Effects of antidepressant exposure on aquatic communities assessed by a combination of morphological identification, functional measurements, environmental DNA metabarcoding and bioassays.

The antidepressant fluoxetine is frequently detected in aquatic ecosystems, yet the effects on aquatic communities and ecosystems are still largely unknown. Therefore the aim of this study is to assess the effects of the long-term application of fluoxetine on key components of aquatic ecosystems including macroinvertebrate-, zooplankton-, phytoplankton- and microbial communities and organic matter decomposition by using traditional and non-traditional assessment methods. For this, we exposed 18 outdoor mesocosms (water volume of 1530 L and 10 cm of sediment) to five different concentrations of fluoxetine (0.2, 2, 20 and 200 µg/L) for eight weeks, followed by an eight-week recovery period. We quantified population and community effects by morphological identification, environmental DNA metabarcoding, in vitro and in vivo bioassays and measured organic matter decomposition as a measure of ecosystem functioning. We found effects of fluoxetine on bacterial, algal, zooplankton and macroinvertebrate communities and decomposition rates, mainly for the highest (200 µg/L) treatment. Treatment-related decreases in abundances were found for damselfly larvae (NOEC of 0.2 µg/L) and Sphaeriidae bivalves (NOEC of 20 µg/L), whereas Asellus aquaticus increased in abundance (NOEC <0.2 µg/L). Fluoxetine decreased photosynthetic activity and primary production of the suspended algae community. eDNA assessment provided additional insights by revealing that the algae belonging to the class Cryptophyceae and certain cyanobacteria taxa were the most negatively responding taxa to fluoxetine. Our results, together with results of others, suggest that fluoxetine can alter community structure and ecosystem functioning and that some impacts of fluoxetine on certain taxa can already be observed at environmentally realistic concentrations.

Assessing ecological responses to exposure to the antibiotic sulfamethoxazole in freshwater mesocosms.

Antibiotics are a contaminant class of worldwide concern as they are frequently detected in aquatic ecosystems. To better understand the impacts of antibiotics on aquatic ecosystems, we conducted an outdoor mesocosm experiment in which aquatic communities were exposed to different concentrations of the antibiotic sulfamethoxazole (0, 0.15, 1.5, 15 and 150 µg/L). These concentrations include mean (0.15 µg/L) and maximum detected concentrations (15 and 150 µg/L) in aquatic ecosystems worldwide. Sulfamethoxazole was applied once a week for eight consecutive weeks to 1530 L outdoor mesocosms in the Netherlands, followed by an eight-week recovery period. We evaluated phytoplankton-, bacterial- and invertebrate responses during and after sulfamethoxazole exposure and assessed impacts on organic matter decomposition. Contrary to our expectations, consistent treatment-related effects on algal and bacterial communities could not be demonstrated. In addition, sulfamethoxazole did not significantly affect zooplankton and macroinvertebrate communities. However, some effects on specific taxa were observed, with an increase in Mesostoma flatworm abundance (NOEC of <0.15 µg/L). In addition, eDNA analyses indicated negative impacts on the insects Odonata at a sulfamethoxazole concentration of 15 µg/L. Overall, environmentally relevant sulfamethoxazole concentration did not result in direct or indirect impairment of entire aquatic communities and ecological processes in our mesocosms. However, several specific macroinvertebrate taxa demonstrated significant (in)direct effects from sulfamethoxazole. Comparison of the results with the literature showed inconsistent results between studies using comparable, environmentally relevant, concentrations. Therefore, our study highlights the importance of testing the ecological impacts of pharmaceuticals (such as sulfamethoxazole) across multiple trophic levels spanning multiple aquatic communities, to fully understand its potential ecological threats.

Airborne antibiotic resistome and human health risk in railway stations during COVID-19 pandemic.

Antimicrobial resistance is recognized as one of the greatest public health concerns. It is becoming an increasingly threat during the COVID-19 pandemic due to increasing usage of antimicrobials, such as antibiotics and disinfectants, in healthcare facilities or public spaces. To explore the characteristics of airborne antibiotic resistome in public transport systems, we assessed distribution and health risks of airborne antibiotic resistome and microbiome in railway stations before and after the pandemic outbreak by culture-independent and culture-dependent metagenomic analysis. Results showed that the diversity of airborne antibiotic resistance genes (ARGs) decreased following the pandemic, while the relative abundance of core ARGs increased. A total of 159 horizontally acquired ARGs, predominantly confering resistance to macrolides and aminoglycosides, were identified in the airborne bacteria and dust samples. Meanwhile, the abundance of horizontally acquired ARGs hosted by pathogens increased during the pandemic. A bloom of clinically important antibiotic (tigecycline and meropenem) resistant bacteria was found following the pandemic outbreak. 251 high-quality metagenome-assembled genomes (MAGs) were recovered from 27 metagenomes, and 86 genera and 125 species were classified. Relative abundance of ARG-carrying MAGs, taxonomically assigned to genus of Bacillus, Pseudomonas, Acinetobacter, and Staphylococcus, was found increased during the pandemic. Bayesian source tracking estimated that human skin and anthropogenic activities were presumptive resistome sources for the public transit air. Moreover, risk assessment based on resistome and microbiome data revealed elevated airborne health risks during the pandemic.

Antibiotics, antibiotic resistance genes and microbial community in grouper mariculture.

Increasing use of feed and medicine in mariculture could cause negative environmental impacts such as habitat modification, microbial disease development and antibiotic resistance. Here we investigated contamination of antibiotics and antibiotic resistance genes (ARGs), and composition of microbial community in grouper mariculture systems in Hainan province, China. Results showed detection of various antibiotic residues with the dominance of fluoroquinolones and tetracyclines in the six grouper cultivation systems. The concentrations of the detected antibiotics in the grouper mariculture water were significantly higher than those in the original seawater. Some of the detected antibiotics such as enrofloxacin, ciprofloxacin, ofloxacin, oxytetracycline and erythromycin in the mariculture water and/or sediment would pose high resistance selection risks. Sulfonamides resistance genes sul1 and sul2 were found to be predominant in water and sediment, while tetracycline resistance genes were prevalent in fish gill and gut. The dominant bacterial phyla in water and sediments were Bacteroides, Actinomycetes, and Proteobacteria, while the dominant ones in fish gill and gut were the Proteobacteria. Genera of Vibrio and Mycobacterium in the core microbiota were important zoonotic pathogens, and there was a significant positive correlation between Vibrio and ARGs. Phyla of Proteobacteria, Actinomyces, and Cyanobacteria were positively correlated to ARGs, indicating that these microorganisms are potential hosts of ARGs. The putative functions of microbiome related to antibiotic resistance and human diseases were significantly higher in fish than in the mariculture environment. This study suggests that mariculture system is a reservoir of ARGs, and the use of antibiotics in mariculture could induce the increase of antibiotic resistance and the prevalence of opportunistic pathogens.

Spread of airborne antibiotic resistance from animal farms to the environment: Dispersal pattern and exposure risk.

Animal farms have been considered as the critical reservoir of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB). Spread of antibiotic resistance from animal farms to the surrounding environments via aerosols has become a growing concern. Here we investigated the dispersal pattern and exposure risk of airborne ARGs (especially in zoonotic pathogens) in the environment of chicken and dairy farms. Aerosol, dust and animal feces samples were collected from the livestock houses and surrounding environments (upwind and downwind areas) for assessing ARG profiles. Antibiotic resistance phenotype and genotype of airborne Staphylococcus spp. was especially analyzed to reveal the exposure risk of airborne ARGs. Results showed that airborne ARGs were detected from upwind (50 m/100 m) and downwind (50 m/100 m/150 m) air environment, wherein at least 30% of bacterial taxa dispersed from the animal houses. Moreover, atmospheric dispersion modeling showed that airborne ARGs can disperse from the animal houses to a distance of 10 km along the wind direction. Clinically important pathogens were identified in airborne culturable bacteria. Genus of Staphylococcus, Sphingomonas and Acinetobacter were potential bacterial host of airborne ARGs. Airborne Staphylococcus spp. were isolated from the environment of chicken farm (n = 148) and dairy farm (n = 87). It is notable that all isolates from chicken-related environment were multidrug-resistance (>3 clinical-relevant antibiotics), with more than 80% of them carrying methicillin resistance gene (mecA) and associated ARGs and MGEs. Presence of numerous ARGs and diverse pathogens in dust from animal houses and the downwind residential areas indicated the accumulation of animal feces origin ARGs in bioaerosols. Employees and local residents in the chick farming environment are exposed to chicken originated ARGs and multidrug resistant Staphylococcus spp. via inhalation. This study highlights the potential exposure risks of airborne ARGs and antibiotic resistant pathogens to human health.

Dissipation of antibiotic resistance genes in manure-amended agricultural soil.

Soil antibiotic resistance due to animal manure application is of great concern in recent years. Little is known about the fate of antibiotic resistance genes (ARGs) in agricultural soils associated with long-term manure application. Here we used soil microcosms to investigate the dissipation of ARGs and the change of bacterial community in agricultural soil originated from a vegetable field which had received 24 years' swine manure application. Soil microcosms were conducted at different soil moistures and with or without biochar over a testing period of two years in lab. Results showed that continuous manure application induced an accumulation of ARGs in soil, wherein the dissipation of ARGs differed from those in non-manure amended soil. ARGs persisted in soils at least two years, although their abundance declined gradually. Meanwhile, soil moisture and biochar had significant impact on the fate of ARGs. ARGs dissipated faster in soil with higher moisture. Biochar amendment contributed to the maintenance of bacterial diversity. Within the two years of simulation experiment, biochar enhanced soil ARG retention as they dissipated slowly in the soil amended with biochar. Succession of microbial community may have sustained the transfer and resilience of ARGs. This study provides insight into the dissipation of antibiotic resistance genes in manure-applied agricultural soil.

Antibiotic resistance genes in surface water and groundwater from mining affected environments.

Mining activities are known to generate a large amount of mine tailings and acid mine drainage which contain varieties of heavy metals. Heavy metals play an important role in co-selection for bacterial antibiotic resistance. However, the characteristics of antibiotic resistance genes (ARGs) in mining-affected water environments are still unclear. Here we investigated the pollution of metals, profiles of ARGs, mobile genetic elements (MGEs) and microbial community in mining-affected surface water and groundwater. The results showed that in the tested water samples, the concentrations of Zn and Mn were the highest, and Ni was the lowest. Higher abundances of ARGs with great proportion of sulfonamides, chloramphenicols and tetracyclines resistance genes were found in mining-affected water when compared with those without mining activities. Additionally, there were positive correlations between heavy metals (especially Ni, Zn and Mn) and these ARGs. Linear regression analysis suggested that MGEs were positively correlated with ARGs. In addition, total phosphorus was correlated with ARGs (p < 0.05). The microbial community was different between the mining-affected water and the reference (p < 0.05). Proteobacteria, Bacteroidetes and Actinobacteria were dominant phyla in the surface water and groundwater. Network analysis showed that many ARGs were significantly associated with these dominant bacteria, which suggested they might be potential hosts for these ARGs. These findings provide a clear evidence that the mining activities in the study area had a significant impact on surface water and groundwater to different degrees.

Trace analysis of 28 antibiotics in plant tissues (root, stem, leaf and seed) by optimized QuEChERS pretreatment with UHPLC-MS/MS detection.

Phytoremediation has proven to be an effective in-situ treatment technique for antibiotic contamination. Due to the immature methods of extracting multi-antibiotics in different plant tissues, the antibiotic absorption and transportation mechanism in the phytoremediation process has yet to be resolved. Therefore, an improved Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) pretreatment with ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection method for 28 antibiotics in different plant tissues (root, stem, leaf and seed) was developed in this study. The optimized method showed satisfactory performance with recoveries for most antibiotics ranging from 70% to 130% (except sulfadoxine with 138 ± 8.84% in root, sulfameter with 68.9 ± 1.87% and sulfadoxine with 141 ± 10.0% in seed). The limits of detection (LODs) of the target compounds in root, stem, leaf and seed were 0.04 ± 0.02 ~ 2.50 ± 1.14 ng/g, 0.05 ± 0.02 ~ 1.78 ± 0.42 ng/g, 0.06 ± 0.01 ~ 2.50 ± 0.14 ng/g and 0.13 ± 0.10 ~ 3.64 ± 0.74 ng/g, respectively. This developed method was successfully applied to the determination of antibiotics in different tissues of hydroponic wetland plants exposed to antibiotics-spiked water for one-month. Sixteen of 28 spiked antibiotics were detected in plant tissue samples. Overall, of these 16 antibiotics, all were detected in root samples (from < LOQ to 1478 ± 353 ng/g), eleven in stem samples (from < LOQ to 425 ± 47.0 ng/g), and nine in leaf samples (from < LOQ to 429 ± 84.5 ng/g). This developed analytical method provided a robust tool for the simultaneous screening and determination of antibiotics in different plant tissues.

[Profiles and Risk of Antibiotic Resistance Genes in Domestic Wells in the Maozhou River Basin].

Intensive use of antibiotics promotes the occurrence and development of antibiotic resistance. Antibiotic resistance genes (ARGs) enter water environments from human and animal sources. Groundwater serves as an important water supply, while the profiles and risk of ARGs in groundwater remain unknown. The abundance and profiles of ARGs in 11 domestic wells in the Maozhou River basin of Shenzhen City were analyzed by high-throughput qPCR. The results showed that a total of 141 ARGs and 8 mobile genetic elements (MGEs) were detected, of which the genes corresponding resistance to sulfonamides, multidrugs, and aminoglycosides were the most abundant. In each well, the number of detected ARGs and MGEs ranged from 48 to 89, with an average of 68. When normalized by the abundance of 16S rRNA genes, it was found that each bacterium carried at least one ARG in the groundwater of W7, W8, and W10, while in W11, each bacterium carried at least four ARGs. Clinically relevant ARGs that code for resistance to glycopeptide (blaSHV, blaTEM, blaCTX, and blaOXA-1), ß-lactams (vanB and vanC-03), or chloramphenicol (floR) were found in groundwater. In addition, the abundance of sulfonamides, multidrugs, aminoglycosides, ß-lactam, and chloramphenicol resistance genes were positively correlated with the abundance of MGEs (P<0.01), suggesting that MGEs may promote the spread of ARGs in groundwater.

Untreated swine wastes changed antibiotic resistance and microbial community in the soils and impacted abundances of antibiotic resistance genes in the vegetables.

Animal waste fertilization is a traditional agricultural practice, which may have adverse effects to soil ecosystem. However, the side-effects of animal waste fertilization on vegetables are less studied. Here we selected a swine farming village for investigation with a nearby village without swine farming as comparison. In the swine farming village, the farmers use untreated swine manure and wastewater as fertilizers for vegetable cultivation. In the reference village, the farmers mainly use commercial organic fertilizers. The objective of this study is to assess the impacts of untreated swine waste fertilization on both soils and vegetables in terms of antibiotics, antibiotic resistance genes (ARGs) and bacterial microbial communities. The results indicate that untreated swine waste fertilization caused both antibiotic and ARG contaminations and changed the microbial community compositions in the soils. Varieties of tetracyclines and related resistance genes were detected especially in swine wastewater treated soils. The soil quality was impacted with the relations to bacterial abundances and microbial geochemical functions. Proteobacteria and Bacteroidetes were prevalent and positively correlated to ARGs in soils, indicating they were potential antibiotic resistant bacteria. Antibiotics and ARGs were detected in vegetables of both villages. The abundances of ARGs were relatively higher in some vegetable samples of the swine farming village than the reference village. In addition, intracellular parasites Rickettsiales with positive correlation to ARGs were prevalent in some vegetables of swine farming village, indicating potential health risks through eating contaminated vegetables. The results of this study suggest that untreated swine wastes may cause adverse effects to not only agricultural soils but also associated vegetables.

Contamination profile of antibiotic resistance genes in ground water in comparison with surface water.

Dissemination of antibiotic resistance genes (ARGs) in the water environment has become an increasing concern. There have been many reports on ARGs in surface water, but little is known about ARGs in groundwater. In this study, we investigated the profiles and abundance of ARGs in groundwater in comparison with those in surface water of Maozhou River using high-throughput quantitative PCR (HT-qPCR). Totally 127 ARGs and 10 MGEs were detected by HT-qPCR, and among them the sulfonamides, multidrug and aminoglycosides resistance genes were the dominant ARG types. According to the results of HT-qPCR, 18 frequently detected ARGs conferring resistance to 6 classes of antibiotics and 3 MGEs were further quantified by qPCR in the wet season and dry season. The absolute abundance ranged from 1.23 × 105 to 8.89 × 106 copies/mL in wet season and from 8.50 × 102 to 2.65 × 106 copies/mL in the dry season, with sul1 and sul2 being the most abundant ARGs. The absolute abundance of ARGs and MGEs has no significant difference between the wet season and dry season while the diversity of ARGs in the dry season was higher than that in the wet season (p < 0.05). Totally 141 and 150 ARGs were detected in the water and sediments of Maozhou River, respectively. A total of 116 ARGs were shared among the groundwater, river water, and sediment, which accounted for 67.1% of all detected genes. Redundancy analysis further demonstrated that the environmental factors contributed 70.7% of the total ARG variations. The findings of large shared ARGs, abundant Total Coliforms and large wastewater burden in the groundwater provide a clear evidence that anthropogenic activities had a significant impact on groundwater.

Swine farming elevated the proliferation of Acinetobacter with the prevalence of antibiotic resistance genes in the groundwater.

Swine farming generates a large amount of wastes containing various contaminants, resulting in environmental contamination and human health problems. Here we investigated the contamination profiles of antibiotics and antibiotic resistance genes (ARGs) as well as microbial community in groundwater of the two villages with or without swine farms, and then assessed the human exposure risks of antibiotics, ARGs and indicator bacteria through drinking groundwater. The results showed that swine farming could lead to enhanced concentration levels of various veterinary antibiotics and ARGs in the groundwater in comparison to the reference village without swine farming. The microbial diversity of groundwater was significantly decreased with predominance of conditional pathogens Acinetobacter (up to 90%) in some wells of the swine farming village. Meanwhile, the abundance of Acinetobacter was significantly correlated to bacterial abundance, ARGs and integrons. The local residents could ingest various antibiotic residues and ARGs as well as pathogens, with daily intake of Acinetobacter up to approximately 10 billion CFU/resident through drinking groundwater contaminated by swine farming. The findings from this study suggest potential health risks of changing gut microbial community and resistome by drinking contaminated groundwater.