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.

Comparison of microbial colonization between natural and plastic substrata in a polluted watershed.

Plastic pollution represents a threat for biological communities and the ecological functions they provide in river ecosystems. In this study, we compared the microbial colonization of two plastics (biodegradable and non-biodegradable) and three natural substrata (leaves, sediment, and rocks) in two study sites of an urbanized watershed differing in their plastic-contamination degree (upstream and downstream). The density and diversity of bacterial, fungal, and algal communities, as well as the extracellular enzymatic activities ß-glucosidase (GLU), N-acetyl-glucosaminidase (NAG), and phosphatase (PHO), were analysed in each substrata and site over a 4-week colonization experiment. Results showed higher microbial densities and enzymatic activities in leaves and sediment compared to plastics and rocks, probably due to the greater availability of organic carbon and nutrients in the former substrata. However, the microbial colonization of the two plastics was only different in the downstream site, where bacterial density and enzymatic activities were higher in the biodegradable plastic compared to the non-biodegradable plastic. Accordingly, the presence of biodegradable plastics would enhance the heterotrophic metabolism in plastic-polluted rivers.

Effect of subtherapeutic and therapeutic sulfamethazine concentrations on transcribed genes and translated proteins involved in Microbacterium sp. C448 resistance and degradation.

Microbacterium sp. C448, isolated from a soil regularly exposed to sulfamethazine (SMZ), can use various sulphonamide antibiotics as the sole carbon source for growth. The basis for the regulation of genes encoding the sulphonamide metabolism pathway, the dihydropteroate synthase sulphonamide target (folP), and the sulphonamide resistance (sul1) genes is unknown in this organism. In the present study, the response of the transcriptome and proteome of Microbacterium sp. C448 following exposure to subtherapeutic (33 µM) or therapeutic (832 µM) SMZ concentrations was evaluated. Therapeutic concentration induced the highest sad expression and Sad production, consistent with the activity of SMZ degradation observed in cellulo. Following complete SMZ degradation, Sad production tended to return to the basal level observed prior to SMZ exposure. Transcriptomic and proteomic kinetics were concomitant for the resistance genes and proteins. The abundance of Sul1 protein, 100-fold more abundant than FolP protein, did not change in response to SMZ exposure. Moreover, non-targeted analyses highlighted the increase of a deaminase RidA and a putative sulphate exporter expression and production. These two novel factors involved in the 4-aminophenol metabolite degradation and the export of sulphate residues formed during SMZ degradation, respectively, provided new insights into the Microbacterium sp. C448 SMZ detoxification process.

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.

Effects of sulfonamide antibiotics on aquatic microbial community composition and functions.

Knowledge on interactions among microbial communities colonizing various streambed substrata (e.g. cobbles, sediment, leaf-litter etc.) is essential when investigating the functioning of stream ecosystems. However, these interactions are often forgotten when assessing the responses of aquatic microbial communities to chemical contamination. Using a stream microcosm approach, the respective impact of two sulfonamide antibiotics (sulfamethoxazole and sulfamethazine) on the ability of microbial heterotrophs to decompose alder leaves was investigated in the presence or absence of periphyton. Our hypothesis suggested that sulfonamides would negatively impair microbial litter decomposition and that periphyton could possibly alleviate this effect by stimulating microbial decomposer activity through a priming effect. Results showed that the presence of periphyton enriched water with oxygen and labile dissolved organic carbon forms. However, these labile organic carbon sources did not stimulate leaf-litter decomposition but mostly decoupled microbial decomposer activity from particulate organic matter to dissolved organic matter through negative priming. Also, the two sulfonamide molecules did not affect the leaf-litter decomposition process but significantly decreased bacterial biomass accrual on leaves. The reduction of bacteria was concomitant with an increase in biomass-specific ß-glucosidase activity and this was attributed to a stress response from bacteria to sulfonamides. Further research looking at microbial interactions would provide for better assessment of chemical contamination effects in communities and processes in stream ecosystems.

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.

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.

Biodegradation and toxicity of a maize herbicide mixture: mesotrione, nicosulfuron and S-metolachlor.

The prediction of chemical mixture toxicity is a major concern regarding unintentional mixture of pesticides from agricultural lands treated with various such compounds. We focused our work on a mixture of three herbicides commonly applied on maize crops within a fortnight, namely mesotrione (ß-triketone), nicosulfuron (sulfonylurea) and S-metolachlor (chloroacetanilide). The metabolic pathways of mesotrione and nicosulfuron were qualitatively and quantitatively determined with a bacterial strain (Bacillus megaterium Mes11). This strain was isolated from an agricultural soil and able to biotransform both these herbicides. Although these pathways were unaffected in the case of binary or ternary herbicide mixtures, kinetics of nicosulfuron disappearance and also of mesotrione and nicosulfuron metabolite formation was strongly modulated. The toxicity of the parent compounds and metabolites was evaluated for individual compounds and mixtures with the standardized Microtox® test. Synergistic interactions were evidenced for all the parent compound mixtures. Synergistic, antagonistic or additive toxicity was obtained depending on the metabolite mixture. Overall, these results emphasize the need to take into account the active ingredient and metabolites all together for the determination of environmental fate and toxicity of pesticide mixtures.

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.

Identification of sulfonylurea biodegradation pathways enabled by a novel nicosulfuron-transforming strain Pseudomonas fluorescens SG-1: Toxicity assessment and effect of formulation.

Nicosulfuron is a selective herbicide belonging to the sulfonylurea family, commonly used on maize culture. A bacterial strain SG-1 was isolated from an agricultural soil previously treated with nicosulfuron. This strain was identified as Pseudomonas fluorescens and is able to quantitatively dissipate 77.5% of nicosulfuron (1mM) at 28°C in the presence of glucose within the first day of incubation. Four metabolites were identified among which ASDM (2-(aminosulfonyl)-N,N-dimethyl-3-pyridinecarboxamide) and ADMP (2-amino-4,6-dimethoxypyrimidine) in substantial proportions, corresponding to the hydrolytic sulfonylurea cleavage. Two-phase dissipation kinetics of nicosulfuron by SG-1 were observed at the highest concentrations tested (0.5 and 1mM) due to biosorption. The extend and rate of formulated nicosulfuron transformation were considerably reduced compared to those with the pure active ingredient (appearance of a lag phase, 30% dissipation after 10days of incubation instead of 100% with the pure herbicide) but the same metabolites were observed. The toxicity of metabolites (standardized Microtox® test) showed a 20-fold higher toxicity of ADMP than nicosulfuron. P. fluorescens strain SG-1 was also able to biotransform two other sulfonylureas (metsulfuron-methyl and tribenuron-methyl) with various novel pathways. These results provide new tools for a comprehensive picture of the sulfonylurea environmental fate and toxicity of nicosulfuron in the environment.

Biotransformation of herbicides by aquatic microbial communities associated to submerged leaves.

Leaf microbial communities possess a large panel of enzymes permitting the breakdown of leaf polymers as well as the transformation of organic xenobiotic compounds present in stream waters. This study aims to assess the potential of leaf microbial communities, exhibiting different exposure histories to pesticides (upstream versus downstream), to biotransform three maize herbicides (mesotrione, S-metolachlor, and nicosulfuron) in single and cocktail molecule exposures. The results showed a high dissipation of nicosulfuron (sulfonylurea herbicide) (from 29.1 ± 10.8% to 66 ± 16.2%, day 40) in both single and cocktail exposures, respectively, but not of mesotrione and S-metolachlor. The formation of nicosulfuron metabolites such as ASDM (2-(aminosulfonyl)-N,N-dimethyl-3-pyridinecarboxamide) and ADMP (2-amino-4,6-dimethoxypyrimidine) and the weak sorption (<0.4%) on the leaf matrix confirmed the transformation of this molecule by leaf microorganisms. In addition, the downstream communities showed a greater ability to transform nicosulfuron than the upstream communities suggesting that the exposure history to pesticides is an important parameter and can enhance the biotransformation potential of leaf microorganisms. After 40-day single exposure to nicosulfuron, the downstream communities were also those experiencing the greatest shifts in fungal and bacterial community diversity suggesting a potential adaptation of microorganisms to this herbicide. Our study emphasizes the importance of leaf microbial communities for herbicide biotransformation in polluted stream ecosystems where fungi could play a crucial role.

Siderophores in Cloud Waters and Potential Impact on Atmospheric Chemistry: Production by Microorganisms Isolated at the Puy de Dôme Station.

A total of 450 bacteria and yeast strains isolated from cloud waters sampled at the puy de Dôme station in France (1465 m) were screened for their ability to produce siderophores. To achieve this, a high-throughput method in 96-well plates was adapted from the CAS (chrome azurol S) method. Notably, 42% of the isolates were siderophore producers. This production was examined according to the phyla of the tested strains and the type of chelating functional groups (i.e., hydroxamate, catechol, and mixed type). The most active bacteria in the clouds belong to the γ-Proteobacteria class, among which the Pseudomonas genus is the most frequently encountered. γ-Proteobacteria are produced in the majority of mixed function siderophores, such as pyoverdines, which bear a photoactive group. Finally, siderophore production was shown to vary with the origin of the air masses. The organic speciation of iron remains largely unknown in warm clouds. Our results suggest that siderophores could partly chelate Fe(III) in cloud waters and thus potentially impact the chemistry of the atmospheric aqueous phase.

Functional and structural characterization of two Bacillus megaterium nitroreductases biotransforming the herbicide mesotrione.

Mesotrione is a selective herbicide belonging to the triketone family, commonly used on maize cultures since 2003. A mesotrione-transforming Bacillus megaterium Mes11 strain isolated from an agricultural soil was used as a model to identify the key enzymes initiating the biotransformation of this herbicide. Two enzymes (called NfrA1 and NfrA2/YcnD) were identified, and functionally and structurally characterized. Both belong to the NfsA FRP family of the nitro-FMN reductase superfamily (type I oxygen-insensitive nitroreductase) and show optimal pH and temperature of 6-6.5 and 23-25°C, respectively. Both undergo a Ping Pong Bi Bi mechanism, with NADPH and NADPH/NADH as cofactors for NfrA1 and NfrA2/YcnD, respectively. It is interesting that both can also reduce various nitro compounds including pesticides, antibiotics, one prodrug and 4-methylsulfonyl-2-nitrobenzoic acid, one of the mesotrione metabolites retrieved from the environment. The present study constitutes the first identification of mesotrione-transforming enzymes. These enzymes (or their corresponding genes) could be used as biomarkers to predict the capacity of ecosystems to transform mesotrione and assess their contamination by both the parent molecule and/or the metabolites.

Soil surface colonization by phototrophic indigenous organisms, in two contrasted soils treated by formulated maize herbicide mixtures.

Soil phototrophic microorganisms, contributors to soil health and food webs, share their particular metabolism with plants. Current agricultural practices employ mixtures of pesticides to ensure the crops yields and can potentially impair these non-target organisms. However despite this environmental reality, studies dealing the susceptibility of phototrophic microorganisms to pesticide mixtures are scarce. We designed a 3 months microcosm study to assess the ecotoxicity of realistic herbicide mixtures of formulated S-metolachlor (Dual Gold Safeneur(®)), mesotrione (Callisto(®)) and nicosulfuron (Milagro(®)) on phototrophic communities of two soils (Limagne vertisol and Versailles luvisol). The soils presented different colonizing communities, with diatoms and chlorophyceae dominating communities in Limagne soil and cyanobacteria and bryophyta communities in Versailles soil. The results highlighted the strong impairment of Dual Gold Safeneur(®) treated microcosms on the biomass and the composition of both soil phototrophic communities, with no resilience after a delay of 3 months. This study also excluded any significant mixture effect on these organisms for Callisto(®) and Milagro(®) herbicides. We strongly recommend carrying on extensive soil studies on S-metolachlor and its commercial formulations, in order to reconsider its use from an ecotoxicological point of view.