altitude, and polar environments included periods of low bacterial air concentrations, thus demonstrating the benefits of the impinger's high flow rate. In conclusion, the impinger described here has many advantages compared with other bioaerosol samplers currently on the market: a potential for long sampling time, a high flow rate, a high sampling and retention efficiency, low costs, and applicability for diverse downstream microbiological and molecular analyses.

Active microorganisms thrive among extremely diverse communities in cloud water.

Clouds are key components in Earth's functioning. In addition of acting as obstacles to light radiations and chemical reactors, they are possible atmospheric oases for airborne microorganisms, providing water, nutrients and paths to the ground. Microbial activity was previously detected in clouds, but the microbial community that is active in situ remains unknown. Here, microbial communities in cloud water collected at puy de Dôme Mountain's meteorological station (1465 m altitude, France) were fixed upon sampling and examined by high-throughput sequencing from DNA and RNA extracts, so as to identify active species among community members. Communities consisted of ~103-104 bacteria and archaea mL-1 and ~102-103 eukaryote cells mL-1. They appeared extremely rich, with more than 28 000 distinct species detected in bacteria and 2 600 in eukaryotes. Proteobacteria and Bacteroidetes largely dominated in bacteria, while eukaryotes were essentially distributed among Fungi, Stramenopiles and Alveolata. Within these complex communities, the active members of cloud microbiota were identified as Alpha- (Sphingomonadales, Rhodospirillales and Rhizobiales), Beta- (Burkholderiales) and Gamma-Proteobacteria (Pseudomonadales). These groups of bacteria usually classified as epiphytic are probably the best candidates for interfering with abiotic chemical processes in clouds, and the most prone to successful aerial dispersion.

Metabolomic study of the response to cold shock in a strain of Pseudomonas syringae isolated from cloud water.

INTRODUCTION: Active microorganisms have been recently discovered in clouds, thus demonstrating the capacity of microorganisms to exist in harsh environments, including exposure to UV and oxidants, osmotic and cold shocks, etc. It is important to understand how microorganisms respond to and survive such stresses at the metabolic level. OBJECTIVES: The objective of this work is to assess metabolome modulation in a strain of Pseudomonas syringae isolated from cloud water and facing temperature downshift from 17 to 5 °C by identifying key molecules and pathways of the response/adaptation to cold shock. METHODS: Bacterial extracts from suspensions of cells grown at 17 °C and further incubated in microcosms at 5 and 17 °C to mimic cloud conditions were analysed by combining LC-MS and NMR; the results were evaluated in comparison to similar suspensions kept at constant temperature. The differences in the metabolome profiles were deciphered using multivariate statistics (PLS-DA). RESULTS: Key cold shock biomarkers were observed, including cryoprotectants (trehalose, glucose, glycerol, carnitine, glutamate), antioxidants (glutathione and carnitine) and their precursors, alkaloids (bellendine and slaframine) and metabolites involved in energy metabolism (ATP, carbohydrates). Furthermore, new short peptides (nine dipeptides and a tetrapeptide) were found that have no known function. CONCLUSIONS: This study shows that in response to cold temperatures, Pseudomonas syringae PDD-32b-74 demonstrates numerous metabolism modifications to counteract the impacts of low temperatures.