Concentrations Of Antibiotic Resistance Genes Research Articles

Longitudinal metagenomic analysis on antibiotic resistome, mobilome, and microbiome of river ecosystems in a sub-tropical metropolitan city.

Rivers play an important role as reservoirs and sinks for antibiotic resistance genes (ARGs). However, it remains underexplored for the resistome and associated mobilome in river ecosystems, and hosts of riverine ARGs particularly the pathogenic ones are rarely studied. This study for the first time conducted a longitudinal metagenomic analysis to unveil the resistome, mobilome, and microbiome in river water, by collecting samples from 16 rivers in Hong Kong over a three-year period and using both short-read and long-read sequencing. Results revealed that aminoglycoside, bacitracin, β-lactam, macrolide lincosamide-streptogramin, and sulfonamide were the predominant ARG types in the river water samples. Riverine ARGs exhibited high spatial variations in abundance and diversity. Environmental factors such as fecal coliform count, Escherichia coli count, 5-day biochemical oxygen demand (BOD5), dissolved oxygen (DO), and total organic carbon (TOC) had a significant correlation to the absolute concentrations of ARGs. Nanopore sequencing was used to reveal the physical genetic linkage of mobile genetic elements (MGEs) with ARGs in river water samples. The results showed that qacEdelta, transposase, integrase, and Tn916 had a high prevalence in ARG-carrying long reads. Host tracking using ARG-carrying reads identified 23 pathogenic bacteria species that harbored ARGs. Some ARGs were shared by different bacterial groups. This study presented a nuanced insight of resistome in river water by a longitudinal metagenomic analysis and deepened our understanding of common and divergent riverine antimicrobial resistant risk across the regional patterns.

Occurrence, removal, and ecological risk of antibiotics and antibiotic resistance genes in township wastewater treatment plants in the upper reaches of the Yangtze River, China.

The excessive use of antibiotics contributes significantly to environmental pollution and the widespread presence of antibiotic resistance genes (ARGs), which poses a serious threat to aquatic ecosystems and human health. Despite being a critical source of antibiotics and ARGs in the environment, research exploring their occurrence and removal characteristics in township wastewater treatment plants (WWTPs) remains limited. This study investigated the abundance and removal efficiencies of 39 antibiotics and 8 ARGs in influent and effluent samples from 40 township WWTPs located in the upper reaches of the Yangtze River. Antibiotics were detected in both influents and effluents, with detection frequencies (DFs) ranging from 2.44% to 100%. In contrast, all targeted ARGs exhibied 100% DFs in both influents and effluents. The average concentrations of antibiotics in influents and effluents were 370.67ng/L and 242.06ng/L, respectively, and the ARGs concentrations were 9.88 × 106 and 7.10 × 105 copies/mL, respectively. Among the antibiotics analyzed, macrolides and fluoroquinolones accounted for over 70% of the total antibiotic concentrations in both influent and effluent samples. Additionally, sul1 and sul2 were identified as the predominant ARGs, constituting more than 99% of the total abundance in both influent and effluent samples. Notably, significant correlations between the ARGs and their respective antibiotics were not consistently observed. The average removal efficiencies for total antibiotics and ARGs by township WWTPs were 34.70% and 93.30%, respectively. Furthermore, all calculated ecological risk values (risk quotient, RQ) for individual antibiotics in effluent samples were below one, except for oxytetracycline, which had RQ values greater than one in three effluents. The total RQs, representing the sum of individual RQs, indicated that 11 effluents presented a high risk (27.50%), while 17 effluents exhibited a moderate risk (42.50%). These findings suggest that township WWTPs may represent a significant source of antibiotics and ARGs in the environment, necessitating further process improvements for their effective elimination.

Effects of aging of polyethylene microplastics and polystyrene nanoplastics on antibiotic resistance gene transfer during primary sludge fermentation

The increasing presence of nano and microplastics (NPs/MPs) in wastewater treatment plants and their inevitable accumulation in the sludge has raised serious concerns in recent years. This study investigated the effects of pristine and aged polyethylene microplastics (PEMPs), polystyrene nanoplastics (PsNPs), and their mixtures on the primary sludge fermentation process. Pristine MPs/NPs (150 μg/L and 2 g/L for PsNPs and PEMPs, respectively) underwent two weeks of weathering in the presence of humic and alginic acids. The results from a batch fermentation experiment (15 days, pH 10) revealed that the exposure to aged PEMPs/PsNPs experienced greater VFA production than pristine samples. Notably, the aged PEMPs/PsNPs mixture showed a 23.12% increase in VFA production over the pristine mixture. The relative abundance and total concentration of antibiotic resistance genes (ARGs) increased in all PEMPs/PsNPs batches compared to the control, with the most significant rise in total ARGs observed in the aged PEMPs sample. Aged PEMPs exhibited a 26.22-fold increase in tetA genes, while aged mix samples showed a 19.68-fold increase in tetM genes compared to their pristine counterparts. Both pristine and aged PEMPs/PsNPs, particularly the aged PEMPs adversely affected the microbial communities at the genus level and altered the microbial structure. Microbial richness and diversity were enhanced in samples exposed to pristine PEMPs/PsNPs and aged PsNPs but decreased in aged PEMPs and in the aged mixture group, suggesting a negative impact of aged polyethylene microplastics on microbial communities. Correlation analysis suggested that phyla Planctomycetes, Proteobacteria, and TM7 are potential hosts of ARGs. These findings manifest the substantial effects of aged nano/microplastics compared to their pristine forms, emphasizing the complex interplay between various forms of PEMPs/PsNPs and microbial dynamics in sludge fermentation processes.

Bioaerosol emission characteristics and potential risks during composting: Focus on pathogens and antimicrobial resistance

In this study, we analyzed bioaerosol emission characteristics and potential risks of antimicrobial resistance (AMR) during composting using the impaction culture method and metagenomic sequencing. The results showed that the highly saturated water vapor in the emission gas mitigated particulate matter emission during the thermophilic period. About the bioaerosols, the airborne aerobic bacterial emissions were suppressed as composting enters the mature period, and the airborne fungi are usually present as single-cell or small-cell aggregates (< 3.3 µm). In addition, the microbial community structure in bioaerosols was stable and independent of composting time. Most importantly, the PM2.5 in bioaerosols contained large amounts of antibiotic resistance genes (ARGs), potential pathogens, and multidrug resistant pathogens, which were diverse and present in high concentrations. Among them, ARGs concentrations encoding 21 antibiotics ranged from – 4.50 to 0.70 ppm/m3 (Log10 ARGs). Among the 89 potential human pathogens detected, Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, and Staphylococcus aureus were the only culturable potentially multidrug resistant pathogens carrying multiple ARGs encoding resistance at high concentrations (– 0.57 to 1.15 ppm/m3 (Log10 ARGs)), and were more likely to persist and multiply in oligotrophic environments. Our findings indicate that composting technology can transfer AMR from solid compost to gas phase and increase the risk of AMR transmission.

Airborne antibiotics, antimicrobial resistance, and bacterial pathogens in a commercial composting facility: Transmission and exposure risk

Multiple elements associated with antimicrobial resistance in compost may escape into the air during the composting process, including antibiotic resistance genes (ARGs), human pathogenic bacteria (HPBs), and even antibiotics. Although antibiotics play a critical role in the evolution of resistance in HPBs, no information is available on airborne antibiotics in composting facilities. In this study, we systematically quantified airborne antibiotics, ARGs, and HPBs in comparison with those in compost. The burden of antibiotics in the air reached 4.17 ± 2.71 × 102 ng/g, significantly higher than that in compost. The concentration of ARGs (102 copies/g) in air also increased compared with that in compost. Concentrations of target contaminants in air were affected by temperature, organic matter, and heavy metals. Co-occurrence network analysis revealed the connectivity and complexity of antibiotics, ARGs, and HPBs were greater in air than in compost. The maximum daily antibiotic intake dose was up to 1.18 × 10−1ng/d/kg, accompanied by considerable inhalation levels of ARGs and HPBs. Our results reveal the severity of airborne antimicrobial resistance (AMR) elements in composting facilities, highlight the non-negligible amount of antibiotics and their co-existence with ARGs and HPBs, and shed light on the potential role of airborne antibiotics in the evolution of environmental AMR.

Occurrence and human exposure risk of antibiotic resistance genes in tillage soils of dryland regions: A case study of northern Ningxia Plain, China

Agricultural soils are important source and sink of antibiotic resistance genes (ARGs). However, little is known about the fate of ARGs in dryland soils, while its human exposure risks were seriously overlooked. Taking the northern Ningxia Plain as a case, this study explored the occurrence of ARGs and its relationship with mobile genetic elements (MGEs), pathogens, and environmental factors. Furthermore, the concentrations of airborne ARGs by soil wind erosion and the human exposure doses of soil ARGs were evaluated. The results showed the abundances of different regions ranged from 4.0 × 105 to 1.6 × 106 copies/g. Soil ARGs are driven by MGEs, but multiply impacted by soil properties, nutrition, and bacterial community. Vibrio metschnikovii, Acinetobacter schindleri, and Serratia marcescens are potential pathogenic hosts for ARGs. Further exploration revealed the concentration of ARGs loaded in dust by soil wind erosion reached more than 105 copies/m3, which were even higher than those found in sewage treatment plants and hospitals. Skin contact is the primary route of ARGs exposure, with a maximum dose of 24071.33 copies/kg/d, which is largely attributed to ARGs loaded in dust. This study bridged the gap on ARGs in dryland soils, and provided reference for human exposure risk assessment of soil ARGs.

Characteristics of air-borne and feces-borne ARGs and microbial community in different livestock farms in China

Livestock farms are hotspots of antibiotic resistance due to the intensive use of antibiotics, in which the characteristics of air-borne and feces-borne antibiotic resistance genes (ARGs) and microbial communities are of great significance. This study delves into the distribution of ARGs and microbial communities across various livestock farms in China, and the correlation of microorganisms between livestock farms and other global environments was investigated. The concentrations of ARGs and mobile genetic elements (MGEs) in air samples were basically at the same level, but those in fecal samples collected from chicken farms were universally higher than those in pig and cattle farms. There was significant ability of ARGs to spread easily among different bacteria in all samples in livestock farms. Additionally, there may be more possible host bacteria of airborne ARGs in chicken farms. In the global-scale analysis of highly similar microbial communities, the database matching with the highest number of similarities to microbial communities collected from livestock farms is genes related to human sources (54.8%). This study advances our understanding of ARG dynamics in different livestock farms and contributes to the development of sustainable livestock management practices.

Antibiotic resistance genes and crAssphage in hospital wastewater and a canal receiving the treatment effluent

Hospital wastewater is a major hotspot for the spread of antimicrobial resistance (AMR) in aquatic ecosystems. This study aimed to investigate the prevalence of antibiotic resistance genes (ARGs) and their correlation with crAssphage in a hospital wastewater treatment plant (HWWTP) and a receiving canal. Water samples were analyzed for 94 ARGs and crAssphage relative to the 16S rRNA using high-throughput quantitative polymerase chain reaction (HT-qPCR). Subsequently, 7 ARGs and crAssphage were selected and quantified using qPCR. The results showed that the detected genes ranged from 79 to 93 out of 95 genes. The raw wastewater (WW) samples had the highest gene diversity compared to the upstream canal, which had less diversity than downstream samples, as determined by HT-qPCR. The blaGES was the most abundant in WW samples, while qacEΔ1, merA, IS6100, tnpA, and IS26 showed high prevalence throughout the treatment processes. The concentrations of intI1, sul1, blaTEM,blaNDM,blaVIM,tetQ, mcr-1, crAssphage, and 16S rRNA, measured using qPCR, were the highest in WW and significantly reduced in treated water samples. Although some water quality parameters, such as total suspended solids and dissolved oxygen, did not significantly differ before and after treatment, removal efficiency ranged from 0.60 to 3.23 log reduction values (LRV). The highest LRV was observed for the tetQ, whereas the mcr-1 had the lowest LRV. Strong positive correlations among the absolute concentrations of ARGs and crAssphage were observed (Spearman's rho = 0.6–1.0), and biochemical oxygen demand correlated with blaTEM and blaVIM (Spearman's rho = 0.6). These results indicate that crAssphage and water quality could reflect the distribution of other ARGs throughout the HWWTP. Further studies are needed to underscore the importance of monitoring ARGs and genetic markers such as crAssphage in HWWTPs and their receiving waters to enhance our understanding of ARG distribution.

Effects of concentrate feeding sequence on VFA production, and cecal microbiota of Dezhou donkeys by metagenomic technology.

Microorganisms residing in the cecum of donkeys are crucial for physiological processes, nutrient metabolism, and immune function. Feeding methods can affect the dynamic balance of animal gut microbiota, thereby affecting indicators such as volatile fatty acids. This study explores suitable feeding methods to promote actual production by changing the feeding order of concentrate. Fifteen Dezhou donkeys with similar age and weight profiles were randomly divided into three groups with the concentrate feeding sequence: fiber-to-concentrate (FC), concentrate-to-fiber (CF), and total mixed ration (TMR). The experiment spanned a duration of 82 days. The analyses conducted were primarily aimed at determining the effects of feeding on gut microbes, primarily using metagenomic sequencing techniques. The experimental findings revealed that the levels of valeric acid were notably higher in the CF and TMR groups compared to the FC group (p < 0.05). These results suggest that the feeding sequence exerts a certain impact on the microbial composition within the cecum of Dezhou donkeys. At the phylum level, the predominant microbiota consisted of Firmicutes and Bacteroidetes, with the CF group displaying a higher relative abundance of Firmicutes compared to both the FC and TMR groups. At the genus level, Prevotella, Bacteroides, and Fibrobacter were the dominant bacterial genera identified in cecum. The functional gene annotation analysis indicated a significantly lower abundance of lacZ (K01190), Por/nifJ (K03737), and ppdK (K01006) genes in CF group relative to the FC and TMR groups (p < 0.05), highlighting their roles in galactose metabolism and glycolysis, respectively. Moreover, the CF group exhibited a higher concentration of antibiotic resistance genes (tetO and tet44) in the gut microbiota compared to the TMR and FC groups (p < 0.05), underscoring the presence of numerous antibiotic resistance genes within the phyla Bacteroidetes, Firmicutes, and Proteobacteria. In conclusion, different precision feed sequences significantly impact the levels of volatile fatty acids in Dezhou fattening donkeys, modify the composition and functional genes of the cecal microbiota, and elucidate the microbial mechanisms influenced by the feeding sequence on the growth and metabolism. These insights are anticipated to provide a foundation for the rational design of precision feed sequences in practical agricultural settings.

Assessment of nucleic acid extraction protocols for antibiotic resistance genes (ARGs) quantification in aircraft wastewater

This study evaluated ten nucleic acid extraction protocols (EP1 to EP10) for measuring five endogenous antibiotic resistance genes (ARGs) in four aircraft wastewater samples (AWW1 to AWW4). The targeted ARGs, including blaCTX-M, blaNDM-1, ermB, qnrS, and tetA, encompassed highly and minimally abundant ARGs. TetA and ermB were consistently detected across four aircraft wastewater samples using the DNeasy Blood and Tissue Kit and the AllPrep PowerViral DNA/RNA kit. QnrS displayed high detection rates with specific extraction protocols and aliquot volumes. Concentrations of ARGs varied across aircraft wastewater samples, with differing extraction protocols influencing quantitative results. The concentrations of tetA, ermB, and qnrS in AWW1 were distinct, while AWW2 to AWW4 exhibited a broader range for tetA, ermB, qnrS, blaCTX-M, and blaNDM-1. EP1 consistently produced the highest concentrations for several ARGs. Collective data analysis revealed varying ARG concentrations across the ten extraction protocols, suggesting the importance of careful extraction protocol selection in ARG monitoring in aircraft wastewater samples. Based on the results, we suggest that a small sample volume (as low as 0.2 mL) may be sufficient for ARG characterization in aircraft wastewater samples. The findings also emphasize the need for considering toilet paper removal without compromising nucleic acid extraction efficiency. The study highlights promising prospects for aircraft wastewater monitoring of ARGs, calling for further investigation into the import and spread of unique ARGs through transport hubs.

Distribution of antibiotic resistance genes and antibiotic residues in drinking water production facilities: Links to bacterial community.

There is a rapid spread of antibiotic resistance in the environment. However, the impact of antibiotic resistance in drinking water is relatively underexplored. Thus, this study aimed to quantify antibiotic resistance genes (ARGs) and antibiotic residues in two drinking water production facilities (NW-E and NW-C) in North West Province, South Africa and link these parameters to bacterial communities. Physicochemical and ARG levels were determined using standard procedures. Residues (antibiotics and fluconazole) and ARGs were quantified using ultra-high performance liquid chromatography (UHPLC) chemical analysis and real-time PCR, respectively. Bacterial community compositions were determined by high-throughput 16S rRNA sequencing. Data were analysed using redundancy analysis and pairwise correlation. Although some physicochemical levels were higher in treated than in raw water, drinking water in NW-E and NW-C was safe for human consumption using the South African Water Quality Guideline (SAWQG). ARGs were detected in raw and treated water. In NW-E, the concentrations of ARGs (sul1, intl1, EBC, FOX, ACC and DHA) were higher in treated water than in raw water. Regarding antimicrobial agents, antibiotic and fluconazole concentrations were higher in raw than in treated water. However, in NW-C, trimethoprim concentrations were higher in raw than in treated water. Redundancy analysis showed that bacterial communities were not significantly correlated (Monte Carlo simulations, p-value >0.05) with environmental factors. However, pairwise correlation showed significant differences (p-value <0.05) for Armatimonas, CL500-29 marine group, Clade III, Dickeya and Zymomonas genera with environmental factors. The presence of ARGs and antibiotic residues in the current study indicated that antibiotic resistance is not only a clinical phenomenon but also in environmental settings, particularly in drinking water niches. Consumption of NW-E and NW-C treated water may facilitate the spread of antibiotic resistance among consumers. Thus, regulating and monitoring ARGs and antibiotic residues in drinking water production facilities should be regarded as paramount.

Integron-associated genes are reliable indicators of antibiotic resistance in wastewater despite treatment- and seasonality-driven fluctuations

The present study aims to characterize the bacterial community, resistome and integron abundance of a municipal wastewater treatment plant (WWTP) over the course of 12 months and evaluate the year-long performance of integron-related genes as potential indicators of antibiotic resistance mechanisms in influents and effluents. For that, total DNA was extracted and subjected to 16S rRNA-targeted metabarcoding, high-throughput (HT) qPCR (48 targets) and standard qPCR (5 targets). Targets included integrase genes, antibiotic resistance genes (ARGs) and putative pathogenic groups. A total of 16 physicochemical parameters determined in the wastewater samples were also considered. Results revealed that the WWTP treatment significantly impacted the bacterial community, as well as the content in ARGs and integrase genes. Indeed, there was a relative enrichment from influent to effluent of 13 pathogenic groups (e.g., Legionella and Mycobacterium) and genes conferring resistance to sulphonamides, aminoglycosides and disinfectants. Effluent samples (n = 25) also presented seasonal differences, with an increase of the total ARGs’ concentration in summer, and differences between winter and summer on relative abundance of sulphonamide and disinfectant resistance mechanisms. From the eight putative integron-related genes selected, all were positively correlated with the total ARGs’ content in wastewater and the relative abundance of resistance to most of the specific antibiotic classes. The genes intI1, blaGES and qacE∆1 were the most strongly correlated with the total concentration of ARGs. Genes blaGES and blaVIM, were better correlated to resistance to beta-lactams, aminoglycosides and tetracyclines. This study supports the use of integron-related genes as powerful indicators of antibiotic resistance in wastewater, being robust despite the variability caused by wastewater treatment and seasonality.

Characterisation of microbial communities and quantification of antibiotic resistance genes in Italian wastewater treatment plants using 16S rRNA sequencing and digital PCR

The spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in humans, animals and environment is a growing threat to public health. Wastewater treatment plants (WWTPs) are crucial in mitigating the risk of environmental contamination by effectively removing contaminants before discharge. However, the persistence of ARB and ARGs even after treatment is a challenge for the management of water system.To comprehensively assess antimicrobial resistance dynamics, we conducted a one-year monitoring study in three WWTPs in central Italy, both influents and effluents. We used seasonal sampling to analyze microbial communities by 16S rRNA, as well as to determine the prevalence and behaviour of major ARGs (sul1, tetA, blaTEM, blaOXA-48, blaCTX-M-1 group, blaKPC) and the class 1 Integron (int1). Predominant genera included in order: Arcobacter, Acinetobacter, Flavobacterium, Pseudarcobacter, Bacteroides, Aeromonas, Trichococcus, Cloacibacterium, Pseudomonas and Streptococcus. A higher diversity of bacterial communities was observed in the effluents compared to the influents. Within these communities, we also identified bacteria that may be associated with antibiotic resistance and pose a significant threat to human health. The mean concentrations (in gene copies per liter, gc/L) of ARGs and int1 in untreated wastewater (absolute abundance) were as follows: sul1 (4.1 × 109), tetA (5.2 × 108), blaTEM (1.1 × 108), blaOXA-48 (2.1 × 107), blaCTX-M-1 group (1.1 × 107), blaKPC (9.4 × 105), and int1 (5.5 × 109). The mean values in treated effluents showed reductions ranging from one to three log. However, after normalizing to the 16S rRNA gene (relative abundance), it was observed that in 37.5 % (42/112) of measurements, the relative abundance of ARGs increased in effluents compared to influents. Furthermore, correlations were identified between ARGs and bacterial genera including priority pathogens. This study improves our understanding of the dynamics of ARGs and provides insights to develop more effective strategies to reduce their spread, protecting public health and preserving the future efficacy of antibiotics.

Screening for Antibiotic Resistance Genes in Bacteria and the Presence of Heavy Metals in the Upstream and Downstream Areas of the Wadi Hanifah Valley in Riyadh, Saudi Arabia.

Valley surface water is considered a focal public health concern owing to the presence of multi-drug-resistant bacteria. The distribution of antimicrobial resistance (AMR) bacteria in the surface water is affected by the presence of multiple factors, including antibiotics coming from wastewater discharge or other contaminant sources such as pharmaceuticals, biocides, and heavy metals. Furthermore, there is evidence suggesting that high levels of antibiotic resistance genes (ARGs) can be transferred within bacterial communities under the influence of heavy metal stress. Hence, the primary aim of this study is to investigate the presence of heavy metals and bacterial ARGs in upstream as well as downstream locations of Wadi Hanifah Valley in Riyadh, Saudi Arabia. Sample collection was conducted at eighteen surface water sites within the valley in total. The selection of ARGs was associated with the most common antibiotics, including β-lactam, tetracycline, erythromycin, gentamicin, sulphonamide, chloramphenicol, vancomycin, trimethoprim, and colistin antibiotics, which were detected qualitatively using polymerase chain reaction (PCR) technology. The tested antibiotic resistance genes (ARGs) included (blaNDM-1 (for the antibiotic class Beta-lactamases), mecA (methicillin-resistant Staphylococcus aureus), tet(M) and tet(B) (for the antibiotic class Tetracycline), ampC (for the antibiotic class Beta-lactamases), vanA (for the antibiotic class vancomycin), mcr-1 (for the antibiotic class colistin), erm(B) (for the antibiotic class erythromycin), aac6'-Ie-aph2-Ia (for the antibiotic class Gentamicin), sulII (for the antibiotic class sulphonamide), catII (for the antibiotic class Chlorophincol), and dfrA1 (for the antibiotic class trimethoprim). Moreover, an assessment of the levels of heavy metals such as lithium (Li), beryllium (Be), chromium (Cr), cobalt (Co), arsenic (As), cadmium (Cd), tin (Sn), mercury (Hg), and lead (Pb) was conducted by using inductively coupled plasma mass spectrometry (ICPMS). According to our findings, the concentrations of sulphonamide, erythromycin, and chloramphenicol ARGs (erm(B), sulII, and catII) were observed to be the most elevated. Conversely, two ARGs, namely mecA and mcr-1, were not detected in the samples. Moreover, our data illustrated a significant rise in ARGs in the bacteria of water samples from the upstream sites as compared with the water samples from the downstream sites of Wadi Hanifah Valley. The mean concentration of Li, Be, Cr, Co, As, Cd, Sn, Hg, and Pb in the water samples was estimated to be 37.25 µg/L, 0.02 µg/L, 0.56 µg/L,0.32 µg/L, 0.93 µg/L, 0.01 µg/L, 200.4 µg/L, 0.027 µg/L, and 0.26 µg/L, respectively, for the selected 18 sites. Furthermore, it was revealed that the concentrations of the screened heavy metals in the water samples collected from various sites did not surpass the maximum limits set by the World Health Organization (WHO). In conclusion, this study offers a concise overview of the presence of heavy metals and ARGs in water samples obtained from the Wadi Hanifah Valley in Riyadh, KSA. Such findings will contribute to the ongoing monitoring and future risk assessment of ARGs spread in surface water.

Spatiotemporal distribution, interactions and toxic effect of microorganisms and ARGs/MGEs from the bioreaction tank in hospital sewage treatment facility

Antibiotic resistance genes (ARGs) can be emitted from wastewater to ambient air and impose unignorable inhalable hazards, which could be exacerbated in antibiotic-concentrated hospital sewage. However, whether the ARG-carrying pathogens are more likely to infect cells remains largely unknown. Here, this study investigated and analyzed the spatiotemporal distribution, interaction, and toxicity of airborne microorganisms and their hosting ARGs in a hospital sewage treatment facility. The average concentration of ARGs/MGEs in sewage of bioreaction tank (BRT-W) was 2.27 × 104 gene copies/L. In the air of bioreaction tank (BRT-A), the average concentration of ARGs/MGEs was 15.86 gene copies/m3. In the four seasons, the ARGs concentration of sewage gradually decreased over time; The concentration of ARGs in the air first decreased and then increased. In spring, the concentration of ARGs/MGEs (qacedelta1-01) in BRT-W was highest (1.05 × 105 gene copies/L); The concentration of ARGs/MGEs (strB) in BRT-A in winter was higher than other seasons (26.18 gene copies/m3). Different from the past, this study also paid attention to the pathogenic potential of ARGs/MGEs in the air. The results of cell experiments showed that the cytotoxicity of drug-resistant Escherichia coli could reach Grade V. This suggested that the longer the drug-resistant E. coli were exposed to cells, the greater the cytotoxicity. Moreover, the cytotoxicity of bacteria increased with the increase in exposure time. In spring, the toxic effect of ARGs/MGEs in sewage of BRT-W was highest. Traceability analysis proved that BRT-W was an essential source of microorganisms and ARGs/MGEs in BRT-A. Furthermore, the combined risk of people exposed to the air of BRT in spring was higher than that in other seasons.

Size effects of microplastics on antibiotic resistome and core microbiome in an urban river

Microplastics (MPs) in aquatic environments provide a new ecological niche that facilitates the attachment of antibiotic-resistance genes (ARGs) and pathogens. However, the effect of particle size on the colonization of antibiotic resistomes and pathogens remains poorly understood. To address this knowledge gap, this study explored the antibiotic resistome and core microbiome on three distinct types of MPs including polyethylene, polypropylene, and polystyrene (PS), with varying sizes of 30, 200, and 3000 μm by metagenomic sequencing. Our finding showed that the ARG abundances of the PS type increased by 4-folds with increasing particle size from 30 to 3000 μm, and significant differences in ARG profiles were found across the three MP types. In addition, the concentrations of ARGs and mobile genetic elements (MGEs) were markedly higher in the MPs than in the surrounding water, indicating their enrichment at these artificial interfaces. Notably, several pathogens such as Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Legionella pneumophila were enriched in MP biofilms, and the co-occurrence of ARGs and virulence factor genes (VFGs)/MGEs suggested the presence of pathogenic antibiotic-resistant microbes with potential mobility. Both redundancy analysis (RDA) and structural equation modeling (SEM) demonstrated that physicochemical properties such as zeta potential, MP size, and contact angle were the most significant contributors to the antibiotic resistome. Strikingly, no significant differences were observed in the health risk scores of the ARG profiles among different sizes and types of MPs. This study expands our knowledge on the impact of MP size on microbial risks, thus enhancing our understanding of the potential health hazards they pose.

Metagenomic absolute quantification of antibiotic resistance genes and virulence factor genes-carrying bacterial genomes in anaerobic digesters

Sewage treatment works have been considered as hotspots for the dissemination of antibiotic resistance genes (ARGs). Anaerobic digestion (AD) has emerged as a promising approach for controlling the spread of ARGs while destroying biomass in sludge. Evaluating the impact of AD on ARG removal relies on the absolute quantification of ARGs. In this study, we quantified the ARG concentrations in both full-scale and lab-scale AD systems using a cellular spike-ins based absolute quantification approach. Results demonstrated that AD effectively removed 68 ± 18 %, 55 ± 12 %, and 57 ± 19 % of total ARGs in semi-continuous AD digesters, with solid retention times of 15, 20, and 25 days, respectively. The removal efficiency of total ARGs increased as the AD process progressed in the batch digesters over 40 days. A significant negative correlation was observed between digestion time and the concentrations of certain ARG types, such as beta-lactam, sulfonamide, and tetracycline. However, certain potential pathogenic antibiotic resistant bacteria (PARB) and multi-resistant high-risk ARGs-carrying populations robustly persisted throughout the AD process, regardless of the operating conditions. This study highlighted the influence of the AD process and its operating parameters on ARG removal, and revealed the broad spectrum and persistence of PARB in AD systems. These findings provided critical insights for the management of microbial hazards.

Linking antibiotic resistance gene patterns with advanced faecal pollution assessment and environmental key parameters along 2300 km of the Danube River

The global spread of antimicrobial resistance (AMR) in the environment is a growing health threat. Large rivers are of particular concern as they are highly impacted by wastewater discharge while being vital lifelines serving various human needs. A comprehensive understanding of occurrence, spread and key drivers of AMR along whole river courses is largely lacking. We provide a holistic approach by studying spatiotemporal patterns and hotspots of antibiotic resistance genes (ARGs) along 2311 km of the navigable Danube River, combining a longitudinal and temporal monitoring campaign. The integration of advanced faecal pollution diagnostics and environmental and chemical key parameters allowed linking ARG concentrations to the major pollution sources and explaining the observed patterns. Nine AMR markers, including genes conferring resistance to five different antibiotic classes of clinical and environmental relevance, and one integrase gene were determined by probe-based qPCR. All AMR targets could be quantified in Danube River water, with intI1 and sul1 being ubiquitously abundant, qnrS, tetM, blaTEM with intermediate abundance and blaOXA-48like, blaCTX−M-1 group, blaCTX−M-9 group and blaKPC genes with rare occurrence. Human faecal pollution from municipal wastewater discharges was the dominant factor shaping ARG patterns along the Danube River. Other significant correlations of specific ARGs were observed with discharge, certain metals and pesticides. In contrast, intI1 was not associated with wastewater but was already established in the water microbiome. Animal contamination was detected only sporadically and was correlated with ARGs only in the temporal sampling set. During temporal monitoring, an extraordinary hotspot was identified emphasizing the variability within natural waters. This study provides the first comprehensive baseline concentrations of ARGs in the Danube River and lays the foundation for monitoring future trends and evaluating potential reduction measures. The applided holistic approach proved to be a valuable methodological contribution towards a better understanding of the environmental occurrence of AMR.

Antibiotic Resistance in a Coastal River in Mississippi, USA – Potential Drivers

Wastewater treatment plants (WWTPs) are major sources of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in water bodies. Most studies on the impact of WWTPs on antibiotic resistance have focused on freshwater systems, with little information on coastal and estuarine waters with variable salinity. This study monitored seasonal levels of ARGs at the effluent and downstream of the Pascagoula— Moss Point WWTP in the lower Pascagoula River, a coastal river in southeastern Mississippi, USA. Surface water samples were collected seasonally at upstream, outflow, and 3 downstream sites from February to November 2016. Bacterial resistance to sulfamethazine, tetracycline, and ciprofloxacin was quantified using selective culture and qPCR. Mixed—effects models were developed to identify potential driving factors of ARG concentrations related to the WWTP and local environmental conditions (salinity, water temperature, and pH). The best model was selected based on the lowest Akaike Information Criterion (AIC) corrected for small sample size. The results show that the genes sul1, sul2, and intI1 were detected, with intI1 having the highest relative concentration. The qPCR analysis suggests a negative relation between ARG levels and temperature and salinity. ARG concentrations peaked immediately downstream of the WWTP and decreased gradually further downstream in some months, but the spatial pattern varied widely between sampling months. The study highlights the complex patterns of environmental ARGs and the importance of accounting for the impact of WWTPs, local environmental factors, and other anthropogenic influences to understand their potential drivers.

Wastewater surveillance for antibiotics and resistance genes in a river catchment: Spatiotemporal variations and the main drivers

Wastewater-based epidemiology (WBE) is used for mining information about public health such as antibiotics resistance. This study investigated the distribution profiles of six types of antibiotic resistance genes (ARGs) in wastewater and rivers in Wuhu City, China. The levels of ARGs found in the Qingyijiang River were significantly higher than other rivers, and were comparable to effluent levels. Among the ARGs, sulfonamides ARGs and intI1 were the predominant in both wastewaters and rivers. Additionally, the concentrations of ARGs were higher on weekends as opposed to weekdays. Their distribution patterns remained consistent inter-week and inter-season using linear regression analysis (p < 0.001). Interestingly, the occurrence levels of ARGs in wastewaters during spring were significantly higher than in autumn, although insignificant in rivers. The apparent removal rate of ARGs in domestic wastewater sources ranged from 61.52–99.29%, except for qepA (-1.91% to 81.09%), whereas the removal rates in mixed domestic and industrial wastewaters showed a marked decrease (-92.94% to 76.67%). A correlation network analysis revealed that azithromycin and erythromycin were key antibiotics, while blaNDM-1, tetM, tetB, and ermB were identified as key ARGs. Sulfonamide and fluoroquinolone antibiotics, and tetracycline and macrolide ARGs were the primary contributors. Linear mixed models demonstrated that socio-economic variables positively impacted the occurrence levels of ARGs, whereas wastewater flow and river runoff were the negative drivers for their concentrations in wastewaters and surface waters, respectively. Overall, this WBE study contributes to the understanding of spatiotemporal profiles and main drivers of the occurrence of ARGs in wastewater and receiving water.