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.

High-throughput,microscopy-based screening and quantification of genetic elements

Synthetic biology relies on the screening and quantification of genetic components to assemble sophisticated gene circuits with specific functions.Microscopy is a powerful tool for characterizing complex cellular phenotypes with increasing spatial and temporal resolution to library screening of genetic elements.Microscopy-based assays are powerful tools for characterizing cellular phenotypes with spatial and temporal resolution and can be applied to large-scale samples for library screening of genetic elements.However,strategies for high-throughput microscopy experiments remain limited.Here,we present a high-throughput,microscopy-based platform that can simultaneously complete the preparation of an 8×12-well agarose pad plate,allowing for the screening of 96 independent strains or experimental conditions in a single experiment.Using this platform,we screened a library of natural intrinsic promoters from Pseudomonas aeruginosa and identified a small subset of robust promoters that drives stable levels of gene expression under varying growth conditions.Additionally,the platform allowed for single-cell measurement of genetic elements over time,enabling the identification of complex and dynamic phenotypes to map genotype in high throughput.We expected that the platform could be employed to accelerate the identification and characterization of genetic elements in various biological systems,as well as to understand the relationship between cellular phenotypes and internal states,including genotypes and gene expression programs.

Construction of retroviral vector carrying HSV tk gene under control of human AFP enhancer core sequence and human pgk promotor *

AIM Tenstruct retroviral vector bringing HSV tk gene under control by human AFP enhancer core sequence and human pgk promotor.

Two Kinds of Calculation Methods in Whole Process and a Best New Comprehensive Figure of Metallic Material Behaviors

Based on the viewpoint that there are similarities to the genetic principles in the mechanics and the engineering fields; based on data in some referents and author＇s theoretical approach, this paper is to consider the microscopic-damage and the macroscopic-damage behaviors of materials that are distinct differences, thereby to propose computing models and methods of subsection calculations in whole process; to consider the behaviors between the short cracks and long ones both of which are always continuous, thereby to propose some computing models and methods of the successive calculations. These computing models refer to formulas of the threshold sizes of cracks （or threshold values of damages）; the propagating rates of cracks; the predicting calculations of lifetime; Particularly, in which it provides a best new comprehensive figure that it could be including mid describing all problems mentioned above. So it may be as a bridge to link the traditional material mechanics, the material discipline and the modem mechanics on fatigue-damage-fracture; perhaps, it can also be as route diagram to guide designs and calculations to some materials and structures. Therefore, above works realize calculations of the strength problems, the growth rate of cracks （damages） mid prediction of lifetime in whole process that would have practical significances.

Recent tectonic stress field research in Shanxi graben system

By comprehension of earthquake focal mechanism solutions and the data of in-situ stress measurements, the tectonic stress field in Shanxi region has been summarized, which indicate that the stress state in this region is different from that of its eastern surrounding regions. The next, by fitting the measured data, the boundary forces that influenced the distribution of the stress field in this region has been studied using inversion method. The inversion results showed the following messages: the effect of the boundary force between the blocks is the main determinative factor for the recent tectonic stress field in Shanxi and the regional material and its property is a secondary factor; the horizontal main stress of tectonic stress field in Shanxi region is consistent with the stretch of fault basins.

Acquisition and dissemination mechanisms of CTXΦ in Vibrio cholerae : New paradigm for dif residents

Vibrio cholerae(V. cholerae) genome is equipped with a number of integrative mobile genetic element(IMGE) like prophages, plasmids, transposons or genomic islands, which provides fitness factors that help the pathogen to survive in changing environmental conditions. Metagenomic analyses of clinical and environmental V. cholerae isolates revealed that dimer resolution sites(dif) harbor several structurally and functionally distinct IMGEs. All IMGEs present in the dif region exploit chromosomally encoded tyrosine recombinases, Xer C and Xer D, for integration. Integration takes place due to site-specific recombination between two specific DNA sequences; chromosomal sequence is called att B and IMGEs sequence is called att P. Different IMGEs present in the att P region have different attP structure but all of them are recognized by Xer C and Xer D enzymes and mediate either reversible or irreversible integration. Cholera toxin phage(CTXΦ), a lysogenic filamentous phage carrying the cholera toxin genes ctx AB, deserves special attention because it provides V. cholerae the crucial toxin and is always present in the dif region of all epidemic cholera isolates. Therefore, understanding the mechanisms of integration and dissemination of CTXΦ, genetic and ecological factors which support CTXΦ integration as well as production of virion from chromosomally integrated phage genome and interactions of CTXΦ with other genetic elements present in the genomes of V. cholerae is important for learning more about the biology of cholera pathogen.

Genetic factors related to the widespread dissemination of ST11 extensively drug-resistant carbapenemase-producing Klebsiella pneumoniae strains within hospital

Background:Carbapenemase-producing Klebsiella pneumoniae(CP-Kp)poses distinct clinical challenges due to extensively drug resistant(XDR)phenotype,and sequence type(ST)11 is the most dominant blaKPC-2-bearing CP-Kp clone in China.The purpose of this current retrospective study was to explore the genetic factors associated with the success of XDR CP-Kp ST11 strains circulated in the intensive care unit(ICU)of a Chinese tertiary hospital.Methods:Six ST11 XDR CP-Kp strains were identified between May and December 2014 and validated by minimum inhibitory concentration examination,polymerase chain reaction,and pyrosequencing.The six ST11 XDR CP-Kp,as well as three multi-drug resistant(MDR)and four susceptible strains,were sequenced using single-molecule real-time method.Comprehensively structural and functional analysis based on comparative genomics was performed to identify genomic characteristics of the XDR ST11 CP-Kp strains.Results:We found that ST11 XDR blaKPC-2-bearing CP-Kp strains isolated from inpatients spread in the ICU of the hospital.Functionally,genes associated with information storage and processing of the ST11 XDR CP-Kp strains were more abundant than those of MDR and susceptible strains,especially genes correlative with mobile genetic elements(MGEs)such as transposons and prophages.Structurally,eleven large-scale genetic regions taken for the unique genome in these ST11 XDR CP-Kp strains were identified as MGEs including transposons,integrons,prophages,genomic islands,and integrative and conjugative elements.Three of them were located on plasmids and eight on chromosomes;five of them were with antimicrobial resistance genes and eight with adaptation associated genes.Notably,a new blaKPC-2-bearingΔΔTn1721-blaKPC-2 transposon,probably transposed and truncated fromΔTn1721-blaKPC-2 by IS903D and ISKpn8,was identified in all six ST11 XDR CP-Kp strains.Conclusion:Our findings suggested that together with clonal spread,MGEs identified uniquely in the ST11 XDR CP-Kp strains might contribute to their formidable adaptability,which facilitated their widespread dissemination in hospital.

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.

Seasonal variations of microbial community and antibiotic resistome in a suburb drinking water distribution system in a northern Chinese city

Antibiotic resistance genes(ARGs)are an emerging issue for drinkingwater safety.However,the seasonal variation of ARGs in drinking water distribution systems(DWDS)is still unclear.This work revealed the tempo-spatial changes of microbial community,ARGs,mobile genetic elements(MGEs)co-occurring with ARGs,ARG hosts in DWDS bulk water by means of metagenome assembly.The microbial community and antibiotic resistome varied with sampling season and site.Temperature,ammonia,chlorite and total plate count(TPC)drove the variations of microbial community structure.Moreover,environmental parameters(total organic carbon(TOC),chlorite,TPC and hardness)shifted antibiotic resistome.ARGs and MGEs co-occurring with ARGs showed higher relative abundance in summer and autumn,which might be attributed to detached pipe biofilm.In particular,ARG-bacitracin and plasmid were the predominant ARG and MGE,respectively.ARG hosts changed with season and site and were more diverse in summer and autumn.In winter and spring,Limnohabitans and Mycobacterium were the major ARG hosts as well as the dominant genera in microbial community.In addition,in summer and autumn,high relative abundance of Achromobacter and Stenotrophomonas were the hosts harboring many kinds of ARGs and MGEs at site in a residential zone(0.4 km from the water treatment plant).Compared with MGEs,microbial community had a greater contribution to the variation of antibiotic resistome.This work gives new insights into the dynamics of ARGs in full-scale DWDS and the underlying factors.

Plant cultivar determined bacterial community and potential risk of antibiotic resistance gene spread in the phyllosphere

The global increased antibiotic resistance level in pathogenic microbes has posed a significant threat to human health.Fresh vegetables have been recognized to be an important vehicle of antibiotic resistance genes(ARGs)from environments to human beings.Phyllosphere ARGs have been indicated to be changed with plant species,yet the influence of plant cultivar on the phyllospheric resistome is still unclear.Here,we detected the ARGs and bacterial communities in the phyllosphere of two cultivars of cilantros and their corresponding soils using high-throughput quantitative PCR technique and bacterial 16S rRNA gene-based high-throughput sequencing,respectively.We further identified the potential bacterial pathogens and analyzed the effects of plant cultivar on ARGs,mobile genetic elements(MGEs),microbiome and potential bacterial pathogens.The results showed that the cultivars did not affect the ARG abundance and composition,but significantly shaped the abundance of MGEs and the composition structure of bacteria in the phyllosphere.The relative abundance of potential bacterial pathogenswas significantly higher in the phyllosphere than that in soils.Mantel test showed that the ARG patterns were significantly correlated to the patterns of potential bacterial pathogens.Our results suggested that the horizontal gene transfer of ARGs in the phyllosphere might be different between the two cultivars of cilantro and highlighted the higher risk of phyllospheric microorganisms compared with those in soils.These findings extend our knowledge on the vegetable microbiomes,ARGs,and potential pathogens,suggesting more agricultural and hygiene protocols are needed to control the risk of foodborne ARGs.

Coastal mudflats as reservoirs of extracellular antibiotic resistance genes: Studies in Eastern China

Despite coastal mudflats serving as essential ecological zones interconnecting terrestrial/freshwater and marine systems,little is known about the profiles of antibiotic resistance genes(ARGs)in this area.In this study,characteristics of typical ARGs,involving both intracellular(iARGs)and extracellular ARGs(eARGs)at different physical states,were explored in over 1000 km of coastal mudflats in Eastern China.Results indicated the presence of iARGs and eARGs at states of both freely present or attached by particles.The abundance of eARGs was significantly higher than that of iARGs(87.3%vs 12.7%),and their dominance was more significant than those in other habitats(52.7%-76.3%).ARG abundance,especially for eARGs,showed an increasing trend(p<0.05)from southern(Nantong)to northern(Lianyungang)coastalmudflats.Higher salinity facilitated the transformation from iARGs to eARGs,and smaller soil particle size was conducive to the persistence of eARGs in northern coastal mudflats.This study addresses the neglected function of coastal mudflats as eARGs reservoirs.

Impact of different organic matters on the occurrence of antibiotic resistance genes in activated sludge

The occurrence of antibiotic resistance genes(ARGs)in various environments has drawn worldwide attention due to their potential risks.Previous studies have reported that a variety of substances can enhance the occurrence and dissemination of ARGs.However,few studies have compared the response of ARGs under the stress of different organicmatters in biologicalwastewater treatment systems.In this study,seven organic pollutantswere added into wastewater treatment bioreactors to investigate their impacts on the ARG occurrence in activated sludge.Based on high-throughput sequencing,it was found that the microbial communities and ARG patterns were significantly changed in the activated sludge exposed to these organic pollutants.Compared with the non-antibiotic refractory organic matters,antibiotics not only increased the abundance of ARGs but also significantly changed the ARG compositions.The increase of Gram-negative bacteria(e.g.,Archangium,Prosthecobacter and Dokdonella)carrying ARGs could be the main cause of ARG proliferation.In addition,significant co-occurrence relationships between ARGs and mobile genetic elements were also observed in the sludge samples,which may also affect the ARG diversity and abundance during the organic matter treatment in the bioreactors.Overall,these findings provide new information for better understanding the ARG occurrence and dissemination caused by organic pollutants in wastewater treatment systems.

Response of antibiotic and heavy metal resistance genes to the co-occurrence of gadolinium and sulfamethoxazole in activated sludge systems

With the increasing use of antibiotics and rare earth elements(REE)during the coronavirus disease(COVID-19)pandemic,the co-occurrence of sulfamethoxazole(SMX)and gadolinium(Gd)has increased in wastewater treatment plants(WWTPs).However,the effects of SMX and Gd exposure on the transmission of antibiotic resistance genes(ARGs)and heavy metal resistance genes(MRGs)remain unknown.This study investigated the impacts of SMX and Gd on the fate of ARGs and MRGs in an activated sludge system.The diversity and relative abundance of ARGs,MRGs,and mobile genetic elements(MGEs)were detected by metagenomic sequencing.The results revealed an increased abundance of ARGs but a decreased abundance of MRGs under the joint effect of SMX and Gd.In addition,Gd alone exerted adverse effects on the proliferation and spread of ARGs and MRGs.However,SMX alone resulted in an increase in the diversity of ARGs and MRGs and promoted the growth of Pseudomonas,Thauera,and Streptomyces in the activated sludge system.Interestingly,a positive correlation was observed between most ARGs and MGEs.These findings provide comprehensive insights into the effects of co-occurring REEs and antibiotics on the fate of ARGs,MRGs,and MGEs,providing evidence to assist in controlling the spread and proliferation of ARGs and MRGs in activated sludge systems.

Collembolans accelerate the dispersal of antibiotic resistance genes in the soil ecosystem

Soils have become an important sink for antibiotic resistance genes(ARGs).To better understand the impacts of ARGs on the soil ecosystem,the transport of ARGs is a basic question.So far,however,the role of soil animals in the dispersal of ARGs is not understood.Here,two treatments(without collembolans and with collembolans)were established,each treatment included unamended and manure-amended soil,and soil samples were collected at 14,28 and 56 days after incubation.The effects of the collembolan Folsomia candida on dispersal of ARGs in the soil ecosystem were explored using high-throughput qPCR combined with Illumina sequencing.As the culture time increased,more shared ARGs and OTUs were detected between the unamended and manured soil,especially in the treatment with collembolans.Vancomycin,aminoglycoside and MLSB genes may have been more readily transported by the collembolan.On the 28th day after incubation,a high abundance of mobile genetic elements(MGEs)was found in the treatment with collembolans.These results clearly reveal that collembolans can accelerate the dispersal of ARGs in the soil ecosystem.Procrustes analysis and the Mantel test both indicate that soil bacterial communities were significantly correlated with ARG profiles.Furthermore,partial redundancy analysis indicates that soil bacterial communities can explain 41.28% of the variation in ARGs.These results suggest that the change of soil microbial community have an important contribution to the dispersal of ARGs by the collembolan.

Metagenomic insights into the profile of antibiotic resistomes in sediments of aquaculture wastewater treatment system

To meet the rapidly growing global demand for aquaculture products,large amounts of antibiotics were used in aquaculture,which might accelerate the evolution of antibiotic-resistant bacteria(ARB)and the propagation of antibiotic genes(ARGs).In our research,we revealed the ARGs profiles,their co-occurrence with mobile genetic elements(MGEs),and potential hosts in sediments of a crab pond wastewater purification system based on metagenomic analysis.The residual antibiotic seems to increase the propagation of ARGs in the crab pond,but there was no clear relationship between a given antibiotic type and the corresponding resistance genes.The effect of aquaculture on sediment was not as profound as that of other anthropogentic activities,but increased the relative abundance of sulfon-amide resistance gene.A higher abundance of MGEs,especially plasmid,increased the po-tential ARGs dissemination risk in crab and purification ponds.Multidrug and sulfonamide resistance genes had greater potential to transfer because they were more frequently carried by MGEs.The horizontal gene transfer was likely to occur among a variety of microorgan-isms,and various ARGs hosts including Pseudomonas,Acinetobacter,Escherichia,and Klebsiella were identified.Bacterial community influenced the composition of ARG hosts,and Pro-teobacteria was the predominant hosts.Overall,our study provides novel insights into the environmental risk of ARGs in sediments of aquaculture wastewater treatment system.

Cell-specific expression of the diphtheria toxin A-chain coding sequence induces cancer cell suicide

OBJECTIVE: To test whether the diphtheria toxin A (DT-A) chain coding sequence linked to murine immunoglobulin Kappa light chain (IgKappa) promoter and enhancer have selective cytocidal effects on IgKappa producing cells. METHODS: The diphtheria toxin A gene or beta galactosidase (beta-gal) gene were linked to a murine IgKappa promoter and enhancer to construct pcDNA3IgKappaDTA or pcDNA3IgKappaLacZ plasmids. These plasmids were transfected into IgKappa producing or non-producing cells by the liposome coated DNA method. Expression of beta-gal activity and effects on cell growth of transfected cells were assessed. RESULTS: The beta-gal gene, under the control of cytomegalovirus (CMV) promoter, can express in all cell lines. Expression of beta-gal under the control of the IgKappa promoter was detected only in the IgKappa producing cell line, CA46. Expression of beta-gal was greatly suppressed when cotransfected with pcDNA3IgKappaDTA in CA46 cells.Cell growth of CA46 cells transfected with pcDNA3IgKappaDTA plasmid was significantly inhibited compared with CA46 cells transfected with pcDNA3IgKappaLacZ. CONCLUSION: Selective killing of IgKappa producing cells can be attained by introducing the diphtheria toxin A gene under the control of IgKappa promoter and enhancer.