Distribution Characteristics Of Virulence Factor Genes And Antibiotic Resistance Genes In Livestock And Poultry Manure Microorganisms In Some Areas Of Wanjiang River Basin

As the number of farms in the Wanjiang River Basin is increasing,a large amount of manure is produced.Livestock manure is one of the important agricultural surface source pollution,in which the main microbiological risk are pathogenic bacteria(PB)and antibioticresistant bacteria(ARB).PB in livestock manure affect the development of livestock farming and directly threaten human and animal health,while antibiotic resistance genes(ARGs)in antibiotic-resistant bacteria can be transmitted to PB through horizontal gene transfer,making pathogenic antibiotic resistant bacteria(PARB).The problem posed by PARB has become a global public health crisis.The pathogenicity of bacterial pathogens depends on the virulence factors they possess,while the type of resistance of ARB depends on the ARGs they carry.Therefore,two important targets for monitoring the microbiological risk of livestock manure are virulence factor genes(VFGs)and ARGs.In this paper,we used metagenomics to investigate the type and abundance of VFGs and ARGs and the correlation between them in 7 chicken manure samples and 14 pig manure samples from some areas of the Wanjiang River basin,identified host microorganisms carrying VFGs and ARGs in livestock and poultry manure,and recovered the draft genomes of bacteria carrying ARGs and VFGs using combined metagenomic binning algorithm.The main studies are as follows.1.Annotation,abundance and correlation of ARGs and VFGs in livestock manureIn this study,a total of 526 ARG subtypes belonging to 29 ARG types were detected in21 livestock fecal samples,and the dominant ARG types were multidrug,aminoglycosides and tetracyclines.The total abundance of ARGs in chicken fecal samples was between 2226 and 3894 TPM,while in pig fecal samples fluctuated more,with a distribution range of 1930 to 9282 TPM.This study also identified core resistome including 128 ARG subtypes in chicken feces microorganisms and core resistome including 51 ARG subtypes in pig feces microorganisms,and principal component analysis revealed significant differences between them.A total of 347 VFG subtypes belonging to 12 VFG types were detected in all samples,and the dominant types were offensive,motility and nonspecific VFG.The relative and absolute abundance of different VFG types were not related to the source of the samples but to the structure of the livestock manure microbial community,as the distribution of virulence genes was concentrated on some PB.Correlation analysis revealed that there was a highly significant positive correlation between virulence genes and drug resistance genes in terms of total abundance and number of subtypes;co-occurrence network analysis showed that aggressive virulence genes were significantly associated with multidrug resistance genes.2.Identification of host bacteria with ARGs and VFGs in livestock and poultry manureTaxonomy annotation of contigs carrying VFGs revealed that Escherichia spp.,Klebsiella spp.,Campylobacter spp.and Pseudomonas spp.carried a high number of virulence genes,for example,in the CA group,Escherichia spp.and Klebsiella spp.carried118 and 72 VFGs,respectively.The relative abundance of Escherichia spp.and Acinetobacter spp.carrying virulence genes accounted for 78.3% and 6.5% of all potential pathogens in swine fecal samples;in chicken fecal samples,the relative abundance of Escherichia spp.,Campylobacter spp.,and Klebsiella spp.were 33.8%,31.1% and 4.9%,respectively.Taxonomy annotation of contigs carrying ARGs revealed that genus such as Escherichia spp.,Klebsiella spp.,E.faecalis spp.and Clostridium spp.carried more resistance genes,for example,in the CA group,Escherichia spp.and Klebsiella spp.carried95 and 91 ARGs,respectively.In swine fecal samples,the relative abundance of Escherichia spp.,Bacillus spp.and Clostridium spp.accounted for 26%,6% and 5% of all PB;in chicken fecal samples,the relative abundance of Bacillus spp.,Clostridium spp.,Cryptobacterium spp.and Escherichia spp.were 8%,7%,5% and 4%,respectively.Note that Escherichia spp.and Klebsiella spp.were the species enriched in VFGs and ARGs,and Escherichia spp.were also the dominant species in terms of abundance.In addition,15 VFGs and 12 ARGs were also detected in E.coli isolated from livestock and poultry manure using multiplex PCR.3.Reconstruction and analysis of the draft genomes of potential pathogenic and antibiotic resistant bacteria in livestock manureIn order to recover the genomes of microorganisms in livestock manure,we first conducted a comprehensive evaluation of 12 origin binner and 3 refine binner in terms of purity,completeness,F1 score,and adjusted Rand index(ARI).The results show that groopm2,metabat2 and maxbin2 have high accuracy,good completeness and relatively low consumption of computational resources,while DASTool can effectively reduce the contamination of binning results and improve the quality of genomes.Then,719 bacterial genomes with at least 60% completeness and at least 10% contamination were successfully recovered using the above four binners,in which the Sclerotinia,Bacteroidetes,Actinomycetes and Proteobacteria were dominant.719 bacterial genomes included 16 genomes of potentially PB,88 genomes of potentially ARB and 10 genomes of PARB.Five of the PARB were E.coli.The average number of VFGs and ARGs carried by the recovered E.coli genomes was 48,including not only all ARG subtypes on E.coli O15:H7 reference genome,but also 13 ARG subtypes were identified,seven of which belonged to the multidrug resistance class.In summary,this study found that livestock and poultry manure microorganisms in the Wanjiang River basin carried a large number and type of VFGs and ARGs,and their abundance was correlated with the microbial community structure.These VFGs and ARGs are enriched on species such as Escherichia spp.and Klebsiella spp.and they should be paid close attention and monitored;while species such as Escherichia spp.,Campylobacter spp.and Synechococcus spp.are potential PB and ARB with high abundance and should be the key targets for the treatment of livestock and poultry manure.E.coli is the most important host of VFGs and ARGs in livestock and poultry manure.The results provide a reference for microbiological risk assessment of livestock manure and pollution control of livestock manure in the Wanjiang River Basin,which is important for ensuring public health safety and sustainable development of livestock and poultry farming industry in the Wanjiang River Basin area.