畜禽粪便中多重耐药细菌及耐药基因的分布特征

Distribution of Multidrug-Resistant Bacteria and Antibiotic-Resistant Genes in Livestock Manures

为了解畜禽粪便中多重抗生素耐药细菌及耐药基因的污染特征,采用微生物培养的方法调查了鸡粪、猪粪中多重耐药细菌的数量,并挑取部分菌株进行16S rDNA鉴定和抗生素敏感性试验;进一步通过高通量测序技术解析多重耐药细菌的群落结构,利用高通量定量PCR对粪便中176种耐药基因的分布情况进行研究.结果表明,不同鸡粪、猪粪中对四环素、环丙沙星和庆大霉素同时耐药的多重耐药细菌比例在7.96%~12.40%;单菌株鉴定和群落结构分析均显示,可培养的多重耐药细菌主要集中在Escherichia(埃希氏杆菌属)、Acinetobacter(不动杆菌属)和Proteus(变形杆菌属)中.与未饲用抗生素的猪粪相比,猪粪样品中耐药基因的总富集倍数达到1.96×10~4~1.54×10~5倍,各类耐药基因的富集情况为:四环素类>β-内酰胺类>MLSB(大环内酯、林可酰胺和链阳性菌素B类)>氨基糖苷类>FCA(氟喹诺酮、喹诺酮、氟苯尼考、氯霉素和酰胺醇类)>磺胺类>万古霉素类.

In the present study,techniques for microbial culture enumeration,16S rDNA gene sequencing for bacterial identification,high-throughput sequencing for the multidrug-resistant bacteria（MRB） communities,and high-throughput quantitative PCR detection for the prevalence and abundance of antibiotic resistance genes（ARGs） were conducted to investigate the distribution characteristics of MRB and ARGs in chicken and swine manures.The results showed that the rates of MRB that were resistant to tetracycline,ciprofloxacin,and gentamycin simultaneously were from 7.96% to 12.40% for the different manure samples.The dominant genera of cultivable MRB were Escherichia,Acinetobacter,and Proteus,both in the chicken and swine manures.High-throughput quantitative PCR results indicated that,compared to the antibiotic free swine manure,the total enrichment of ARGs increased by 1.96×10~4-1.54×10~5 times that in the swine manure samples,with different antibiotics following the sequence of tetracyclinesβ-lactamsMLSB（macrolide,lincosamide,and streptogramin B）aminoglycosidesFCA（fluoroquinolone,quinolone,florfenicol,chloramphenicol,and amphenicol）〉sulfonamides〉vancomycins.