Antibiotic Resistome Research Articles

Reforestation substantially changed the soil antibiotic resistome and its relationships with metal resistance genes, mobile genetic elements, and pathogens

Revealing the effects of reforestation on soil antibiotic resistome is essential for assessing ecosystem health, yet related studies remain scarce. Here, to determine the responses of the soil antibiotic resistome to reforestation, 30 pairs of cropland and forest soil samples were collected from southwestern China, a region with high environmental heterogeneity. All the forests had been derived from croplands more than one decade ago. The diversity and abundance of soil antibiotic resistance genes (ARGs), metal resistance genes (MRGs), mobile genetic elements (MGEs), and pathogens were determined by metagenomic sequencing and real-time PCR. The results showed that reforestation significantly increased soil microbial abundance and the contents of Cu, total carbon, total nitrogen, total organic carbon, and ammonium nitrogen. Nevertheless, it decreased the contents of soil Zn, Ba, nitrate nitrogen, and available phosphorus. The main soil ARGs identified in this region were vancomycin, multidrug, and bacitracin resistance genes. Reforestation significantly increased the soil ARG abundance by 62.58%, while it decreased the ARG richness by 16.50%. Reforestation exerted no significant effects on the abundance of heavy metal resistance genes and pathogens, but it doubled the abundance of MGEs. Additionally, reforestation substantially decreased the co-occurrence frequencies of ARGs with MRGs and pathogens. In contrast, the correlation between ARGs and MGEs was greatly enhanced by reforestation. Similarly, the correlations between soil ARG abundance and environmental factors were also strengthened by reforestation. These findings suggest that reforestation can substantially affect the soil antibiotic resistome and exerts overall positive effects on soil health by decreasing ARG richness, providing critical information for assessing the effects of “grain for green” project on soil health.

Combined effects of microplastics and chlortetracycline on the intestinal barrier, gut microbiota, and antibiotic resistome of Muscovy ducks (Cairina moschata)

Antibiotics and microplastics (MPs) have become critical concerns worldwide because of their increasing amount and ecological risks to ecosystems. However, how MPs exposure affects the bioaccumulation and risks of antibiotics in waterfowls remains poorly understood. In this study, Muscovy ducks were exposed to single and combined contamination with polystyrene MPs and chlortetracycline (CTC) for 56 days, and the effects of MPs on CTC bioaccumulation and their risks in duck intestines were investigated. MPs exposure reduced the bioaccumulation of CTC in the intestine and liver of ducks and increased their fecal CTC excretion. MPs exposure caused severe oxidative stress, inflammatory response, and intestinal barrier damages. Microbiome analysis showed that MPs exposure induced microbiota dysbiosis by increasing the abundance of Streptococcus and Helicobacter, the increase of which may exacerbate intestinal damages. Co-exposure to MPs and CTC alleviated the intestinal damage by regulating the gut microbiome. Metagenomic sequencing revealed that the combined exposure to MPs and CTC increased the abundance of Prevotella, Faecalibacterium, and Megamonas and incidence of total antibiotic resistance genes (ARGs), especially tetracycline ARGs subtypes in the gut microbiota. The results obtained herein provide new insights into the potential risks of polystyrene MPs and antibiotics in waterfowls living in aquatic environments.

Antibiotic resistomes in face-mask biofilm along an urban river: Multiple drivers and co-occurrence with human opportunistic pathogens

Discarded face masks from the global COVID-19 pandemic have contributed significantly to plastic pollution in surface water, whereas their potential as a reservoir for aquatic pollutants is not well understood. Herein, we conducted a field experiment along a human-impacted urban river, investigating the variations of antibiotic resistance genes (ARGs), pathogens, and water-borne contaminants in commonly-used face masks. Results showed that high-biomass biofilms formed on face masks selectively enriched more ARGs than stone biofilm (0.08–0.22 vs 0.07–0.15 copies/16 S rRNA gene copies) from bulk water, which mainly due to unique microbial communities, enhanced horizontal gene transfer, and selective pressure of accumulated contaminants based on redundancy analysis and variation partitioning analysis. Several human opportunistic pathogens (e.g., Acinetobacter, Escherichia-Shigella, Bacillus, and Klebsiella), which are considered potential ARG carriers, were also greatly concentrated in face-mask biofilms, imposing a potential threat to aquatic ecological environment and human health. Moreover, wastewater treatment plant effluents, as an important source of pollutants to urban rivers, further aggravated the abundances of ARGs and opportunistic pathogens in face-mask biofilms. Our findings demonstrated that discarded face masks provide a hotspot for the proliferation and spread of ARGs and pathogens in urban water, highlighting the urgent requirement for implementing stricter regulations in face mask disposal.

Deciphering the dynamics of metal and antibiotic resistome profiles under different metal(loid) contamination levels

Metal(loid) contaminations pose considerable threats to ecological security and public health, yet little is known about the dynamics of metal resistance genes (MRGs) and antibiotic resistance genes (ARGs) under different metal(loid) contamination levels. Here, we provided a systematic investigation of MRGs and ARGs in three zones (Zones I, II, and III) with different metal(loid) contamination levels across an abandoned sewage reservoir. More diverse MRGs and ARGs were detected from the high-contaminated Zone I and the moderate-contaminated Zone II, while the abundant MGEs (mobile genetic elements) potentially enhanced the horizontal gene transfer potential and the resistome diversity in Zone I. Particularly, resistome hosts represented by Thiobacillus, Ramlibacter, and Dyella were prevalent in Zone II, promoting the vertical gene transfer of MRGs and ARGs. The highest health risk of ARGs was predicted for Zone I (about 7.58% and 0.48% of ARGs classified into Rank I and Rank II, respectively), followed by Zone II (2.11% and 0%) and Zone III (0% and 0%). However, the ARGs co-occurring with MRGs might exhibit low proportions and low health risks (all were Rank IV) in the three zones. Overall, these findings uncovered the dynamic responses of resistomes and their hosts to different metal(loid) contamination levels, contributing to formulating accurate management and bioremediation countermeasures for various metal(loid) contaminated environments.

Swine farm groundwater is a hidden hotspot for antibiotic-resistant pathogenic Acinetobacter

Acinetobacter is present in the livestock environment, but little is known about their antibiotic resistance and pathogenic species in the farm groundwater. Here we investigated antibiotic resistance of Acinetobacter in the swine farm groundwater (JZPG) and residential groundwater (JZG) of a swine farming village, in comparison to a nearby (3.5 km) non-farming village (WTG) using metagenomic and culture-based approaches. Results showed that the abundance of antibiotic resistome in some JZG and all JZPG (~3.4 copies/16S rRNA gene) was higher than that in WTG (~0.7 copies/16S rRNA gene), indicating the influence of farming activities on both groundwater types. Acinetobacter accounted for ~95.7% of the bacteria in JZG and JZPG, but only ~8.0% in WTG. They were potential hosts of ~95.6% of the resistome in farm affected groundwater, which includes 99 ARG subtypes against 23 antibiotic classes. These ARGs were associated with diverse intrinsic and acquired resistance mechanisms, and the predominant ARGs were tetracyclines and fluoroquinolones resistance genes. Metagenomic binning analysis elucidated that non-baumannii Acinetobacter including A. oleivorans, A. beijerinckii, A. seifertii, A. bereziniae and A. modestus might pose environmental risks because of multidrug resistance, pathogenicity and massive existence in the groundwater. Antibiotic susceptibility tests showed that the isolated strains were resistant to multiple antibiotics including sulfamethoxazole (resistance ratio: 96.2%), levofloxacin (42.5%), gatifloxacin (39.0%), ciprofloxacin (32.6%), tetracycline (32.0%), doxycycline (29.0%) and ampicillin (12.0%) as well as last-resort polymyxin B (31.7%), colistin (24.1%) and tigecycline (4.1%). The findings highlight potential prevalence of groundwater-borne antibiotic-resistant pathogenic Acinetobacter in the livestock environment.

The effect of synthetic silver nanoparticles on the antibiotic resistome and the removal efficiency of antibiotic resistance genes in a hybrid filter system treating municipal wastewater

Engineered nanoparticles, including silver nanoparticles (AgNPs), are released into the environment mainly through wastewater treatment systems. Knowledge of the impact of AgNPs on the abundance and removal efficiency of antibiotic resistance genes (ARGs) in wastewater treatment facilities, including constructed wetlands (CWs), is essential in the context of public health. This study evaluated the effect of increased (100-fold) collargol (protein-coated AgNPs) and ionic Ag+ in municipal wastewater on the structure, abundance, and removal efficiency of the antibiotic resistome, integron-integrase genes, and pathogens in a hybrid CW using quantitative PCR and metagenomic approaches. The abundance of ARGs in wastewater and the removal efficiency of ARGs in the hybrid system were significantly affected by higher Ag concentrations, especially with collargol treatment, resulting in an elevated ARG discharge of system effluent into the environment. The accumulated Ag in the filters had a more profound effect on the absolute and relative abundance of ARGs in the treated water than the Ag content in the water. This study recorded significantly enhanced relative abundance values for tetracycline (tetA, tetC, tetQ), sulfonamide (sul1, sul2), and aminoglycoside (aadA) resistance genes, which are frequently found on mobile genetic elements in collargol- and, to a lesser extent, AgNO3-treated subsystems. Elevated plasmid and integron-integrase gene levels, especially intI1, in response to collargol presence indicated the substantial role of AgNPs in promoting horizontal gene transfer in the treatment system. The pathogenic segment of the prokaryotic community was similar to a typical sewage community, and strong correlations between pathogen and ARG proportions were recorded in vertical subsurface flow filters. Furthermore, the proportion of Salmonella enterica was positively related to the Ag content in these filter effluents. The effect of AgNPs on the nature and characteristics of prominent resistance genes carried by mobile genetic elements in CWs requires further investigation.

Exploring antibiotic resistance load in paddy-upland rotation fields amended with commercial organic and chemical/slow release fertilizer.

Agricultural fertilization caused the dissemination of antibiotic resistance genes (ARGs) in agro-ecological environment, which poses a global threat to crop-food safety and human health. However, few studies are known about the influence of different agricultural fertilization modes on antibiotic resistome in the paddy-upland rotation soils. Therefore, we conducted a field experiment to compare the effect of different fertilization (chemical fertilizer, slow release fertilizer and commercial organic fertilizer replacement at various rates) on soil antibiotic resistome in paddy-upland rotation fields. Results revealed that a total of 100 ARG subtypes and 9 mobile genetic elements (MGEs) occurred in paddy-upland rotation soil, among which MDR-ARGs, MLSB-ARGs and tet-ARGs were the dominant resistance determinants. Long-term agricultural fertilization remarkably facilitated the vertical accumulation of ARGs, in particular that bla ampC and tetO in relative abundance showed significant enrichment with increasing depth. It's worth noting that slow release fertilizer significantly increased soil ARGs, when comparable to manure with 20% replacing amount, but chemical fertilizer had only slight impact on soil ARGs. Fertilization modes affected soil microbial communities, mainly concentrated in the surface layer, while the proportion of Proteobacteria with the highest abundance decreased gradually with increasing depth. Furthermore, microbial community and MGEs were further proved to be essential factors in regulating the variability of ARGs of different fertilization modes by structural equation model, and had strong direct influence (λ = 0.61, p < 0.05; λ = 0. 55, p < 0.01). The results provided scientific guidance for reducing the spreading risk of ARGs and control ARG dissemination in agricultural fertilization.

Long-term field application of manure induces deep selection of antibiotic resistomes in leaf endophytes of Chinese cabbage

Antibiotic resistomes in leaf endophytes of vegetables threaten human health through the food chain. However, little is known about the ability of long-term manure fertilization to impact the deep selection of antibiotic resistance genes (ARGs) in leaf endophytes of vegetables planted in different types of soils. Here, by high-throughput quantitative PCR, we characterized the ARGs of leaf endophytes of Chinese cabbage (Brassica pekinensis (Lour.) Rupr.) grown in long-term (14 year) manure-amended acidic, neutral and calcareous soils. In total, 87 ARGs and 4 mobile genetic elements (MGEs) were detected in all the samples. Manure fertilization significantly increased the ARG numbers and normalized abundance in leaf endophytes, especially in acidic soil. Moreover, in acidic soil, manure application also led to a higher increase in the normalized abundance of opportunist and specialist ARGs, and more opportunist and specialist ARGs posed a high risk according to their risk ranks. Random forest analysis revealed that Proteobacteria and MGEs were the major drivers affecting the normalized abundance of opportunist and specialist ARGs in both acidic and neutral soils, respectively. In calcareous soil, Cyanobacteria and Actinobacteria were the most important contributors. Collectively, this study expands our knowledge about the deep selection of plant resistomes under long-term manure application.

Disparate toxicity mechanisms of parabens with different alkyl chain length in freshwater biofilms: Ecological hazards associated with antibiotic resistome

As emerging organic pollutants, parabens are of global concern because of their ubiquitous presence and adverse effects. However, few researchers have addressed the relationship between parabens' structural features and toxicity mechanisms. This study conducted theoretical calculations and laboratory exposure experiments to uncover the toxic effects and mechanisms of parabens with different alkyl chains in freshwater biofilms. The result demonstrated that parabens' hydrophobicity and lethality increased with their alkyl-chain length, whereas the possibility of chemical reactions and reactive sites were unchanged despite the alkyl-chain length alteration. Due to the hydrophobicity variation, parabens with different alkyl-chain presented different distribution patterns in cells of freshwater biofilms and consequently induced distinct toxic effects and led to diverse cell death modes. The butylparaben with longer alkyl-chain preferred to stay in the membrane and altered membrane permeability by non-covalent interaction with phospholipid, which caused cell necrosis. The methylparaben with shorter alkyl-chain preferred to enter into the cytoplasm and influence mazE gene expression by chemically reacting with biomacromolecules, thereby triggering apoptosis. The different cell death patterns induced by parabens contributed to different ecological hazards associated with antibiotic resistome. Compared with butylparaben, methylparaben was more likely to spread ARGs among microbial communities despite its lower lethality.

Phyllosphere antibiotic resistome in a natural primary vegetation across a successional sequence after glacier retreat

The spread of antibiotic-resistance genes (ARGs) has posed a significant threat to human health over the past decades. Despite the fact that the phyllosphere represents a crucial pool of microorganisms, little is known about the profile and drivers of ARGs in less human interference natural habitats. In order to minimize the influence of environmental factors, here we collected leaf samples from the early-, middle- and late-successional stages across a primary vegetation successional sequence within 2 km, to investigate how the phyllosphere ARGs develop in natural habitats. Phyllosphere ARGs were determined using high-throughput quantitative PCR. Bacterial community and leaf nutrient content were also measured to assess their contribution to the phyllosphere ARGs. A total of 151 unique ARGs were identified, covering almost all recognized major antibiotic classes. We further found that there was some stochastic and a core set of the phyllosphere ARGs during the plant community succession process, due to the fluctuant phyllosphere habitat and specific selection effect of plant individuals. The ARG abundance significantly decreased due to the reduction of the phyllosphere bacterial diversity, community complexity, and leaf nutrient content during the plant community succession process. While the closer links between soil and fallen leaf resulted in a higher ARG abundance in leaf litter than in fresh leaf. In summary, our study reveals that the phyllosphere harbors a broad spectrum of ARGs in the natural environment. These phyllosphere ARGs are driven by various environmental factors, including the plant community composition, host leaf properties, and the phyllosphere microbiome.

Mitigation of antibiotic resistome in swine manure by black soldier fly larval conversion combined with composting

The increasing prevalence of antibiotic resistance genes (ARGs) in animal manure has attracted considerable attention because of their potential contribution to the development of multidrug resistance worldwide. Insect technology may be a promising alternative for the rapid attenuation of ARGs in manure; however, the underlying mechanism remains unclear. This study aimed to evaluate the effects of black soldier fly (BSF, Hermetia illucens [L.]) larvae conversion combined with composting on ARGs dynamics in swine manure and to uncover the mechanisms through metagenomic analysis. Compared to natural composting (i.e. without BSF), BSFL conversion combined with composting reduced the absolute abundance of ARGs by 93.2 % within 28 days. The rapid degradation of antibiotics and nutrient reformulation during BSFL conversion combined with composting indirectly altered manure bacterial communities, resulting in a lower abundance and richness of ARGs. The number of main antibiotic-resistant bacteria (e.g., Prevotella, Ruminococcus) decreased by 74.9 %, while their potential antagonistic bacteria (e.g., Bacillus, Pseudomonas) increased by 128.7 %. The number of antibiotic-resistant pathogenic bacteria (e.g., Selenomonas, Paenalcaligenes) decreased by 88.3 %, and the average number of ARGs carried by each human pathogenic bacterial genus declined by 55.8 %. BSF larvae gut microbiota (e.g., Clostridium butyricum, C. bornimense) could help reduce the risk of multidrug-resistant pathogens. These results provide insight into a novel approach to mitigate multidrug resistance from the animal industry in the environment by using insect technology combined with composting, in particular in light of the global “One Health” requirements.

Unveiling the threshold values of organic and oxytetracycline loadings for nitrification recovery of a full-scale pharmaceutical wastewater treatment system

Nitrification is a crucial step in removing nitrogen from wastewater, but it often fails in industrial wastewater treatment due to the lack of knowledge regarding the impacts of environmental factors on nitrifying bacteria. This study aimed to recover the nitrification performance of a full-scale pharmaceutical wastewater treatment system inhibited by high organic and oxytetracycline (OTC) loadings. A bench-scale nitrifying reactor was used to treat OTC production wastewater under various organic loading rates (OLRs) and influent OTC concentrations, and the derived threshold values were used to guide the optimization of the full-scale system. Changes in physicochemical and biological parameters including antibiotic resistome were monitored to track the recovery process. After simultaneously reducing the OLR and influent OTC concentration to 0.111±0.025 g·COD/g·MLSS/d and 9.69±1.54 mg/L, respectively, the abundances of amoA gene and Nitrobacter quickly increased to (4.87±1.88) × 107 and (1.07±0.41) × 107 copies/L, and did not change much thereafter. Partial nitrification (about 50%) occurred 18 days later, while it took another 18 days to achieve complete nitrification. Batch nitrification test revealed that the adsorbed OTC at a concentration above 1,630 mg·OTC/kg·MLSS could inhibit the nitrification performance, which should be the main reason responsible for the slow recovery of complete nitrification considering the relatively high adsorbed OTC in activated sludge (from 23,280±2204 to 2936±221 mg·OTC/g·MLSS). The relative abundances of resistome and high-risk resistant determinants decreased by 63.2% and 67.8% along with the recovery process. This study deepened our understanding of the impacts of environmental factors on nitrification, and could guide the optimization of the pharmaceutical wastewater treatment processes.

Mariculture affects antibiotic resistome and microbiome in the coastal environment

Antibiotics are increasingly used and released into the marine environment due to the rapid development of mariculture, resulting in spread of antibiotic resistance. The pollution, distribution, and characteristics of antibiotics, antibiotic resistance genes (ARGs) and microbiomes have been investigated in this study. Results showed that 20 antibiotics were detected in Chinese coastal environment, with predominance of erythromycin-H2O, enrofloxacin and oxytetracycline. In coastal mariculture sites, antibiotic concentrations were significantly higher than in control sites, and more types of antibiotics were detected in the South than in the North of China. Residues of enrofloxacin, ciprofloxacin and sulfadiazine posed high resistance selection risks. β-Lactam, multi-drug and tetracycline resistance genes were frequently detected with significantly higher abundance in the mariculture sites. Of the 262 detected ARGs, 10, 26, and 19 were ranked as high-risk, current-risk, future-risk, respectively. The main bacterial phyla were Proteobacteria and Bacteroidetes, of which 25 genera were zoonotic pathogens, with Arcobacter and Vibrio in particular ranking in the top10. Opportunistic pathogens were more widely distributed in the northern mariculture sites. Phyla of Proteobacteria and Bacteroidetes were the potential hosts of high-risk ARGs, while the conditional pathogens were associated with future-risk ARGs, indicating a potential threat to human health.

A global perspective on compositions, risks, and ecological genesis of antibiotic resistance genes in biofilters of drinking water treatment plants

Antibiotic resistance genes (ARGs) in biofilters of drinking water treatment plants (DWTPs) are regarded to be a remarkable potential health risk to human. A global survey on ARGs in biofilters may help evaluate their risk features as a whole. This study aims to explore the compositions, risks, and ecological genesis of ARGs in the biofilters of DWTPs. In total, 98 metagenomes of DWTP biofilters were collected from Sequence Read Archive (SRA) of National center for Biotechnology Information (NCBI), and the main ARG types were recognized, with multidrug, bacitracin, and beta-lactam as the first three types. Source water types (surface water vs. groundwater) were found to significantly influence antibiotic resistome, overpassing biofilter media and locations. Although ARG abundances of surface water biofilters were approximately five times higher than that of groundwater biofilters, the risk pattern of ARGs was highly similar between surface water biofilters and groundwater biofilters, and up to 99.61% of the ARGs on average belong to the least risk and unassessed ranks, and only 0.23% the highest risk rank. Monobactam biosynthesis pathway and prodigiosin biosynthesis pathway, two antibiotics biosynthesis pathways, were observed to be positively correlated with several ARG types and total ARG abundance in samples of surface water and groundwater biofilters, respectively, suggesting their potential roles in ecological genesis of ARGs. Overall, the results of this study would deepen our understanding of the ARG risks in biofilters of DWTPs and shed light on their ecological genesis inside.

Deep sequencing reveals comprehensive insight into the prevalence, mobility, and hosts of antibiotic resistance genes in mangrove ecosystems

Mangrove receives aquaculture wastewater and urban sewage, and thus is a potential reservoir for antibiotic resistance genes (ARGs). However, there is a dearth of a comprehensive profile of ARGs in mangrove ecosystems. We used metagenomic techniques to uncover the occurrence, host range, and potential mobility of ARGs in six mangrove ecosystems in southeastern China. Based on deep sequencing data, a total of 348 ARG subtypes were identified. The abundant ARGs were associated with acriflavine, bacitracin, beta-lactam, fluoroquinolone, macrolide-lincosamide-streptogramin, and polymyxin. Resistance genes tetR, aac(6′)-Iae, aac(3)-IXa, vanRA, vanRG, and aac(3)-Ig were proposed as ARG indicators in mangrove ecosystems that can be used to evaluate the abundance of 100 other co-occurring ARGs quantitatively. Remarkably, 250 of 348 identified ARG subtypes were annotated as mobile genetic elements-associated ARGs, indicating a high potential risk of propagation of ARGs in mangrove ecosystems. By surveying the distribution of ARGs in 6281 draft genomes, more than 42 bacterial phyla were identified as the putative hosts of the ARGs. Among them, 21.97% were potentially multidrug-resistant hosts, including human and animal opportunistic pathogens. This research adds to our understanding of the distribution and spread of antibiotic resistomes in mangrove ecosystems, helping improve ARG risk assessment and management worldwide.

Elevated CO2 alleviated the dissemination of antibiotic resistance genes in sulfadiazine-contaminated soil: A free-air CO2 enrichment study

Climate change affects soil microbial communities and their genetic exchange, and subsequently modifies the transfer of antibiotic resistance genes (ARGs) among bacteria. However, how elevated CO2 impacts soil antibiotic resistome remains poorly characterized. Here, a free-air CO2 enrichment system was used in the field to investigate the responses of ARGs profiles and bacterial communities to elevated CO2 (+200 ppm) in soils amended with sulfadiazine (SDZ) at 0, 0.5 and 5 mg kg−1. Results showed that SDZ exposure induced the co-occurrence of beta-lactamase and tetracycline resistance genes, and SDZ at 5 mg kg−1 enhanced the abundance of aminoglycoside, sulfonamide and multidrug resistance genes. However, elevated CO2 weakened the effects of SDZ at 0.5 mg kg−1 following an observed reduction in the total abundance of ARGs and mobile genetic elements. Additionally, elevated CO2 significantly decreased the abundance of vancomycin resistance genes and alleviated the stimulation of SDZ on the dissemination of aminoglycoside resistance genes. Correlation analysis and structural equation models revealed that elevated CO2 could directly influence the spread of ARGs or impose indirect effects on ARGs by affecting soil properties and bacterial communities. Overall, our results furthered the knowledge of the dissemination risks of ARGs under future climate scenarios.

The gastrointestinal antibiotic resistome in pediatric leukemia and lymphoma patients.

Most children with leukemia and lymphoma experience febrile neutropenia. These are treated with empiric antibiotics that include β-lactams and/or vancomycin. These are often administered for extended periods, and the effect on the resistome is unknown. We examined the impact of repeated courses and duration of antibiotic use on the resistome of 39 pediatric leukemia and lymphoma patients. Shotgun metagenome sequences from 127 stool samples of pediatric oncology patients were examined for abundance of antibiotic resistance genes (ARGs) in each sample. Abundances were grouped by repeated courses (no antibiotics, 1-2 courses, 3+ courses) and duration (no use, short duration, long and/or mixed durationg) of β-lactams, vancomycin and "any antibiotic" use. We assessed changes in both taxonomic composition and prevalence of ARGs among these groups. We found that Bacteroidetes taxa and β-lactam resistance genes decreased, while opportunistic Firmicutes and Proteobacteria taxa, along with multidrug resistance genes, increased with repeated courses and/or duration of antibiotics. Efflux pump related genes predominated (92%) among the increased multidrug genes. While we found β-lactam ARGs present in the resistome, the taxa that appear to contain them were kept in check by antibiotic treatment. Multidrug ARGs, mostly efflux pumps or regulators of efflux pump genes, were associated with opportunistic pathogens, and both increased in the resistome with repeated antibiotic use and/or increased duration. Given the strong association between opportunistic pathogens and multidrug-related efflux pumps, we suggest that drug efflux capacity might allow the opportunistic pathogens to persist or increase despite repeated courses and/or duration of antibiotics. While drug efflux is the most direct explanation, other mechanisms that enhance the ability of opportunistic pathogens to handle environmental stress, or other aspects of the treatment environment, could also contribute to their ability to flourish within the gut during treatment. Persistence of opportunistic pathogens in an already dysbiotic and weakened gastrointestinal tract could increase the likelihood of life-threatening blood borne infections. Of the 39 patients, 59% experienced at least one gastrointestinal or blood infection and 60% of bacteremia's were bacteria found in stool samples. Antimicrobial stewardship and appropriate use and duration of antibiotics could help reduce morbidity and mortality in this vulnerable population.

Extracellular DNA includes an important fraction of high-risk antibiotic resistance genes in treated wastewaters

Wastewater treatment plants are among the main hotspots for the release of antibiotic resistance genes (ARGs) into the environment. ARGs in treated wastewater can be found in the intracellular DNA (iDNA) and in the extracellular DNA (eDNA). In this study, we investigated the fate and the distribution (either in eDNA or in iDNA) of ARGs in the treated wastewaters pre and post-disinfection by shotgun metagenomics. The richness of the intracellular resistome was found to be higher than the extracellular one. However, the latter included different high risk ARGs. About 11% of the recovered metagenome assembled genomes (MAGs) from the extracted DNA was positive for at least one ARG and, among them, several were positive for more ARGs. The high-risk ARG bacA was the most frequently detected gene among the MAGs. The disinfection demonstrated to be an important driver of the composition of the antibiotic resistomes. Our results demonstrated that eDNA represents an important fraction of the overall ARGs, including a number of high-risk ARGs, which reach the environment with treated wastewater effluents. The studied disinfections only marginally affect the whole antibiotic resistome but cause important shifts from intracellular to extracellular DNA, potentially threating human health.

Effect of composted pig manure, biochar, and their combination on antibiotic resistome dissipation in swine wastewater-treated soil

The prevalence of antibiotic resistance genes (ARGs), owing to irrigation using untreated swine wastewater, in vegetable-cultivated soils around swine farms poses severe threats to human health. Furthermore, at the field scale, the remediation of such soils is still challenging. Therefore, here, we performed field-scale experiments involving the cultivation of Brassica pekinensis in a swine wastewater-treated soil amended with composted pig manure, biochar, or their combination. Specifically, the ARG and mobile genetic element (MGE) profiles of bulk soil (BS), rhizosphere soil (RS), and root endophyte (RE) samples were examined using high-throughput quantitative polymerase chain reaction. In total, 117 ARGs and 22 MGEs were detected. Moreover, we observed that soil amendment using composted pig manure, biochar, or their combination decreased the absolute abundance of ARGs in BS and RE after 90 days of treatment. However, the decrease in the abundance of ARGs in RS was not significant. We also observed that the manure and biochar co-application showed a minimal synergistic effect. To clarify this observation, we performed network and Spearman correlation analyses and used structure equation models to explore the correlations among ARGs, MGEs, bacterial composition, and soil properties. The results revealed that the soil amendments reduced the abundances of MGEs and potential ARG-carrying bacteria. Additionally, weakened horizontal gene transfer was responsible for the dissipation of ARGs. Thus, our results indicate that composted manure application, with or without biochar, is a useful strategy for soil nutrient supplementation and alleviating farmland ARG pollution, providing a justification for using an alternative to the common agricultural practice of treating the soil using only untreated swine wastewater. Additionally, our results are important in the context of soil health for sustainable agriculture.

Coal-source acid mine drainage reduced the soil multidrug-dominated antibiotic resistome but increased the heavy metal(loid) resistome and energy production-related metabolism

A recent global scale study found that mining-impacted environments have multi-antibiotic resistance gene (ARG)-dominated resistomes with an abundance similar to urban sewage but much higher than freshwater sediment. These findings raised concern that mining may increase the risk of ARG environmental proliferation. The current study assessed how typical multimetal(loid)-enriched coal-source acid mine drainage (AMD) contamination affects soil resistomes by comparing with background soils unaffected by AMD. Both contaminated and background soils have multidrug-dominated antibiotic resistomes attributed to the acidic environment. AMD-contaminated soils had a lower relative abundance of ARGs (47.45 ± 23.34 ×/Gb) than background soils (85.47 ± 19.71 ×/Gb) but held high-level heavy metal(loid) resistance genes (MRGs, 133.29 ± 29.36 ×/Gb) and transposase- and insertion sequence-dominated mobile genetic elements (MGEs, 188.51 ± 21.81 ×/Gb), which was 56.26 % and 412.12 % higher than background soils, respectively.Procrustes analysis showed that the microbial community and MGEs exerted more influence on driving heavy metal(loid) resistome variation than antibiotic resistome. The microbial community increased energy production-related metabolism to fulfill the increasing energy needs required by acid and heavy metal(loid) resistance. Horizontal gene transfer (HGT) events primarily exchanged energy- and information-related genes to adapt to the harsh AMD environment. These findings provide new insight into the risk of ARG proliferation in mining environments.