Functional analysis, diversity, and distribution of the ean cluster responsible for 17ß-estradiol degradation in sphingomonads.

17ß-estradiol (E2) is a natural endocrine disruptor that is frequently detected in surface and groundwater sources, thereby threatening ecosystems and human health. The newly isolated E2-degrading strain Sphingomonas colocasiae C3-2 can degrade E2 through both the 4,5-seco pathway and the 9,10-seco pathway; the former is the primary pathway supporting the growth of this strain and the latter is a branching pathway. The novel gene cluster ean was found to be responsible for E2 degradation through the 4,5-seco pathway, where E2 is converted to estrone (E1) by EanA, which belongs to the short-chain dehydrogenases/reductases (SDR) superfamily. A three-component oxygenase system (including the P450 monooxygenase EanB1, the small iron-sulfur protein ferredoxin EanB2, and the ferredoxin reductase EanB3) was responsible for hydroxylating E1 to 4-hydroxyestrone (4-OH-E1). The enzymatic assay showed that the proportion of the three components is critical for its function. The dioxygenase EanC catalyzes ring A cleavage of 4-OH-E1, and the oxidoreductase EanD is responsible for the decarboxylation of the ring A-cleavage product of 4-OH-E1. EanR, a TetR family transcriptional regulator, acts as a transcriptional repressor of the ean cluster. The ean cluster was also found in other reported E2-degrading sphingomonads. In addition, the novel two-component monooxygenase EanE1E2 can open ring B of 4-OH-E1 via the 9,10-seco pathway, but its encoding genes are not located within the ean cluster. These results refine research on genes involved in E2 degradation and enrich the understanding of the cleavages of ring A and ring B of E2.IMPORTANCESteroid estrogens have been detected in diverse environments, ranging from oceans and rivers to soils and groundwater, posing serious risks to both human health and ecological safety. The United States National Toxicology Program and the World Health Organization have both classified estrogens as Group 1 carcinogens. Several model organisms (proteobacteria) have established the 4,5-seco pathway for estrogen degradation. In this study, the newly isolated Sphingomonas colocasiae C3-2 could degrade E2 through both the 4,5-seco pathway and the 9,10-seco pathway. The novel gene cluster ean (including eanA, eanB1, eanC, and eanD) responsible for E2 degradation by the 4,5-seco pathway was identified; the novel two-component monooxygenase EanE1E2 can open ring B of 4-OH-E1 through the 9,10-seco pathway. The TetR family transcriptional regulator EanR acts as a transcriptional repressor of the ean cluster. The cluster ean was also found to be present in other reported E2-degrading sphingomonads, indicating the ubiquity of the E2 metabolism in the environment.

Bacterial community assembly and antibiotic resistance genes in soils exposed to antibiotics at environmentally relevant concentrations.

Understanding how bacterial community assembly and antibiotic resistance genes (ARGs) respond to antibiotic exposure is essential to deciphering the ecological risk of anthropogenic antibiotic pollution in soils. In this study, three loam soils with different land management (unmanured golf course, dairy-manured pasture, and swine-manured cornfield) were spiked with a mixture of 11 antibiotics at the initial concentration of 100 and 1000 µg kg-1 for each antibiotic and incubated over 132 days, mimicking a scenario of pulse disturbance and recovery in soils, with unspiked soil samples as the control treatment. The Infer Community Assembly Mechanisms by Phylogenetic-bin-based null model (iCAMP) analysis demonstrated that drift and dispersal limitation contributed to 57%-65% and 16%-25%, and homogeneous selection 12%-16% of soil bacterial community assembly. Interestingly, antibiotic exposure to 1000 µg kg-1 level significantly increased the contribution of drift to community assembly, largely due to the positive response from Acidobacteria-6 in the golf course and pasture soils and from Chthoniobacteraceae in the cornfield soil to the antibiotic exposure. However, ARG abundance and diversity in the three soils exhibited antibiotics-independent temporal fluctuations, but were associated with the changes in soil bacterial communities over time. This study provides the first insight into the relative contributions of different bacterial community assembly processes in soils upon antibiotic exposure at environmentally relevant concentrations.

Unrecognized risk of perfluorooctane sulfonate in promoting conjugative transfers of bacterial antibiotic resistance genes.

Antibiotic resistance is a major global health crisis facing humanity, with horizontal gene transfer (HGT) as a principal dissemination mechanism in the natural and clinical environments. Perfluoroalkyl substances (PFASs) are emerging contaminants of global concern due to their high persistence in the environment and adverse effects on humans. However, it is unknown whether PFASs affect the HGT of bacterial antibiotic resistance. Using a genetically engineered Escherichia coli MG1655 as the donor of plasmid-encoded antibiotic resistance genes (ARGs), E. coli J53 and soil bacterial community as two different recipients, this study demonstrated that the conjugation frequency of ARGs between two E. coli strains was (1.45 ± 0.17) × 10-5 and perfluorooctane sulfonate (PFOS) at environmentally relevant concentrations (2-50 µg L-1) increased conjugation transfer between E. coli strains by up to 3.25-fold. Increases in reactive oxygen species production, cell membrane permeability, biofilm formation capacity, and cell contact in two E. coli strains were proposed as major promotion mechanisms from PFOS exposure. Weighted gene co-expression network analysis of transcriptome data identified a series of candidate genes whose expression changes could contribute to the increase in conjugation transfer induced by PFOS. Furthermore, PFOS also generally increased the ARG transfer into the studied soil bacterial community, although the uptake ability of different community members of the plasmid either increased or decreased upon PFOS exposure depending on specific bacterial taxa. Overall, this study reveals an unrecognized risk of PFOS in accelerating the dissemination of antibiotic resistance. IMPORTANCE Perfluoroalkyl substances (PFASs) are emerging contaminants of global concern due to their high persistence in the environment and adverse health effects. Although the influence of environmental pollutants on the spread of antibiotic resistance, one of the biggest threats to global health, has attracted increasing attention in recent years, it is unknown whether environmental residues of PFASs affect the dissemination of bacterial antibiotic resistance. Considering PFASs, often called "forever" compounds, have significantly higher environmental persistence than most emerging organic contaminants, exploring the effect of PFASs on the spread of antibiotic resistance is more environmentally relevant and has essential ecological and health significance. By systematically examining the influence of perfluorooctane sulfonate on the antibiotic resistance gene conjugative transfer, not only at the single-strain level but also at the community level, this study has uncovered an unrecognized risk of PFASs in promoting conjugative transfers of bacterial antibiotic resistance genes, which could be incorporated into the risk assessment framework of PFASs.

Natural Solar Irradiation Produces Fluorescent and Biodegradable Nanoplastics.

Nanoplastics (NPs) have raised global concern owing to their potential health effects. Herein, after simulated and natural solar irradiation, polyethylene, polypropylene, polystyrene, and poly(vinyl chloride) nanoplastics (PVC NPs) were observed to exhibit enhanced fluorescence, particularly PVC NPs. Furthermore, the role of photoaged NPs as a potential fluorescence indicator was evaluated by exposing a model aquatic organism Daphnia magna to these NPs. Our results revealed that photoaged NPs exhibited strong fluorescence owing to the generation of conjugated π bonds, which can achieve π-π* electron transition with low energy consumption. Photogenerated fluorescence also enabled the photoaged NPs to act as efficient fluorescent tracers, which can help track NP migration in various organisms. The results of two-photon laser confocal scanning microscopy revealed that the photoaged NPs could translocate across biological barriers and accumulate in extraintestinal tissues in addition to being ingested and excreted. Moreover, compared with pristine NPs, the photoaged NPs underwent biodegradation more easily, probably because of increased hydrophilicity due to photogenerated oxygen-containing moieties. Therefore, in addition to producing fluorescent NPs without the attachment of external fluorescent dyes, the natural photoaging process can promote the migration and degradation of photoaged NPs in food chains.

Influence of organic cosolvents on hexabromobenzene degradation in solution by montmorillonite-templated subnanoscale zero-valent iron.

Organic cosolvents are commonly used to increase the dissolution of poorly water-soluble organic pollutants into aqueous solutions during environmental remediation. In this study, the influences of five organic cosolvents on hexabromobenzene (HBB) degradation catalyzed by one typical reactive material montmorillonite-templated subnanoscale zero-valent iron (CZVI) were investigated. The results demonstrated that all cosolvents promoted HBB degradation but the degree of promotion was different for different cosolvents, which was associated with inconsistent solvent viscosities, dielectric constant properties, and the extent of interactions between cosolvents with CZVI. Meanwhile, HBB degradation was highly dependent on the volume ratio of cosolvent to water, which increased in the range of 10%-25% but persistently decreased in the range of more than 25%. This might be due to the fact that the cosolvents increased HBB dissolution at low concentrations but reduced the protons supplied by water and the contact between HBB with CZVI at high concentrations. In addition, the freshly-prepared CZVI had higher reactivity to HBB than the freeze-dried CZVI in all water-cosolvent solutions, probably because freeze-drying reduced the interlayer space of CZVI and thus the contact probability between HBB and active reaction sites. Finally, the CZVI-catalyzed HBB degradation mechanism was proposed as the electron transfer between zero-valent iron and HBB, which led to the formation of four debromination products. Overall, this study provides helpful information for the practical application of CZVI in the remediation of persistent organic pollutants in the environment.

Bacterial Community under the Influence of Microplastics in Indoor Environment and the Health Hazards Associated with Antibiotic Resistance Genes.

Selectively colonized microbial communities and enriched antibiotic resistance genes (ARGs) in (micro)plastics in aquatic and soil environments make the plastisphere a great health concern. Although microplastics (MPs) are distributed in indoor environments in high abundance, information on the effect of MPs on a microbial community in an indoor environment is lacking. Here, we detected polymers (containing MPs and natural polymers), bacterial communities, and 18 kinds of ARGs in collected indoor dust samples. A significant correlation by Procrustes analysis between bacterial community composition and the abundance of MPs was observed, and correlation tests and redundancy analysis identified specific associations between MP polymers and bacterial taxa, such as polyamide and Actinobacteria. In addition, the abundance of MPs showed a positive correlation with the relative abundance of the ARGs (to 16S RNA), while natural polymers, such as cellulosics, showed positive correlations with the absolute abundance of ARGs and 16S rRNA. Simulated experiments verified that significantly higher bacterial biomasses and ARGs were observed on the surface of cotton, hair, and wool than on MPs, while a higher relative abundance of ARGs was detected on MPs. However, a significantly higher amount of ARG was found on MPs of poly(lactic acid), the biodegradable plastics with the highest yield. In addition to the plastisphere in water and soil environments, MPs in an indoor environment may also affect the bacterial community and specifically enrich ARGs. Moreover, degradable MPs and nondegradable MPs may result in different health hazards due to their distinct effects on bacterial community.

Antibiotic Resistance Gene-Carrying Plasmid Spreads into the Plant Endophytic Bacteria using Soil Bacteria as Carriers.

Applications of animal manure and treated wastewater could enrich antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the plant microbiome. However, the mechanistic studies of the transmission of ARB and ARGs from the environment to plant endophytic bacteria were few. Herein, a genetically engineered fluorescent Escherichia coli harboring a conjugative RP4 plasmid that carries three ARGs was used to trace its spread into Arabidopsis thaliana interior in a tetracycline-amended hydroponic system in the absence or presence of a simulated soil bacterial community. Confocal microscope observation demonstrated that E. coli was internalized into plant tissues and the carried RP4 plasmid was transferred into plant endophytic bacteria. More importantly, we observed that soil bacteria inhibited the internalization of E. coli but substantially promoted RP4 plasmid spread into the plant microbiome. The altered RP4-carrying bacterial community composition in the plant microbiome and the increased core-shared RP4-carrying bacteria number between plant interior and exterior in the presence of soil bacteria collectively confirmed that soil bacteria, especially Proteobacteria, might capture RP4 from E. coli and then translocate into plant microbiome, resulting in the increased RP4 plasmid spread in the plant endophytes. Overall, our findings provided important insights into the dissemination of ARB and ARGs from the environment to the plant microbiome.

Ionic Liquid Enriches the Antibiotic Resistome, Especially Efflux Pump Genes, Before Significantly Affecting Microbial Community Structure.

An expanding list of chemicals may permeabilize bacterial cells and facilitate horizontal gene transfer (HGT), which enhances propagation of antibiotic resistance genes (ARGs) in the environment. Previous studies showed that 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF6]), an ionic liquid, can facilitate HGT of some ARGs among bacteria. However, the dynamic response of a wider range of ARGs and associated mobile genetic elements (MGEs) in different environments is unknown. Here, we used metagenomic tools to study shifts of the resistome and microbiome in both sediments and freshwater microcosms exposed to [BMIm][PF6]. Exposure for 16 h to 0.1 or 1.0 g/L significantly enriched more than 207 ARG subtypes primarily encoding efflux pumps in freshwater microcosms as well as cultivable antibiotic-resistant bacteria. This resistome enrichment was attributed to HGT facilitated by MGEs (428 plasmids, 61 integron-integrase genes, and 45 gene cassettes were enriched) as well as to HGT-related functional genes. Interestingly, resistome enrichment occurred fast (within 16 h) after [BMIm][PF6] exposure, before any significant changes in bacterial community structure. Similar ARG enrichment occurred in sediment microcosms exposed to [BMIm][PF6] for 28 d, and this longer exposure affected the microbial community structure (e.g., Proteobacteria abundance increased significantly). Overall, this study suggests that [BMIm][PF6] releases could rapidly enrich the antibiotic resistome in receiving environments by increasing HGT and fortuitously selecting for efflux pump genes, thus contributing to ARG propagation.

Interactions of Gaseous 2-Chlorophenol with Fe3+-Saturated Montmorillonite and Their Toxicity to Human Lung Cells.

The interactions of gaseous 2-chlorophenol with Fe3+-saturated montmorillonite particles in a gas-solid system were investigated to simulate the reactions of mineral dusts with volatile organic pollutants in the atmosphere. Results suggested that Fe3+-saturated montmorillonite mediated the dimerization of gaseous 2-chlorophenol to form hydroxylated polychlorinated biphenyl, hydroxylated polychlorinated diphenyl ether, and hydroxylated polychlorinated dibenzofuran. The toxicity of Fe3+-montmorillonite particles to A549 human lung epithelial cells before and after interaction with 2-chlorophenol was examined to explore their adverse impact on human health. Based on cell morphological analysis, cytotoxicity tests, and Fourier-transform infrared imaging spectra, surface-catalyzed reactions of Fe3+-montmorillonite with 2-chlorophenol increased the toxicity of montmorillonite particle on A549 cells. This was supported by increased cellular membrane permeability, the release of extracellular lactate dehydrogenase, and cell damages on cellular DNA, proteins, and lipids. Since mineral dusts are important components of particulate matter, our results help to understand the interactions of volatile organic pollutants with particulate matter in the atmosphere and their adverse impacts on human health.

Bioavailability of Soil-Sorbed Tetracycline to Escherichia coli under Unsaturated Conditions.

Increasing concentrations of anthropogenic antibiotics in soils are partly responsible for the proliferation of bacterial antibiotic resistance. However, little is known about how soil-sorbed antibiotics exert selective pressure on bacteria in unsaturated soils. This study investigated the bioavailability of tetracycline sorbed on three soils (Webster clay loam, Capac sandy clay loam, and Oshtemo loamy sand) to a fluorescent Escherichia coli bioreporter under unsaturated conditions using agar diffusion assay, microscopic visualization, and model simulation. Tetracycline sorbed on the soils could be desorbed and become bioavailable to the E. coli cells at matric water potentials of -2.95 to -13.75 kPa. Bright fluorescent rings were formed around the tetracycline-loaded soils on the unsaturated agar surfaces, likely due to radial diffusion of tetracycline desorbed from the soils, tetracycline uptake by the E. coli cells, and its inhibition on E. coli growth, which was supported by the model simulation. The bioavailability of soil-sorbed tetracycline was much higher for the Oshtemo soil, probably due to faster diffusion of tetracycline in coarse-textured soils. Decreased bioavailability of soil-sorbed tetracycline at lower soil water potential likely resulted from reduced tetracycline diffusion in soil pore water at smaller matric potential and/or suppressed tetracycline uptake by E. coli at lower osmotic potential. Therefore, soil-sorbed tetracycline could still exert selective pressure on the exposed bacteria, which was influenced by soil physical processes controlled by soil texture and soil water potential.

Influence of Dissolved Organic Matter on Tetracycline Bioavailability to an Antibiotic-Resistant Bacterium.

Complexation of tetracycline with dissolved organic matter (DOM) in aqueous solution could alter the bioavailability of tetracycline to bacteria, thereby alleviating selective pressure for development of antibiotic resistance. In this study, an Escherichia coli whole-cell bioreporter construct with antibiotic resistance genes coupled to green fluorescence protein was exposed to tetracycline in the presence of DOM derived from humic acids. Complexation between tetracycline and DOM diminished tetracycline bioavailability to E. coli, as indicated by reduced expression of antibiotic resistance genes. Increasing DOM concentration resulted in decreasing bioavailability of tetracycline to the bioreporter. Freely dissolved tetracycline (not complexed with DOM) was identified as the major fraction responsible for the rate and magnitude of antibiotic resistance genes expressed. Furthermore, adsorption of DOM on bacterial cell surfaces inhibited tetracycline diffusion into the bioreporter cells. The magnitude of the inhibition was related to the amount of DOM adsorbed and tetracycline affinity for the DOM. These findings provide novel insights into the mechanisms by which the bioavailability of tetracycline antibiotics to bacteria is reduced by DOM present in water. Agricultural lands receiving livestock manures commonly have elevated levels of both DOM and antibiotics; the DOM could suppress the bioavailability of antibiotics, hence reducing selective pressure on bacteria for development of antibiotic resistance.

Spatial distribution of two acaricides and five neonicotinoids in beehives and surrounding environments in China.

Managed bees commonly suffer from cross-contamination with acaricides and neonicotinoids, posing robust threats to bee population health. However, their residual characteristics and spatial distribution in beehives and surrounding environments are poorly understood. This study detected two common acaricides and five neonicotinoids in 240 beehive samples and 44 surrounding environmental samples collected from 25 Chinese provinces. The results showed that 40.0% of the honey samples contained acaricides and 83.1% contained neonicotinoids. Neonicotinoid concentrations in honey were geographically distinguished by the "Hu Huanyong line", and concentrations of neonicotinoids in honey from eastern areas were 2.65-fold higher than those in honey from western areas. Compared to the approved acaricide amitraz, the banned acaricide coumaphos was detected more frequently in honey and was positively correlated with that quantified in the paired pollen samples. Although coumaphos was identified in only three soil samples, lower coumaphos residues in honey might be associated with persistent pollution in the surrounding environment. Conversely, neonicotinoids were detected at higher levels in honey than in the pollen and soil, demonstrating that the neonicotinoid residues in honey have a cumulative effect. This study contributes to a better understanding of the pesticide contamination scenarios that underlie the exposure risks of bees.

NaClO Co-selects antibiotic and disinfectant resistance in Klebsiella pneumonia: Implications for the potential risk of extensive disinfectant use during COVID-19 pandemic.

The inappropriate use of antibiotics is widely recognized as the primary driver of bacterial antibiotic resistance. However, less attention has been given to the potential induction of multidrug-resistant bacteria through exposure to disinfectants. In this study, Klebsiella pneumonia, an opportunistic pathogen commonly associated with hospital and community-acquired infection, was experimentally exposed to NaClO at both minimum inhibitory concentration (MIC) and sub-MIC levels over a period of 60 days. The result demonstrated that NaClO exposure led to enhanced resistance of K. pneumonia to both NaClO itself and five antibiotics (erythromycin, polymyxin B, gentamicin, tetracycline, and ciprofloxacin). Concurrently, the evolved resistant strains exhibited fitness costs, as evidenced by decreased growth rates. Whole population sequencing revealed that both concentrations of NaClO exposure caused genetic mutations in the genome of K. pneumonia. Some of these mutations were known to be associated with antibiotic resistance, while others had not previously been identified as such. In addition, 11 identified mutations were located in the virulence factors, demonstrating that NaClO exposure may also impact the pathogenicity of K. pneumoniae. Overall, this study highlights the potential for the widespread use of NaClO-containing disinfectants during the COVID-19 pandemic to contribute to the emergence of antibiotic-resistant bacteria. ENVIRONMENTAL IMPLICATION: Considering the potential hazardous effects of disinfectant residues on environment, organisms and biodiversity, the sharp rise in use of disinfectants during COVID-19 pandemic has been considered highly likely to cause worldwide secondary disasters in ecosystems and human health. This study demonstrated that NaClO exposure enhanced the resistance of K. pneumonia to both NaClO and five antibiotics (erythromycin, polymyxin B, gentamicin, tetracycline, and ciprofloxacin), highlighting the widespread use of NaClO-containing disinfectants during the COVID-19 pandemic may increase the emergence of antibiotic-resistant bacteria in the environment.

Dissemination of antibiotic resistance genes from aboveground sources to groundwater in livestock farms.

Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are prevalent in various environments on livestock farms, including livestock waste, soil, and groundwater. Contamination of groundwater by ARB and ARGs in livestock farms is a growing concern as it may have potentially huge risks to human health. However, the source of groundwater-borne ARB and ARGs in animal farms remains largely unknown. In this study, different types of samples including groundwater and its potential contamination sources from aboveground (pig feces, wastewater, and soil) from both working and abandoned swine feedlots in southern China were collected and subjected to metagenomic sequencing and ARB isolation. The source tracking based on metagenomic analysis revealed that 56-95 % of ARGs in groundwater was attributable to aboveground sources. Using metagenomic assembly, we found that 45 ARGs predominantly conferring resistance to aminoglycosides, sulfonamides, and tetracyclines could be transferred from the aboveground sources to groundwater, mostly through plasmid-mediated horizontal gene transfer. Furthermore, the full-length nucleotide sequences of sul1, tetA, and TEM-1 detected in ARB isolates exhibited the close evolutionary relationships between aboveground sources and groundwater. Some isolated strains of antibiotic-resistant Pseudomonas spp. from aboveground sources and groundwater had the high similarity (average nucleotide identity > 99 %). Notably, the groundwater-borne ARGs were identified as mainly carried by bacterial pathogens, potentially posing risks to human and animal health. Overall, this study underscores the dissemination of ARGs from aboveground sources to groundwater in animal farms and associated risks.

Uptake of the Plant Agriculture-Used Antibiotics Oxytetracycline and Streptomycin by Cherry Radish─Effect on Plant Microbiome and the Potential Health Risk.

Antibiotics are used to control certain bacterial diseases in plant agriculture. Understanding antibiotic uptake by edible vegetables after application and associated risks on plant microbiome and human health is critical. In this study, oxytetracycline and streptomycin, the two most commonly used antibiotics in plant agriculture, were applied to cherry radish via continuous soil drenching to study their translocations into plant tissues, influence on radish microbiome, and the potential health risk to mice. The results demonstrated that oxytetracycline induced hormesis in radish plants and both antibiotics were translocated into the leaves, fruits, and roots of radishes from the soil, with significantly higher plant uptake of streptomycin than oxytetracycline. Interestingly, the proportion of culturable oxytetracycline or streptomycin-resistant bacteria in the antibiotic-accumulated radish tissues was significantly higher than that in the antibiotic-free radish tissues, although both bacterial and fungal communities in different radish tissues were not affected by the accumulated antibiotics, demonstrating that antibiotic application could enrich antibiotic resistance in the plant microbiome. Feeding mice with antibiotics-accumulated radish tissues did not show significant effects on the weight and blood glucose levels of mice. Overall, this study provides important insights into the risk of using antibiotics in plant agriculture.

Unraveling the influence of human fecal pollution on antibiotic resistance gene levels in different receiving water bodies using crAssphage indicator gene.

Discharged wastewater treatment plant (WWTP) effluents can contaminate receiving water bodies with human feces and alter the abundance of antibiotic resistance genes (ARGs). In this study, we examined the co-occurrence of ARGs, human fecal pollution indicator crAssphage, and antibiotics in human feces and a series of connected receiving water bodies affected by human feces, including water from different treatment units of a WWTP, river, lake, and tap waters. Results showed that crAssphage was detected in 68.2 % of the studied water bodies, confirming widespread human fecal contamination. Both ARG and crAssphage abundances exhibited a distance-decay effect from the emission source to the receiving environment. Interestingly, the detected ARG abundance in the water bodies was significantly correlated with crAssphage abundance but not with the residual antibiotic concentration, demonstrating that the presence of ARG could largely be explained by the extent of fecal pollution, with no clear signs of antibiotic selection. In addition, 14 ARGs co-shared by human feces and water bodies were significantly correlated with crAssphage. Furthermore, a close evolutionary relationship was observed between the blaTEM-1 gene from human feces and aquatic environments. These results imply a potential ARG exchange between human feces and receiving water bodies. Overall, this study provides important insights into the distribution and sources of ARGs in water bodies affected by human fecal contamination.

Response of viable bacteria to antibiotics in aerobic granular sludge: Resistance mechanisms and behaviors, bacterial communities, and driving factors.

The assessment of antimicrobial resistance (AMR) risk by DNA-based techniques mainly relies on total bacterial DNA. In this case, AMR risk recognition is restricted to the genotype level, lacking crucial phenotypic information, such as the distribution of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in dead and viable bacteria. This limitation hinders the recognition of AMR behavior. Herein, based on propidium monoazide (PMA) shielding method, this work firstly quantified the intracellular ARGs/MGEs in viable and dead bacteria, and the impact of viable bacteria composition on the formation of intracellular/extracellular polymeric substance-related /cell-free ARGs (i/e/cARGs) and MGEs (i/e/cMGEs) in aerobic granular sludge (AGS). The shielding efficiency of PMA against dead bacteria was optimized to be as high as 97.5% when the MLSS of AGS was 2.0 g/L. Under antibiotic stimulation, 29.0% ∼ 49.0% of iARGs/iMGEs were carried by viable bacteria, and the remaining proportion were carried by dead bacteria. 18 out of the top 20 dominant genera showed a change in abundance by more than 1% after PMA treatment. 29 viable hosts were identified to associate with 52 iARGs, of which 28 and 15 hosts were also linked to 40 eARGs and 26 cARGs. Also, partial least-squares path model and variance partitioning analysis disclosed that viable bacteria and i/e/cMGEs had a positive effect on i/e/cARGs, with both contributing as much as 64.5% to the total ARGs enrichment. These results better visualized the AMR risk carried by viable bacteria and the categories of viable hosts. This work provides a novel insight into analyzing the actual AMR risk and viable hosts, helping to the reduction and control of AMR in wastewater treatment plants.

Tetracycline accumulation in biofilms enhances the selection pressure on Escherichia coli for expression of antibiotic resistance.

Microorganisms are present as either biofilm or planktonic species in natural and engineered environments. Little is known about the selection pressure emanating from exposure to sub-minimal inhibitory concentration of antibiotics on planktonic vs. biofilm bacteria. In this study, an E. coli bioreporter was used to develop biofilms on glass and high-density polyethylene (HDPE) surfaces, and compared with the corresponding planktonic bacteria in antibiotic resistance expression when exposed to a range of µg/L levels of tetracycline. The antibiotic resistance-associated fluorescence emissions from biofilm E. coli reached up to 1.6 times more than those from planktonic bacteria. The intensively developed biofilms on glass surfaces caused the embedded bacteria to experience higher selection pressure and express more antibiotic resistance than those on HDPE surfaces. The temporal pattern of fluorescence emissions from biofilm E. coli was consistent with the biofilm-developing processes during the experimental period. The increased expression of antibiotic resistance from biofilm bacteria could be attributed to the high affinity of tetracycline with extracellular polymeric substances (EPS). The enhanced accumulation of tetracycline in biofilms could exert higher selection pressure on the embedded bacteria. These results suggest that in many natural and engineered systems the higher antibiotic resistance in biofilm bacteria could be attributed partially to the retention antibiotics by the EPS in biofilms.

Halide salts induced the photodegradation of a fat-burning compound 2, 4-dinitrophenol by iron-montmorillonite.

Natural montmorillonite clay and anthropogenic organic pollutants frequently coexist in the estuarine environment where freshwater from rivers mixes with saltwater from the ocean. In this environment, the sharply changed aqueous chemistry especially salt content could significantly alter the photochemical behaviors of pollutants. However, this process was rarely investigated. In this study, the photodegradation of a representative anthropogenic weight-loss compound 2,4-dinitrophenol in the presence of Fe3+-montmorillonite and different halide salts was systematically investigated. Results show that 2,4-dinitrophenol was resistant to photodegradation by Fe3+-montmorillonite alone, but the presence of NaCl, NaBr, and sea salts in the system can evoke significant 2,4-dinitrophenol degradation. The enhancement effect was further elucidated as the replacement reaction between the clay associated Fe3+ and Na + which leads to the release of more interlayer Fe3+ from montmorillonite, resulting in increased production of high active hydroxyl radicals (˙OH) that can substantially damage 2,4-dinitrophenol molecule. In addition, halogen radicals from the reaction of halide ions with ˙OH were also confirmed to participate in 2,4-dinitrophenol degradation. Overall, this study implied that the changed salty condition in the estuarine water could induce the rapid transformation of organic pollutants that move from freshwater and have relatively stable photochemical properties.

NaCl salinity enhances tetracycline bioavailability to Escherichia coli on agar surfaces.

Soil salinity is a worldwide problem and is damaging soil functions. Meanwhile, increasing amounts of anthropogenic antibiotics are discharged to agricultural soils. Little is known about how soil salinity (e.g., NaCl) could influence the bioavailability of antibiotics to bacteria. In this study, a tetracycline-responsive Escherichia coli bioreporter grew on the surfaces of agar microcosms at the same tetracycline concentration (200 µg/L), but various NaCl concentrations (0.5-19.2 g/L) with estimated osmotic potential of -0.18 to -1.80 MPa, and agar content (0.3%-5%) with estimated intrinsic permeability of 38 to 32,928 nm2. These agar microcosms mimicked very fine textured soils with a range of NaCl salinity. Increasing agar content lowered the intrinsic permeability hence decreasing tetracycline bioavailability to E. coli, due likely to the reduced mass transfer of tetracycline via water flow. Intriguingly, tetracycline bioavailability increased with increasing NaCl concentration which caused the increase in osmotic stress. This is contradictory to the notion that osmotic stress reduces bacterial chemical uptake. Further analysis of E. coli membrane integrity demonstrated that the enhanced tetracycline bioavailability to bacteria could result from the compromised cell membranes and enhanced membrane permeability at higher NaCl salinity. Overall, this study suggests that high soil salinity (NaCl) may enhance the selection pressure exerted by antibiotics on bacteria.