Total Antibiotic Resistance Genes Research Articles

Horizontal gene transfer plays a crucial role in the development of antibiotic resistance in an antibiotic-free shrimp farming system

Antibiotic selective pressure in aquaculture systems often results in the antibiotic resistance genes (ARGs) proliferation. Nonetheless, a paucity of data exists concerning the mechanisms of ARGs development in aquaculture systems without the influences of antibiotics. This study utilized metagenomic approaches to elucidate the dynamics and transfer mechanisms of ARGs throughout the aquaculture of Pacific white shrimp. A marked change in the resistome was observed throughout the aquaculture without antibiotics. The total ARGs relative abundance increased from 0.05 to 0.33 by day 90 of cultivation, with even higher in mixed wastewater (0.44). Both bacterial communities and mobile genetic elements play pivotal roles in the development of ARGs. Metagenome-assembled genomes showed enrichment of environmentally intrinsic ARGs on chromosomes including macB and mdtK. The plasmid-mediated horizontal transfer was recognized as a principal factor contributing to the rise of ARGs, particularly for tetG and floR, and this led to an escalation of resistance risk, peaking at a risks core of 35.43 on day 90. This study demonstrates that horizontal gene transfer plays a crucial role in ARGs development without antibiotic pressure, which can provide a theoretical foundation for controlling ARGs proliferation in aquaculture systems.

Dynamics of antibiotic resistance genes during manure composting: Reduction in herbivores manure and accumulation in carnivores

The elevated levels of antibiotic resistance genes (ARGs) in livestock manure represent a significant threat to both the environment and human health. Composting has been recognized as an effective strategy to mitigate the abundance of ARGs in manure. However, notable rebounds in ARGs abundance have been observed during this process. This study explored the changes in ARGs abundance and the underlying influencing factors during the composting of carnivore (chicken and pig) and herbivore (sheep and cow) manures, along with mushroom residues. The findings revealed that the total relative abundance of ARGs increased by 6.96 and 10.94 folds in chicken and pig manure composts, respectively, whereas it decreased by a remarkable 91.72% and 98.37% in sheep and cow manure composts. Nitrogen content emerged as the primary physicochemical factors governing the abundance of ARGs in chicken and pig manure composts. Conversely, carbon content played a pivotal role in determining ARGs abundance in chicken and pig manure composts. Furthermore, the presence of dominant hosts, such as Corynebacterium, Bacillus, and Clostridium, along with emerging bacteria like Thermobifida, Saccharomonospora, and Actinomadura, contributed significantly to the enrichment of total ARGs, including tetG, tetO, tetX, and sul2, in chicken and pig manure composts. The coexistence of these genes with mobile genetic elements and a plethora of host bacteria, coupled with their high abundance, renders them particularly high-risk ARGs. On the other hand, the observed decrease in the abundance of total ARGs in sheep and cow manure composts can be attributed to the decline in the population of host bacteria, specifically Atopostipes, Psychrobacter, and Corynebacterium. Collectively, these results provide crucial insights into the management of ARGs risks and offer essential theoretical support for enhancing the safe utilization of organic fertilizer in agriculture.

Horizontal gene transfer plays a crucial role in the development of antibiotic resistance in an antibiotic-free shrimp farming system

Antibiotic selective pressure in aquaculture systems often results in the antibiotic resistance genes (ARGs) proliferation. Nonetheless, a paucity of data exists concerning the mechanisms of ARGs development in aquaculture systems without the influences of antibiotics. This study utilized metagenomic approaches to elucidate the dynamics and transfer mechanisms of ARGs throughout the aquaculture of Pacific white shrimp. A marked change in the resistome was observed throughout the aquaculture without antibiotics. The total ARGs relative abundance increased from 0.05 to 0.33 by day 90 of cultivation, with even higher in mixed wastewater (0.44). Both bacterial communities and mobile genetic elements play pivotal roles in the development of ARGs. Metagenome-assembled genomes showed enrichment of environmentally intrinsic ARGs on chromosomes including macB and mdtK. The plasmid-mediated horizontal transfer was recognized as a principal factor contributing to the rise of ARGs, particularly for tetG and floR, and this led to an escalation of resistance risk, peaking at a risks core of 35.43 on day 90. This study demonstrates that horizontal gene transfer plays a crucial role in ARGs development without antibiotic pressure, which can provide a theoretical foundation for controlling ARGs proliferation in aquaculture systems. Environmental ImplicationGiven the excessive use of antibiotics, antibiotic resistance genes (ARGs) are emerging as a new form of hazardous pollutant. Previous studies have indicated that incorporating antibiotics in aquaculture can result in the accumulation of ARGs. In contrast, this study examines an aquaculture system unaffected by antibiotics, expressly exploring the impact of horizontal gene transfer on the accumulation and risk propagation of ARGs during the aquaculture process, aiming to provide a theoretical reference for the control of ARGs in the aquaculture industry.

Mitigated dissemination of antibiotic resistance genes by nanoscale zero-valent iron and iron oxides during anaerobic digestion: Roles of microbial succession and regulation

Nanoscale zero-valent iron (ZVI) and the oxides have been documented as an effective approach for mitigating the dissemination of antibiotic resistance genes (ARGs) during anaerobic digestion (AD). However, the mechanism of ARGs dissemination mitigated by nanoscale ZVI and iron oxides remain unclear. Here, we investigated the influencing mechanisms of nanoscale ZVI and iron oxides on ARGs dissemination during AD. qPCR results indicated that nanoscale ZVI and iron oxides significantly declined the total ARGs abundances, and the strongest inhibiting effect was observed by 10 g/L nanoscale ZVI. Mantel test showed ARGs distribution was positively correlated with physiochemical properties, integrons and microbial community, among which microbial community primarily contributed to ARGs dissemination (39.74%). Furthermore, redundancy and null model analyses suggested the dominant and potential ARGs host was Fastidiosipila, and homogeneous selection in the determinism factors was the largest factor for driving Fastidiosipila variation, confirming the inhibition of Fastidiosipila was primary reason for mitigating ARGs dissemination by nanoscale ZVI and iron oxides. These results were related to the inhibition of ARGs transfer related functions. This work provides novel evidence for mitigating ARGs dissemination through regulating microbial succession and regulation induced by ZVI and iron oxides.

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.

Assessing intracellular and extracellular distribution of antibiotic resistance genes in the commercial organic fertilizers

Compost-based organic fertilizers often contain high levels of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). Previous studies focused on quantification of total ARGs and MGEs. For a more accurate risk assessment of the dissemination risk of antibiotic resistance, it is necessary to quantify the intracellular and extracellular distribution of ARGs and MGEs. In the present study, extracellular ARGs and MGEs (eARGs and eMGEs) and intracellular ARGs and MGEs (iARGs and iMGEs) were separately analyzed in 51 commercial composts derived from different raw materials by quantitative polymerase chain reaction (qPCR) and metagenomic sequencing. Results showed that eARGs and eMGEs accounted for 11–56% and 4–45% of the total absolute abundance of ARGs and MGEs, respectively. Comparable diversity, host composition and association with MGEs were observed between eARGs and iARGs. Contents of high-risk ARGs were similar between eARGs and iARGs, with high-risk ARGs in the two forms accounting for 6.7% and 8.2% of the total abundances, respectively. Twenty-four percent of the overall ARGs were present in plasmids, while 56.7% of potentially mobile ARGs were found to be associated with plasmids. Variation partitioning analysis, null model and neutral community model indicated that the compositions of both eARGs and iARGs were largely driven by deterministic mechanisms. These results provide important insights into the cellular distribution of ARGs in manure composts that should be paid with specific attention in risk assessment and management.

Oxygen mediated mobilization and co-occurrence of antibiotic resistance in lab-scale bioreactor using metagenomic binning.

Sub-lethal levels of antibiotic stimulate bacteria to generate reactive oxygen species (ROS) that promotes emergence and spread of antibiotic resistance mediated by mobile genetic elements (MGEs). Nevertheless, the influence of dissolved oxygen (DO) levels on mobility of antibiotic resistance genes (ARGs) in response to ROS-induced stress remains elusive. Thus, the study employs metagenomic assembly and binning approaches to decipher mobility potential and co-occurrence frequency of ARGs and MGEs under hyperoxic (5.5-7 mgL- 1), normoxic (2.5-4 mgL- 1), and hypoxic (0.5-1 mgL- 1) conditions in lab-scale bioreactor for 6 months. Among 163 high-quality metagenome-assembled genomes (MAGs) recovered from 13 metagenomes, 42 MAGs harboured multiple ARGs and were assigned to priority pathogen group. Total ARG count increased by 4.3 and 2.5% in hyperoxic and normoxic, but decreased by 0.53% in hypoxic conditions after 150 days. On contrary, MGE count increased by 7.3-1.3% in all the DO levels, with only two ARGs showed positive correlation with MGEs in hypoxic compared to 20 ARGs under hyperoxic conditions. Opportunistic pathogens (Escherichia, Klebsiella, Clostridium, and Proteus) were detected as potential hosts of ARGs wherein co-localisation of critical ARG gene cassette (sul1, dfr1,adeF, and qacC) were identified in class 1 integron/Tn1 family transposons. Thus, enhanced co-occurrence frequency of ARGs with MGEs in pathogens suggested promotion of ARGs mobility under oxidative stress. The study offers valuable insights into ARG dissemination and hosts dynamics that is essential for controlling oxygen-related stress for mitigating MGEs and ARGs in the environment.

To what extent do water reuse treatments reduce antibiotic resistance indicators? A comparison of two full-scale systems

Water reuse is an essential strategy for reducing water demand from conventional sources, alleviating water stress, and promoting sustainability, but understanding the effectiveness of associated treatment processes as barriers to the spread of antibiotic resistance is an important consideration to protecting human health. We comprehensively evaluated the reduction of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) in two field-operational water reuse systems with distinct treatment trains, one producing water for indirect potable reuse (ozone/biologically-active carbon/granular activated carbon) and the other for non-potable reuse (denitrification-filtration/chlorination) using metagenomic sequencing and culture. Relative abundances of total ARGs/clinically-relevant ARGs and cultured ARB were reduced by several logs during primary and secondary stages of wastewater treatment, but to a lesser extent during the tertiary water reuse treatments. In particular, ozonation tended to enrich multi-drug ARGs. The effect of chlorination was facility-dependent, increasing the relative abundance of ARGs when following biologically-active carbon filters, but generally providing a benefit in reduced bacterial numbers and ecological and human health resistome risk scores. Relative abundances of total ARGs and resistome risk scores were lowest in aquifer samples, although resistant Escherichia coli and Klebsiella pneumoniae were occasionally detected in the monitoring well 3-days downgradient from injection, but not 6-months downgradient. Resistant E. coli and Pseudomonas aeruginosa were occasionally detected in the nonpotable reuse distribution system, along with increased levels of multidrug, sulfonamide, phenicol, and aminoglycoside ARGs. This study illuminates specific vulnerabilities of water reuse systems to persistence, selection, and growth of ARGs and ARB and emphasizes the role of multiple treatment barriers, including aquifers and distribution systems.

Effect of wheat crops on the persistence and attenuation of antibiotic resistance genes in soil after swine wastewater application

Swine wastewater (SW) application introduces antibiotic resistance genes (ARGs) into farmland soils. However, ARG attenuation in SW-fertigated soils, especially those influenced by staple crops and soil type, remains unclear. This study investigated twelve soil ARGs and one mobile genetic element (MGE) in sandy loam, loam, and silt loam soils before and after SW application in wheat-planted and unplanted soils. The results revealed an immediate increase in the abundance of ARGs in soil by two orders of magnitude above background levels following SW application. After SW application, the soil total ARG abundance was attenuated, reaching background levels at 54 days; However, more individual ARGs were detected above the detection limit than pre-application. Among the 13 genes, acc(6′)-lb, tetM, and tetO tended to persist in the soil during wheat harvest. ARG half-lives were up to four times longer in wheat-planted soils than in bare soils. Wheat planting decreased the persistence of acc(6′)-lb, ermB, ermF, and intI2 but increased the persistence of others such as sul1 and sul2. Soil type had no significant impact on ARG and MGE fates. Our findings emphasize the need for strategic SW application and the consideration of crop cultivation effects to mitigate ARG accumulation in farmland soils.

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.

Effect of UV pretreatment on the source control of floR during subsequent biotreatment of florfenicol wastewater.

UV photolysis has been recommended as an alternative pretreatment method for the elimination of antibacterial activity of antibiotics against the indicator strain, but the pretreated antibiotic intermediates might not lose their potential to induce antibiotic resistance genes (ARGs) proliferation during subsequent biotreatment processes. The presence of florfenicol (FLO) in wastewater seriously inhibits the metabolic performance of anaerobic sludge microorganisms, especially the positive correlation between UV irradiation doses and ATP content, while it did not significantly affect the organics utilization ability and protein biosynthetic process of aerobic microorganisms. After sufficient UV pretreatment, the relative abundances of floR from genomic or plasmid DNA in subsequent aerobic and anaerobic biotreatment processes both decreased by two orders of magnitude, maintained at the level of the groups without FLO selective pressure. Meanwhile, the abundances of floR under anaerobic condition were always lower than that under aerobic condition, suggesting that anaerobic biotreatment systems might be more suitable for the effective control of target ARGs. The higher abundance of floR in plasmid DNA than in genome also indicated that the potential transmission risk of mobile ARGs should not be ignored. In addition, the relative abundance of intI1 was positively correlated with floR in its corresponding genomic or plasmid DNA (p < 0.05), which also increased the potential horizontal transfer risk of target ARGs. This study provides new insights into the effect of preferential UV photolysis as a pretreatment method for the enhancement of metabolic performance and source control of target ARGs in subsequent biotreatment processes. KEY POINTS: • Sufficient UV photolytic pretreatment efficiently controlled the abundance of floR • A synchronous decrease in abundance of intI1 reduced the risk of horizontal transfer • An appreciable abundance of floR in plasmid DNA was a potential source of total ARGs.

Impacts of irrigation with treated livestock wastewater on the accumulation characteristic of ARGs in the farmland soil: a case study in Hohhot, China.

Irrigation with treated livestock wastewater (TWW) is a promising strategy for reusing resources. However, TWW irrigation might introduce antibiotic resistant genes (ARGs) into the soil, posing environmental risks associated with antibiotic resistance. This study focuses on investigating the influence of irrigation amounts and duration on the fate of ARGs and identifies key factors driving their changes. The results showed that there were 13 ARGs in TWW, while only 5 ARGs were detected in irrigated soil. That is some introduced ARGs from TWW could not persistently exist in the soil. After 1-year irrigation, an increase in irrigation amount from 0.016t/m2 to 0.048t/m2 significantly enhanced the abundance of tetC by 29.81%, while ermB and sul2 decreased by 45.37% and 76.47%, respectively (p < 0.01). After 2-year irrigation, the abundance of tetC, ermB, ermF, dfrA1, and total ARGs significantly increased (p < 0.05) when the irrigation amount increased. The abundances of ARGs after 2-year irrigation were found to be 2.5-34.4 times higher than 1 year. Obviously, the irrigation years intensified the positive correlation between ARGs abundance and irrigation amount. TetC and ermF were the dominant genes resulting in the accumulation of ARGs. TWW irrigation increased the content of organic matter and total nitrogen in the soil, which affected microbial community structure. The changes of the potential host were the determining factors driving the ARGs abundance. Our study demonstrated that continuous TWW irrigation for 2 years led to a substantial accumulation of ARGs in soil.

Dynamics of antibiotic resistance genes and the association with bacterial community during pig manure composting with chitin and glucosamine addition.

In modern ecological systems, the overuse and misuse of antibiotics have escalated the prevalence of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), positioning them as emerging environmental contaminants. Notably, composting serves as a sustainable method to recycle agricultural waste into nutrient-rich fertilizer while potentially reducing ARGs and MGEs. This study conducted a 47-day composting experiment using pig manure and corn straw, supplemented with chitin and N-Acetyl-D-glucosamine, to explore the impact of these additives on the dynamics of ARGs and MGEs, and to unravel the interplay between these genetic elements and microbial communities in pig manure composting. Results showed that adding 5% chitin into composting significantly postponed thermophilic phase, yet enhanced the removal efficiency of total ARGs and MGEs by over 20% compared to the control. Additionally, the addition of N-Acetyl-D-glucosamine significantly increased the abundance of tetracycline-resistant and sulfonamide-resistant genes, as well as MGEs. High-throughput sequencing revealed that N-Acetyl-D-glucosamine enhanced bacterial α-diversity, providing diverse hosts for ARGs and MGEs. Resistance mechanisms, predominantly efflux pumps and antibiotic deactivation, played a pivotal role in shaping the resistome of composting process. Co-occurrence network analysis identified the key bacterial phyla Proteobacteria, Firmicutes, Gemmatimonadota, and Myxococcota in ARGs and MGEs transformation and dissemination. Redundancy analysis indicated that physicochemical factors, particularly the carbon-to-nitrogen ratio emerged as critical variables influencing ARGs and MGEs. The findings lay a foundation for the developing microbial regulation method to reduce the risks of ARGs in animal manure composts.

Long-term occurrence, resistance risk and chaotic characteristics of antibiotic resistance genes in sludge anaerobic digestion system

The long-term occurrence, dynamics and risk of antibiotic resistance genes (ARGs) in anaerobic digestion (AD) of excess sludge (ES) are not fully understood. Therefore, 13-month metagenomic monitoring was carried out in a full-scale AD plant. The highest ARG abundance and risk scores were observed in spring. AD achieved a 35 % removal rate for the total ARG abundance, but the risk score of AD sludge was not always lower than ES samples, because of the higher proportion of Rank I ARGs in AD sludge. ARGs showed less obvious patterns under linear models compared with microbial community, implying their chaotic dynamics, which was further confirmed by nonlinearity tests. Empirical dynamic modeling performed better than the autoregressive integrated moving average model for ARG dynamics, especially for those with simple and nonlinear dynamics. This study highlighted spring for its higher ARG abundance and risk, and recommended nonlinear models for revealing the dynamics of ARGs.

Temporal variations of antimicrobial resistance genes in aerosols: A one-year monitoring at the puy de Dôme summit (Central France)

The recent characterization of antibiotic resistance genes (ARGs) in clouds evidenced that the atmosphere actively partakes in the global spreading of antibiotic resistance worldwide. Indeed, the outdoor atmosphere continuously receives large quantities of particles of biological origins, emitted from both anthropogenic or natural sources at the near Earth's surface. Nonetheless, our understanding of the composition of the atmospheric resistome, especially at mid-altitude (i.e. above 1000 m a.s.l.), remains largely limited. The atmosphere is vast and highly dynamic, so that the diversity and abundance of ARGs are expected to fluctuate both spatially and temporally. In this work, the abundance and diversity of ARGs were assessed in atmospheric aerosol samples collected weekly between July 2016 and August 2017 at the mountain site of puy de Dôme (1465 m a.s.l., central France). Our results evidence the presence of 33 different subtypes of ARGs in atmospheric aerosols, out of 34 assessed, whose total concentration fluctuated seasonally from 59 to 1.1 × 105 copies m−3 of air. These were heavily dominated by genes from the quinolone resistance family, notably the qepA gene encoding efflux pump mechanisms, which represented >95 % of total ARGs concentration. Its abundance positively correlated with that of bacteria affiliated with the genera Kineococcus, Neorhizobium, Devosia or Massilia, ubiquitous in soils. This, along with the high abundance of Sphingomonas species, points toward a large contribution of natural sources to the airborne ARGs. Nonetheless, the increased contribution of macrolide resistance (notably the erm35 gene) during winter suggests a sporadic diffusion of ARGs from human activities. Our observations depict the atmosphere as an important vector of ARGs from terrestrial sources. Therefore, monitoring ARGs in airborne microorganisms appears necessary to fully understand the dynamics of antimicrobial resistances in the environment and mitigate the threats they may represent.

The fate of antibiotic resistance genes during anaerobic digestion of sewage sludge with ultrasonic pretreatment.

This study investigated the effect of ultrasonic (US) pretreatment at three different contact times (30, 45, and 60 min) with a power of 240 W and frequency of 40 kHz on the fate of antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and enteric pathogens during anaerobic digestion (AD) of sludge. By using real time-qPCR, three MGEs (int1, int2, and tnpA) and seven ARGs (sul1, sul2, tetW, tetA, tetO, ermF, and aac(6')-lb) were quantified that have serious human health impacts and represent the most widely used antibiotics (tetracycline, sulfonamide, macrolide, and aminoglycoside). Results indicated that US pretreatment under different contact times improved the removal of ARGs and MGEs. Compared to 30 and 45 min of US pretreatment, 60 min of US pretreatment resulted in a higher reduction of ARGs with total ARG reduction of 41.70 ± 1.13%. Furthermore, the relative abundance of ARGs and MGEs after US pretreatment was reduced more effectively in anaerobic reactors than in a control AD without US pretreatment. The total ARGs and MGEs removal efficiency of control AD was 44.07 ± 0.72% and 63.69 ± 1.43%, and if US pretreatment at different times were applied, the total ARGs and MGEs removal efficiency of the whole pretreatment AD process improved to 59.71 ± 2.76-68.54 ± 1.58% and 69.82 ± 2.15-76.84 ± 0.22%. The highest removal of total ARGs (68.54 ± 1.58%) and MGEs (76.84 ± 0.22%) was achieved after AD with US pretreatment at 45 min. However, US pretreatment and AD with US pretreatment were not effective in inactivation of enteric pathogens (total coliforms and E. coli), suggesting that posttreatment is needed prior to land application of sludge to reduce the level of enteric pathogens. There was no detection of the studied ARGs and MGEs in the enteric pathogens after US pretreatment in subsequent AD. According to this study, long contact times of US pretreatment can mitigate ARGs and MGEs in AD processes, offering valuable insight into improving environmental safety and sustainable waste management. Additionally, the study highlights the need to investigate posttreatment techniques for reducing enteric pathogens in AD effluent, a crucial consideration for agricultural use and environmental protection.

Investigating antimicrobial resistance determinants and micropollutants in urban sewage treatment plants of India: Occurrence, removal and ecotoxicological risk

Environmental contamination caused by emerging pollutants (EPs) and their associated ecological risk have received limited attention in India. Thus, this study aims to address this gap by investigating the occurrence and removal of antibiotic resistant bacteria (ARB), antibiotic resistance genes (ARGs) and pharmaceutical and personal care products (PPCPs) in two sewage treatment plants (STPs) in New Delhi. The study also assessed the ecological risk associated with PPCPs in aquatic environments. Among the targeted ARB and ARGs, erythromycin resistant bacteria and ermB gene, respectively were the most ubiquitous pollutants in the influent of both STPs. In the final effluent, there was a reduction ranging from 0.8 log to 2.8 log units for most ARB, while removal of the majority of ARGs was less than 1 log unit. The secondary treatment demonstrated better removal of total ARG concentrations compared to the tertiary treatment methods employed in both STPs. Regarding PPCPs, metformin was quantified in both influent and effluent samples at a concentration exceeding 19,000 ng/L, indicating its persistence in wastewater. High concentrations of ARB, ARGs, and PPCPs were observed in undigested biosolids, while low to moderate removal of these pollutants was observed after anaerobic digestion. To assess the ecological risk, a risk quotient (RQ) assessment was conducted. The results revealed that compounds such as erythromycin, terbinafine, 17-α ethinylestradiol (EE2), 17-β estradiol (E2), diclofenac, triclosan, carbamazepine, and mefenamic acid exhibited high risks in the effluent. Additionally, EE2 and E2 showed higher risk levels in the digested biosolids, indicating potential risks to the aquatic ecosystem.

Assessing impacts of municipal wastewater treatment plant upgrades on bacterial hazard contributions to the receiving urban river using SourceTracker

Upgrading municipal wastewater treatment plants (MWTPs) has been implemented in many megacities of China to reduce the discharges of nutrients and other pollutants and improve water quality of highly urbanized rivers. However, the contribution of MWTP discharge to bacterial hazards in the receiving rivers after upgrades has been largely unknown. In this study, high-throughput sequencing and shotgun metagenomics were applied to investigate the changes in the abundance, composition, potential risks, and contributions of bacteria and antibiotic resistance genes (ARGs) from effluent to receiving river after upgrading the third-largest MWTP in China with denitrification biofilters, ultrafiltration, ozonation, and disinfection processes. The annual loadings of total nitrogen and 27 types of pharmaceuticals were reduced by 42.4% ± 13.2% and 46.2% ± 15.4%, respectively. Bacterial biomass decreased from (3.58 ± 0.49) to (1.23 ± 0.27) × 107 16S rRNA gene copies/mL, and identified biomarkers in effluent and downstream shifted due to the adopted processes. Opportunistic pathogen bacteria downstream were also reduced. Although the relative abundance of total ARGs in MWTP effluent increased from 1.10 ± 0.02 to 2.19 ± 0.03 copies/16S rRNA gene after upgrades, that of total and high-risk ARGs downstream showed no significant difference. More importantly, the Bayesian-based SourceTracker method provided valuable insight by revealing that the contributions of MWTP discharge to downstream bacteria (from 44.2% ± 1.5%–31.4% ± 0.9%) and ARGs (from 61.2% ± 5.3%–47.6% ± 4.1%) were significantly reduced following the upgrades, indicating upgrading MWTP showed integrated benefits to the bacterial hazards in the receiving river. This study provides useful information for better control of bacterial hazard risks and operational strategy for the improvement of the urban aquatic ecosystem.

Deterministic effect of oxygen level variation on shaping antibiotic resistome

An increase in acquisition of antibiotic resistance genes (ARGs) by pathogens under antibiotic selective pressure poses public health threats. Sub-inhibitory antibiotics induce bacteria to generate reactive oxygen species (ROS) dependent on dissolved oxygen (DO) levels, while molecular connection between ROS-mediated ARG emergence through DNA damage and metabolic changes remains elusive. Thus, the study investigates antibiotic resistome dynamics, microbiome shift, and pathogen distribution in hyperoxic (5–7 mg L−1), normoxic (2–4 mg L−1), and hypoxic (0.5–1 mg L−1) conditions using lab-scale bioreactor. Composite inoculums in the reactor were designed to represent comprehensive microbial community and AR profile from selected activated sludge. RT-qPCR and metagenomic analysis showed an increase in ARG count (100.98 ppm) with enrichment of multidrug efflux pumps (acrAB, mexAB) in hyperoxic condition. Conversely, total ARGs decreased (0.11 ppm) under hypoxic condition marked by a major decline in int1 abundance. Prevalence of global priority pathogens increased in hyperoxic (22.5%), compared to hypoxic (0.9%) wherein major decrease were observed in Pseudomonas, Shigella, and Borrelia. The study observed an increase in superoxide dismutase (sodA, sodB), DNA repair genes (nfo, polA, recA, recB), and ROS (10.4 µmol L−1) in adapted biomass with spiked antibiotics. This suggests oxidative damage that facilitates stress-induced mutagenesis providing evidence for observed hyperoxic enrichment of ARGs. Moreover, predominance of catalase (katE, katG) likely limit oxidative damage that deplete ARG breeding in hypoxic condition. The study proposes a link between oxygen levels and AR development that offers insights into mitigation and intervention of AR by controlling oxygen-related stress and strategic selection of bacterial communities.

Temporal dynamics of microbial composition and antibiotic resistome in fermentation bed culture pig farms across various ages

The discharge from pig farms presents significant challenges to the environment and human health, specifically regarding the dissemination of antimicrobial resistance (AMR). Fermentation bed culture has emerged as an increasingly popular and environmentally friendly pig farming model in China, as it minimizes the release of harmful substances into the environment. However, there remains a limited understanding of the occurrence and dynamics of microbiome and antibiotic resistome in fermentation bed culture. Herein, we collected fermentation bed materials (FBM) from four fermentation bed culture pig farms with varying service ages and investigated their bacterial communities, antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), metal resistance genes (MRGs) and potential antibiotic-resistant bacterial hosts through metagenomics. Pseudomonadota, Actinomycetota, Bacteroidota and Bacillota were identified as the dominant phyla present in the FBM. In total, we detected 258 unique ARGs in the FBM samples, with 79 core ARGs shared by all FBM samples, accounting for 95 % of the total ARG abundance. Our results revealed significant variations in microbial communities and ARG profiles across varying service ages of FBM. Compared to long-term FBW, short-term FBM exhibited higher numbers and abundances of ARGs, MRGs and MGEs, along with higher levels of potential bacterial pathogens and high-risk ARGs. Further analysis of metagenome-assembled genome (MAG) indicated that the putative hosts of ARGs primarily belonged to Pseudomonadota, Actinomycetota and Bacillota. Alarmingly, among the 80 recovered ARG-carrying MAGs, 23 MAGs encoded multi-resistance, including clinically significant species that require urgent attention. Overall, this study provided valuable insights into the temporal patterns of antibiotic resistome and bacterial communities within FBM, enhancing our understanding of FBM in pig farming. The findings could potentially contribute to the development of effective strategies for evaluating and regulating fermentation bed culture practices in pig farming.