粪肥对土壤细菌四环素耐药性接合转移的影响及其机制

Manure has become a reservoir of antibiotic resistant bacteria and resistant genes, its long-term application to agricultural soils would increase the antibiotic resistant pollution and promote the dissemination of antibiotic resistances in the environment. Conjugative transfer of resistance genes are believed to be the most convenient and effective mode in the adaptation of bacteria to antibiotics pressure, but the influence and mechanism of long-term manure application to the soil bacteria in transferring its resistance genes to other recipient bacteria by conjugative elements (e.g. plasmids) remains ambiguous. By conventional microbiology methods and molecular techniques, combining field sampling with laboratory research, we will systematically compare and analysis the bacteria community resistant to tetracycline in agricultural soil after long-term application of manure or chemical fertilizer, and its potential for transferring resistance genes to human enteric bacteria. On the basis of pinpointing the CFU numbers and community features of the tetracycline resistance bacteria, transfer frequencies of the resistance gene in each bacteria community will be measured by filter-mating method, and the tetracycline genes and transferable elements carried by these bacteria will also be detected, quantified and positioned. Our purpose is to explore the impact and mechanism of manure application to the soil bacteria in transferring its resistance genes to human enteric bacteria, which will contribute scientific basis in estimating the risk of the resistance gene pollution and dissemination to the ecological system during manure application.

粪肥中含有大量抗性细菌和抗性基因，粪肥长期施用引起的抗性基因污染和转移扩散风险是广泛关注的热点。接合转移是细菌耐药性转移效率最高的方式，深入研究施用粪肥对土壤细菌耐药性接合转移的影响和机制对于控制耐药性在环境中的传播和扩散具有指导意义。本项目拟采用传统微生物和现代分子生物学技术，结合野外采样和实验室研究，对长期施用粪肥或化肥土壤中的四环素抗性细菌的种群分布和耐药性转移能力开展系统的比较和研究。在明确长期施肥土壤中四环素抗性细菌数量及其群落特征的基础上；通过filter-mating方法，揭示粪肥长期施用对细菌四环素耐药性接合转移能力的影响；对细菌携带的可移动遗传因子和抗性基因进行检测、位点分析和定量表征，探明施用粪肥对土壤细菌四环素耐药性接合转移的影响机制。项目研究成果可为评价粪肥农用过程中抗生素抗性基因的生态风险提供科学依据。

本项目利用江苏常熟农田生态系统国家野外科学观测站长期施肥水旱轮作土壤为研究对象，采用传统微生物和现代分子生物学技术，对长期施用新鲜猪粪的土壤（HF）和长期施用化肥的土壤（CF）中四环素抗性细菌（TRB）的种群分布和耐药性转移能力开展系统的比较和研究。研究结果表明：（1）长期施用新鲜猪粪显著增加了土壤中TRB的数量、种类和多样性，使土壤中的优势TRB类群由Chryseobacterium转变为Bacillus。（2）TRB携带的TRG主要位于基因组上，部分TRB携带质粒且质粒上均能检测到TRG。长期施用猪粪增加了TRB基因组和质粒上携带的TRG种类，改变了TRB携带的优势TRG类型；此外，可移动遗传因子（MGE）的检出率也增加，其中intI1和IS1111为优势MGE。长期施用粪肥增加了土壤中携带多种TRG或MGE的TRB比例，这或许是长期施用粪肥后土壤中TRB数量、比例和多样性增加的主要原因之一。（3）虽然分离的TRB携带多种TRG和MGE，但是在缓冲液中或滤膜上，即使存在不同浓度的抗生素选择压力，TRB菌株也不能将其携带的TRG转移给抗生素敏感型沙门氏菌。然而，在自然条件下，粪肥或土壤中的TRG或携带TRG的质粒却能够转移到沙门氏菌中。本项目研究成果可为评价粪肥农用过程中抗生素抗性基因的生态风险提供科学依据，也为粪肥无害化处理的必要性提供了理论依据。

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