蝇蛆肠道微生物菌群对猪粪残留抗生素的降解及抗性基因的影响

Housefly larvae gut microbiome may attenuate veterinary antibiotics and alter the resistome in swine manure

摘要:

[目的]过度使用抗生素作为动物饲料添加剂，导致畜禽粪便已成为抗生素抗性基因的主要蓄积库，为了研究蝇蛆（Musca domestica）对猪粪中残留抗生素及抗性基因的影响，本文动态采集了实际农场条件下蝇蛆转化过程中猪粪堆体及虫体样本。[方法]利用qPCR、液相色谱-电喷雾质谱、同位素内标法、Illumina高通量测序以及局部相似性研究蝇蛆生物转化过程中残留抗生素降解效能及相关抗性基因组变化的微生物生态机制。[结果]6 d周期内，猪粪中四环素、土霉素、金霉素、强力霉素、磺胺嘧啶、诺氟沙星、氧氟沙星、环丙沙星以及恩诺沙星等9种残留抗生素含量显著下降，累积减量为34.3%–58.1%，每日减量百分比介于7.8%–57.4%之间。猪粪中共检测到的158种抗性基因，其中有118种大幅衰减，衰减量平均达79.3%；23种抗性基因存在富集现象，富集倍数平均为3.48。在蝇蛆肠道的作用下，粪源微生物群落中Bacteroidetes相对丰度下降，Proteobacteria相对丰度增加，尤其是Ignatzschineria增幅最大。网络分析发现，抗性基因的增减与微生物群落的变化显著相关，与抗性基因衰减相关的微生物主要属于Clostridiales和Bacteroidales，而与抗性基因富集相关的微生物主要为Alcaligenaceae、[Weeksellaceae]及Bacillales。[结论]蝇蛆可有效削减猪粪中的残留抗生素及防控抗性基因向环境扩散。

Abstract:

[Objective] The overuse of antibiotics as veterinary feed additives is contributing to a significant reservoir of antibiotic resistance in livestock manure. A housefly larvae (Musca domestica) vermireactor has been increasingly adopted for swine manure value-added bioconversion and pollution control. In order to investigate its efficiency on attenuations of antibiotic and the associated resistome during manure vermicomposting, we implemented dynamic sampling during a typical cycle (6 days). [Methods] High-capacity qPCR, liquid chromatography-electrospray ionization tandem mass spectrometry, the isotope-labeled internal standard method and Illumina Hiseq sequencer were used to explore the effectiveness of antibiotic degradation and microbial mechanisms on dynamics of the associated resistome of full-scale larvae manure vermicomposting. [Results] Nine antibiotics (tetracycline, oxytetracycline, chlortetracycline, doxycycline, sulfadiazine, norfloxacin, ofloxacin, ciprofloxacin and enrofloxacin) were dramatically removed. The cumulative removal of these tested antibiotics reached 34.3%-58.1%, along with the daily reduction percentages ranging from 7.8% to 57.4%. In total, 113 of 158 antibiotic resistance genes (ARGs) were significantly attenuated (by 79.3%), while 23 were significantly enriched (3.48-fold) following vermicomposting. The manure-borne bacterial community showed a decrease in the relative abundance of Bacteroidetes, and an increase in Proteobacteria, specifically Ignatzschineria, following gut transit, while ARG attenuation was significantly correlated with changes in microbial community structure. Bacteria correlated with attenuated ARGs mainly belonged to order Clostridiales and Bacteroidales. Enriched ARGs, were mostly correlated with family Alcaligenaceae,[Weeksellaceae] and order Bacillales. [Conclusion] Housefly larvae vermicomposting is an efficient way to attenuate antibiotics and ARGs in swine manure within the 6-day treatment.