Epilithic Biofilms Research Articles

Presence and antimicrobial susceptibility profile of Salmonella spp. isolated from fish farms

Salmonella spp. are important foodborne pathogens often associated with infectious outbreaks caused by consuming contaminated food. Although these microorganisms are widely distributed in several animal production chains, there are few records of their occurrence in aquaculture. This study aimed to evaluate the presence and antimicrobial resistance of Salmonella spp. isolated from fish farms. Samples were collected from three fish farms, covering water, fish, and epilithic biofilm. Salmonella isolates were identified by PCR, and serotyping was performed using the slide agglutination method, according to the Kaufman-White system. Susceptibility to antimicrobial agents was assessed using the Kirby and Bauer disk diffusion method. The presence of class 1 and 2 integrons was determined by PCR. Ninety samples were examined, of which 13 (14.44%) were positive for S. enterica, of which 5 strains were isolated from lagoon water, 3 from fish, and 5 from epilithic biofilm. Among the samples, the serotypes S. Minnesota, S. Panamá, and S. Anatum were identified. Antimicrobial resistance rates were highest for sulfonamides (92.30%), trimethoprim (84.61%), tetracycline (46.15%), and streptomycin (46.15%). Multiple antibiotic resistance was confirmed in 84.61% of the isolates, with 100% presenting integron class 1 and 7.69% integron class 2. This study demonstrated a high prevalence of multiresistant bacteria in fish farms, which reinforces public health concerns due to the use of antibiotics and the risk associated with food from farmed freshwater fish. Given these results, the need for new external research into control and prevention strategies for Salmonella spp. is evident. in aquaculture, as well as greater regulation in fish production chains.

Temporal changes in the morphological and microbial diversity of biofilms on the surface of a submerged stone in the Danube River

Epilithic biofilms are ubiquitous in large river environments and are crucial for biogeochemical processes, but their community structures and functions remain poorly understood. In this paper, the seasonal succession in the morphological structure and the taxonomic composition of an epilithic bacterial biofilm community at a polluted site of the Danube River were followed using electron microscopy, high-throughput 16S rRNA gene amplicon sequencing and multiplex/taxon-specific PCRs. The biofilm samples were collected from the same submerged stone and carried out bimonthly in the littoral zone of the Danube River, downstream of a large urban area. Scanning electron microscopy showed that the biofilm was composed of diatoms and a variety of bacteria with different morphologies. Based on amplicon sequencing, the bacterial communities were dominated by the phyla Pseudomonadota and Bacteroidota, while the most abundant archaea belonged to the phyla Nitrososphaerota and Nanoarchaeota. The changing environmental factors had an effect on the composition of the epilithic microbial community. Critical levels of faecal pollution in the water were associated with increased relative abundance of Sphaerotilus, a typical indicator of “sewage fungus”, but the composition and diversity of the epilithic biofilms were also influenced by several other environmental factors such as temperature, water discharge and total suspended solids (TSS). The specific PCRs showed opportunistic pathogenic bacteria (e.g. Pseudomonas spp., Legionella spp., P. aeruginosa, L. pneumophila, Stenotrophomonas maltophilia) in some biofilm samples, but extended spectrum β-lactamase (ESBL) genes and macrolide resistance genes could not be detected.

Seasonal variation and human impacts of the river biofilm bacterial communities in the Shiting River in southeastern China.

Bacterial communities in epilithic biofilm plays an important role in biogeochemistry processes in freshwater ecosystems. Nevertheless, our understanding of the geographical and seasonal variations of the composition of bacterial communities in the biofilm of gravels on river bed is still limited. Various anthropogenic activities also influence the biofilm bacteria in gravel rivers. By taking the Shiting River in the upper Yangtze River basinin Sichuan Province as an example, we studied the geographical and seasonal variations of epilithic bacteria and the impacts of weirs and other human activities (e.g., sewage pollution). The river has experienced severe degradation since the Ms 8.0 Wenchuan Earthquake, and weirs were constructed to prevent bed erosion.We collected epilithic biofilms samples at 17 sites along ~ 30km river reach of the Shiting River in the autumn of 2021 and the summer of 2022, respectively. We applied 16S rRNA gene high-throughput sequencing technology and Functional Annotation of Prokaryotic Taxa (FAPROTAX) to analyze the seasonal and biogeographic patterns and potential functions of the biofilm bacterial communities. The results showed that epilithic bacteria from the two surveys exhibited variation in community composition, bacterial diversity and potential functions. The bacteria samples collected in the autumn have much higher alpha diversity and richness than those collected in the summer. Bacterial richness and diversity were lower downstream of the weirs than upstream. Low diversity was observed at a sampling site influenced by sewage inflow, which contains high level of nitrogen-related chemicals.

Polyphasic description of Ciimarium marinum gen. et sp. nov. (Prochlorococcaceae, Synechococcales): a new picocyanobacterial taxon from the Portuguese coastal ecosystem

In this paper, we describe Ciimarium marinum gen. et sp. nov. (Prochlorococcaceae, Synechococcales) based on 16S rRNA gene phylogenies, 16S-23S ITS secondary structures, ultrastructure (TEM), morphological, pigments and ecological data of “Synechococcus” sp. LEGE 11466. The strain was isolated from a sample collected at Leixões Harbour, Portugal (41°11’08.1”N 8°43’08.8”W) on September 26th, 2011, by scraping an epilithic biofilm under 10-13m depth, less than 1km offshore. Bayesian Inference and Maximum Likelihood phylogenies demonstrate that the Ciimarium sequence is placed in a monophyletic clade with strong phylogenetic support (BI=1, ML=100), within the Prochlorococcaceae cluster. The type strain LEGE 11466 is phylogenetically distant from other reference strains of Cyanobacteria and is grouped with Uncultured “Synechococcus” MLS1228cl3 , “Synechococcus” MLCB, “Cyanobium” ATX 6A2 ATX-6A2-C45 and “Synechococcus” MBIC10613.. The secondary structures of 16S-23S ITS corroborate with the separation of Ciimarium from all the Prochlorococcaceae genera. Also, they show that MLCB and ATX 6A2 ATX-6A2-C45 may be different species from the type LEGE 11466. The ultrastructure analysis shows parietal thylakoids, in agreement with the family description. Morphologically the genus is indistinguishable from other Prochlorococcaceae genera. The pigments analysis shows Zeaxanthin and β-carotene as the main pigments, and also a low concentration of phycocyanin. Finally, our phylogenies indicate that a revision of the Prochlorococcaceae family is necessary, as there are still many Synechococcus-like Cyanobacteria strains that should be described as new genera in the future.

Effects of large dams on the aquatic food web along a coastal stream with high sediment loads

The contribution of two basal energy sources – detrital organic matter and primary producers – as part of aquatic food webs varies typically along river continua. A host of barriers to river flow increase the water residence time and sediment and nutrient retention in reservoirs worldwide, and potentially alter the balance between detritus-based and algae-based energy pathways in the downstream food webs. We explored this issue on the Sélune River (Normandy, France), a small coastal stream that drains an agricultural catchment with high sediment runoff. Seasonal measurements of the following parameters were compared upstream and downstream of the reservoirs of two large dams (16 m and 36 m high): sediment fluxes, nutrient and chlorophyll a concentrations, algal communities in the epilithic biofilm (taxonomic composition, biomass and growth), and benthic invertebrate communities (abundance and trophic guild structure). As anticipated, annual sediment fluxes were much lower downstream of the reservoirs, where significant decreases in water turbidity, phosphate and silicate concentrations were recorded. A higher chlorophyll a concentration in water and a higher contribution of pelagic algae taxa to the photosynthetic biofilm suggested drifting and deposition of reservoir-borne phytoplankton downriver. Photosynthetic biofilm growth was higher downstream of the reservoirs in spring and fall, and so was the abundance of herbivores in the invertebrate community, notably scrapers and algae eaters. Energy pathways within riverine food webs were traced using stable isotope analyses of carbon (C) and nitrogen in the tissues of aquatic consumers (invertebrates and fish). Mixing models revealed a discontinuity in the origin of the C entering the food webs along the river continuum, confirming a greater contribution of algal C to aquatic consumers downstream of the reservoirs. These results illustrate mechanisms whereby large reservoirs can modulate C flow in food webs along a small coastal river with high sediment loads, and make it possible to anticipate the effects of dam removal on the future river ecosystem.

Impacts of substrate properties and aquatic nutrient concentrations on the relative abundance of nitrifying/denitrifying genes and the associated microbes in epilithic biofilms.

Substrates like sand or gravels and aquatic nutrient concentrations of rivers are highly heterogeneous, influencing the abundance of functional genes in epilithic biofilms where nitrification-denitrification processes take place. To analyze how the relative abundance of nitrifying/denitrifying genes and the associated microbes changes with the physical properties of substrates and aquatic concentrations of nutrients, this paper utilized metagenomics to comprehensively characterize these functional genes (i.e., amoA, hao, and nxrB involved in nitrification, and napA, narG, nirS, norB, and nosZ associated with denitrification) from epilithic biofilms collected along the Shitingjiang River in Southwest China and further obtained the relative abundance of major nitrifiers and denitrifiers. The results show that substrate size most significantly affects the relative abundance of hao and norB by altering the hydrodynamic conditions. In sampling sites with high heterogeneity in substrate size distribution, the relative abundance of most denitrifying genes is also higher. The carbon-nitrogen ratio negatively correlates with the relative abundance of all the nitrifying genes, while ammonium, total inorganic carbon, and total organic carbon concentrations positively affect the relative abundance of amoA and nxrB. As to the relative abundance of nitrifiers and denitrifiers, mainly belonging to phyla Proteobacteria and Actinobacteria, substrate heterogeneity and the aquatic concentrations of nutrients have greater influences than substrate size. Also, the substrate heterogeneity exerted positive influence on functional species of Pseudogemmobacter bohemicus and Paracoccus zhejiangensis. Considering the genes' functions and the dominant species linked to denitrification, nitrous oxide is more likely to occur in rivers with higher heterogeneity and larger substrates.

Temporal patterns of stream biofilm in a mountain catchment: one-year monthly samplings across streams of the Orobic Alps (Northern Italy)

Periphyton is the dominant primary producer in mountain streams and sustains the higher trophic levels. While certain periphyton groups, particularly diatoms, have received extensive study, the comprehensive characterization of the entire community has been largely neglected. This study aims to investigate the temporal pattern of biofilm in mountain streams characterized by different water thermal regimes. A one-year quantitative campaign, involving monthly samplings, was conducted in five subalpine streams in Northern Italy’s Orobic Alps to collect epilithic biofilm from a wide surface area. The total biomass was quantified and the periphyton was analyzed both for composition (diatoms, green algae, cyanobacteria, and red algae) and for pigments. Disturbance, water temperature, physico-chemical conditions, nutrients, substrate diversity, and light availability were assessed concurrently with biofilm samplings. Results show sharp biofilm variations over months. In all sites, the disturbance was the primary factor reducing biomass and pigment content. Annually, all sites experienced similar turnover in periphyton composition mainly associated with light and water temperature. Overall, the study indicates that frequent quantitative investigations of biofilm help understand intra-annual variations and identify key drivers. Such information is useful to understand the ecosystem processes and the food web dynamics.

Modeling in-stream biogeochemical processes at catchment scale: Coupling SWAT and RIVE models

Representation of biogeochemical in-stream processes is complex, and requires use of models to understand factors controling the transfer of contaminants on watershed scale. The biogeochemical model RIVE was integrated into the SWAT model and was used, instead of the current QUAL2E subroutine, to calculate biogeochemical in-stream processes. The coupled model was applied to an 89-km section of the Garonne watershed, downstream of Toulouse, for the 2000–2010 period, with a daily time-step. An epilithic biofilm equation was added to represent the river's algae communities, dominated by drifted benthic diatoms. The model matched observed data for major variables of water quality. Dominant processes for nitrogen, phosphorus, silica and organic suspended matter were assessed. The aim of coupling RIVE to the SWAT model was to improve the biogeochemical representation of the river's in-stream ecological processes linked to C, N, P and Si elements and dissolved oxygen, with minimal calibration.

Epilithic biofilms provide large amounts of nitrogen to tropical mountain landscapes.

We show that epilithic biofilms are a relevant nitrogen (N) source in a rocky mountain range in Brazil. During different seasons, we quantified nitrate, ammonium, dissolved organic N (DON) and total dissolved N (TDN) leached by a simulated short rain event. We quantified the epilithic autotrophic biomass by taxonomic groups and its correlation with leached N. We hypothesized that leached N would be correlated to heterocystous cyanobacteria biomass since they are more efficient N2 fixers. We estimated a landscape N supply of 8.5 kg.ha-1 .year-1 considering the mean precipitation in the region. TDN in leachate was mainly composed of DON (83.8% ± 22%), followed by nitrate (12.1% ± 3%) and ammonium (5% ± 5%). The autotrophic epilithic community was mainly composed of non-heterocystous (Gloeocapsopsis) and heterocystous cyanobacteria (Scytonema and Stigonema), except for a site more commonly affected by fire events that showed a dominance of Chlorophyta. Biogeochemical upscaling was facilitated by the fact that N leaching was not different among sites or related to autotrophic epilithic biomass or assemblage composition. In conclusion, the capacity of epilithic biofilms to provide N to surrounding systems is an ecosystem service that underscores the necessity to conserve them and their habitats.

Differential effects of wastewater treatment plant effluents on the antibiotic resistomes of diverse river habitats.

Wastewater treatment plants (WWTPs) are key sources of antimicrobial resistance genes (ARGs) that could influence the resistomes of microbial communities in various habitats of the receiving river ecosystem. However, it is currently unknown which habitats are most impacted and whether ARGs, like certain chemical contaminants, could be accumulated or enriched in the river ecosystem. We conducted a systematic metagenomic survey on the antibiotic resistomes of WWTP effluent, four riverine habitats (water, suspended particles, sediment, epilithic biofilm), and freshwater amphipod gut microbiomes. The impact of WWTP effluent on the downstream habitats was assessed in nine Swiss rivers. While there were significant differences in resistomes across habitats, the wastewater resistome was more similar to the resistome of receiving river water than to the resistomes of other habitats, and river water was the habitat most strongly impacted by the WWTPs effluent. The sulfonamide, beta-lactam, and aminoglycoside resistance genes were among the most abundant ARGs in the WWTP effluents, and especially aadA, sul1, and class A beta-lactamase genes showed significantly increased abundance in the river water of downstream compared to upstream locations (p < 0.05). However, this was not the case for the sediment, biofilm, and amphipod gut habitats. Accordingly, evidence for accumulation or enrichment of ARGs through the riverine food web was not identified. Our study suggests that monitoring riverine antimicrobial resistance determinants could be conducted using "co-occurrence" of aadA, sul1, and class A beta-lactamase genes as an indicator of wastewater-related pollution and should focus on the water as the most affected habitat.

Epilithic biofilms as a discriminating matrix for long-term and growing season pesticide contamination in the aquatic environment: Emphasis on glyphosate and metabolite AMPA

The indiscriminate use of pesticides represents high ecological risk in aquatic systems. Recently, the inclusion of epilithic biofilms as a reactive matrix has shown potential in diagnosing the health of water resources. The objective of this study was to use multiple matrices (water, suspended sediments, and biofilms) to discriminate contamination degrees in catchments with long and recent history of intensive pesticide use and to monitor growing season pesticides transfer to watercourses. Two catchments were monitored: one representative of “modern agriculture” in a subtropical environment, and another representative of recent agricultural expansion over the Pampa Biome in subtropical Brazil. Glyphosate and AMPA were accumulated in the biofilms and were detected at all sites and at all monitoring times, in concentrations ranging from 195 to 7673 μg kg−1 and from 225 to 4180 μg kg−1, respectively. Similarly, the fungicide tebuconazole has always been found in biofilms. The biofilms made it possible to discriminate the long-term history of pesticide use in the catchments and even to identify the influx pulses of pesticides immediately after their application to crops, which was not possible with active water sampling and even with suspended sediment monitoring. It is strongly recommended that, in regions with intensive cultivation of soybeans and other genetically modified crops, the presence of glyphosate and its metabolite AMPA be permanently monitored, a practice still very scarce in the literature.

Epilithic biofilms of the Eastern Caspian (Aktau region, Kazakhstan) under conditions of falling sea level

Communities of microorganisms that form biofilms on the rocky shores of the Middle Caspian are capable of maintaining the integrity of the coastal landscape in the conditions of sea retreat associated with the ongoing drop in sea level since 1995. Algal-bacterial biofilms developing on rocky substrates of the East Caspian coast within the City of Aktau were studied in autumn (October 2022) and spring (April 2023) seasons. Communities in biotopes with different structure were investigated. It has been established that the most stable and diverse communities with a pronounced vertical structure live in the areas with high sheer cliffs on capes. On flat areas of the coast, which have undergone recent shallowing and are protected from surfing waves by ridges of large stones, less diverse cenoses, demonstrating clear spatial organization. In the shallow open areas of the shore, which are subject to intense wave action, live communities of two types were observed. Both cenoses contained a relatively small number of species, but differed in structure and potential for protecting the coast from erosion. The first of them live in biotopes outside the impact of urban domestic sewage, and are able to protect coastal marl from destruction due to the development of stable, rapidly regenerating complex structural elements. The latter were noted in the conditions of along-shore spreading of untreated domestic wastewater from new city developments located close to the coast. Such biofilms were no longer able to protect marls from being destroyed by waves, and took the form of constantly renewed patches with minimum diversity of components and unstable spatial organization.

Bacterial diversity and co‐occurrence patterns differ across a world‐wide spatial distribution of habitats in glacier ecosystems

Abstract Glaciers cover nearly 10% of the Earth's surface and are unique biomes dominated by microbial communities that support key ecosystem processes. The melting of glaciers is among the most conspicuous consequences of global climate change, with impacts on microbial ecology and associated biogeochemistry. However, we are still missing an integrative understanding of microbial biodiversity from divergent habitats associated with glaciers. Here, we compiled global microbiome metadata from 93 representative glaciers over 180 locations and used random forest, microbial ecological networks and structural equation modelling to evaluate the biodiversity and environmental factors associated with the glacier microbiomes of seven contrasting habitats: water, epilithic biofilm, cryoconite, mat, ice, sediment and permafrost soil. The results showed that microbial diversity largely changed across habitats, with the highest in permafrost soil, followed by sediment, ice, mat, cryoconite, water and epilithic biofilm. More importantly, we provided critical evidence that the environmental and climatic factors associated with the microbiomes of glaciers varied with glacier habitats. Microbial diversity in water was highly correlated with latitude, cryoconite microbial diversity was significantly (p = 0.01) correlated with pH, and permafrost soil and sediment microbial diversity were mainly explained by temperature (17.05% and 13.37% respectively). Using ecological association network analysis, we identified some tightly linked common microbial taxa (e.g. Proteobacteria and Bacteroidetes) that were present in all the habitats and were vulnerable to climatic factors, such as temperature and precipitation. This study demonstrated that microbial diversity, drivers and co‐occurrence patterns differ among glacier habitats globally, and diverse habitat‐dependent glacier microbiomes could serve as early warning sentinels for the study of life on glaciers and its potential future in a warming world. Read the free Plain Language Summary for this article on the Journal blog.

Does sterol availability in a forested headwater stream constitute a nutritional constraint for macroinvertebrates?

ABSTRACT Headwater streams are characterised by predominantly heterotrophic functioning resulting from leaf litter input, but autochthonous primary production can also contribute to energy fluxes and the supply of nutrients. While much work has focused on the role of polyunsaturated fatty acids as essential nutrients, less attention has been paid to the significance of sterols in stream food webs. Yet these molecules are essential to arthropods that can only synthesise cholesterol from a limited range of dietary sterols. In a headwater stream, we tracked the transfer of dominant sterols from allochthonous and autochthonous resources to 5 benthic macroinvertebrates. Despite their formal functional feeding group, all the taxa tended to be opportunistic omnivores that relied on all available resources. These behaviours could be attributed to nutritional constraints in the stream food web, but these constraints were not related to the sterol supply from the different basal sources. Dominant sterols from detrital sources (leaf litter, fine benthic organic matter [FBOM]) and primary producers (epilithic biofilms, bryophytes) were all Δ5-sterols (β-sitosterol, campesterol, stigmasterol, epibrassicasterol). The levels of cholesterol detected in macroinvertebrates, as well as the presence of desmosterol, indicate efficient dealkylation activity of these Δ5-sterols. Leaf litter has consistently shown high β-sitosterol/stigmasterol ratios, giving it greater nutritional value than usually accepted. But bryophytes, and especially epilithic biofilms, contained cholesterol, providing a direct supply to invertebrates, thereby promoting their growth. Detrital particles (FBOM) colonised by microalgae could be the best-balanced diet to avoid sterol deficiencies due to their cholesterol content and high β-sitosterol/stigmasterol ratios.

Evidence of Bacterial Community Coalescence between Freshwater and Discharged tpm-Harboring Bacterial Taxa from Hospital and Domestic Wastewater Treatment Plants among Epilithic Biofilms.

The ability of WWTP outflow bacteria at colonizing rock surfaces and contributing to the formation of river epilithic biofilms was investigated. Bacterial community structures of biofilms (b-) developing on rocks exposed to treated wastewaters (TWW) of a hospital (HTWW) and a domestic (DTWW) clarifier, and to surface waters of the stream located at 10 m, 500 m, and 8 km from the WWTP outlet, were compared. Biofilm bacterial contents were analyzed by cultural approaches and a tpm-based DNA metabarcoding analytical scheme. Co-occurrence distribution pattern analyses between bacterial datasets and eighteen monitored pharmaceuticals were performed. Higher concentrations of iohexol, ranitidine, levofloxacin, and roxithromycin were observed in the b-HTWW while atenolol, diclofenac, propranolol, and trimethoprim were higher in the b-DTWW. MPN growth assays showed recurrent occurrences of Pseudomonas aeruginosa and Aeromonas caviae among these biofilms. An enrichment of multi-resistant P. aeruginosa cells was observed in the hospital sewer line. P. aeruginosa MPN values were negatively correlated to roxithromycin concentrations. The tpm DNA metabarcoding analyses confirmed these trends and allowed an additional tracking of more than 90 species from 24 genera. Among the recorded 3082 tpm ASV (amplicon sequence variants), 41% were allocated to the Pseudomonas. Significant differences through ANOSIM and DESeq2 statistical tests were observed between ASV recovered from b-HTWW, b-DTWW, and epilithic river biofilms. More than 500 ASV were found restricted to a single sewer line such as those allocated to Aeromonas popoffii and Stenotrophomonas humi being strictly found in the b-HTWW file. Several significant correlations between tpm ASV counts per species and pharmaceutical concentrations in biofilms were recorded such as those of Lamprocystis purpurea being positively correlated with trimethoprim concentrations. A tpm source tracking analysis showed the b-DTWW and b-HTWW tpm ASV to have contributed, respectively, at up to 35% and 2.5% of the epilithic river biofilm tpm-taxa recovered downstream from the WWTP outlet. Higher contributions of TWW taxa among epilithic biofilms were recorded closer to the WWTP outlet. These analyses demonstrated a coalescence of WWTP sewer communities with river freshwater taxa among epilithic biofilms developing downstream of a WWTP outlet.

Community structures and biodeterioration processes of epilithic biofilms imply the significance of micro-environments

Epilithic biofilms colonising outdoor stone monuments can intensify the deterioration processes of the stone materials and pose great challenges to their protection. In this study, biodiversity and community structures of the epilithic biofilms colonising the surfaces of five outdoor stone dog sculptures were characterised by high-throughput sequencing. Although they are exposed to the same envrionment in a small yard, the analysis of their biofilm populations revealed high biodiversity and species richness as well as great differences in community compostions. Interestingly, populations responsible for pigment production (e.g., Pseudomonas, Deinococcus, Sphingomonas and Leptolyngbya) and for nitrogen (e.g., Pseudomonas, Bacillus, and Beijerinckia) and sulfur cycling (e.g., Acidiphilium) were the core common taxa in the epilithic biofilms, suggesting the potential biodeterioration processes. Furthermore, significant positive corrolections of metal elements rich in stone with biofilm communities showed that epilithic biofilms could take in minerals of stone. Importantly, geochemical properties of soluble ions (higher concentration of SO42− than NO3−) and slightly acidic micro-environments on the surfaces suggest corrosion of biogenic sulfuric acids as a main mechanism of biodeterioration of the sculptures. Interestingly, relative abundacne of Acidiphilium showed a positive correlation with acidic micro-environments and SO42− concentrations, implying they could be an indicator of sulfuric acid corrosion. Together, our findings support that micro-environments are inportant to community assembly of epilithic biofilms and the biodeterioration processes involved.

Epilithic biofilms, POCIS, and water samples as complementary sources of information for a more comprehensive view of aquatic contamination by pesticides and pharmaceuticals in southern Brazil

Spatial-temporal monitoring of the presence of pesticides and pharmaceuticals in water requires rigor in the choice of matrix to be analyzed. The use of matrices, isolated or combined, may better represent the real state of contamination. In this sense, the present work contrasted the effectiveness of using epilithic biofilms with active water sampling and with a passive sampler—POCIS. A watershed representative of South American agriculture was monitored. Nine sites with different rural anthropic pressures (natural forest, intensive use of pesticides, and animal waste), and urban areas without sewage treatment, were monitored. Water and epilithic biofilms were collected during periods of intensive pesticide and animal waste application. After the harvest of the spring/summer crop, a period of low agrochemical input, the presence of pesticides and pharmaceuticals was monitored using the POCIS and epilithic biofilms. The spot water sampling leads to underestimation of the level of contamination of water resources as it does not allow discrimination of different anthropic pressures in rural areas. The use of endogenous epilithic biofilms as a matrix for the analysis of pesticides and pharmaceuticals is a viable and highly recommended alternative to diagnose the health of water sources, especially if associated with the use of POCIS.

Diversity and Composition of Culturable Microorganisms and Their Biodeterioration Potentials in the Sandstone of Beishiku Temple, China.

Microbial colonization on stone monuments leads to subsequent biodeterioration; determining the microbe diversity, compositions, and metabolic capacities is essential for understanding biodeterioration mechanisms and undertaking heritage management. Here, samples of epilithic biofilm and naturally weathered and exfoliated sandstone particles from different locations at the Beishiku Temple were collected to investigate bacterial and fungal community diversity and structure using a culture-based method. The biodeterioration potential of isolated fungal strains was analyzed in terms of pigmentation, calcite dissolution, organic acids, biomineralization ability, and biocide susceptibility. The results showed that the diversities and communities of bacteria and fungi differed for the different sample types from different locations. The population of culturable microorganisms in biofilm samples was more abundant than that present in the samples exposed to natural weathering. The environmental temperature, relative humidity, and pH were closely related to the variation in and distribution of microbial communities. Fungal biodeterioration tests showed that isolated strains four and five were pigment producers and capable of dissolving carbonates, respectively. Their biomineralization through the precipitation of calcium oxalate and calcite carbonate could be potentially applied as a biotechnology for stone heritage consolidation and the mitigation of weathering for monuments. This study adds to our understanding of culturable microbial communities and the bioprotection potential of fungal biomineralization.

Presence of pharmaceuticals and bacterial resistance genes in river epilithic biofilms exposed to intense agricultural and urban pressure

The continuous discharge of pharmaceutical compounds into the aquatic environment has raised concerns over the contamination of water resources. Urban activities and intensive animal breeding are important sources of contamination. The accumulation of antibiotics may lead to the transfer or alternatively maintain the presence of resistance genes in natural microbial communities existing in epilithic biofilms. The objective of this study was to evaluate the pharmaceutical contamination levels and the presence of resistance genes in biofilms from a South Brazilian watershed. The Guaporé watershed exhibits a high diversity of land use, including agricultural and urban areas with differing levels of anthropogenic pressure. Seventeen sites along the Guaporé watershed were monitored. Biofilm samples were collected in two seasons (winter and summer), and the pharmaceutical concentration and quantity of resistance genes were analyzed. All monitored sites were contaminated with pharmaceuticals. Agricultural activities contribute through transferring pharmaceuticals derived from the application of animal waste to agricultural fields. The most contaminated site (pharmaceuticals and bacterial resistance genes) was located in an urban area exposed to high pressure. Decreases in the contamination of biofilms were also observed, exemplifying processes of natural attenuation in the watershed. The quality of the biofilms sampled throughout the watershed served as a useful tool to understand and monitor environmental pollution.

Glacier-Fed Stream Biofilms Harbor Diverse Resistomes and Biosynthetic Gene Clusters.

Antimicrobial resistance (AMR) is a universal phenomenon the origins of which lay in natural ecological interactions such as competition within niches, within and between micro- to higher-order organisms. To study these phenomena, it is crucial to examine the origins of AMR in pristine environments, i.e., limited anthropogenic influences. In this context, epilithic biofilms residing in glacier-fed streams (GFSs) are an excellent model system to study diverse, intra- and inter-domain, ecological crosstalk. We assessed the resistomes of epilithic biofilms from GFSs across the Southern Alps (New Zealand) and the Caucasus (Russia) and observed that both bacteria and eukaryotes encoded twenty-nine distinct AMR categories. Of these, beta-lactam, aminoglycoside, and multidrug resistance were both abundant and taxonomically distributed in most of the bacterial and eukaryotic phyla. AMR-encoding phyla included Bacteroidota and Proteobacteria among the bacteria, alongside Ochrophyta (algae) among the eukaryotes. Additionally, biosynthetic gene clusters (BGCs) involved in the production of antibacterial compounds were identified across all phyla in the epilithic biofilms. Furthermore, we found that several bacterial genera (Flavobacterium, Polaromonas, Superphylum Patescibacteria) encode both atimicrobial resistance genes (ARGs) and BGCs within close proximity of each other, demonstrating their capacity to simultaneously influence and compete within the microbial community. Our findings help unravel how naturally occurring BGCs and AMR contribute to the epilithic biofilms mode of life in GFSs. Additionally, we report that eukaryotes may serve as AMR reservoirs owing to their potential for encoding ARGs. Importantly, these observations may be generalizable and potentially extended to other environments that may be more or less impacted by human activity. IMPORTANCE Antimicrobial resistance is an omnipresent phenomenon in the anthropogenically influenced ecosystems. However, its role in shaping microbial community dynamics in pristine environments is relatively unknown. Using metagenomics, we report the presence of antimicrobial resistance genes and their associated pathways in epilithic biofilms within glacier-fed streams. Importantly, we observe biosynthetic gene clusters associated with antimicrobial resistance in both pro- and eukaryotes in these biofilms. Understanding the role of resistance in the context of this pristine environment and complex biodiversity may shed light on previously uncharacterized mechanisms of cross-domain interactions.