Abundance Of Antibiotic Resistance Genes Research Articles

The Role of Wastewater Treatment Plants in Dissemination of Antibiotic Resistance: Source, Measurement, Removal and Risk Assessment.

Antibiotic Resistance Genes (ARGs) are contaminants of emerging concern with marked potential to impact public and environmental health. This review focusses on factors that influence the presence, abundance, and dissemination of ARGs within Wastewater Treatment Plants (WWTPs) and associated effluents. Antibiotic-Resistant Bacteria (ARB) and ARGs have been detected in the influent and the effluent of WWTPs worldwide. Different levels of wastewater treatment (primary, secondary, and tertiary) show different degrees of removal efficiency of ARGs, with further differences being observed when ARGs are captured as intracellular or extracellular forms. Furthermore, routinely used molecular methodologies such as quantitative polymerase chain reaction or whole genome sequencing may also vary in resistome identification and in quantifying ARG removal efficiencies from WWTP effluents. Additionally, we provide an overview of the One Health risk assessment framework, as well as future strategies on how WWTPs can be assessed for environmental and public health impact.

Prolonged hospitalization signature and early antibiotic effects on the nasopharyngeal resistome in preterm infants

Respiratory pathogens, commonly colonizing nasopharynx, are among the leading causes of death due to antimicrobial resistance. Yet, antibiotic resistance determinants within nasopharyngeal microbial communities remain poorly understood. In this prospective cohort study, we investigate the nasopharynx resistome development in preterm infants, assess early antibiotic impact on its trajectory, and explore its association with clinical covariates using shotgun metagenomics. Our findings reveal widespread nasopharyngeal carriage of antibiotic resistance genes (ARGs) with resistomes undergoing transient changes, including increased ARG diversity, abundance, and composition alterations due to early antibiotic exposure. ARGs associated with the critical nosocomial pathogen Serratia marcescens persist up to 8–10 months of age, representing a long-lasting hospitalization signature. The nasopharyngeal resistome strongly correlates with microbiome composition, with inter-individual differences and postnatal age explaining most of the variation. Our report on the collateral effects of antibiotics and prolonged hospitalization underscores the urgency of further studies focused on this relatively unexplored reservoir of pathogens and ARGs.

One Health Threat of Treated Wastewater Discharge in Urban Ohio Rivers: Implications for Surface Water and Fish Gut Microbiome and Resistome.

Wastewater treatment plants (WWTPs) are thought to be a major disseminating source of antibiotic resistance (AR) to the environment, establishing a crucial connection between human and environmental resistome. The objectives of this study were to determine how wastewater effluents impact microbiome and resistome of freshwater and fish, and identify potential AR-carrying clinically relevant pathogens in these matrices. We analyzed wastewater influent and effluent from four WWTPs in three metropolitan areas of Ohio, USA via shotgun metagenomic sequencing. We also sequenced river water and fish guts from three reaches (upstream, at the WWTP outfall, and downstream). Notably, we observed a decline in microbiome diversity and AR gene abundance from wastewater to the receiving river. We also found significant differences by reach and trophic level (diet) in beta-diversity of the fish gut microbiomes. SourceTracker revealed that 0.443 and 0.248 more of the of the fish gut microbiome was sourced from wastewater effluent in fish from the outfall and downstream locations, respectively, compared to upstream fish. Additionally, AR bacteria of public health concern were annotated in effluent and river water samples, indicating potential concern for human exposure. In summary, our findings show the continued role of wastewater as a significant AR reservoir and underscores the considerable impact of wastewater discharge on aquatic wildlife, which highlights the One Health nature of this issue.

Tebuconazole exacerbates co-occurrence and horizontal transfer of antibiotic resistance genes

As one of the most widely used pesticides in the global fungicide market, tebuconazole has become heavily embedded in soil along with antibiotic resistance genes (ARGs). However, it remains unclear whether the selective pressure produced by tebuconazole affects ARGs and their horizontal transfer. In this experiment, we simulated a tebuconazole-contaminated soil ecosystem and observed changes in the abundance of ARGs and mobile genetic element (MGEs) due to tebuconazole exposure. We also established a plasmid RP4-mediated conjugative transfer system to investigate in depth the impact of tebuconazole on the horizontal transfer of ARGs and its mechanism of action. The results showed that under tebuconazole treatment at concentrations ranging from 0 to 10 mg/L, there was a gradual increase in the frequency of plasmid conjugative transfer, peaking at 10 mg/L which was 7.93 times higher than that of the control group, significantly promoting horizontal transfer of ARGs. Further analysis revealed that the conjugative transfer system under tebuconazole stress exhibited strong ability to form biofilm, and the conjugative transfer frequency ratio of biofilm to planktonic bacteria varied with the growth cycle of biofilm. Additionally, scanning electron microscopy and flow cytometry demonstrated increased cell membrane permeability in both donor and recipient bacteria under tebuconazole stress, accompanied by upregulation of ompA gene expression controlling cell membrane permeability. Furthermore, enzyme activity assays indicated significant increases in CAT, SOD activity, and GSH content in recipient bacteria under tebuconazole stress. Moreover, expression levels of transmembrane transporter gene trfAp as well as genes involved in oxidative stress and SOS response were found to be correlated with the frequency of plasmid conjugative transfer.

Metagenomic profiling uncovers microbiota and antibiotic resistance patterns across human, chicken, pig fecal, and soil environments

The ongoing and progressive evolution of antibiotic resistance presents escalating challenges for the clinical management and prevention of bacterial infections. Understanding the makeup of resistance genomes and accurately quantifying the current abundance of antibiotic resistance genes (ARGs) are crucial for assessing the threat of antimicrobial resistance (AMR) to public health. This comprehensive study investigated the distribution and diversity of bacterial community composition, ARGs, and virulence factors (VFs) across human, chicken, pig fecal, and soil microbiomes in various provinces of China. As a result, multidrug resistance was identified across all samples. Core ARGs primarily related to multidrug, MLS (Macrolides-Lincosamide-Streptogramins), and tetracycline resistance were characterized. A significant correlation between ARGs and bacterial taxa was observed, especially in soil samples. Probiotic strains such as Lactobacillus harbored ARGs, potentially contributing to the dissemination of antibiotic resistance. We screened subsets of ARGs from samples from different sources as indicators to assess the level of ARGs contamination in samples, with high accuracy. These results underline the complex relationship between microbial communities, resistance mechanisms, and environmental factors, emphasizing the importance of continued research and monitoring to better understand these dynamics.

Comparison of Chinese medicine additives for antibiotic resistance risks in the Wuding chickens under high stocking density: Advantages and drawbacks

Traditional Chinese medicines (TCMs) with anti-pathogenic properties have been increasingly utilized in animal husbandry as substitutes for antibiotics. However, lingering uncertainties persist regarding the mobility, bacterial hosts, and potential human hazards associated with antibiotic resistance genes (ARGs) in animal husbandry when TCMs are introduced. In this study, cecal digesta from Wuding chickens subjected to varying stocking densities (moderate and high) and treated with different TCMs (dihydroartemisinin, berberine hydrochloride, and oligomeric proanthocyanidins) were collected and subjected to metagenomics-based analysis. A total of 280 ARG subtypes were identified, with multidrug and aminoglycoside resistance genes being the most diverse and abundant. The primary antibiotic resistance bacteria identified belonged to the phyla of Firmicutes (38.68%), Bacteroidetes (22.74%), and Proteobacteria (12.71%); however, some pathogenic antibiotic-resistant bacteria (PARB) were also identified across different samples. Surprisingly, high stocking density was not observed to propagate ARGs, which could be due to the restricted usage of antibiotics. Furthermore, higher proportions of mobile genetic elements and some multidrug-resistant PARB (∼4 times) were observed in high-stocking-density chickens fed berberine hydrochloride than in high-stocking-density chickens fed other TCMs, which could facilitate the horizontal gene transfer (HGT) of ARGs and pose potential human health risks. In contrast, dihydroartemisinin reduced the total abundance of ARGs and the resistome risks to human health (∼10% decrease compared to control). Moreover, rank I ARGs with higher human health risk and the risk index based on ARG risk also ranked lowest (p < 0.05) in high-stocking-density chickens fed dihydroartemisinin. The reduction effect of dihydroartemisinin was achieved by regulating the cecal microbiome of broilers and inhibiting HGT (reducing the contribution from 17.7% to 4.0%). This study provides metagenomic evidence of the development of ARGs in broiler breeding environments. The findings highlight the distinct characteristics of TCMs as feed additives on regulating the antibiotic resistome during broiler farming and demonstrate dihydroartemisinin as an effective additive complying with the environmental safety.

Unveiling the overlooked threat: antibiotic resistance in groundwater near an abandoned sulfuric acid plant in Xingyang, China.

Groundwater near a sulfuric acid plant in Xingyang, Henan, China was sampled from seven distinct sites to explore the prevalence of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). Results showed that genes aadA, blaCTX-M, tetA, qnrA, and sul1 were detected with 100% frequency followed by aac(6')-Ib (85.71%), ermB (85.71%), and tetX (71.42%). Most abundant ARGs were sul1 in LSA2 (1.15 × 1011 copies/mL), tetA in LSA6 (4.95 × 1010 copies/mL), aadA in LSA2 (4.56 × 109 copies/mL), blaCTX-M in LSA4 (1.19 × 109 copies/mL), and ermB in LSA5 (1.07 × 109 copies/mL). Moreover, in LSA2, intl1 as a marker of class 1 integron emerged as the most abundant gene as part of MGE (2.25 × 1011 copies/mL), trailed by ISCR1 (1.57 × 109 copies/mL). Environmental factors explained 81.34% of ARG variations, with a strong positive correlation between the intl2 and blaCTX-M genes, as well as the ISCR1 gene and qnrA, tetA, intl2, and blaCTX-M. Furthermore, the intI1 gene had a strong positive connection with the aadA, tetA, and sul1 genes. Moreover, the aac(6')-Ib gene was associated with As, Pb, Mg, Ca, and HCO3-. The intl2 gene was also shown to be strongly associated with Cd. Notably, network analysis highlighted blaCTX-M as the most frequently appearing gene across networks of at least five genera. Particularly, Lactobacillus, Plesiomonas, and Ligilactobacillus demonstrated correlations with aadA, qnrA, blaCTX-M, intI2, and ISCR1. Based on 16S rRNA sequencing, the dominant phyla were Proteobacteria, Firmicutes, Bacteroidota, Acidobacteriota, and Actinobacteriota, with dominant genera including Pseudomonas, Ligilactobacillus, Azoarcus, Vogesella, Streptococcus, Plesiomonas, and Ferritrophicum. These findings enhance our understanding of ARG distribution in groundwater, signaling substantial contamination by ARGs and potential risks to public health.

Combined use of digestate and inorganic fertilizer alleviates the burden of class 1 integrons in perennial ryegrass rhizosphere without compromising aerial biomass production.

Antimicrobial resistance (AMR) is one of the main global health challenges. Anaerobic digestion (AD) can significantly reduce the burden of antibiotic resistance genes (ARGs) in animal manures. However, the reduction is often incomplete. The agronomic use of digestates requires assessments of their effects on soil ARGs. The objective of this study was to assess the effect of digestate on the abundance of ARGs and mobile genetic elements (MGEs) in the rhizosphere of ryegrass (Lolium perenne L.) and to determine whether half-dose replacement of digestate with urea (combined fertilizer) can be implemented as a safer approach while maintaining a similar biomass production. A greenhouse assay was conducted during 190days under a completely randomized design with two experimental factors: fertilizer type (unfertilized control and fertilized treatments with equal N dose: digestate, urea and combined fertilizer) and sampling date (16 and 148days after the last application). The results indicated that the digestate significantly increased the abundance of clinical class 1 integrons (intI1 gene) relative to the unfertilized control at both sampling dates (P < 0.05), while the combined fertilizer only increased them at the first sampling. Sixteen days after completing the fertilization scheme only the combined fertilizer and urea significantly increased the biomass production relative to the control (P < 0.05). Additionally, by the end of the assay, the combined fertilizer showed significantly lower levels of the macrolide-resistance gene ermB than digestate and a cumulative biomass similar to urea or digestate. Overall, the combined fertilizer can alleviate the burden of integrons and ermB while simultaneously improving biomass production.

Fluoranthene slow down sulfamethazine migration in soil via π-π interaction to increase the abundance of antibiotic resistance genes

Sulfonamide antibiotics and polycyclic aromatic hydrocarbons (PAHs) often coexist in soil, leading to compound pollution through various pathways. This study focuses on sulfamethazine (SMZ) and PAHs (fluoranthene) as the subject for compound pollution research. Using a soil-groundwater simulation system, we investigated the migration characteristics of SMZ under coexistence with fluoranthene (Fla) and observed variations in the abundance of antibiotic resistance genes (ARGs). Through molecular docking simulations and isothermal adsorption experiments, we discovered that Fla bound with SMZ via π−π interactions, resulting in a 20.9% increase in the SMZ soil-water partition coefficient. Under compound conditions, the concentration of SMZ in surface soil could reach 1.4 times that of SMZ added alone, with an 13.4% extension in SMZ half-life. The deceleration of SMZ's vertical migration rate placed additional stress on surface soil microbiota, leading to a proliferation of ARGs by 66.3%–125.8%. Moreover, under compound pollution, certain potential hosts like Comamonadaceae and Gemmatimonas exhibited a significant positive correlation with resistance genes such as sul 1 and sul 2. These findings shed light on the impact of PAHs on sulfonamide antibiotic migration and the abundance of ARGs. They also provide theoretical insights for the development of technologies aimed at mitigating compound pollution in soil.

Preference and regulation mechanism mediated via mobile genetic elements for antibiotic and metal resistomes during composting amended with nano ZVI loaded on biochar

This study assessed the effectiveness of nano zero-valent iron loaded on biochar (BC-nZVI) during swine manure composting. BC-nZVI significantly reduced the abundance of antibiotic resistance genes (ARGs), metal resistance genes (MRGs), and mobile genetic elements (MGEs). BC-nZVI modified the preference of MGEs to carry ARGs and MRGs, and the corrosion products of BC-nZVI could destroy cell structure, hinder electron transfer between cells, and weaken the association between ARGs, MRGs, and host bacteria. Functional genes analysis revealed that BC-nZVI down-regulated the abundance of genes affecting the transmission and metabolism of ARGs and MRGs, including type IV secretion systems, transporter systems, two-component systems, and multidrug efflux pumps. Furthermore, the BC-nZVI decreased genes related to flagella and pili production and cell membrane permeability, thereby hindering the transfer of ARGs, MRGs, and MGEs in the environment. Redundancy analysis demonstrated that changes in the microbial community induced by BC-nZVI were pivotal factors impacting the abundance of ARGs, MRGs, and MGEs. Overall, this study confirmed the efficacy of BC-nZVI in reducing resistance genes during swine manure composting, offering a promising environmental strategy to mitigate the dissemination of these contaminants.

Profiles of Antibiotic Resistance Genes in the Coastal Aquatic Environment of Xiamen City

The coastal areas and the adjacent islands are the hotspots of human economic and social activities, including urbanization, industrialization, and agricultural practices, which have profound impacts on the ecological environment of the coastal environment. Antibiotic resistance genes （ARGs）, as emerging contaminants, have become hot topics in water ecological security and public concern. However, the profiles of antibiotic resistome in the costal water remain largely unknown, impeding resistome risk assessment associated with coastal environments. In this study, the high-throughput quantitative PCR technique was used to investigate the abundance and distribution of ARGs in the coastal environment of Xiamen City. Combined with the 16S rDNA gene amplicon sequencing method, the structure and composition of the microbial community in a water environment were investigated, and the influencing factors and associated mechanism of ARGs in seawater were deeply explained. The results of this study showed that a total of 187 ARGs were detected in the coastal water environment, and the abundance level was up to 1.29×1010 copies·L-1. Multidrug resistance, aminoglycosides, and β lactamase resistance genes were the three main classes of antibiotic resistance genes in the water environment of the Xiamen coastal zone. On the whole, the profile of ARGs was of high abundance, great diversity, and common co-existence, and the coastal water environment was an important hot area and reservoir for antibiotic resistance genes. Twenty-two microbes, including Nautella, Candidatus, Tenacibaculum, Rubripirellula, and Woeseia, were potential carriers of the corresponding 16 antibiotic resistance genes. The mobile genetic elements （MGEs） and microbial community structure accounted for 93.9% of the variation in environmental resistance genes in water. Therefore, microbial community and its mobile genetic elements were the most important driving forces for the occurrence and evolution of ARGs in coastal waters. Based on the results, it is implied that the environmental antibiotic resistance genes in the waters near Xiamen Island have potential risks to water ecological security and human health and highlight the necessity for comprehensive surveillance of ARGs associated with microbial contamination in the coastal aquatic environment.

Insight into the effect of CaO2 addition on the prevalence and removal of antibiotic resistance genes during anaerobic sludge fermentation under ambient conditions

This study investigated the effect of CaO2 and its related hydrolysis products on the prevalence and removal of antibiotic resistance genes (ARGs) during anaerobic fermentation of waste activated sludge (WAS) under ambient conditions. Comprehensive analyses were conducted to elucidate the mechanisms underlying the effects of CaO2 addition, focusing on the microbial community structure, host bacteria, cell membrane permeability and the interaction between ARGs and environmental factors. Results showed the alkalinity, oxidation and calcium of CaO2 synergistically contributed to the elimination of ARGs. The top ARGs, such as sulI, sulII, tetC, tetX and ereA, were effectively reduced by 43.9–84.9 % with CaO2 addition during the ambient anaerobic fermentation. Ca(OH)2 treatment also shows good effectiveness in removing typical ARGs, followed by H2O2 treatment, while NaOH treatment increases ARGs abundance and raise the risk of their transmission. Among the influencing factors, the abundance and diversity of microbial community, organic matters, calcium and pH were the primary factors for ARGs migration. The effective reduction of Proteobacteria was an important reason for the removal of typical ARGs, particularly sulfonamide resistance genes. CaO2 also downregulated the relative abundance of autoinducer and functional genes related to outer membrane proteins (OMPs) and ARGs dissemination, thereby inhibiting ARGs transmission. This work provides a deep insight into the advantage of CaO2 in reducing ARGs, thus expanding its potential application in sludge treatment and ARGs elimination.

Optimising microbial processes with nano-carbon/selenite materials: An eco-friendly approach for antibiotic resistance mitigation in broiler manure

The presence of antibiotic resistance genes (ARGs) in animal excrement has been a significant threat to global public health, necessitating sustainable animal husbandry practices to mitigate agriculture’s environmental impact. This study introduces two innovative nano-carbon/selenite materials: carbon-based selenite (Cse) and graphene-based selenite (Gse), synthesized using nano-carbon powder and graphene as carriers. These materials, with their high specific surface area, stability, and adsorption capabilities, were hypothesized to modulate microbial structure in broiler manure, potentially reducing ARGs pollution risk through effects on nitrogen metabolism and DNA replication. The order of ARGs abundance in broiler manure was as follows: control (CON) group > Cse group > Gse group, supporting the hypothesis. The top five antibiotic classes made up over 80 % of total gene abundances, with the multidrug class being the most prevalent, reaching 40.69 % and 40.48 % in Cse and Gse groups, respectively. PCA and LEfSe analyses revealed different ARG subtypes and biomarkers in the groups, showing the impact of these materials on ARG subtypes. Notably, the study demonstrated that Gse, utilizing graphene, exhibited greater efficacy in inhibiting essential microbial DNA replication genes compared to Cse. Additionally, the research identified associations between microbial DNA replication genes and ARGs, indicating a potential relationship between DNA replication and cellular reactions to antibiotics. Furthermore, both Cse and Gse were found to significantly impact nitrogen cycling pathways, possibly through their distinct characteristics that could alter key gene abundance. The findings expand our understanding of the genetic mechanisms underlying biogeochemical processes at a microscopic level. The study also reveals new ideas for mitigating potential hazards of ARGs in livestock waste to human well-being and ecological stability arising.

Variability of faecal microbiota and antibiotic resistance genes in flocks of migratory gulls and comparison with the surrounding environment

Gulls commonly rely on human-generated waste as their primary food source, contributing to the spread of antibiotic-resistant bacteria and their resistance genes, both locally and globally. Our understanding of this process remains incomplete, particularly in relation to its potential interaction with surrounding soil and water. We studied the lesser black-backed gull, Larus fuscus, as a model to examine the spatial variation of faecal bacterial communities, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) and its relationship with the surrounding water and soil. We conducted sampling campaigns within a connectivity network of different flocks of gulls moving across functional units (FUs), each of which represents a module of highly interconnected patches of habitats used for roosting and feeding. The FUs vary in habitat use, with some gulls using more polluted sites (notably landfills), while others prefer more natural environments (e.g., wetlands or beaches). Faecal bacterial communities in gulls from flocks that visit and spend more time in landfills exhibited higher richness and diversity. The faecal microbiota showed a high compositional overlap with bacterial communities in soil. The overlap was greater when compared to landfill (11%) than to wetland soils (6%), and much lower when compared to bacterial communities in surrounding water (2% and 1% for landfill and wetland water, respectively). The relative abundance of ARGs and MGEs were similar between FUs, with variations observed only for specific families of ARGs and MGEs. When exploring the faecal carriage of ARGs and MGEs in bird faeces relative to soil and water compartments, gull faeces were enriched in ARGs classified as High-Risk. Our results shed light on the complex dynamics of antibiotic resistance spread in wild bird populations, providing insights into the interactions among gull movement and feeding behavior, habitat characteristics, and the dissemination of antibiotic resistance determinants across environmental reservoirs.

Unraveling the Microbial Symphony: Impact of Antibiotics and Probiotics on Infant Gut Ecology and Antibiotic Resistance in the First Six Months of Life.

We aimed to examine the effects of antibiotic and probiotic usage on the gut microbiota structure and the presence of antibiotic-resistance genes (ARGs) in infants during the first six months of life. Questionnaires and fecal samples were collected within three days of birth, two months, and six months to assess antibiotic and probiotic exposure. Gut microbiotas were sequenced via 16S rRNA, and ARGs were conducted by qPCR, including beta-lactam (mecA, blaTEM), tetracycline (tetM), fluoroquinolone (qnrS), aminoglycoside (aac(6')-Ib), and macrolide (ermB). Infants were categorized by antibiotic and probiotic usage and stratified by delivery mode, microbial composition, and ARG abundances were compared, and potential correlations were explored. A total of 189 fecal samples were analyzed in this study. The gut microbiota diversity (Chao1 index) was significantly lower in the "only probiotics" (PRO) group compared to the "neither antibiotics nor probiotics" (CON) group at six months for the CS stratification (p = 0.029). Compositionally, the abundance of core genus Bifidobacterium_pseudocatenulatum was less abundant for the antibiotic during delivery (IAP) group than that in the CON group within the first three days (p = 0.009), while core genus Enterococcus_faecium was more abundant in the PRO than that in the CON group (p = 0.021) at two months. ARGs were highly detected, with Enterococcus hosting tetM and Escherichia associated with blaTEM within three days of birth, though no correlation was found between Bifidobacterium and ARGs. These findings emphasized the critical importance of carefully managing antibiotic and probiotic exposures in early life, with implications for promoting lifelong health through preserving a healthy infant gut ecosystem.

Occurrence and distribution of antibiotic resistance genes in urban rivers with black-odor water of Harbin, China

Antibiotic resistance gene contamination in polluted rivers remains a widely acknowledged environmental issue. This study focused on investigating the contamination conditions of antibiotic resistance genes (ARGs) in Harbin’s urban black-odor rivers, specifically Dongfeng Ditch and Hejia Ditch. The research employed a SmartChip Real-Time PCR System to explore the types, abundance, and distribution of ARGs in diverse habitats, such as surface water and sediment. Additionally, the study examined the correlation of ARGs with mobile genetic elements (MGEs) and various environmental factors. It was found that antibiotic resistance genes were prevalent in both water and sediment within the black-odor ditches. The dominant types of ARGs identified included aminoglycoside, sulfonamide, multidrug-resistant, and β-lactam ARGs. Notably, the top four ARGs, in terms of relative abundance, were sul1, fox5, qacEdelta1-01 and aadA1. Most categories of ARGs have significant positive connections with MGEs, indicating that the enrichment and spreading of ARGs in rivers are closely related to MGEs. Based on the correlation analysis, it is found that environmental factors such as dissolved oxygen (DO), ammonia nitrogen (NH4–N), and phosphate (PO4–P) played a substantial role in influencing the variations observed in ARGs. By employing a risk assessment framework based on the human association, host pathogenicity, and mobility of ARGs, the identification of seven high-risk ARGs was achieved. In addition, it is important to assess the environmental risk of ARGs from multiple perspectives (abundance,detection rateand mobility). This study provides a significant reference regarding the presence of ARGs contamination in urban inland black-odor rivers, essential for assessing the health risks associated with ARGs and devising strategies to mitigate the threat of antibiotic resistance.

Disinfectant polyhexamethylene guanidine triggered simultaneous efflux pump antibiotic- and metal-resistance genes propagation during sludge anaerobic digestion

The environmental transmission of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) exerted devastating threats to global public health, and their interactions with other emerging contaminants (ECs) have raised increasing concern. This study investigated that the abundances of ARGs and MRGs with the predominant type of efflux pump were simultaneously increased (8.4–59.1%) by disinfectant polyhexamethylene guanidine (PHMG) during waste activated sludge (WAS) anaerobic digestion. The aggregation of the same microorganisms (i.e., Hymenobacter and Comamonas) and different host bacteria (i.e., Azoarcus and Thauera) were occurred upon exposure to PHMG, thereby increasing the co-selection and propagation of MRGs and ARGs by vertical gene transfer. Moreover, PHMG enhanced the process of horizontal gene transfer (HGT), facilitating their co-transmission by the same mobile genetic elements (20.2–223.7%). Additionally, PHMG up-regulated the expression of critical genes (i.e., glnB, trpG and gspM) associated with the HGT of ARGs and MRGs (i.e., two-component regulatory system and quorum sensing) and exocytosis system (i.e., bacterial secretion system). Structural equation model analysis further verified that the key driver for the simultaneous enrichment of ARGs and MRGs under PHMG stress was microbial community structure. The study gives new insights into the aggravated environmental risks and mechanisms of ECs in sludge digestion system, providing guidance for subsequent regulation and control of ECs.

Parachlorometaxylenol stress caused multidrug-type antibiotic resistance genes proliferation via simultaneously reshaping microbial community and interfering metabolic traits during wastewater treatment process

Despite biological wastewater treatment processes (e.g., sequencing batch reactors (SBR)) being able to reduce the dissemination of antibiotic resistance genes (ARGs), the variation of ARGs under exogenous pollutant stress is an open question. This work investigated the impacts of para-chloro-meta-xylenol (PCMX, typical antibacterial contaminants) on ARGs spread in long-term SBR operation. Although the SBR process inherently decreased ARGs abundance, the presence of PCMX substantially amplified both the prevalence (mainly multidrug) and abundance of total ARGs (1.17-fold of the control). Further analysis demonstrated that PCMX disintegrated sludge structures as well as increased membrane permeability, facilitating the release of mobile genetic elements and subsequent horizontal transfer of ARGs. In addition, PCMX selectively enriched potential ARG hosts, notably Nitrospira and Candidatus Accumulibacter, which predominantly served as multidrug ARG hosts. Concurrently, the self-adaptive functions of ARGs hosts in the PCMX-exposed SBR system were activated via quorum sensing, two-component regulatory system, ATP-binding cassette transporters, and bacterial secretion system. The upregulation of these metabolic pathways also contributed to the dissemination of ARGs.

Earthworm gut digestion drives the transfer behavior of antibiotic resistance genes in layers of extracellular polymeric substances during vermicomposting of dewatered sludge

Gut digestion by earthworms (GDE) is a crucial step in vermicomposting, affecting the fate of antibiotic resistance genes (ARGs) in vermicompost sludge. The extracellular polymeric substance (EPS) matrix of sludge is an important space for ARG transfer. However, the effect of GDE on EPS-associated ARGs remains unclear. Therefore, this study explored the role of GDE in driving the transfer of ARGs within different EPS layers in sludge. For this, the changes in intracellular ARGs and EPS-associated ARGs in sludge were analyzed after 5 days of the GDE process. The results showed that after the GDE process, both nitrate and dissolved organic carbon significantly increased in all EPS layers of sludge, while the proteins and polysaccharides only enhanced in soluble and loosely bound EPS of sludge. In addition, a 7.0% decrease in bacterial diversity was recorded after the GDE process, with a functional bacterial community structure emerging. Moreover, the absolute abundance of total ARGs and mobile genetic elements decreased by 90.71% and 61.83%, respectively, after the GDE process. Intracellular ARGs decreased by 92.1%, while EPS-associated ARGs increased by 4.9%, indicative of intracellular ARG translocation into the EPS during the GDE process. Notably, the ARGs exhibited significant enrichment in both the soluble and loosely bound EPS, whereas they were reduced in the tightly bound EPS. The structural equation modeling revealed that the GDE process effectively mitigated the ARG dissemination risk by modulating both the EPS structure and microenvironment, with the organic structure representing a primary factor influencing ARGs in the EPS.

Fate of antibiotic resistance genes and EPS defence mechanisms during simultaneous denitrification and methanogenesis, coupled with the biodegradation of multiple antibiotics under zinc stress

High-strength nitrogen and antibiotics-containing wastewater can be efficiently eliminated by simultaneous denitrification and methanogenesis (SDM). Heavy metals and antibiotics are two critical factors that can lead to horizontal transfer of antibiotic resistance genes (ARGs), which can be simultaneously detected in wastewater. Unfortunately, the impacts of heavy metals on SDM and antibiotic biodegradation have not been fully elucidated. Herein, the effects of SDM and multiple antibiotics biodegradation, extracellular polymeric substances (EPSs) and protein response mechanisms, and ARG fate under Zn(II) stress were comprehensively evaluated. The results indicated that a high level of Zn(II) (≥5 mg/L) stress significantly decreased the degradation rate of multiple antibiotics and suppressed denitrification and methanogenesis. In addition, Zn(II) exposure prompted the liberation of proteins from microbes into the EPSs, and the combination of EPSs with small molecules quenched the original fluorescent components and destroyed the protein structure. The dominant proteins can bind to both Zn(II) and multiple antibiotics through several types of chemical interactions, including metallic and hydrogen bonds, hydrophobic interactions, and salt bridges, relieving the toxicity of harmful substances. Moreover, metagenomic sequencing revealed that the abundance of zinc resistance genes (Zn-RGs), ARGs (mainly tetracyclines), and mobile genetic elements (MGEs) increased under Zn(II) stress. Mantel test illustrated that the ARGs mecD, tetT, and tetB(60) were most affected by MGEs. Moreover, molecular network analysis revealed that several MGEs can bridge metal resistance genes (MRGs) and ARGs, facilitating the horizontal transfer of ARGs. This study provides theoretical guidance for the environmental risk control of antibiotics-containing wastewater treated by an SDM system.