奶牛养殖场堆积粪便中病原微生物多样性及耐药基因丰度研究

Diversity Research of Pathogenic Microorganisms and Abundance of Antibiotic Resistance Gene of Piled Manure in Dairy Farm

为了探讨堆积粪便和周围土壤中微生物相对丰度,分析粪便堆积和周围土壤中病原微生物和抗生素耐药基因的分布及富集情况,本研究收集宁夏规模化奶牛养殖场3份堆积粪便和3份堆积区域周围土壤样本,采用宏基因组测序获得样本全基因组信息,使用NCBI-NR数据库进行分类学注释和分析,通过PHI数据库对样本内病原微生物和毒力基因进行注释分析,通过ARDB数据库对样本内抗生素耐药基因进行注释分析。结果表明,在堆积粪便和土壤中共注释到39个门、76个纲、167个目、369个科、1103个属、3264个种,其中细菌在堆积粪便及土壤样品中分别占97.49%和98.63%,为绝对优势。奶牛养殖场堆积粪便和周围土壤微生物群落主要由变形菌门、拟杆菌门和放线菌门等39个门组成。堆积粪便中病原微生物的种类、毒力基因相对丰度均高于土壤中相对丰度(657961.00±391617.24;155553.67±43038.27,P<0.01)。土壤中病原微生物相对丰度(relative abundances,RAs)在不同土壤深度中无差异,而堆积粪便中34个病原微生物属RAs均随粪便堆积深度的增加而增大,优势种主要为铜绿假单胞菌(Pseudomonas aeruginosa)、沙门氏菌(Salmonella enterica)、金黄色葡萄球菌(Staphylococcus aureus)、肺炎链球菌(Streptococcus pneumoniae)、爱德华氏菌(Edwardsiella ictaluri)、稻瘟病菌(Magnaporthe oryzae)和稻白叶枯黄杆菌(Xanthomonas oryzae)。堆积粪便中共注释到所属49类抗生素78种优势耐药基因(相对丰度>0.5%),高于土壤中的49种耐药基因(所属31类抗生素),其中29种耐药基因的相对丰度随堆积深度上调。主要包括链阳霉素A(vatb基因)、林可酰胺类抗生素/大环内脂类抗生素/链阳霉素B(tlrc,cara,oleb,srmb基因)、万古霉素(vanug基因)。可见牛奶养殖粪便的堆积会促进病原微生物、毒力基因、耐药基因的富集,并可能对周围土壤造成污染。

To investigate the relative abundance of microorganisms,analysis distribution and enrichment of pathogenic microorganisms and antibiotic resistance genes in piled manure and surrounding soil,3 piled manure and 3 surrounding soil specimens were collected in dairy farm in Ningxia.The DNA samples were exam based on metagenomic sequencing;Contigs were estimated by taxonomy annotation using NCBI-NR database;PHI database was used to detect abundance of pathogens and virulence genes;ARDB database was used to detect abundance of antibiotic resistance genes(ARGs).The results show that 39 phyla,76 classes,167 orders,369 families,1103 genera and 3264 species were obtained in 6 samples.Bacteria absolute superiority and accounted for 97.49%and 98.63%in piled manure and soil samples,respectively.The microbial communitys in dairy farms are mainly composed of 39 phylum such as Proteobacteria,Bacteroidetes and Actinomycetes.RAs of pathogenic microorganisms and virulence genes in manure was higher than in soil(657961.00±391617.24:155553.67±43038.27,P<0.01).There was no difference in the RAs of pathogens and virulence genes at different soil depths,while the RAs of 34 pathogens in the manure increased with the increase of the piled depth.The dominant species including Pseudomonas aeruginosa,Salmonella enterica,Staphylococcus aureus,Streptococcus pneumoniae,Edwardsiella ictaluri,Magnaporthe oryzae and Xanthomonas oryzae.Total of 78 dominant ARGs root in 49 antibiotics(RAs>0.5%)were observed in piled manure,which were higher than 49 ARGs root in 31 antibiotics in the soil.Among them,the RAs of 29 ARGs were increased with the piled depth.It mainly includes streptomycin A(vatb),lincomycin/macrolide antibiotic/streptomycin B(tlrc,cara,oleb,srmb),vancomycin(vanug).Waste manure piled in dairy farm will promote the accumulation of pathogens,virulence genes,ARGs,and may cause pollution to the surrounding soil.