兽用抗生素胁迫下猪粪堆肥对抗生素抗性基因的影响机制解析

The application of veterinary antibiotics in livestock breeding industry has caused significant pollution problem of antibiotic resistance genes (ARGs), which directly affects the recycle of livestock manure. Compost is the key technology to achieve planting-breeding technology, which can influence the concentration of ARGs, but the effect and mechanism is not clear. Therefore, this project uses three antibiotics commonly used in the pig husbandry, including doxycycline, tilmicosin and enrofloxacin as model antibiotic, collect the pig manure to obtain the concentration of antibiotics, corresponding ARGs and dominate microorganism from pigs treated with antibiotics. In addition, the composting test is carried out on the basis of pig manure to find out the dominant bacteria which carried resistance genes and their succession rule by using 16SrRNA and 18SrRNA sequencing, metagenome sequencing and quantitative PCR, etc. Furthermore, to confirm the effect of mobile genetic genes (MGEs) on horizontal transfer of ARGs and find out the key influence factor of the concentration of AGRs during piggery manure composting by combining the redundancy and association analysis of the AGRs concentration, veterinary antibiotics and its active metabolites, microbial community structure and physicochemical properties, etc. This project has great significance for controlling and reducing the environmental pollution of ARGs to promote the recycle of pig manure.

兽用抗生素在养殖业的应用带来了不容忽视的抗生素抗性基因污染，直接影响畜禽粪便的资源化利用前景。堆肥作为畜禽粪便资源化利用的主推接口技术，可影响抗生素抗性基因，但效果不一且机制未明。因此本项目以养猪业常用的强力霉素、替米考星和恩诺沙星为研究对象，通过猪粪收集试验获得含兽用抗生素及抗生素抗性基因的猪粪，定位治疗用药后猪粪中携带抗性基因的优势菌群；并以此猪粪为基质进行堆肥试验，综合16S rRNA、18S rRNA和宏基因组测序等技术，定位堆肥中携带抗性基因的优势菌群，揭示其演替规律，并结合抗生素抗性基因、兽用抗生素及其活性代谢产物、堆肥理化性状等指标，通过冗余分析，建立兽用抗生素、微生物群落结构、堆肥理化性状与抗生素抗性基因间的关联，解析兽用抗生素胁迫下猪粪堆肥对抗生素抗性基因的影响机制。本项目对于控制和减少抗生素抗性基因环境污染，促进猪粪的资源化利用具有重要意义。

兽用抗生素在养殖业的应用带来了不容忽视的抗生素抗性基因污染，直接影响畜禽粪便的资源化利用前景。堆肥作为畜禽粪便资源化利用的主推技术，可影响抗生素抗性基因，但效果不一且机制未明。本项目以养猪业常用的强力霉素、替米考星和恩诺沙星为研究对象，通过猪粪收集试验获得含兽用抗生素及抗生素抗性基因的猪粪，定位治疗用药后猪粪中携带抗性基因的优势菌群；并以此猪粪为基质进行堆肥试验，综合16S rRNA、宏基因组测序和epicPCR等技术，定位堆肥中携带抗性基因的优势菌群，揭示其演替规律，解析兽用抗生素胁迫下猪粪堆肥对抗生素抗性基因的影响机制。结果表明，经饲强力霉素未显著影响猪粪中四环素类ARGs的总相对丰度，但经饲高浓度强力霉素通过改变猪粪微生物群落结构，使tetW、tetA和tolC的潜在宿主菌减少，从而导致其绝对丰度显著降低了17.60%-75.04%；厚壁菌门相对丰度的降低是堆肥对四环素类ARGs去除的关键因素，但高浓度强力霉素残留时厚壁菌门、放线菌门的丰度增加显著阻碍了堆肥对四环素类ARGs的消减。按规范使用恩诺沙星不会增加猪粪中氟喹诺酮类ARGs的残留风险；堆肥对猪粪中qnrS和qnrD等胞内氟喹诺酮类ARGs丰度的去除率为91.13%-99.06%，但未有效去除胞外氟喹诺酮类ARGs；优势菌属链球菌属和乳酸菌属的减少是胞内氟喹诺酮类ARGs丰度下降的主要原因之一。经饲替米考星可显著改变猪粪微生物群落结构，且替米考星胁迫可导致猪粪中多重耐药类ARGs tolC通过int1和int2在优势微生物之间水平传播，从而使tolC绝对丰度增加；堆肥对ermC等MLSB类ARGs、mefA等大环内酯类ARGs、acrA等多重耐药ARGs和intl2等可移动遗传元件的去除率范围为59.24%-94.60%，但ermA的相对丰度在堆肥结束后增加了32.50%。综上所述，本项目明确了猪粪堆肥通过优势菌群的演替从而影响ARGs的消减效果，解析了三种兽用抗生素胁迫对猪粪堆肥中ARGs的影响机制，为开发猪粪源ARGs的控制和消减技术提供参考，对促进畜禽粪便的资源化利用具有重要意义。