宁夏养鸡场粪污和周边土壤中抗生素及抗生素抗性基因分布特征

Distribution Characteristics of Antibiotics and Antibiotic Resistance Genes in Manure and Surrounding Soil of Poultry Farm in Ningxia

畜禽粪便中的抗生素及其抗性基因(ARGs)具有潜在的生态风险.为揭示宁夏养鸡场鸡粪和养殖场周边土壤中抗生素和ARGs的分布特征,以宁夏12家不同规模蛋鸡养殖场中鸡粪、周边及施用鸡粪的土壤为研究对象,采用超高效液相色谱-串联质谱法和高通量荧光定量法进行调查研究.结果表明:(1)鸡粪中四环素类、氨基糖苷类和磺胺类是优势抗生素.不同养殖期鸡粪中抗生素种类和含量不同,育雏期抗生素种类较多和平均含量较高,初产期相反.(2)周边土壤中仅距离养鸡场20 m处检测到少量抗生素,养殖场对周边土壤中抗生素的分布影响较小;施用鸡粪的土壤中喹诺酮类抗生素含量明显增加.(3)鸡粪中共检测到ARGs亚型132~168种,主要是氨基糖苷类和四环素类ARGs.育成期ARGs数目最多,初产期最少;育雏期各类ARGs总丰度最高,终产期相反.所有养殖期鸡粪中共存ARGs有110种.(4)养鸡场周边及施用鸡粪的土壤中共检测到ARGs亚型23~105种,其中氨基糖苷类ARGs数目最多,其次是多药类.养殖场周边土壤中ARGs数目和相对丰度随距养殖场距离的增加而逐渐减少;施用鸡粪的土壤中ARGs数目和相对丰度都明显增高,但低于距离养殖场20 m的土壤.(5)鸡粪中β-内酰胺酶类、氨基糖苷类和大环内酯类-林肯酰胺类-链阳性菌素B类(MLSB)ARGs都存在水平移动风险;土壤中氯霉素类ARGs存在水平移动风险.鸡粪中ARGs与其对应抗生素含量间相关性不显著.(6)不同类型ARGs之间具有相关的共现性:鸡粪中氨基糖苷类ARGs相对丰度与β-内酰胺酶类和可移动元件(MGEs)、多药类与万古霉素类等均呈显著正相关;土壤中氨基糖苷类ARGs相对丰度与四环素类、万古霉素类、磺胺类和MLSB类、四环素类与MLSB类等呈极显著正相关.土壤中各类ARGs相对丰度间的共现性明显强于鸡粪.本研究可为养鸡场选址、蛋鸡规模化养殖抗生素种类及剂量的选择和鸡粪施用提供理论依据.

Antibiotics and antibiotic resistance genes(ARGs) in livestock and poultry manure pose potential ecological risks.In order to understand the distribution characteristic of antibiotics and ARGs in manure and surrounding soils of poultry farms in Ningxia,the poultry manure and relative soil samples were collected from 12 layers of different poultry breeding farms.The compositions of antibiotics and ARGs in the samples were analyzed using UPLC-MS/MS and HT-qPCR.The results showed that:(1) tetracycline,aminoglycoside,and sulfonamide were the dominant antibiotics in poultry manure.The types and contents of antibiotics in poultry manure were different in different breeding periods.There were more types of antibiotics in the brooding period,the average content was high,and the initial stage showed the opposite trend.(2) A small amount of antibiotics was detected in the surrounding soil only 20 m away from the poultry farm,and the poultry farm had little effect on the distribution of antibiotics in the surrounding soil.The content of quinolone in the soils with poultry manure application was significantly higher than that in the control and surrounding soil.(3) We detected 132-168 ARGs in poultry manure,and the number of aminoglycosides and tetracycline was higher.The relative abundance of ARGs in the rearing period was highest,and the initial stage showed the opposite trend.The total relative abundance of ARGs in the brooding period was highest,but the terminal period showed the opposite.There were 110 ARGs in poultry manure during all breeding periods.(4) There were 23-105 ARGs in the soils,and the number of aminoglycoside was highest,followed by multidrug ARGs.The poultry farm had a great effect on the number and relative abundance of ARGs in the surrounding soil.For example,the number and relative abundance of ARGs in the surrounding soil of poultry farms gradually decreased with the increase in the distance from the poultry farms.The number and relative abundance of ARGs in the soil with applied poultry manure were significantly increased;however,these values were lower than those in the soil 20 m away from the poultry farm.(5) β-lactamases,aminoglycosides,and macrolide lincosamide-streptogramin B(MLSB) ARGs were all at risk of horizontal movement in manure,and chloramphenicol ARGs were at risk of horizontal movement in soil.Correlation analysis showed that the relative abundance of aminoglycoside,tetracycline,sulfonamide,β-lactamase,and MLSB were not significantly correlated with their contents.(6) Different types of ARGs had related co-occurrence phenomena,such as the positive correlation between the relative abundance of ARGs in poultry manure,and aminoglycoside and β-lactamases,MGEs,multidrugs and vancomycins.The relative abundances of ARGs in soil,aminoglycoside and tetracyclines,vancomycins,sulfonamides,and MLSBs;tetracyclines and MLSBs;etc.,all showed a significant positive correlation.In short,the co-occurrence among the relative abundance of ARGs in soil was significantly stronger than that in poultry manure.These results could provide the theoretical basis for the site selection of poultry farms,the selection of antibiotic types and dosages for large-scale breeding of laying hens,and the application of poultry manure.