Core and variable antimicrobial resistance genes in the gut microbiomes of Chinese and European pigs

Monitoring the prevalence of antimicrobial resistance genes(ARGs)is vital for addressing the global crisis of antibiotic-resistant bacterial infections.Despite its importance,the characterization of ARGs and microbiome structures,as well as the identification of indicators for routine ARG monitoring in pig farms,are still lacking,particularly concerning variations in antimicrobial exposure in different countries or regions.Here,metagenomics and random forest machine learning were used to elucidate the ARG profiles,microbiome structures,and ARG contamination indicators in pig manure under different antimicrobial pressures between China and Europe.Results showed that Chinese pigs exposed to high-level antimicrobials exhibited higher total and plasmid-mediated ARG abundances compared to those in European pigs(P<0.05).ANT(6)-Ib,APH(3')-IIIa,and tet(40)were identified as shared core ARGs between the two pig populations.Furthermore,the core ARGs identified in pig populations were correlated with those found in human populations within the same geographical regions.Lactobacillus and Prevotella were identified as the dominant genera in the core microbiomes of Chinese and European pigs,respectively.Forty ARG markers and 43 biomarkers were able to differentiate between the Chinese and European pig manure samples with accuracies of 100%and 98.7%,respectively.Indicators for assessing ARG contamination in Chinese and European pigs also achieved high accuracy(r=0.72-0.88).Escherichia flexneri in both Chinese and European pig populations carried between 21 and 37 ARGs.The results of this study emphasize the importance of global collaboration in reducing antimicrobial resistance risk and provide validated indicators for evaluating the risk of ARG contamination in pig farms.

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.

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.

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.

Preliminary Study on the Treatment Efficiency of Pasteurized Lime Thermal Alkaline Hydrolysis for Excess Activated Sludge and Reduction of Tetracycline Resistance Genes

Thermal alkaline hydrolysis is a common pretreatment method for the utilization of excess activated sludge(EAS).Owing to strict environment laws and need for better energy utilization,new methods were developed in this study to improve the efficiency of pretreatment method.Direct thermal hydrolysis(TH),pasteurized thermal hydrolysis(PTH),and alkaline pasteurized thermal hydrolysis(PTH+CaO and PTH+NaOH)methods were used to treat EAS.Each method was compared and analyzed in terms of dissolution in ammonium nitrogen(NH_(4)^(+)-N)and soluble COD(SCOD)in EAS.Furthermore,the removal of tetracycline resistance genes(TRGs)and class 1 transposon gene intI1 from EAS was investigated.The NH_(4)^(+)-N and SCOD concentrations in EAS treated by PTH were 1.24 and 2.58 times higher than those of TH.However,the removal efficiency of total TRGs and intI1 between the groups was comparable.The SCOD concentration of the PTH+NaOH group was 4.37 times higher than that of the PTH group,and the removal efficiency of total TRGs was increased by 9.52%compared with that by PTH.The NH_(4)^(+)-N and SCOD concentrations of the PTH+CaO group could reach 85.04%and 92.14%of the PTH+NaOH group,but the removal efficiency of total TRGs by PTH+CaO was 19.78%lower than that by PTH+NaOH.Thus,to reduce the financial cost in actual operation,lime(CaO)can be used instead of a strong alkali(NaOH),and pasteurized steam at 70℃ instead of conventional high-temperature heating to treat EAS.This study provides a reference for the development of alkaline hydrolysis under moderate temperatures along with the removal of TRGs in EAS.

Effects of Microplastics on Expression of Resistance Genes and Virulence Genes of Vibrio alginolyticus

[Objectives]To study the effects of microplastics on antibiotic resistance genes and virulence genes of Vibrio alginolyticus,so as to provide a certain reference for controlling marine pollution,curbing the spread of environmental antibiotic resistance genes and virulence genes,formulating environmental policies,and maintaining food safety.[Methods]After adding V.alginolyticus into the artificial seawater,they were divided into three groups,namely blank control group(BLK),polyvinyl chloride microplastic group(PVC group)and polyvinyl alcohol microplastic group(PVA group).Aerated culture experiments were carried out,and the effects of microplastics on the expression of resistance genes and virulence genes of V.alginolyticus were studied by PCR and qPCR methods.[Results]The presence of microplastics significantly changed the resistance gene structure of V.alginolyticus.Compared with the control group,the cfxA and cfr resistance genes were detected in the microplastic group.However,only PVC group detected blaZ resistance gene,and only PVA group did not detect aaC resistance gene.In addition,compared with the control group,the expressions of virulence genes in the microplastic group were all down-regulated(P<0.01).[Conclusions]This study provides some reference for curbing the spread of environmental antibiotic resistance genes and virulence genes,formulating environmental policies,and maintaining food safety,but the specific mechanisms of drug resistance and virulence need further research.

Molecular Screening of Rice Cultivated in Benin for the Identification of Xanthomonas oryzae Pv. oryzae and Bacterial Leaf Blight Resistance Genes

One of the most devastating diseases of rice worldwide is bacterial blight (BLB) caused by Xanthomonas oryzae pv. Oryzae (Xoo). In Benin, Xoo was first described in 2013 on wild rice Oryzae longistaminata. So far, no study has been done on Beninese Xoo strains. We do not know whether the pathogen has already passed into the rice varieties grown, or if they are exposed to other bacteria. Whereas the use of resistant varieties, carrying resistance genes, is the only highly effective and environmentally friendly way to control this disease, no information is available on these Xoo resistance genes in rice varieties grown in Benin apart from the one we recently. This study aims to identify Beninese Xoo strains, causing BLB and screen rice varieties grown in Benin for the main resistance genes. Diseased rice leaves showing typical symptoms of fire blight collected from different rice fields in the three phytogeographic areas of Benin were analyzed by PCR for Xoo-specific sequence identification. Furthermore, seventy-five collected rice accessions were screened to identify xa5, Xa7, xa13, and Xa21 resistance genes to Xoo. The results reveal that Xanthomonas oryzae was identified in two fields in Banikouara and one in Malanville. On the other hand, Sphingomonas sp. has been identified in several other rice fields in Benin. Forty-seven of seventy-five rice accessions examined (62.66%) carried Xoo resistance genes with 3 (4%) and 40 (53.33%) of xa5 and Xa21 respectively. None of the accessions had either Xa7 or xa13 resistance genes. Three accessions possess both xa5 and Xa21 genes. Isogenic lines IRBB60 and IRBB21, supposed to be a positive control, presented a Xoo sensitivity allele. These results indicate that Xoo has moved from the wild rice variety to the cultivated variety in northern Benin and varietal improvement programs must be implemented with varieties having several resistance genes for the efficient response against a possible BLB pandemic in Benin.

Postulation of Seedlings Resistance Genes to Yellow Rust in Commercial Wheat Cultivars from Yunnan Province in China

The objective of this study was to characterize yellow （stripe） rust resistance gene（s） in 52 commercial wheat cultivars from Yunnan Province in China, and to provide information for their rational deployment in field. Seedlings of wheat cultivars were inoculated with 25 differential isolates ofPuccinia striiformis from foreign and home to postulate resistance genes to yellow rust, and then validated by pedigree. There were 10 probable resistance genes characterized in these cultivars, in which, Yr9 was most commonly postulated to be present in thirteen cultivars. Yr21, the second, was present in four cultivars. Yr8, the third, were present in three cultivars. Yr6, Yrl 7 and Yr26, the fourth, was present in two cultivars respectively. The other gene（s） such as, Yr2＋YrA, Yr7 and Yr27, were only present in single cultivar（s）; unknown gene（s） or gene（s） combination（s） were present in 22 cultivars. One cultivar （Yunmai 42） had no resistance gene tested in this study. Cultivars such as Yunmai 52, Mian 1971-98, Kunmai 4, and Yunmai 56 carried effective genes and can be popularized mainly; Yr9 should be planted with other Yr genes. In the meantime other effective genes should be introduced to realize gene diversity for controlling wheat yellow rust. Yunmai 42 should be reduced to avoid rust breakout. Unknown gene cultivars should be utilized and be researched deeply.

Marker-assisted pyramiding of soybean resistance genes R_(SC4),R_(SC8),and R_(SC14Q) to soybean mosaic virus

Soybean mosaic virus （SMV） is one of the major viral pathogens affecting soybean crops worldwide. Three SMV resistance genes, Rsc4, Rsc8, and Rsc14Q, have been identified and mapped on soybean chromosomes 14, 2, and 13 from Dabaima, Kefeng 1, and Qihuang 1 cultivars, respectively. Soybean cultivar Nannong 1138-2 is widely grown in the Yangtze River Valley of China. In this study, crosses were made between Qihuang l^Kefeng 1 and DabaimaxNannong 1138-2. Ten simple sequence repeat （SSR） markers linked to three resistance loci （Rsc4, Rsc8, and Rsc^4Q） were used to assist pyramided breeding. Pyramided families containing three resistance loci （Rsc4, Rsc8, and Rsc14Q） were evaluated by inoculating them with 21 SMV strains from China. Results indicated that the 10 markers can be used effectively to assist the selection of resistant individuals containing Rsc4, Rsc8, and Rsc14Q. A total of 53 F6 plants were confirmed to contain three homozygous alleles conferring resistance to SMV. Five F7 homozygous pyramided families exhibited resistance to 21 strains of SMV and showed desirable agronomic traits using dual selection. The strategy of pyramiding resistance gene derived from different varieties has practical breeding value in providing broad-spectrum resistance against the existing strains of SMV in China.

Seafood as a Reservoir of Gram-negative Bacteria Carrying Integrons and Antimicrobial Resistance Genes in Japan

PCR and DNA sequencing were used to screen and characterize integrons and resistance genes in Gram-negative bacteria isolated from seafood products in Japan.A total of 215 Gram-negative bacteria were isolated from local and imported seafood samples collected from retail markets in Hiroshima Prefecture.Class 1 integrons containing gene cassettes encoding resistance to trimethoprim

Cytogenetic Mapping of Disease Resistance Genes and Analysis of Their Distribution Features on Chromosomes in Maize

Cytogenetic maps of four clusters of disease resistance genes were generated by ISH of the two RFLP markers tightly linked to and flanking each of maize resistance genes and the cloned resistance genes from other plant species onto maize chromosomes, combining with data published before. These genes include Helminthosporium turcium Pass resistance genes Ht1, Htn1 and Ht2, Helminthosporium maydis Nisik resistance genes Rhm1 and Rhm2, maize dwarf mosaic virus resistance gene Mdm1, wheat streak mosaic virus resistance gene Wsm1, Helminthosporium carbonum ULLstrup resistance gene Hml and the cloned Xanthomonas oryzae pv. Oryzae resistance gene Xa21 of rice, Cladosporium fulvum resistance genes Cf-9 and Cf-2.1 of tomato,and Pseudomonas syringae resistance gene RPS2 of Arabidopsis. Most of the tested disease resistance genes located on the four chromosomes, i.e., chromosomes1, 3, 6 and 8, and they closely distributed at the interstitial regions of these chromosomal long arms with percentage distances ranging 31.44(±3.72)-72.40(±3.25) except for genes Rhm1, Rhm2, Mdm1 and Wsm1 which mapped on the satellites of the short arms of chromosome6. It showed that the tested RFLP markers and genes were duplicated or triplicated in maize genome. Homology and conservation of disease resistance genes among species, and relationship between distribution features and functions of the genes were discussed. The results provide important scientific basis for deeply understanding structure and function of disease resistance genes and breeding in maize.

Response to Comment on "The role of wildlife (wild birds) in the global transmission of antimicrobial resistance genes"

DEAR EDITOR,Since our first identification of plasmid-mediated colistin resistance gene mcr-1 in 2015 （Liu et al., 2016）, it has been described in human clinics, domestic animals, foods, and the environment worldwide （Schwarz ＆ Johnson, 2016）. Although it is still rare, the emergence of mcr-I in wild animals is of great concern. We summarized two previous reports on mcr-1 in wild birds from Lithuania and Argentina to describe its emergence and characteristics in wildlife and highlight the potentially important role of wild animals, particularly birds, in its global transmission （Wang et al., 2017）. The first detection of mcr-1 in wildlife in Asia was identified in an extended-spectrum β- lactamase-producing Escherichia coil strain isolated from Eurasian coot （Fulica atra）,

Screening and identification of candidate Fusarium wilt-resistance genes from pumpkin

Pumpkin(Cucurbita moschata)has been widely used as cucumber(Cucumis sativus L.)rootstock to defend against Fusarium wilt(FW)and increase cucumber yields and profits.However,the resistance genes and mechanisms underlying the FW tolerance in pumpkin are poorly understood.Here we analyzed the transcriptome of pumpkin inoculated with the cucumber FW causal agent Fusarium oxysporum f.sp.cucumerinum(Foc),and obtained 3152 and 4735 upregulated genes induced by Foc at 24h after Foc inoculation compared with at 0h and 24h non-inoculated control,respectively.Next,404 common differentially expressed genes(DEGs)were screened using the criterion log_(2) FPKM(fold change)≥2.In total,206 of 404 DEGs were predominantly expressed in roots,which is the first tissue that Foc contacts and invades.140 DEGs were selected and classified into four groups(pathogenesis resistance,secondary metabolism-related,transcription factor and signal binding)based on their functional descriptions.Then,29 genes having high expression levels were selected to investigate the expression patterns induced by a Foc inoculation.Among them,16 genes were significantly induced by Foc and showed high expression levels at various treatment time points.These candidate genes may act as positive regulators of FW resistance in pumpkin and provide effective resources for improving cucumber FW resistance through breeding programs.

Comment on ＂The role of wildlife (wild birds)in the global transmission of antimicrobial resistance genes＂

DEAR EDITOR, We read with interest the article by Wang and colleagues regarding the role of wildlife in the transmission of antimicrobial resistance （AMR） （Wang et al., 2017）. Although we appreciate the efforts in reviewing this important topic, we would like to comment on some statements that we believe are not up-to- date or properly cited.

Molecular Marker-Assisted Backcrossing of Anthracnose Resistance Genes into Common Beans (<i>Phaseolus vulgaris</i>L.) Varieties

Anthracnose, caused by Colletotrichum lindemuthianum, is a major disease of common bean and results in high yield loss. Due to the high degree of pathogenic variability of the fungus and the continual emergence of new races, genetic resistance in the host is not durable. Gene pyramiding using Marker Assisted Selection (MAS) is proposed as a viable approach to improve the durability of major genes conditioning resistance to anthracnose. In this study a common bean line Urugezi x AND 1062 susceptible to anthracnose but already improved for Pythium root rot resistance was improved for anthracnose resistance through a backcross breeding program. Genotypic selection was done in Rubilizi laboratory in Kigali, Rwanada whereas phenotypic selection was conducted in an anthracnose hotspot at Rwerere, a research Centre of the Rwanda Agricultural and Animal Resources Development Board (RAB). Analysis of variance for effect of bean varieties and anthracnose isolates on disease expression showed significant differences (p < 0.001) among varieties and isolates and for the interaction between isolates and varieties. Developed BC2F1 plants were 41% of them resistant and 59% susceptible to anthracnose. However, the observed proportion of 26 resistants and 37 susceptible in BC2F1 plants didn’t fit the goodness of fit of the expected proportion of 75 resistants to 25 susceptible. Only 41% of BC2F1 plants inherited the resistance genes and were phenotypically resistant. Presence of SCAR-markers, SAB3 and SBB14, in the developed resistant lines h suggested successful resistance transfer of anthracnose resistance genes.

Transgenic Cotton and Disease Resistance Genes

Success in conventional breeding for resistance to mycotoxin-producing or other phytopathogenic fungi is dependent on the availability of resistance gene(s) in the germplasm.Even when it is available,breeding for disease-resistant crops is very time consuming,especially in perennial crops such

Development of herbicide resistance genes and their application in rice

Rice is one of the most important food crops in the world.Weeds seriously affect the rice yield and grain quality.In recent years,there are tremendous progresses in the research and application of herbicideresistant genes in rice worldwide.This article reviews the working mechanisms of six herbicides(glyphosate,glufosinate,acetolactate synthase inhibitor herbicides,acetyl-Co A carboxylase inhibitor herbicides,hydroxyhenylpyruvate dioxygenase(HPPD)inhibitor herbicides and dinitroaniline herbicides),the resistance mutations of the corresponding herbicide-target genes,and the herbicide detoxification mechanisms by non-target genes.Examples are provided on herbicide-resistant rice materials obtained by transformation of exogenous resistance genes,by artificial mutagenesis and mutant screening,and by modifying the target genes through gene editing.This paper also introduces the current application of herbicide-resistant rice,points out problems that may be caused by utilization of herbicide resistant rice and solutions to the problems,and discusses the future prospects for the development of herbicideresistant rice.

Strategy for Use of Rice Blast Resistance Genes in Rice Molecular Breeding

Rice blast is one of the most destructive diseases affecting rice production worldwide.The development and rational use of resistant varieties has been the most effective and economical measure to control blast.In this review,we summarized the cloning and utilization of rice blast resistance genes,such as Pi1,Pi2,Pi9,Pi54,Pigm and Piz-t.We concluded that three main problems in the current breeding of rice blast resistance are:availability of few R(resistance)genes that confer resistance to both seedling and panicle blast,the resistance effect of pyramided lines is not the result of a simple accumulation of resistance spectrum,and only a few R genes have been successfully used for molecular breeding.Therefore,novel utilization strategies for rice blast R genes in molecular breeding were proposed,such as accurately understanding the utilization of R genes in main modern rice varieties,creating a core resistant germplasm with excellent comprehensive traits,screening and utilizing broadspectrum and durable resistance gene combinations.Lastly,the trends and possible development direction of blast resistance improvement were also discussed,including new genes regulating resistance identified via GWAS(genome-wide association study)and improving rice blast resistance using genetic editing.

Genetic Analysis and Preliminary Mapping of Two Recessive Resistance Genes to Brown Planthopper, Nilaparvata lugens Stl in Rice

An F2 population derived from the cross of WB01, an introgression line resistant to brown planthopper (BPH) originated from Oryza rufipogon Griff. and a susceptible indica variety 9311, was developed for genetic analysis and gene mapping. The population with 303 F2:3 families was genotyped by 141 simple sequence repeat (SSR) markers and used for gene mapping. Two softwares, Mapmaker/Exp 3.0 and Windows QTL Cartographer V2.0 were applied to detect QTLs. Totally, two QTLs resistant to BPH, named temporarily as bph22(t) and bph23(t), were identified to locate on chromosomes 4 and 8, individually had LOD values of 2.92 and 3.15, and explained 11.3% and 14 .9% of the phenotypic variation, respectively.