Mobile Antibiotic Resistance Genes Research Articles

Cryptic environmental conjugative plasmid recruits a novel hybrid transposon resulting in a new plasmid with higher dispersion potential.

Cryptic conjugative plasmids are extrachromosomal DNA molecules without antibiotic-resistance genes (ARGs). Environmental bacteria carrying cryptic plasmids with a high conjugation rate threaten public health because they can capture clinically relevant ARGs and rapidly spread them to pathogenic bacteria. However, the mechanism to recruit ARG by cryptic conjugative plasmids in environmental bacteria has not been observed experimentally. Here, we document the first translocation of a transposon with multiple clinically relevant ARGs to a cryptic environmental conjugative plasmid. The new multidrug-resistant conjugative plasmid has a conjugation rate that is two orders of magnitude higher than the original plasmid that carries the ARG (i.e., the new plasmid from the environment can spread ARG more than two orders of magnitude faster). Our work illustrates the importance of studying the mobilization of ARGs in environmental bacteria. It sheds light on how cryptic conjugative plasmids recruit ARGs, a phenomenon at the root of the antibiotic crisis.

Effect of polyethylene microplastics on antibiotic resistance genes: A comparison based on different soil types and plant types

Microplastics (MPs) and antibiotic resistance genes (ARGs) are two types of contaminants that are widely present in the soil environment. MPs can act as carriers of microbes, facilitating the colonization and spread of ARGs and thus posing potential hazards to ecosystem safety and human health. In the present study, we explored the microbial networks and ARG distribution characteristics in different soil types (heavy metal (HM)-contaminated soil and agricultural soil planted with different plants: Bidens pilosa L., Ipomoea aquatica F., and Brassica chinensis L.) after the application of MPs and evaluated environmental factors, potential microbial hosts, and ARGs. The microbial communities in the three rhizosphere soils were closely related to each other, and the modularity of the microbial networks was greater than 0.4. Moreover, the core taxa in the microbial networks, including Actinobacteriota, Proteobacteria, and Myxococcota, were important for resisting environmental stress. The ARG resistance mechanisms were dominated by antibiotic efflux in all three rhizosphere soils. Based on the annotation results, the MP treatments induced changes in the relative abundance of microbes carrying ARGs, and the G1–5 treatment significantly increased the abundance of MuxB in Verrucomicrobia, Elusimicrobia, Actinobacteria, Planctomycetes, and Acidobacteria. Path analysis showed that changes in MP particle size and dosage may indirectly affect soil enzyme activities by changing pH, which affects microbes and ARGs. We suggest that MPs may provide surfaces for ARG accumulation, leading to ARG enrichment in plants. In conclusion, our results demonstrate that MPs, as potentially persistent pollutants, can affect different types of soil environments and that the presence of ARGs may cause substantial environmental risks.

Co-localization of antibiotic resistance genes is widespread in the infant gut microbiome and associates with an immature gut microbial composition

BackgroundIn environmental bacteria, the selective advantage of antibiotic resistance genes (ARGs) can be increased through co-localization with genes such as other ARGs, biocide resistance genes, metal resistance genes, and virulence genes (VGs). The gut microbiome of infants has been shown to contain numerous ARGs, however, co-localization related to ARGs is unknown during early life despite frequent exposures to biocides and metals from an early age.ResultsWe conducted a comprehensive analysis of genetic co-localization of resistance genes in a cohort of 662 Danish children and examined the association between such co-localization and environmental factors as well as gut microbial maturation. Our study showed that co-localization of ARGs with other resistance and virulence genes is common in the early gut microbiome and is associated with gut bacteria that are indicative of low maturity. Statistical models showed that co-localization occurred mainly in the phylum Proteobacteria independent of high ARG content and contig length. We evaluated the stochasticity of co-localization occurrence using enrichment scores. The most common forms of co-localization involved tetracycline and fluoroquinolone resistance genes, and, on plasmids, co-localization predominantly occurred in the form of class 1 integrons. Antibiotic use caused a short-term increase in mobile ARGs, while non-mobile ARGs showed no significant change. Finally, we found that a high abundance of VGs was associated with low gut microbial maturity and that VGs showed even higher potential for mobility than ARGs.ConclusionsWe found that the phenomenon of co-localization between ARGs and other resistance and VGs was prevalent in the gut at the beginning of life. It reveals the diversity that sustains antibiotic resistance and therefore indirectly emphasizes the need to apply caution in the use of antimicrobial agents in clinical practice, animal husbandry, and daily life to mitigate the escalation of resistance.CR8ihftTez1HgYaufYhgHyVideo

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.

Promoter regulatory mode evolution enhances the high multidrug resistance of tmexCD1-toprJ1.

Antibiotic resistance could rapidly emerge from acquiring the mobile antibiotic resistance genes, which are commonly evolved from an intrinsic gene. The emergence of the plasmid-borne mobilized efflux pump gene cluster tmexCD1-toprJ1 renders the last-resort antibiotic tigecycline ineffective, although its evolutionary mechanism remains unclear. In this study, we investigate the regulatory mechanisms of the progenitor NfxB-MexCD-OprJ, a chromosomally encoded operon that does not mediate antibiotic resistance in the wild-type version, and its homologs, TNfxB1-TMexCD1-TOprJ1 mediating high-level tigecycline resistance, and TNfxB3-TMexCD3-TOprJ1. Mechanistic studies demonstrated that in nfxB-mexCD-oprJ, MexCD expression was under a weaker promoter, PmexC and inhibited by a strong repressor NfxB. For tmexCD1-toprJ1, TMexCD1 was highly expressed owing to the presence of a strong promoter, PtmexC1, and an inactive suppressor, TNfxB1, with a T39R mutation that rendered it unable to bind to promoter DNA. In tnfxB3-tmexCD3-toprJ1b, TMexCD3 expression was intermediate because of the local regulator TNfxB3, which binds to two inverted repeat sequences of PtmexC. Additionally, TNfxB3 exhibited lower protein expression and weaker DNA binding affinity than its ancestor NfxB, together with their promoter activities difference explaining the different expression levels of tmexCD-toprJ homologs. Distinct fitness burdens on these homologs-carrying bacteria were observed due to the corresponding expression level, which might be associated with their global prevalence. In summary, our data depict the mechanisms underlying the evolution and dissemination of an important mobile antibiotic resistance gene from an intrinsic chromosomal gene.IMPORTANCEAs antibiotic resistance seriously challenges global health, tigecycline is one of the few effective drugs in the pipeline against infections caused by multidrug-resistant pathogens. Our previous work identified a novel tigecycline resistance efflux pump gene cluster tmexCD1-toprJ1 in animals and humans, together with its various variants, a rising clinical concern. Herein, this study focused on how the local regulation modes of tmexCD1-toprJ1 evolved to a highly expressed efflux pump. Through comparative analysis between three tnfxB-tmexCD-toprJ homologs and their progenitor nfxB-mexCD-oprJ, modes, we demonstrated the evolutionary dynamics from a chromosomal silent gene to an active state. We found the de-repression of the local regulator and an increase of the promoter activity work together to promote a high production of drug efflux machines and enhance multidrug resistance. Our findings revealed that TMexCD1-TOprJ1 adopts a distinct evolutionary path to achieve higher multidrug resistance, urgently needing tight surveillance.

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.

Composting reduces the risks of resistome in beef cattle manure at the transcriptional level.

Transcriptomic evidence is needed to determine whether composting is more effective than conventional stockpiling in mitigating the risk of resistome in livestock manure. The objective of this study is to compare composting and stockpiling for their effectiveness in reducing the risk of antibiotic resistance in beef cattle manure. Samples collected from the center and the surface of full-size manure stockpiling and composting piles were subject to metagenomic and metatranscriptomic analyses. While the distinctions in resistome between stockpiled and composted manure were not evident at the DNA level, the advantages of composting over stockpiling were evident at the transcriptomic level in terms of the abundance of antibiotic resistance genes (ARGs), the number of ARG subtypes, and the prevalence of high-risk ARGs (i.e., mobile ARGs associated with zoonotic pathogens). DNA and transcript contigs show that the pathogen hosts of high-risk ARGs included Escherichia coli O157:H7 and O25b:H4, Klebsiella pneumoniae, and Salmonella enterica. Although the average daily temperatures for the entire composting pile exceeded 55°C throughout the field study, more ARG and ARG transcripts were removed at the center of the composting pile than at the surface. This work demonstrates the advantage of composting over stockpiling in reducing ARG risk in active populations in beef cattle manure.IMPORTANCEProper treatment of manure before land application is essential to mitigate the spread of antibiotic resistance in the environment. Stockpiling and composting are two commonly used methods for manure treatment. However, the effectiveness of composting in reducing antibiotic resistance in manure has been debated. This work compared the ability of these two methods to reduce the risk of antibiotic resistance in beef cattle manure. Our results demonstrate that composting reduced more high-risk resistance genes at the transcriptomic level in cattle manure than conventional stockpiling. This finding not only underscores the effectiveness of composting in reducing antibiotic resistance in manure but also highlights the importance of employing RNA analyses alongside DNA analyses.

NO3− as an electron acceptor elevates antibiotic resistance gene and human bacterial pathogen risks in managed aquifer recharge (MAR): A comparison with O2

Managed aquifer recharge (MAR) stands out as a promising strategy for ensuring water resource sustainability. This study delves into the comparative impact of nitrate (NO3−) and oxygen (O2) as electron acceptors in MAR on water quality and safety. Notably, NO3−, acting as an electron acceptor, has the potential to enrich denitrifying bacteria, serving as hosts for antibiotic resistance genes (ARGs) and enriching human bacterial pathogens (HBPs) compared to O2. However, a direct comparison between NO3− and O2 remains unexplored. This study assessed risks in MAR effluent induced by NO3− and O2, alongside the presence of the typical refractory antibiotic sulfamethoxazole. Key findings reveal that NO3− as an electron acceptor resulted in a 2 times reduction in dissolved organic carbon content compared to O2, primarily due to a decrease in soluble microbial product production. Furthermore, NO3− significantly enriched denitrifying bacteria, the primary hosts of major ARGs, by 747%, resulting in a 66% increase in the overall abundance of ARGs in the effluent of NO3− MAR compared to O2. This escalation was predominantly attributed to horizontal gene transfer mechanisms, as evidenced by a notable 78% increase in the relative abundance of mobile ARGs, alongside a minor 27% rise in chromosomal ARGs. Additionally, the numerous denitrifying bacteria enriched under NO3− influence also belong to the HBP category, resulting in a significant 114% increase in the abundance of all HBPs. The co-occurrence of ARGs and HBPs was also observed to intensify under NO3− influence. Thus, NO3− as an electron acceptor in MAR elevates ARG and HBP risks compared to O2, potentially compromising groundwater quality and safety.

Horizontal transmission of a multidrug-resistant IncN plasmid isolated from urban wastewater

Wastewater treatment plants (WWTPs) are considered reservoirs of antibiotic resistance genes (ARGs). Given that plasmid-mediated horizontal gene transfer plays a critical role in disseminating ARGs in the environment, it is important to inspect the transfer potential of transmissible plasmids to have a better understanding of whether these mobile ARGs can be hosted by opportunistic pathogens and should be included in One Health’s considerations. In this study, we used a fluorescent-reporter-gene based exogenous isolation approach to capture extended-spectrum beta-lactamases encoding mobile determinants from sewer microbiome samples that enter an urban water system (UWS) in Denmark. After screening and sequencing, we isolated a ∼73 Kbp IncN plasmid (pDK_DARWIN) that harboured and expressed multiple ARGs. Using a dual fluorescent reporter gene system, we showed that this plasmid can transfer into resident urban water communities. We demonstrated the transfer of pDK_DARWIN to microbiome members of both the sewer (in the upstream UWS compartment) and wastewater treatment (in the downstream UWS compartment) microbiomes. Sequence similarity search across curated plasmid repositories revealed that pDK_DARWIN derives from an IncN backbone harboured by environmental and nosocomial Enterobacterial isolates. Furthermore, we searched for pDK_DARWIN sequence matches in UWS metagenomes from three countries, revealing that this plasmid can be detected in all of them, with a higher relative abundance in hospital sewers compared to residential sewers. Overall, this study demonstrates that this IncN plasmid is prevalent across Europe and an efficient vector capable of disseminating multiple ARGs in the urban water systems.

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.

Effects of treatments and distribution on microbiome and antibiotic resistome from source to tap water in three Chinese geographical regions based on metagenome assembly

Antibiotic resistance genes (ARGs) represent emerging environmental pollutants that present health risks. Drinking water supply systems (DWSSs), including sources to tap water, play crucial roles in the dissemination and propagation of ARGs. However, there was a paucity of knowledge on the relative abundance, diversity, mobility, and pathogenic hosts of ARGs in DWSSs from source to tap. Therefore, the effects of treatments and distributions on the microbial community and ARGs from three geographical regions (downstream areas of the Yellow, Yangtze, and Pearl Rivers) were elucidated in the present study. Treatment processes lowered the complexity of the microbial community network, whereas transportation increased it. The assembly mechanisms of the microbial community and antibiotic resistome were primarily driven by stochastic processes. Distribution greatly increased the contribution of stochastic processes. Multidrug ARGs (for example, multidrug transporter and adeJ) and bacitracin ARG (bacA) were the primary mobile ARGs in drinking water, as identified by the metagenomic assembly. Achromobacter xylosoxidans, Acinetobacter calcoaceticus, and Acinetobacter junii harbored diverse multidrug ARGs and mobile genetic elements (MGEs) (recombinases, integrases, and transposases) as potential pathogens and were abundant in the disinfected water. Environmental factors, including pH, chlorine, latitude, longitude, and temperature, influenced the ARG abundance by directly regulating the MGEs and microbial community diversity. This study provides critical information on the fate, mobility, host pathogenicity, and driving factors of ARGs in drinking water, which is conducive to ARG risk assessment and management to provide high-quality drinking water to consumers.

Prioritization of Critical Factors for Surveillance of the Dissemination of Antibiotic Resistance in Pseudomonas aeruginosa: A Systematic Review.

Pseudomonas aeruginosa is the primary opportunistic human pathogen responsible for a range of acute and chronic infections; it poses a significant threat to immunocompromised patients and is the leading cause of morbidity and mortality for nosocomial infections. Its high resistance to a diverse array of antimicrobial agents presents an urgent health concern. Among the mechanisms contributing to resistance in P. aeruginosa, the horizontal acquisition of antibiotic resistance genes (ARGs) via mobile genetic elements (MGEs) has gained recognition as a substantial concern in clinical settings, thus indicating that a comprehensive understanding of ARG dissemination within the species is strongly required for surveillance. Here, two approaches, including a systematic literature analysis and a genome database survey, were employed to gain insights into ARG dissemination. The genome database enabled scrutinizing of all the available sequence information and various attributes of P. aeruginosa isolates, thus providing an extensive understanding of ARG dissemination within the species. By integrating both approaches, with a primary focus on the genome database survey, mobile ARGs that were linked or correlated with MGEs, important sequence types (STs) carrying diverse ARGs, and MGEs responsible for ARG dissemination were identified as critical factors requiring strict surveillance. Although human isolates play a primary role in dissemination, the importance of animal and environmental isolates has also been suggested. In this study, 25 critical mobile ARGs, 45 critical STs, and associated MGEs involved in ARG dissemination within the species, are suggested as critical factors. Surveillance and management of these prioritized factors across the One Health sectors are essential to mitigate the emergence of multidrug-resistant (MDR) and extensively resistant (XDR) P. aeruginosa in clinical settings.

Insights into the reduction of antibiotic-resistant bacteria and mobile antibiotic resistance genes by black soldier fly larvae in chicken manure

The increasing prevalence of antibiotic-resistant bacteria (ARB) from animal manure has raised concerns about the potential threats to public health. The bioconversion of animal manure with insect larvae, such as the black soldier fly larvae (BSFL, Hermetia illucens [L.]), is a promising technology for quickly attenuating ARB while also recycling waste. In this study, we investigated BSFL conversion systems for chicken manure. Using metagenomic analysis, we tracked ARB and evaluated the resistome dissemination risk by investigating the co-occurrence of antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and bacterial taxa in a genetic context. Our results indicated that BSFL treatment effectively mitigated the relative abundance of ARB, ARGs, and MGEs by 34.9%, 53.3%, and 37.9%, respectively, within 28 days. Notably, the transferable ARGs decreased by 30.9%, indicating that BSFL treatment could mitigate the likelihood of ARG horizontal transfer and thus reduce the risk of ARB occurrence. In addition, the significantly positive correlation links between antimicrobial concentration and relative abundance of ARB reduced by 44.4%. Moreover, using variance partition analysis (VPA), we identified other bacteria as the most important factor influencing ARB, explaining 20.6% of the ARB patterns. Further analysis suggested that antagonism of other bacteria on ARB increased by 1.4 times, while nutrient competition on both total nitrogen and crude fat increased by 2.8 times. Overall, these findings provide insight into the mechanistic understanding of ARB reduction during BSFL treatment of chicken manure and provide a strategy for rapidly mitigating ARB in animal manure.

Hazardous risk of antibiotic resistance genes: Host occurrence, distribution, mobility and vertical transmission from different environments to corn silage

Antibiotic resistance genes (ARGs) are one of the emerging contaminants posing a great deal of hazardous risk to public health. This study employed metagenomics and deciphered the potential risk of the antibiotic resistome and their vertical transfer to ensiled whole-crop corn silage harvested from six climate zones: 1. Warm temperate-fully humid-hot summer (Cfa), 2. Arid-desert-cold arid (BWk), 3. Snow-desert-cold summer (Dwc), 4. Snow-desert-hot summer (Dwa), 5. Arid-steppe-cold arid (BSk), and 6. Equatorial-desert (Aw) based on the Köppen-Geiger climate classification in China. The findings demonstrate a high diversity of ARGs, which is related to the drug classes of tetracycline, ciprofloxacin, lincosamide, fosfomycin, and beta lactam. Resistome variations are mostly related to variations in microbial composition and fermentation characteristics of the silages from different climate zones, which are indirectly influenced by environmental conditions. The most dominating ARGs in corn silage were tetM, acrA, H-NS, lnuA, emrR, and KpnG, which is primarily hosted by Klebsiella and Lactobacilli. There were 5 high-risk ARGs (tetM, bacA, SHV-1, dfrA17, and QnrS1) in silage from different climate zones, and the tetM was the most prevalent high-risk ARG. However, throughout the ensiling process, the abundance of ARGs, and mobile ARGs were reduced. The resistome contamination in silage from Tibet (Dwc) with high altitude and harsh environment was relatively low due to the low variety and abundance of ARGs, the low abundance of mobile ARGs and high-risk ARGs. In addition, most of the bacteria responsible for the silage fermentation were also found to be the hosts to the ARGs, although their abundance decreased after 90 d of silage fermentation. Hence, we alert the existence of ARGs-related biosafety risk in silages and call for more attention to the silage ARGs, their hosts, and mobile genetic elements in order to curtail their possible risk to public health.

Anthropogenic contributions to antibiotic resistance gene pollution in household drinking water revealed by machine-learning-based source-tracking

Although the presence of antibiotic resistance genes (ARGs) in drinking water and their potential horizontal gene transfer to pathogenic microbes are known to pose a threat to human health, their pollution levels and potential anthropogenic sources are poorly understood. In this study, broad-spectrum ARG profiling combined with machine-learning-based source classification SourceTracker was performed to investigate the pollution sources of ARGs in household drinking water collected from 95 households in 47 cities of eight countries/regions. In total, 451 ARG subtypes belonging to 19 ARG types were detected with total abundance in individual samples ranging from 1.4 × 10−4 to 1.5 × 10° copies per cell. Source tracking analysis revealed that many ARGs were highly contributed by anthropogenic sources (37.1%), mainly wastewater treatment plants. The regions with the highest detected ARG contribution from wastewater (∼84.3%) used recycled water as drinking water, indicating the need for better ARG control strategies to ensure safe water quality in these regions. Among ARG types, sulfonamide, rifamycin and tetracycline resistance genes were mostly anthropogenic in origin. The contributions of anthropogenic sources to the 20 core ARGs detected in all of the studied countries/regions varied from 36.6% to 84.1%. Moreover, the anthropogenic contribution of 17 potential mobile ARGs identified in drinking water was significantly higher than other ARGs, and metagenomic assembly revealed that these mobile ARGs were carried by diverse potential pathogens. These results indicate that human activities have exacerbated the constant input and transmission of ARGs in drinking water. Our further risk classification framework revealed three ARGs (sul1, sul2 and aadA) that pose the highest risk to public health given their high prevalence, anthropogenic sources and mobility, facilitating accurate monitoring and control of anthropogenic pollution in drinking water.

Flooding drives the temporal turnover of antibiotic resistance gene in manure-amended soil–water continuum

Rice paddy soil is a hotspot of antibiotic resistance genes (ARGs) due to the application of organic fertilizers. However, the temporal dynamics of ARGs in rice paddy soil and its flooded water during the growing season remain underexplored. In this study, a microcosm experiment was conducted to explore the ARG profiles in a long term (130 days) flooded two-phase manure-amended soil–water system. By using high-throughput quantitative PCR array, a total of 23–98 and 34–85 ARGs were detected in the soil and overlying water, respectively. Regression analysis exhibited significant negative correlations between ARG profile similarities and flooding duration, indicating that flooding significantly altered the resistome (P < 0.001). This finding was validated by the increased ARG abundance in the soil and the overlying water, for example, after 130 days flooding, the abundance of ARGs in CK soil was increased from 0.03 to 1.20 copies per 16S rRNA. The PCoA analysis further suggested pig manure application resulted in distinct ARG profiles in the soil–water continuum compared with those of the non-amended control (Adonis, P < 0.05). The Venn diagram showed that all ARGs detected in the pig manure were present in the treated soil. Twelve ARGs (e.g., sul1) were shared among the pig manure, manure-amended soil, and overlying water, indicating that certain manure- or soil-borne ARGs were readily dispersed from the soil to the overlying water. Moreover, the enhanced relationships between the ARGs and mobile genetic elements in pig manure applied soil–water continuum indicate that the application of organic matter could accelerate the emergence and dissemination of ARGs. These findings suggested that flooding represents a crucial pathway for dispersal of ARGs from the soil to the overlying water. Identification of highly mobile ARGs in the soil–water continuum is essential for assessing their potential risk to human health and promoting the development of sustainable agricultural practices to mitigate their spread.

Machine learning and metagenomics reveal shared antimicrobial resistance profiles across multiple chicken farms and abattoirs in China.

China is the largest global consumer of antimicrobials and improving surveillance methods could help to reduce antimicrobial resistance (AMR) spread. Here we report the surveillance of ten large-scale chicken farms and four connected abattoirs in three Chinese provinces over 2.5 years. Using a data mining approach based on machine learning, we analysed 461 microbiomes from birds, carcasses and environments, identifying 145 potentially mobile antibiotic resistance genes (ARGs) shared between chickens and environments across all farms. A core set of 233 ARGs and 186 microbial species extracted from the chicken gut microbiome correlated with the AMR profiles of Escherichia coli colonizing the same gut, including Arcobacter, Acinetobacter and Sphingobacterium, clinically relevant for humans, and 38 clinically relevant ARGs. Temperature and humidity in the barns were also correlated with ARG presence. We reveal an intricate network of correlations between environments, microbial communities and AMR, suggesting multiple routes to improving AMR surveillance in livestock production.

Occurrence, fate and potential health risks of antibiotic resistomes in a constructed wetlands-reservoir ecosystem for drinking water source improvement

The development of effective and feasible engineering technologies to control the transmission of antibiotic resistance genes (ARGs) and pathogenic antibiotic-resistant bacteria (PARB) form drinking water sources is urgently needed for ensuring drinking water safety. In this study, metagenomic analysis was applied to systematically explore the full profiles, removal, and potential health risks of antibiotic resistomes in a large constructed wetlands-reservoir ecosystem (CWs-R) for drinking water source improvement. A total of 343 ARG subtypes belonging to 18 ARG types were identified from water and sediment samples in the CWs-R ecosystem, with an average abundance of 0.339 copies/cell, and bacitracin and multidrug resistance genes were the predominant ARG types in the water and sediment, respectively. The CWs-R ecosystem showed an excellent removal efficiency of ARGs and mobile genetic elements (MGEs) in water, with the total removal rate reaching 64.82 % and 77.09 %, respectively, among which the emergent plant zone and ecological storage unit played major roles. The metagenomic assembly tracked many mobile ARGs and opportunistic pathogens in the CWs-R ecosystem and identified 19 contigs as ARG-carrying pathogens, including Staphylococcus aureus, Salmonella enterica, Escherichia coli, and Klebsiella pneumonia. Overall, the CWs-R ecosystem has an important role in reducing the potential public health risks posed by antibiotic resistomes in drinking water sources but still cannot fully eliminate them. Therefore, we further classified water and sediment samples in the CWs-R ecosystem and identified potential ARGs and PARB indicators based on the metagenomic analysis results by considering the potential for horizontal transfer of ARGs to opportunistic pathogens. Taken together, this work demonstrates the CWs-R ecosystem as an economical and feasible engineering technology to reduce the dissemination of antibiotic resistomes in the drinking water source, provides useful information for monitoring and controlling antibiotic resistance in similar water sources, and ensures biosafety of drinking water.

Antibiotic resistome associated with inhalable bioaerosols from wastewater to atmosphere: Mobility, bacterial hosts, source contributions and resistome risk

Antibiotic resistome can be carried by the bioaerosols and propagate from wastewater treatment plants (WWTPs) to the atmosphere, but questions remain regarding their mobility, bacterial hosts, source, and resistome risk. Here, fine particulate matter (PM2.5) was collected within and around a large WWTP and analyzed by the metagenomic assembly and binning. PM2.5 was discovered with increasing enrichment of total antibiotic resistance genes (ARGs), potentially mobile ARGs, and antibiotic-resistant bacteria (ARB) along the WWTP-downwind-upwind gradient. Some ARGs were found to be flanked by certain mobile genetic elements and generally mediated by plasmids in WWTP-PM2.5. Totally, 198 metagenome assembled genomes assigning to seven phyla were identified as the ARB, and a contig-based analysis indicated that 32 pathogens were revealed harboring at least two ARGs. Despite disparate aerosolization potentials of ARGs or ARB at different WWTP units, high resistome risks were found, along with the dominant contribution of wastewater for airborne ARGs (44.79–62.82%) and ARB (35.03–40.10%). Among the detected WWTP matrices, the sludge dewatering room was characterized by the highest resistome risk associated with PM2.5. This study underscores the dispersion of ARGs and ARB from WWTPs to the atmosphere and provides a reference for managing risks of antibiotic resistance.

A survey on computational strategies for genome-resolved gut metagenomics.

Recovering high-quality metagenome-assembled genomes (HQ-MAGs) is critical for exploring microbial compositions and microbe-phenotype associations. However, multiple sequencing platforms and computational tools for this purpose may confuse researchers and thus call for extensive evaluation. Here, we systematically evaluated a total of 40 combinations of popular computational tools and sequencing platforms (i.e. strategies), involving eight assemblers, eight metagenomic binners and four sequencing technologies, including short-, long-read and metaHiC sequencing. We identified the best tools for the individual tasks (e.g. the assembly and binning) and combinations (e.g. generating more HQ-MAGs) depending on the availability of the sequencing data. We found that the combination of the hybrid assemblies and metaHiC-based binning performed best, followed by the hybrid and long-read assemblies. More importantly, both long-read and metaHiC sequencings link more mobile elements and antibiotic resistance genes to bacterial hosts and improve the quality of public human gut reference genomes with 32% (34/105) HQ-MAGs that were either of better quality than those in the Unified Human Gastrointestinal Genome catalog version 2 or novel.