RESUMEN

BACKGROUND: Environmental reservoirs of antibiotic resistance pose a threat to human and animal health. Aquatic biofilms impacted by wastewater effluent (WW) are known environmental reservoirs for antibiotic resistance; however, the relative importance of biotic factors and abiotic factors from WW on the abundance of antibiotic resistance genes (ARGs) within aquatic biofilms remains unclear. Additionally, experimental evidence is limited within complex aquatic microbial communities as to whether genes bearing low sequence similarity to validated reference ARGs are functional as ARGs. RESULTS: To disentangle the effects of abiotic and biotic factors on ARG abundances, natural biofilms were previously grown in flume systems with different proportions of stream water and either ultrafiltered or non-ultrafiltered WW. In this study, we conducted deep shotgun metagenomic sequencing of 75 biofilm, stream, and WW samples from these flume systems and compared the taxonomic and functional microbiome and resistome composition. Statistical analysis revealed an alignment of the resistome and microbiome composition and a significant association with experimental treatment. Several ARG classes exhibited an increase in normalized metagenomic abundances in biofilms grown with increasing percentages of non-ultrafiltered WW. In contrast, sulfonamide and extended-spectrum beta-lactamase ARGs showed greater abundances in biofilms grown in ultrafiltered WW compared to non-ultrafiltered WW. Overall, our results pointed toward the dominance of biotic factors over abiotic factors in determining ARG abundances in WW-impacted stream biofilms and suggested gene family-specific mechanisms for ARGs that exhibited divergent abundance patterns. To investigate one of these specific ARG families experimentally, we biochemically characterized a new beta-lactamase from the Planctomycetota (Phycisphaeraceae). This beta-lactamase displayed activity in the cleavage of cephalosporin analog despite sharing a low sequence identity with known ARGs. CONCLUSIONS: This discovery of a functional planctomycete beta-lactamase ARG is noteworthy, not only because it was the first beta-lactamase to be biochemically characterized from this phylum, but also because it was not detected by standard homology-based ARG tools. In summary, this study conducted a metagenomic analysis of the relative importance of biotic and abiotic factors in the context of WW discharge and their impact on both known and new ARGs in aquatic biofilms. Video Abstract.

Asunto(s)

Biopelículas , Metagenómica , Ríos , Aguas Residuales , beta-Lactamasas , Biopelículas/efectos de los fármacos , Aguas Residuales/microbiología , beta-Lactamasas/genética , Ríos/microbiología , Microbiota/efectos de los fármacos , Bacterias/genética , Bacterias/clasificación , Bacterias/efectos de los fármacos , Bacterias/aislamiento & purificación , Antibacterianos/farmacología , Planctomycetales/genética , Planctomycetales/efectos de los fármacos , Metagenoma , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo

RESUMEN

Antibiotics are ubiquitously found in natural surface waters and cause great harm to aquatic organisms. Stream biofilm is a complex and active community composed of algae, bacteria, fungi and other microorganisms, which mainly adheres to solid substances such as rocks and sediments. The durability and diverse structural and metabolic characteristics of biofilms make them a representative of microbial life in aquatic micrecosystems and can reflect major ecosystem processes. Microorganisms and extracellular polymeric substances in biofilms can adsorb and actively accumulate antibiotics. Therefore, biofilms are excellent biological indicators for detecting antibiotic in polluted aquatic environments, but the biotransformation potential of stream biofilms for antibiotics has not been fully explored in the aquatic environment. The characteristics of stream biofilm, such as high abundance and activity of bacterial community, wide contact area with pollutants, etc., which increases the opportunity of biotransformation of antibiotics in biofilm and contribute to bioremediation to improve ecosystem health. Recent studies have demonstrated that both exposure to high and sub-minimum inhibitory concentrations of antibiotics may drive the development of antibiotic resistance genes (ARGs) in natural stream biofilms, which are susceptible to the effects of antibiotic residues, microbial communities and mobile genetic elements, etc. On the basis of peer-reviewed papers, this review explores the distribution behavior of antibiotics in stream biofilms and the contribution of biofilms to the acquisition and spread of antibiotic resistance. Considering that antibiotics and ARGs alter the structure and ecological functions of natural microbial communities and pose a threat to river organisms and human health, our research findings provide comprehensive insights into the migration, transformation, and bioavailability of antibiotics in biofilms.

Asunto(s)

Antibacterianos , Ríos , Humanos , Antibacterianos/farmacología , Ríos/microbiología , Ecosistema , Bacterias/genética , Genes Bacterianos , Biopelículas

RESUMEN

Bacteria in stream biofilms contribute to stream biogeochemical processes and are potentially sensitive to the substantial levels of pollution entering urban streams. To examine the effects of contaminants on stream biofilm bacteria in situ, we exposed growing biofilms to experimental additions of nutrients [nitrogen (N), phosphorus (P), and iron (Fe)], pharmaceuticals (caffeine and diphenhydramine), nutrients plus pharmaceuticals, or no contaminants using contaminant exposure substrates (CES) in three catchments in northern Utah. We performed our study at montane and urban sites to examine the influence of existing pollution on biofilm response. We identified bacterial core communities (core) for each contaminant treatment at each land-use type (e.g., nutrient addition montane bacterial core, nutrient addition urban bacterial core, pharmaceutical addition montane bacterial core) by selecting all taxa found in at least 75% of the samples belonging to each specific grouping. Montane and urban land-use distinguished bacterial cores, while nutrients and pharmaceuticals had subtle, but nonetheless distinct effects. Nutrients enhanced the dominance of already abundant copiotrophs [i.e., Pseudomonadaceae (Gammaproteobacteria) and Comamonadaceae (Betaproteobacteria)] within bacterial cores at montane and urban sites. In contrast, pharmaceuticals fostered species-rich bacterial cores containing unique contaminant-degrading taxa within Pseudomonadaceae and Anaerolineaceae (Chloroflexi). Surprisingly, even at urban sites containing ambient pharmaceutical pollution, pharmaceutical additions increased bacterial core richness, specifically within DR-16 (Betaproteobacteria), WCHB1-32 (Bacteroidetes), and Leptotrichiaceae (Fusobacteria). Nutrients exerted greater selective force than pharmaceuticals in nutrient plus pharmaceutical addition treatments, creating bacterial cores more closely resembling those under nutrient rather than pharmaceutical addition, and promoting unique Oscillatoriales (Cyanobacteria) taxa in urban streams. Our results show that additions of N, P, and Fe intensified the dominance of already abundant copiotrophs, while additions of caffeine and diphenhydramine enabled unique taxa associated with contaminant degradation to participate in bacterial cores. Further, biofilm bacteria at urban sites remained sensitive to pharmaceuticals commonly present in waters, suggesting a dynamic interplay among pharmaceutical pollution, bacterial diversity, and contaminant degradation.

RESUMEN

Stream biofilms have been shown to be among the most sensitive indicators of environmental stress in aquatic ecosystems and several endpoints have been developed to measure biofilm adverse effects caused by environmental stressors. Here, we compare the effects of long-term exposure of stream biofilms to diuron, a commonly used herbicide, on several traditional ecotoxicological endpoints (biomass growth, photosynthetic efficiency, chlorophyll-a content, and taxonomic composition), with the effects measured by recently developed methods [community structure assessed by flow cytometry (FC-CS) and measurement of extracellular polymeric substances (EPS)]. Biofilms grown from local stream water in recirculating microcosms were exposed to a constant concentration of 20 µg/L diuron over a period of 3 weeks. During the experiment, we observed temporal variation in photosynthetic efficiency, biomass, cell size, presence of decaying cells and in the EPS protein fraction. While biomass growth, photosynthetic efficiency, and chlorophyll-a content were treatment independent, the effects of diuron were detectable with both FC and EPS measurements. This demonstrates that, at least for our experimental setup, a combination of different ecotoxicological endpoints can be important for evaluating biofilm environmental stress and suggests that the more recent ecotoxicological endpoints (FC-CS, EPS protein content and humic substances) can be a useful addition for stream biofilm ecotoxicological assessment.