Mobile genetic elements facilitate the transmission of antibiotic resistance genes in multidrug-resistant Enterobacteriaceae from duck farms

Multidrug-resistant(MDR)Enterobacteriaceae critically threaten duck farming and public health.The phenotypes,genotypes,and associated mobile genetic elements(MGEs)of MDR Enterobacteriaceae isolated from 6 duck farms in Zhejiang Province,China,were investigated.A total of 215 isolates were identified as Escherichia coli(64.65%),Klebsiella pneumoniae(12.09%),Proteus mirabilis(10.23%),Salmonella(8.84%),and Enterobacter cloacae(4.19%).Meanwhile,all isolates were resistant to at least two antibiotics.Most isolates carried tet(A)(85.12%),blaTEM(78.60%)and sul1(67.44%)resistance genes.Gene co-occurrence analysis showed that the resistance genes were associated with IS26 and integrons.A conjugative IncFII plasmid pSDM004 containing all the above MGEs was detected in Proteus mirabilis isolate SDM004.This isolate was resistant to 18 antibiotics and carried the blaNDM-5 gene.MGEs,especially plasmids,are the primary antibiotic resistance gene transmission route in duck farms.These findings provide a theoretical basis for the rational use of antibiotics in farms which are substantial for evaluating public health and food safety.

Performance Parameters:Demobilization Antibiotic Resistant Bacteria(ARB)and Carrying Genes(ARG)in Wastewater Disinfection

The UV irradiation is used for removing Antibiotic Resistant Bacteria(ARB)and Antibiotic Resistance Genes(ARG)from wastewater treatment.Bacteriophages are viruses that infect within bacteria,are recognized for bacterial control.The influence of some parameters in quantification and performance influencing of pathogen demobilization could be considered in disinfection of wastewater.The comparison of Polyvalent phage(NE1)versus Coliphage(NE4)in suppressing a bacterium Escherichia coli(NDM-1:b-lactam-resistant)with UV irradiation was observed the efficacy in reduction of cells in the disinfection and parameter process.The results with the effect of UV-C irradiation on NDM-1 infected with 1%of NE4 showed a decrease of cells from 8×10^(6)to 2×10^(5)in 60 min with UV-C dose.The NDM1(E.coli)was infected with 1%of NE4(Polyvalent Phage)under magnetic stirring for 1 h,the cells count was 8×10^(6).After 1 h in UV-C e×posure,the cells number reached 3×10^(5).The NDM1 that was e×posed in 1 h of UV-C irradiation and then was infected with 1%of NE4.Cells counting were done 24 h after this procedure.These cells were e×posed in UV-C and showed a reduction in the number of cells from 1×10^(8)to 4×10^(5)after 60 min.The results indicate that bacteriophages can mitigate bacteria species,and combined the conventional water disinfection technologies that can support the microbial safety control strategies.

Trifunctional Cu-Mesh/Cu_(2)O@FeO Nanoarrays for Highly Efficient Degradation of Antibiotic, Inactivation of Antibiotic-Resistant Bacteria, and Damage of Antibiotics Resistance Genes

Trifunctional Cu-mesh/Cu_(2)O@FeO nanoarrays heterostructure is designed and fabricated by integrating CuCu_(2)O@FeO nanoarrays onto Cu-mesh(CM)via an in situ growth and phase transformation process.It is successfully applied to efficiently mitigate the antibiotic pollution,including degradation of antibiotics,inactivation of antibiotic-resistant bacteria(ARB),and damage of antibiotics resistance genes(ARGs).Under visible-light irradiation,CM/CuCu_(2)O@FeO nanoarrays exhibit a superior degradation efficiency on antibiotics(e.g.,up to 99%in 25 min for tetracycline hydrochloride,TC),due to the generated reactive oxygen species(ROS),especially the dominant·O^(2−).It can fully inactivate E.coli(HB101)with initial number of~108 CFU mL^(−1) in 10 min,which is mainly attributed to the synergistic effects of 1D nanostructure,dissolved metal ions,and generated ROS.Meanwhile,it is able to damage ARGs after 180 min of photodegradation,including tetA(vs TC)of 3.3 log 10,aphA(vs kanamycin sulfate,KAN)of 3.4 log 10,and tnpA(vs ampicillin,AMP)of 4.4 log 10,respectively.This work explores a green way for treating antibiotic pollution under visible light.

Analysis of antibiotic resistance genes and their effects on marine environment

The application of overgrowth antibiotic resistance genes in marine research was discussed.Using the China National Knowledge Infrastructure(CNKI)network database to retrieve papers on resistance genes(n=30627)and antibiotic resistance genes(n=3277),the published trends,subject headings and other aspects of bibliometrics were analyzed.Literature in this field has increased rapidly in the past 38 years.55.3%of the papers were published in the last ten years,31.2%of the papers were published in the last five years.The relevance of research topics has increased in recent years,the current research mainly focus on antibiotics,resistance gene,antibiotic resistance gene,and drug resistance.Literature analysis is helpful to understand the overall development trend of overgrowth antibiotic resistance genes in marine research.

UV-Based Advanced Oxidation Processes for Antibiotic Resistance Control: Efficiency, Influencing Factors, and Energy Consumption

Antibiotic resistant bacteria(ARB)with antibiotic resistance genes(ARGs)can reduce or eliminate the effectiveness of antibiotics and thus threaten human health.The United Nations Environment Programme considers antibiotic resistance the first of six emerging issues of concern.Advanced oxidation processes(AOPs)that combine ultraviolet(UV)irradiation and chemical oxidation(primarily chlorine,hydrogen peroxide,and persulfate)have attracted increasing interest as advanced water and wastewater treatment technologies.These integrated technologies have been reported to significantly elevate the efficiencies of ARB inactivation and ARG degradation compared with direct UV irradiation or chemical oxidation alone due to the generation of multiple reactive species.In this study,the performance and underlying mechanisms of UV/chlorine,UV/hydrogen peroxide,and UV/persulfate processes for controlling ARB and ARGs were reviewed based on recent studies.Factors affecting the process-specific efficiency in controlling ARB and ARGs were discussed,including biotic factors,oxidant dose,UV fluence,pH,and water matrix properties.In addition,the cost-effectiveness of the UV-based AOPs was evaluated using the concept of electrical energy per order.The UV/chlorine process exhibited a higher efficiency with lower energy consumption than other UV-based AOPs in the wastewater matrix,indicating its potential for ARB inactivation and ARG degradation in wastewater treatment.Further studies are required to address the trade-off between toxic byproduct formation and the energy efficiency of the UV/chlorine process in real wastewater to facilitate its optimization and application in the control of ARB and ARGs.

Fate and Behavior of Tetracycline Resistance Genes in Activated Carbon Adsorption

The accessibility of tetracycline resistance gene (tetG) into the pores of activated carbon (AC), as well as the impact of the pore size distribution (PSD) of AC on the uptake capacity of tetG, were investigated using eight types of AC (four coal-based and four wood-based). AC showed the capability to admit tetG and the average reduction of tetG for coal-based and wood-based ACs at the AC dose of 1 g·L-1 was 3.12 log and 3.65 log, respectively. The uptake kinetic analysis showed that the uptake of the gene followed the pseudo-second-order kinetics reaction, and the uptake rate constant for the coal-based and wood-based ACs was in the range of 5.97 × 10-12 - 4.64 × 10-9 and 7.02 × 10-11 - 1.59 × 10-8 copies·mg-1·min-1, respectively. The uptake capacity analysis by fitting the obtained experiment data with the Freundlich isotherm model indicated that the uptake constant (KF) values were 1.71 × 103 - 8.00 × 109 (copies·g-1)1-1/n for coal-based ACs and 7.00 × 108 - 3.00 × 1010 (copies·g-1)1-1/n for wood-based ones. In addition, the correlation analysis between KF values and pore volume as well as pore surface at different pore size regions of ACs showed that relatively higher positive correlation was found for pores of 50 - 100 Å, suggesting ACs with more pores in this size region can uptake more tetG. The findings of this study are valuable as reference for optimizing the adsorption process regarding antibiotic resistance-related concerns in drinking water treatment.

Identification and characterization of integron mediated antibiotic resistance in pentachlorophenol degrading bacterium isolated from the chemostat

A bacterial consortium was developed by continuous enrichment of microbial population isolated from sediment core of pulp and paper mill effluent in mineral salts medium（MSM） supplemented with pentachlorophenol（PCP） as sole source of carbon and energy in the chemostat.The consortia contained three bacterial strains.They were identified as Escherichia coli,Pseudomonas aeruginosa and Acinetobacter sp.by 16S rRNA gene sequence analysis.Acinetobacter sp.readily degraded PCP through the formation of tetrachloro-p-hydroquinone（TecH）,2-chloro-1,4-benzenediol and products of ortho ring cleavage detected by gas chromatograph/mass spectrometer（GC-MS）.Out of the three acclimated PCP degrading bacterial strains only one strain,Acinetobacter sp.showed the presence of integron gene cassette as a marker of its stability and antibiotic resistance.The strain possessed a 4.17 kb amplicon with 22 ORF＇s.The plasmid isolated from the Acinetobacter sp.was subjected to shotgun cloning through restriction digestion by BamHI,HindIII and SalI,ligated to pUC19 vector and transformed into E.coli XLBlue1α,and finally selected on MSM containing PCP as sole source of carbon and energy with ampicillin as antibiotic marker.DNA sequence analysis of recombinant clones indicated homology with integron gene cassette and multiple antibiotic resistance genes.

Extended role for insertion sequence elements in the antibiotic resistance of Bacteroides

The Bacteroides species are important micro-organisms, both in the normal physiology of the intestines and as frequent opportunistic anaerobic pathogens, with a deeply-rooted phylogenetic origin endowing them with some interesting biological features. Their prevalence in anaerobic clinical specimens is around 60%-80%, and they display the most numerous and highest rates of antibiotic resistance among all pathogenic anaerobes. In these antibiotic resistance mechanisms there is a noteworthy role for the insertion sequence(IS) elements, which are usually regarded as representatives of ‘selfish' genes; the IS elements of Bacteroides are usually capable of up-regulating the antibiotic resistance genes. These include the cep A(penicillin and cephalosporin), cfx A(cephamycin), cfi A(carbapenem), nim(metronidazole) and erm F(clindamycin) resistance genes. This is achieved by outwardoriented promoter sequences on the ISs. Although some representatives are well characterized, e.g., the resistance gene-IS element pairs in certain resistant strains, open questions remain in this field concerning a better understanding of the molecular biology of theantibiotic resistance mechanisms of Bacteroides, which will have clinical implications.

Enhanced removal of antibiotic resistance genes during chicken manure composting after combined inoculation of Bacillus subtilis with biochar

This study explored the combined effects of Bacillus subtilis inoculation with biochar on the evolution of bacterial communities,antibiotic resistance genes(ARGs),and mobile genetic elements(MGEs)during the composting of chicken manure.The results showed that B.subtilis inoculation combined with biochar increased bacterial abundance and diversity as well as prolonged the compost thermophilic period.Promoted organic matter biodegradation and facilitated the organic waste compost humification process,reduced the proliferation of ARGs by altering the bacterial composition.Firmicutes and Actinobacteriota were the main resistant bacteria related to ARGs and MGEs.The decrease in ARGs and MGEs was associated with the reduction in the abundance of related host bacteria.Compost inoculation with B.subtilis and the addition of biochar could promote nutrient transformation,reduce the increase in ARGs and MGEs,and increase the abundance of beneficial soil taxa.

Occurrence and possible sources of antibiotic resistance genes in seawater of the South China Sea

Antibiotic resistance genes(ARGs)might have great effect on ecological security and human health.Oceans are important reservoirs that receive tremendous amounts of pollutants globally.However,information on the proliferation of ARGs in seawater is still limited.This study performed field sampling to investigate the occurrence and distribution of ARGs in seawater of the South China Sea,which is the deepest and largest sea in China.The results showed that the total absolute abundances of ARGs in seawater samples ranged from 2.1×10^(3)to 2.3×10^(4)copies/mL,with an of 5.0×10^(3)copies/mL and a range of 2.2×10^(3)–1.8×10^(4)copies/mL for those with mobile genetic elements(MGEs).Genes resistant to multidrug,aminoglycoside,tetracycline,and fluoroquinolone antibiotics accounted for 77.3%–88.6%of total ARGs in seawater.Proteobacteria and Cyanobacteria represented 32.1%–56.2%and 30.4%–49.5%of microbial community,respectively.Prochlorococcus_MIT9313 and Clade_la were the prevalent genera in seawater of the South China Sea.Complex co-occurrence relationship existed among ARGs,MGEs,and bacteria.Anthropogenic activities had critical influence on ARGs and MGEs.Hospital wastewater,wastewater treatment plant effluent,sewage,aquaculture tailwater,and runoff were determined as the important sources of ARGs in seawater of the South China Sea based on positive matrix factorization analysis.

Removal of antibiotic-resistant genes during drinking water treatment:A review

Once contaminate the drinking water source,antibiotic resistance genes(ARGs)will propagate in drinking water systems and pose a serious risk to human health.Therefore,the drinking water treatment processes(DWTPs)are critical to manage the risks posed by ARGs.This study summarizes the prevalence of ARGs in raw water sources and treated drinking water worldwide.In addition,the removal efficiency of ARGs and related mechanisms by different DWTPs are reviewed.Abiotic and biotic factors that affect ARGs elimination are also discussed.The data on presence of ARGs in drinking water help come to the conclusion that ARGs pollution is prevalent and deserves a high priority.Generally,DWTPs indeed achieve ARGs removal,but some biological treatment processes such as biological activated carbon filtration may promote antibiotic resistance due to the enrichment of ARGs in the biofilm.The finding that disinfection and membrane filtration are superior to other DWTPs adds weight to the advice that DWTPs should adopt multiple disinfection barriers,as well as keep sufficient chlorine residuals to inhibit re-growth of ARGs during subsequent distribution.Mechanistically,DWTPs obtain direct and inderect ARGs reduction through DNA damage and interception of host bacterias of ARGs.Thus,escaping of intracellular ARGs to extracellular environment,induced by DWTPs,should be advoided.This review provides the theoretical support for developping efficient reduction technologies of ARGs.Future study should focus on ARGs controlling in terms of transmissibility or persistence through DWTPs due to their biological related nature and ubiquitous presence of biofilm in the treatment unit.

Prevalence of class 1 integron and its gene cassettes carrying antibiotic resistance genes in drinking water treatment and distribution systems

Class 1 integrons are vital mobile genetic elements involved in the environmental transmission of antibiotic resistance genes(ARGs).However,knowledge about the diversity and abundance of class 1 integrons and gene cassettes during drinking water treatment and distribution is still limited.In this study,we aimed to uncover the prevalence of class 1 integrons in the drinking water treatment and distribution systems with the combination of culture-dependent and culture-independent methods.Further,we applied the nanopore sequencing method to characterize the diversity and arrangement of ARGs carried by class 1 integron-associated gene cassettes.A total of 42 isolates were inti-positive among the 208 strains isolated from drinking water,which tended to confer multi-drug resistance compared with intll-negative isolates.The absolute abundance of the intl1 average i.15×10^(9) copies/L in the source water and underwent the most significant reduction of over 99.9% after liquid chiorine disinfection.Furthermore,nanopore sequencing revealed that the class 1 integron-associated gene cassettes carried 51 subtypes of ARGs in drinking water,mainly conferring resistance to aminoglycosides and trimethoprim.The treatment processes,especially liquid chlorine disinfection,reduced most of the ARGs carried by gene cassettes,though some of the ARG subtypes persisted along the treatment and distribution ike aac(6)-Il,aadA,and dfrB2.The antibiotic resistance gene cassette array laac(6')-Ⅱ arr was most frequently detected,especially in the chlorinated water.This study underlined that drinking water was potential reservoir for integron-mediated ARGs transfer,indicating that the health risks of resistance gene cassettes in class 1 integrons deserved urgent attention.

Distribution characteristics and removal rate of antibiotics and antibiotic resistance genes in different treatment processes of two drinking water plants

Emerging pollutants,such as antibiotics and antibiotic-resistance genes,are becoming increasingly important sources of safety and health concerns.Drinking water safety,which is closely related to human health,should receive more attention than natural water body safety.However,minimal research has been performed on the efficacy of existing treatment processes in water treatment plants for the removal of antibiotics and antibiotic resistance genes.To address this research gap,this study detected and analyzed six main antibiotics and nine antibiotic resistance genes in the treatment processes of two drinking water plants in Wuhan.Samples were collected over three months and then detected and analyzed using ultra-high-performance liquid chromatography-tandem mass spectrometry and fluorescence quantitation.The total concentrations of antibiotics and antibiotic resistance genes in the influent water of the two water plants were characterized as December>March>June.The precipitation and filtration processes of the Zou Maling Water Plant and Yu Shidun Water Plant successfully removed the antibiotics.The ozone-activated carbon process increased the removal rate of most antibiotics to 100%.However,a large amount of antibiotic resistance gene residues remained in the effluents of the two water plants.The experiments demonstrated that the existing ozone-activated carbon processes could not effectively remove antibiotic resistance genes.This study provides a reference for the optimization of drinking water treatment processes for antibiotics and antibiotic resistance gene removal.

Health risk ranking of antibiotic resistance genes in the Yangtze River

Antibiotic resistance is an escalating global health concern,exacerbated by the pervasive presence of antibiotic resistance genes(ARGs)in natural environments.The Yangtze River,the world's third-longest river,traversing areas with intense human activities,presents a unique ecosystem for studying the impact of these genes on human health.Here,we explored ARGs in the Yangtze River,examining 204 samples from six distinct habitats of approximately 6000 km of the river,including free-living and particle-associated settings,surface and bottom sediments,and surface and bottom bank soils.Employing shotgun sequencing,we generated an average of 13.69 Gb reads per sample.Our findings revealed a significantly higher abundance and diversity of ARGs in water-borne bacteria compared to other habitats.A notable pattern of resistome coalescence was observed within similar habitat types.In addition,we developed a framework for ranking the risk of ARG and a corresponding method for calculating the risk index.Applying them,we identified water-borne bacteria as the highest contributors to health risks,and noted an increase in ARG risks in particle-associated bacteria correlating with heightened anthropogenic activities.Further analysis using a weighted ARG risk index pinpointed the ChengdueChongqing and Yangtze River Delta urban agglomerations as regions of elevated health risk.These insights provide a critical new perspective on ARG health risk assessment,highlighting the urgent need for strategies to mitigate the impact of ARGs on human health and to preserve the ecological and economic sustainability of the Yangtze River for future human use.

Nanoplastics promote the dissemination of antibiotic resistance genes and diversify their bacterial hosts in soil

The wide application of plastics has led to the ubiquitous presence of nanoplastics and microplastics in terrestrial environments.However,few studies have focused on the mechanism underlying the effects of plastic particles on soil microbiomes and resistomes,especially the differences between nanoplastics and microplastics.This study investigated the microbiome and resistome in soil exposed to polystyrene microplastics(mPS)or nanoplastics(nPS)through 16S rRNA and shotgun metagenomic sequencing.Distinct microbial communities were observed between mPS and nPS exposure groups,and nPS exposure significantly changed the bacterial composition even at the lowest amended rate(0.01%,w/w).The abundance of antibiotic resistance genes(ARGs)in nPS exposure(1%)was 0.26 copies per cell,significantly higher than that in control(0.21 copies per cell)and mPS exposure groups(0.21 copies per cell).It was observed that nanoplastics,bacterial community,and mobile genetic elements(MGEs)directly affected the ARG abundance in nPS exposure groups,while in mPS exposure groups,only MGEs directly induced the change of ARGs.Streptomyces was the predominant host for multidrug in the control and mPS exposure,whereas the primary host was changed to Bacillus in nPS exposure.Additionally,exposure to nPS induced several bacterial hosts to exhibit possible multi-antibiotic resistance characteristics.Our results indicated that the effects of plastic particles on the soil microbial community were size-dependent,and nano-sized plastic particles exhibited more substantial impacts.Both microplastics and nanoplastics promoted ARG transfer and diversified their bacterial hosts.These findings bear implications for the regulation of plastic waste and ARGs.

Evaluating human exposure to antibiotic resistance genes

Antibiotic resistance is an escalating global concern,leading to millions of annual fatalities.Antibiotic resistance genes(ARGs)present in bacteria equip them to withstand the effects of antibiotics.Intra-and interspecific ARGs transmission through horizontal gene transfer further exacerbates resistance dissemination.The presence of ARGs in the environment heightens the probability of human exposure via direct inhalation,ingestion,or contact with polluted air,food,or water,posing substantial biosafety and health hazards.Consequently,ARGs represent a critical focal point in public health and environmental safety and are classified as emerging contaminants.This perspective underscores the necessity to assess ARG exposure within the One Health framework and to accord greater attention to the mitigation strategies and tactics associated with ARGs.

Characteristics of microbial communities and antibiotic resistance genes in typical rivers of the western Qinghai Lake basin

Environmental factors and anthropogenic activity are key factors that shape the distribution of bacteria and antibiotic resistance genes(ARGs)in natural environments.However,few studies have focused on the occurrence of bacteria and ARGs in remote and pristine environments.In this study,the distribution of bacteria and ARGs in two typical rivers of the western Qinghai Lake basin was investigated.Results showed that cold-resistant Planomicrobium sp.was the predominant genus due to the low temperature,followed by unclassified_f_Planococcaceae.High nitrogen nutrients increased the abundance and diversity of the bacteria community and denitrification was the dominant means of bacterial nitrate reduction.Tetracycline resistance genes including tetA,tetB,and tetC were the dominant ARGs in the western Qinghai Lake basin,ranging from 2.30×10^(3)to 1.91×10^(8)copies/L,while the low abundances of intI1,and ARGs such as sul1 indicated low anthropogenic activity in the western Qinghai Lake basin.Finally,the strong positive correlation between ARGs and intI1 highlights the potential transmission risk of ARGs through cross-or co-selection by horizontal gene transfer.Our study emphasized the adaptation of bacteria to the environment and the facilitation of anthropogenic activity in the propagation of ARGs in natural environments.

A preliminary investigation on the occurrence and distribution of antibiotic resistance genes in the Beijiang River, South China

The occurrence of antibiotic resistance genes （ARGs） was investigated and quantified in 20 water samples collected in the Beijiang River, South China. Sulfonamide- and tetracycline-resistant bacteria were present in 17 and 14 of the collected 20 samples. For sulfonamide ARGs, sulI and sulII were frequently observed in the Beijiang River. The levels of sulI were higher than sulII （p 〈 0.05）, with the mean values of （1.41 ± 1.12） × 10-2 and （1.58 ± 1.71） × 10-3 copies/16S rDNA, respectively. For tetracycline ARGs, tetG had the highest frequency, 100%, followed by tetA （85%）, tetO （85%）, tetC （70%）, tetX （60%）, tetM （40%） and tetQ （20%）, while tetE and tetS were not detected in all the samples from the Beijiang River. On the other hand, tetC had the highest concentration, ranging from 8.30 × 10-2 to 13.20 copies/16S rDNA. The poor correlation between ARGs and antibiotic concentrations revealed that the self-amplification and persistence of ARGs were the reasons that made ARGs exist in the water environment even though the antibiotic selecting pressure was absent. Because so few field measurements have been conducted for investigating the levels of ARGs in rivers in South China, this study provides an important insight on better understanding the occurrence and spread of ARGs in such an ecosystem.

Characterizing the antibiotic resistance genes in a river catchment： Influence of anthropogenic activities

Previous studies on environmental antibiotics resistance genes（ARGs） have focused on the pollution sources such as wastewater treatment plants, aquaculture and livestock farms,etc. Few of them had addressed this issue in a regional scale such as river catchment. Hence,the occurrence and abundances of 23 ARGs were investigated in surface water samples collected from 38 sites which located from the river source to estuary of the Beijiang River.Among them, 11 ARGs were frequently detected in this region and 5 ARGs（sul Ⅰ, sul Ⅱ, tet B,tet C, and tet W） were selected for their distribution pattern analysis. The abundances of the selected ARGs were higher in the upstream（8.70 × 10^6 copies/ng DNA） and downstream areas（3.17 × 10^6 copies/ng DNA） than those in the midstream areas（1.23 × 10^6 copies/ng DNA）, which was positively correlated to the population density and number of pollution sources. Pollution sources of ARGs along the Beijiang River not only had a great impact on the abundances and diversity, but also on the distribution of specific ARGs in the water samples. Both sul Ⅰ and sul Ⅱ were likely originated from aquaculture farms and animal farms,tet W gene was possibly associated with the mining/metal melting industry and the electric waste disposal and tet C gene was commonly found in the area with multiple pollution sources.However, the abundance of tet B was not particularly related to anthropogenic impacts. These findings highlight the influence of pollution sources and density of population on the distribution and dissemination of ARGs at a regional scale.

Film mulching reduces antibiotic resistance genes in the phyllosphere of lettuce

Phyllosphere is an important reservoir of antibiotic resistance genes(ARGs),but the transfer mechanism of ARGs from soil and air to phyllosphere remains unclear.This study demonstrated that soil-air-phyllosphere was the dominant ARG transfer pathway,and blocking it by film mulching can reduce typical phyllosphere ARGs in lettuce by 80.7%-98.7%(89.5%on average).To further eliminate phyllosphere ARGs in lettuce grown with film mulching,the internal soil-endosphere-phyllosphere transfer pathway deserves more attention.We analyzed the ARG hosts and the resistome in lettuce rhizosphere and phyllosphere with film mulching via hybrid Illumina-Nanopore sequencing.Pseudomonas sp.7SR1 was more abundant than other ARG hosts,accounting for 1.0%and 47.1%of the total bacteria in rhizosphere and phyllosphere,respectively.The species has flagella that can promote mobility and can excrete extracellular polymeric substances and/or surfactant-like microbial products,which benefits its colonization in the phyllosphere.Impeding the migration of Pseudomonas sp.7SR1 via the soil-endosphere-phyllosphere pathway would be effective to further reduce ARGs in phyllosphere.Multidrug resistant genes were predominant in phyllosphere(40.3%of the total),and 87.6%of the phyllosphere ARGs were located on chromosomes,indicating relatively low horizontal gene transfer(HGT)potentials.This study provides insights into the transfer mechanism,hosts,and control strategies of phyllosphere ARGs in typical plants.