Toward a Universal Unit for Quantification of Antibiotic Resistance Genes in Environmental Samples.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

(Poly)phenol characterisation in white and red cardoon stalks: could the sous-vide technique improve their bioaccessibility?

This study aims to evaluate whether sous-vide cooking better preserves the (poly)phenol content and profile of red and white cardoon stalks versus traditional boiling, both before and after simulated oral-gastro-intestinal digestion. Thirty one (poly)phenols were quantified in red and white cardoon by HPLC-MS/MS, phenolic acids being >95%, and 5-caffeoylquinic and 1,5-dicaffeoylquinic acids the major ones. Although both varieties showed a similar profile, raw red cardoon had 1.7-fold higher (poly)phenol content than raw white cardoon. Culinary treatments decreased (poly)phenol content, but sous-vide cooked cardoon had a greater content than the boiled one, suggesting a protective effect. After gastrointestinal digestion, (poly)phenol bioaccessibility of boiled and sous-vide cooked cardoon (52.6-90.5%) was higher than that of raw samples (0.2-0.7%), although sous-vide system no longer played a protective effect compared to boiling. In summary, red cardoon was a richer source of bioaccessible (poly)phenols than white cardoon, even sous-vide cooked or boiled.

DNA damage and DNA protection from digested raw and griddled green pepper (poly)phenols in human colorectal adenocarcinoma cells (HT-29).

PURPOSE: To determine whether (poly)phenols from gastrointestinal-digested green pepper possess genoprotective properties in human colon cells and whether the application of a culinary treatment (griddling) on the vegetable influences the potential genoprotective activity. METHODS: (Poly)phenols of raw and griddled green pepper (Capsicum annuum L.) submitted to in vitro-simulated gastrointestinal digestion were characterized by LC-MS/MS. Cytotoxicity (MTT, trypan blue and cell proliferation assays), DNA damage and DNA protection (standard alkaline and formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay) of different concentrations of (poly)phenolic extracts were assessed in colon HT-29 cells. RESULTS: A total of 32 (poly)phenolic compounds were identified and quantified in digested raw and griddled green pepper. Twenty of them were flavonoids and 12 were phenolic acids. Griddled pepper doubled the (poly)phenol concentration compared to raw; luteolin 7-O-(2-apiosyl)-glucoside and quercitrin constituted the major (poly)phenols in both extracts. Raw and griddled pepper (poly)phenolic extracts impaired cell proliferation and induced low levels of Fpg-sensitive sites, in a dose-dependent manner, even at a non-cytotoxic concentration. None of the concentrations tested induced DNA strand breaks or alkaline labile sites. Nor did they show significant genoprotection against the DNA damage induced by H2O2 or KBrO3. CONCLUSIONS: Green pepper (poly)phenols did not show genoprotection against oxidatively generated damage in HT-29 cells at simulated physiological concentrations, regardless of the application, or not, of a culinary treatment (griddling). Furthermore, high concentrations of (poly)phenolic extracts induced a slight pro-oxidant effect, even at a non-cytotoxic concentration.

Development of a Novel High-Density [3H]Hypoxanthine Scintillation Proximity Assay To Assess Plasmodium falciparum Growth.

The discovery and development of new antimalarial drugs are becoming imperative because of the spread of resistance to current clinical treatments. The lack of robustly validated antimalarial targets and the difficulties with the building in of whole-cell activity in screening hits are hampering target-based approaches. However, phenotypic screens of structurally diverse molecule libraries are offering new opportunities for the identification of novel antimalarials. Several methodologies can be used to determine the whole-cell in vitro potencies of antimalarial hits. The [(3)H]hypoxanthine incorporation assay is considered the "gold standard" assay for measurement of the activity of antimalarial compounds against intraerythrocytic forms of Plasmodium falciparum However, the method has important limitations, as the assay is not amenable for high-throughput screening since it remains associated with the 96-well plate format. We have overcome this drawback by adapting the [(3)H]hypoxanthine incorporation method to a 384-well high-density format by coupling a homogeneous scintillation proximity assay (SPA) and thus eliminating the limiting filtration step. This SPA has been validated using a diverse set of 1,000 molecules, including both a representative set from the Tres Cantos Antimalarial Set (TCAMS) of compounds and molecules inactive against whole cells. The results were compared with those from the P. falciparum lactate dehydrogenase whole-cell assay, another method that is well established as a surrogate for parasite growth and is amenable for high-throughput screening. The results obtained demonstrate that the SPA-based [(3)H]hypoxanthine incorporation assay is a suitable design that is adaptable to high-throughput antimalarial drug screening and that maintains the features, robustness, and reliability of the standard filtration hypoxanthine incorporation method.

A high-throughput fluorescence-based assay for Plasmodium dihydroorotate dehydrogenase inhibitor screening.

Plasmodium dihydroorotate dehydrogenase (DHODH) is a mitochondrial membrane-associated flavoenzyme that catalyzes the rate-limiting step of de novo pyrimidine biosynthesis. DHODH is a validated target for malaria, and DSM265, a potent inhibitor, is currently in clinical trials. The enzyme catalyzes the oxidation of dihydroorotate to orotate using flavin mononucleotide (FMN) as cofactor in the first half of the reaction. Reoxidation of FMN to regenerate the active enzyme is mediated by ubiquinone (CoQD), which is the physiological final electron acceptor and second substrate of the reaction. We have developed a fluorescence-based high-throughput enzymatic assay to find DHODH inhibitors. In this assay, the CoQD has been replaced by a redox-sensitive fluorogenic dye, resazurin, which changes to a fluorescent state on reduction to resorufin. Remarkably, the assay sensitivity to find competitive inhibitors of the second substrate is higher than that reported for the standard colorimetric assay. It is amenable to 1536-well plates with Z' values close to 0.8. The fact that the human enzyme can also be assayed in the same format opens additional applications of this assay to the discovery of inhibitors to treat cancer, transplant rejection, autoimmune diseases, and other diseases mediated by rapid cellular growth.

Development of two novel high-throughput assays to quantify ubiquitylated proteins in cell lysates: application to screening of new anti-malarials.

BACKGROUND: The ubiquitin proteasome system (UPS) is one of the main proteolytical pathways in eukaryotic cells and plays an essential role in key cellular processes such as cell cycle, stress response, signal transduction, and transcriptional regulation. Many components of this pathway have been implicated in diverse pathologies including cancer, neurodegeneration and infectious diseases, such as malaria. The success of proteasome inhibitors in clinical trials underlines the potential of the UPS in drug discovery. METHODS: Plasmodium falciparum, the malaria causative pathogen, has been used to develop two assays that allow the quantification of the parasite protein ubiquitylation levels in a high-throughput format that can be used to find new UPS inhibitors. RESULTS: In both assays tandem ubiquitin binding entities (TUBEs), also known as ubiquitin traps, have been used to capture ubiquitylated proteins from cell lysates. The primary assay is based on AlphaLISA technology, and the orthogonal secondary assay relies on a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) system. A panel of well-known proteasome inhibitors has been used to validate both technologies. An excellent correlation was obtained between these biochemical assays and the standard whole cell assay that measures parasite growth inhibition. CONCLUSIONS: The two assays presented can be used in a high-throughput format to find new UPS inhibitors for P. falciparum and could help to identify new targets within this system. This methodology is also applicable to other cellular contexts or pathologies.

In vitro studies on the stability in the proximal gastrointestinal tract and bioaccessibility in Caco-2 cells of chlorogenic acids from spent coffee grounds.

Spent coffee grounds are a potential commercial source of substantial amounts of chlorogenic acids (CGAs). The aim of this study was to evaluate the stability of spent coffee CGAs using in vitro simulated gastroduodenal digestion and to investigate their potential absorption using an in vitro Caco-2 model of human small intestinal epithelium. During in vitro digestion, lactones were partially degraded while caffeoylquinic and feruloylquinic acids were much more stable. Transport and metabolism studies showed that 1% of the total CGAs were absorbed and transported from the apical to the basolateral side of a Caco-2 cell monolayer after 1 h. Lactones and coumaroylquinic acids showed the rate of highest absorption. Caco-2 cells possessed low metabolic activity. In conclusion, spent coffee extracts contain large amounts of CGAs, which remained bioaccessible across the intestinal barrier, albeit to a relatively low degree.

Bacterial dynamics of the plastisphere microbiome exposed to sub-lethal antibiotic pollution.

BACKGROUND: Antibiotics and microplastics are two major aquatic pollutants that have been associated to antibiotic resistance selection in the environment and are considered a risk to human health. However, little is known about the interaction of these pollutants at environmental concentrations and the response of the microbial communities in the plastisphere to sub-lethal antibiotic pollution. Here, we describe the bacterial dynamics underlying this response in surface water bacteria at the community, resistome and mobilome level using a combination of methods (next-generation sequencing and qPCR), sequencing targets (16S rRNA gene, pre-clinical and clinical class 1 integron cassettes and metagenomes), technologies (short and long read sequencing), and assembly approaches (non-assembled reads, genome assembly, bacteriophage and plasmid assembly). RESULTS: Our results show a shift in the microbial community response to antibiotics in the plastisphere microbiome compared to surface water communities and describe the bacterial subpopulations that respond differently to antibiotic and microplastic pollution. The plastisphere showed an increased tolerance to antibiotics and selected different antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs). Several metagenome assembled genomes (MAGs) derived from the antibiotic-exposed plastisphere contained ARGs, virulence factors, and genes involved in plasmid conjugation. These include Comamonas, Chryseobacterium, the opportunistic pathogen Stenotrophomonas maltophilia, and other MAGs belonging to genera that have been associated to human infections, such as Achromobacter. The abundance of the integron-associated ciprofloxacin resistance gene aac(6')-Ib-cr increased under ciprofloxacin exposure in both freshwater microbial communities and in the plastisphere. Regarding the antibiotic mobilome, although no significant changes in ARG load in class 1 integrons and plasmids were observed in polluted samples, we identified three ARG-containing viral contigs that were integrated into MAGs as prophages. CONCLUSIONS: This study illustrates how the selective nature of the plastisphere influences bacterial response to antibiotics at sub-lethal selective pressure. The microbial changes identified here help define the selective role of the plastisphere and its impact on the maintenance of environmental antibiotic resistance in combination with other anthropogenic pollutants. This research highlights the need to evaluate the impact of aquatic pollutants in environmental microbial communities using complex scenarios with combined stresses. Video Abstract.

Industrial and culinary treatments applied to Piquillo pepper (Capsicum annuum cv. Piquillo) impact positively on (poly)phenols' bioaccessibility and gut microbiota catabolism.

Thermal treatments applied to plant-based foods prior to consumption might influence (poly)phenols' bioaccessibility and the metabolization of these compounds by the gut microbiota. In the present research, the impact of industrial (grilling and canning) and culinary (microwaving and frying) treatments on the bioaccessibility and colonic biotransformations of (poly)phenols from Piquillo pepper (Capsicum annum cv. Piquillo) were evaluated by in vitro gastrointestinal digestion and colonic fermentation models and HPLC-ESI-MS/MS. The application of industrial treatments impacted positively on (poly)phenols' bioaccessibility compared to raw pepper. Microwaving also exerted a positive effect on (poly)phenols' bioaccessibility compared to canning whereas the addition of oil for frying seemed to negatively affect (poly)phenols' release from the food matrix. Throughout the 48 hours of the colonic fermentation process (poly)phenolic compounds were catabolized into different (poly)phenol derivatives whose formation was also positively affected by industrial and culinary treatments. Based on the concentration and time of appearance of these derivatives, catabolic pathways of (poly)phenols from Piquillo pepper were proposed. The major (poly)phenol derivatives identified (3-(3'-hydroxyphenyl)propanoic acid, 4-hydroxy-3-methoxyphenylacetic acid and benzene-1,2-diol) are considered of great interest for the study of their bioactivity and the potential effect on human health.

Emerging contaminants: A One Health perspective.

Environmental pollution is escalating due to rapid global development that often prioritizes human needs over planetary health. Despite global efforts to mitigate legacy pollutants, the continuous introduction of new substances remains a major threat to both people and the planet. In response, global initiatives are focusing on risk assessment and regulation of emerging contaminants, as demonstrated by the ongoing efforts to establish the UN's Intergovernmental Science-Policy Panel on Chemicals, Waste, and Pollution Prevention. This review identifies the sources and impacts of emerging contaminants on planetary health, emphasizing the importance of adopting a One Health approach. Strategies for monitoring and addressing these pollutants are discussed, underscoring the need for robust and socially equitable environmental policies at both regional and international levels. Urgent actions are needed to transition toward sustainable pollution management practices to safeguard our planet for future generations.

LC-MS/MS Analysis Elucidates the Different Effects of Industrial and Culinary Processing on Total and Individual (Poly)phenolic Compounds of Piquillo Pepper (Capsicum annuum cv. Piquillo).

Pepper constitutes an important source of (poly)phenols, mainly flavonoids. Nevertheless, heat treatments applied prior to consumption may have an impact on these antioxidants, and thus may also affect their potential bioactivity. In this study, the effect of industrial and culinary treatments on the total and individual (poly)phenolic content of Piquillo pepper (Capsicum annuum cv. Piquillo) was thoroughly evaluated by high-performance liquid chromatography coupled to tandem mass spectrometry. A total of 40 (poly)phenols were identified and quantified in raw pepper. Flavonoids (10 flavonols, 15 flavones, and 2 flavanones) were the major compounds identified (62.6%). Among the 13 phenolic acids identified in raw samples, cinnamic acids were the most representative. High temperatures applied and subsequent peeling during industrial grilling drastically decreased the total (poly)phenolic content from 2736.34 to 1099.38 µg/g dm (59.8% reduction). In particular, flavonoids showed a higher reduction of 87.2% after grilling compared to nonflavonoids which only decreased by 14%. Moreover, 9 nonflavonoids were generated during grilling, modifying the (poly)phenolic profile. After culinary treatments, specifically frying, (poly)phenols appear to be better released from the food matrix, enhancing their extractability. Overall, industrial and culinary treatments differently affect both the total and individual (poly)phenolic compounds of pepper and, despite the reduction, they might also positively influence their bioaccessibility.

Sub-inhibitory gentamicin pollution induces gentamicin resistance gene integration in class 1 integrons in the environment.

Antibiotics at sub-inhibitory concentrations are often found in the environment. Here they could impose selective pressure on bacteria, leading to the selection and dissemination of antibiotic resistance, despite being under the inhibitory threshold. The goal of this study was to evaluate the effects of sub-inhibitory concentrations of gentamicin on environmental class 1 integron cassettes in natural river microbial communities. Gentamicin at sub-inhibitory concentrations promoted the integration and selection of gentamicin resistance genes (GmRG) in class 1 integrons after only a one-day exposure. Therefore, sub-inhibitory concentrations of gentamicin induced integron rearrangements, increasing the mobilization potential of gentamicin resistance genes and potentially increasing their dissemination in the environment. This study demonstrates the effects of antibiotics at sub-inhibitory concentrations in the environment and supports concerns about antibiotics as emerging pollutants.

Impact of in situ solar irradiation on snow bacterial communities and functional potential.

Polar regions are increasingly exposed to ultraviolet light due to ozone depletion. Snowpacks contain photochemically active particles that, when irradiated, can lead to the production and accumulation of reactive species that can induce oxidative stress on snow microorganisms. This could generate a selective pressure on snowpack bacteria. In this study, snow microcosms were buried in a snowpack at Ny-Ålesund (Svalbard), either exposed to solar irradiation or incubated in the dark for 10 days, and the bacterial response to solar irradiation was evaluated in situ using a metagenomics approach. Solar irradiation induced a significant decrease in bacterial abundance and richness. Genes involved in glutathione synthesis, sulphur metabolism, and multidrug efflux were significantly enriched in the light, whereas genes related to cell wall assembly and nutrient uptake were more abundant in the dark. This is the first study demonstrating the response of snow bacterial communities to solar irradiation in situ and providing insights into the mechanisms involved. Our research shows that polar sun irradiation is sufficiently intense to impose a selective pressure on snow bacteria and supports the concern that increased ultraviolet exposure due to anthropogenic activities and climatic change could drive critical changes in the structure and functioning of snow bacterial communities.

Evolution and interaction of microbial communities in mangrove microbial fuel cells and first description of Shewanella fodinae as electroactive bacterium.

Understanding exoelectrogenic bacteria mechanisms and their interactions in complex biofilm is critical for the development of microbial fuel cells (MFCs). In this article, assumptions concerning the benefits of the complex sediment microbial community for electricity production were explored with both the complex microbial community and isolates identified as Shewanella. Analysis of the microbial community revealed a strong influence of the sediment community on anodes and electrolytes compared to that of only water. Moreover, while Pelobacteraceae-related genera were dominant in our MFCs instead of Desulfuromonas and Geobacter as usually reported, the electroactive Shewanella algae and Shewanella fodinae were isolated and cultivated from the anodic biofilm. S. fodinae, described for the first time as an electroactive bacterium to the best of our knowledge, led to a maximal current density of 3.6 A/m2 set as 0.3 V/SCE in a three-electrode set-up fed with lactate. S. algae, in a complex medium containing several available substrates, showed several preferential oxidative behaviors including a diauxic behavior. In pure culture and under our conditions, S. fodinae and S. algae were not able to use acetate as a sole electron donor. However, their presence in our acetate-fed MFCs and the adaptive behavior of S. algae hint a syntrophic interaction between the bacteria to optimize the use of the substrate in a complex environment.

Health associations of liver enzymes and inflammatory scores with urinary citrus flavonoid metabolites.

Background: Dietary flavonoid intake is associated with a reduced risk of some cardiometabolic disorders, attributed in part to their claimed anti-inflammatory activity. Our aim was to investigate the potential association between specific urine flavonoid metabolites, liver enzymes, and inflammatory status in individuals with metabolic syndrome (MetS). Methods: In this cross-sectional study, clinical and dietary data from 267 participants, aged 55 to 75 years, participating in the PREDIMED Plus study (PREvención con DIeta MEDiterránea) were analyzed. At the baseline, spot urine samples were collected and seven urinary flavonoid metabolites were quantified using ultra-performance liquid chromatography coupled to triple quadrupole mass spectrometry (UPLC-Q-q-Q MS). Liver enzymes, inflammatory scores, and urinary flavonoid concentrations were inverse normally transformed. Results: Adjusted linear regression models showed an inverse association between urinary citrus flavanone concentrations and gamma-glutamyl transferase (GGT) (all p-values <0.05). Naringenin 7'-GlcUA was significantly associated with a lower aggregate index of systemic inflammation (AISI) (Bper 1SD = -0.14; 95% CI: -0.27 to -0.02; p-value = 0.025) and systemic inflammation index (SII) (Bper 1SD = -0.14; 95% CI: -0.27 to -0.02; p-value = 0.028). To investigate the relationship between flavanone subclasses and GGT levels, we fitted a score of citrus-flavanones, and subjects were stratified into quartiles. The highest values of the citrus-flavanone score (per 1-SD increase) were associated with lower GGT levels (Bper 1SD = -0.41; 95% CI: -0.74 to -0.07), exhibiting a linear trend across quartiles (p-trend = 0.015). Conclusion: This cross-sectional study showed that higher urinary excretion of citrus-flavanone metabolites was associated with lower GGT levels in subjects diagnosed with MetS and obesity.

Discovery and biochemical characterization of Plasmodium thioredoxin reductase inhibitors from an antimalarial set.

Plasmodium falciparum is the most prevalent and deadly species of the human malaria parasites, and thioredoxin reductase (TrxR) is an enzyme involved in the redox response to oxidative stress. Essential for P. falciparum survival, the enzyme has been highlighted as a promising target for novel antimalarial drugs. Here we report the discovery and characterization of seven molecules from an antimalarial set of 13533 compounds through single-target TrxR biochemical screens. We have produced high-purity, full-length, recombinant native enzyme from four Plasmodium species, and thioredoxin substrates from P. falciparum and Rattus norvegicus. The enzymes were screened using a unique, high-throughput, in vitro native substrate assay, and we have observed selectivity between the Plasmodium species and the mammalian form of the enzyme. This has indicated differences in their biomolecular profiles and has provided valuable insights into the biochemical mechanisms of action of compounds with proven antimalarial activity.

In vivo study of the bioavailability and metabolic profile of (poly)phenols after sous-vide artichoke consumption.

Artichokes are a rich source of (poly)phenols, mainly caffeoylquinic acids, but little is known about their bioavailability from this source. This study investigated the absorption, metabolism and excretion of (poly)phenols after sous-vide artichoke consumption (5776 µmol of (poly)phenols) by healthy volunteers. Seventy-six (poly)phenol metabolites were identified by UHPLC-MS/MS using authentic standards, including acyl-quinic acids plus C6-C3, C6-C1, C6-C2, C6-C1-N, C6-C0 metabolites, and their phase-II conjugates. The major metabolites were 3'-methoxy-4'-hydroxycinnamic acid, 3'-methoxycinnamic acid-4'-sulfate, and 4'-hydroxycinnamic acid-3'-sulfate, which appeared early in plasma (Tmax < 4 h); plus 3-(3'-methoxy-4'-hydroxyphenyl)propanoic acid, 3-(4'-methoxyphenyl)propanoic acid-3'-glucuronide, 3-(3'-hydroxyphenyl) propanoic acid and hippuric acids, which appeared later (Tmax > 6 h). The 24 h urinary recovery averaged 8.9% (molar basis) of the (poly)phenols consumed. Hepatic beta-oxidation of 3',4'-dihydroxycinnamic acid and methylated conjugates occurred, but was limited (<0.04%). 3'-Methylation exceeded 4'-methylation and interindividual variability was high, especially for gut microbial metabolites (up to 168-fold).

Sequencing Depth Has a Stronger Effect than DNA Extraction on Soil Bacterial Richness Discovery.

Although Next-Generation Sequencing techniques have increased our access to the soil microbiome, each step of soil metagenomics presents inherent biases that prevent the accurate definition of the soil microbiome and its ecosystem function. In this study, we compared the effects of DNA extraction and sequencing depth on bacterial richness discovery from two soil samples. Four DNA extraction methods were used, and sequencing duplicates were generated for each DNA sample. The V3-V4 region of the 16S rRNA gene was sequenced to determine the taxonomical richness measured by each method at the amplicon sequence variant (ASV) level. Both the overall functional richness and antibiotic resistance gene (ARG) richness were evaluated by metagenomics sequencing. Despite variable DNA extraction methods, sequencing depth had a greater influence on bacterial richness discovery at both the taxonomical and functional levels. Sequencing duplicates from the same sample provided access to different portions of bacterial richness, and this was related to differences in the sequencing depth. Thus, the sequencing depth introduced biases in the comparison of DNA extraction methods. An optimisation of the soil metagenomics workflow is needed in order to sequence at a sufficient and equal depth. This would improve the accuracy of metagenomic comparisons and soil microbiome profiles.

Gentamicin at sub-inhibitory concentrations selects for antibiotic resistance in the environment.

Antibiotics released into the environment at low (sub-inhibitory) concentrations could select for antibiotic resistance that might disseminate to the human microbiome. In this case, low-level anthropogenic sources of antibiotics would have a significant impact on human health risk. In order to provide data necessary for the evaluation of this risk, we implemented river water microcosms at both sub-inhibitory and inhibitory concentrations of gentamicin as determined previously based on bacterial growth in enriched media. Using metagenomic sequencing and qPCR/RT-qPCR, we assessed the effects of gentamicin on water bacterial communities and their resistome. A change in the composition of total and active communities, as well as a gentamicin resistance gene selection identified via mobile genetic elements, was observed during a two-day exposure. We demonstrated the effects of sub-inhibitory concentrations of gentamicin on bacterial communities and their associated resistome in microcosms (simulating in situ conditions). In addition, we established relationships between antibiotic dose and the magnitude of the community response in the environment. The scope of resistance selection under sub-inhibitory concentrations of antibiotics and the mechanisms underlying this process might provide the basis for understanding resistance dispersion and associated risks in relatively low impacted ecosystems.

Environmental and Anthropogenic Factors Shape the Snow Microbiome and Antibiotic Resistome.

Winter tourism can generate environmental pollution and affect microbial ecology in mountain ecosystems. This could stimulate the development of antibiotic resistance in snow and its dissemination through the atmosphere and through snow melting. Despite these potential impacts, the effect of winter tourism on the snow antibiotic resistome remains to be elucidated. In this study, snow samples subjected to different levels of anthropogenic activities and surrounding forest were obtained from the Sudety Mountains in Poland to evaluate the impact of winter tourism on snow bacteria using a metagenomic approach. Bacterial community composition was determined by the sequencing of the V3-V4 hypervariable region of the 16S rRNA gene and the composition of the antibiotic resistome was explored by metagenomic sequencing. Whereas environmental factors were the main drivers of bacterial community and antibiotic resistome composition in snow, winter tourism affected resistome composition in sites with similar environmental conditions. Several antibiotic resistance genes (ARGs) showed a higher abundance in sites subjected to human activities. This is the first study to show that anthropogenic activities may influence the antibiotic resistome in alpine snow. Our results highlight the need to survey antibiotic resistance development in anthropogenically polluted sites.