畜禽粪便源芽孢杆菌多样性及对铜耐受性研究

Diversity and copper tolerance of Bacillus strains from livestock and poultry feces

芽孢杆菌制剂广泛应用于畜禽养殖和粪污处理等领域,铜作为饲料添加剂用于畜禽养殖,导致畜禽粪便中大量残留,在此压力下芽孢杆菌等会产生铜耐受性,由于芽孢杆菌的抗逆性,这种耐受性可由可移动遗传元件介导在环境中传播.本研究采集了多个规模化养殖场的牛粪、鸡粪和猪粪,从粪便中分离芽孢杆菌,通过PCR技术、最小抑菌浓度测定和全基因组测序方法,了解畜禽粪便中芽孢杆菌对铜耐受性.研究发现:(1)分离出芽孢杆菌属23种芽孢杆菌共235株.分离数量前三分别是枯草芽孢杆菌(Bacillus subtilis)77株,占总数的32.77%;蜡样芽孢杆菌(B.cereus)31株,占比13.19%;贝莱斯芽孢杆菌(B.velezensis)28株,占比11.91%.(2)芽孢杆菌对铜耐受表型和基因型研究发现,铜对芽孢杆菌的MIC集中在200~400 mg·L^(-1),占总数235株的77.87%(n=183),铜耐受能力较高的菌株能够达到500~600 mg·L^(-1),占总数的16.17%(n=38),5株MIC达到600 mg·L^(-1)的菌株全部来自于猪粪.检测了6种铜耐受基因,占比前三的基因分别是pco C、pco D、cop A,比例分别是30.21%(n=71)、29.79%(n=70)、22.98%(n=54),pco A、cop B分别为15.74%(n=37)、15.32%(n=36),检出率最低的为pco R(n=15,P=6.38%).由6种检测到的基因形成了42种携带组合情况,pco系统铜耐受基因组合情况共计39种,携带cop系统铜耐受基因的组合情况仅3种.(3)筛选出不含质粒介导铜耐受基因(pco系统)的高温耐受菌株,进行全基因组测序,分析了菌株携带耐受基因情况及铜耐药基因环境特征.本研究为畜禽粪便堆肥发酵中菌种安全利用提供了依据.

Bacillus preparations are widely used in livestock breeding,fecal sewage treatment and other fields.Copper is used as a feed additive in livestock breeding,resulting in a large number of residues in livestock and poultry feces.The copper pressure may select copper tolerant Bacillus strains.The copper tolerance of the Bacillus strains can be transmitted via mobile genetic elements in the environment.We collected cow dung,chicken manure and pig manure from large-scale farms,isolated Bacillus from the feces,and analyzed the copper tolerance of the Bacillus isolates by determining the minimum inhibitory concentration(MIC)of copper,and by using PCR technology and whole genome sequencing.The results showed that:(1)A total of 235 Bacillus strains,belonging to 23 Bacillus species,were isolated,including 77 B.subtilis strains,accounting for 32.77%of the isolates,31 B.cereus strains,accounting for 13.19%,and 28 B.velezensis strains,accounting for 11.91%.(2)The MIC of copper in Bacillus strains was in the range of 200~400 mg·L^(-1);the 183 copper tolerant strains accounted for 77.87%of the total 235 strains.The strains with high copper tolerance had MIC from 500 mg·L^(-1)to 600 mg·L^(-1),and accounted for 16.17%of all isolates(n=38).The five strains with the MIC of 600 mg·L^(-1)were all from pig manure.Six types of copper tolerance genes were detected.The genes pco C,pco D and cop A were detected in 30.21%(n=71),29.79%(n=70)and22.98%(n=54)of the strains,respectively.The pco A and cop B genes were detected in 15.74%(n=37)and 15.32%(n=36)of the strains,respectively.The gene pco R was detected in 6.38%(n=15)of the strains.The strains carried the genes in 42 different gene combinations,among which39 gene combinations included pco type copper resistance genes and only 3 included cop type genes.(3)The high temperature resistant strains without plasmid-mediated copper tolerance gene(pco system)were screened.The types of tolerance genes the strains carried were analyzed by whole genome sequencing,and the environmental characteristics of copper tolerance genes were also analyzed.This study provides a basis for the safe utilization of Bacillus strains in livestock manure composting.