猪圈发酵床中细菌的分离鉴定及其特征研究
|
摘要
目的:研究猪圈发酵床分离细菌的系统发育树及生理生化特征。方法:采用R2A培养基对猪发酵床上层垫料进行分离菌株培养,将培养得到的单个菌落培养于固体培养基中,获得纯培养菌株;肉眼观察培养基中细菌的生长情况,电镜下观察细菌的形态、大小和排列特点,采用16S rRNA基因序列扩增、测序,邻接法构建16S rRNA序列的系统发育树,将分离到的猪圈发酵床中的细菌与文献中获知的初期发酵床优势细菌的标准菌株进行生理生化特征的比对。结果:在R2A培养基中分离得到3株细菌SZDIS-1-1、GZDIS-1和SZDIS-2,鉴定为微杆菌属细菌;与文献中获知的4种标准菌株一样,培养得到的3种细菌镜下均为杆状,过氧化氢酶、吲哚试验均为阴性,能够利用麦芽糖作为生长碳源;药敏试验显示,分离得到的3株细菌对阿米卡星、头孢呋辛、利福平、妥布霉素、青霉素和环丙沙星等抗生素比较敏感,其中头孢呋辛抑菌效果最佳。结论:在猪圈发酵床首次分离得到3株菌株,并构建系统发育树。
Objective: To investigate phylogenetic tree and physiological-biochemical characteristic of microbial identification from sawdust bed in a pigpen. Methods: The bacteria were isolated and cultured by R2 A solid media,and cultured individual bacteria colony was further cultured in solid culture medium to obtain pure culture strains. Observing bacteria growth condition in culture medium with naked eye,morphological features,size and sequencing features were observed under electron microscope. Characteristics of the cultured bacteria was analyzed by using 16 S rRNA gene NASBA and sequencing,and buiding phylogenetic tree of 16 S rRNA sequence by neighbor-joining method. Comparing physiological-biochemical characteristics of isolates in the sawdust bed of pigpen and the dominant strain of the type species in reference. Results: Three bacteria strains isolated on the R2 A solid medium,SZDIS-1-1,GZDIS-1 and SZDIS-2,were identified as Microbacterium by gene sequences analysis. Identical with the 4 standard strains from reference,the three cultured bacteria was in the form of rod under microscope. Peroxidase and Benzazole experiment showed negative,it could use malt sugar as growth source. The drug sensitive test showed that the three strains were sensitive to Amikacin,Cefuroxime,Rifampicin,Tobramycin,Penicillin,Ciprofloxacin,of which Cefuroxime showed the best bacteria inhibition effect. Conclusions: Three strains isolated from sawdust bed in pigpen established phylogenetic tree.
Objective: To investigate phylogenetic tree and physiological-biochemical characteristic of microbial identification from sawdust bed in a pigpen. Methods: The bacteria were isolated and cultured by R2 A solid media,and cultured individual bacteria colony was further cultured in solid culture medium to obtain pure culture strains. Observing bacteria growth condition in culture medium with naked eye,morphological features,size and sequencing features were observed under electron microscope. Characteristics of the cultured bacteria was analyzed by using 16 S rRNA gene NASBA and sequencing,and buiding phylogenetic tree of 16 S rRNA sequence by neighbor-joining method. Comparing physiological-biochemical characteristics of isolates in the sawdust bed of pigpen and the dominant strain of the type species in reference. Results: Three bacteria strains isolated on the R2 A solid medium,SZDIS-1-1,GZDIS-1 and SZDIS-2,were identified as Microbacterium by gene sequences analysis. Identical with the 4 standard strains from reference,the three cultured bacteria was in the form of rod under microscope. Peroxidase and Benzazole experiment showed negative,it could use malt sugar as growth source. The drug sensitive test showed that the three strains were sensitive to Amikacin,Cefuroxime,Rifampicin,Tobramycin,Penicillin,Ciprofloxacin,of which Cefuroxime showed the best bacteria inhibition effect. Conclusions: Three strains isolated from sawdust bed in pigpen established phylogenetic tree.
引文
[1]李文春.猪胆管的生物力学特性及作为组织工程胆管的可行性[D].武汉:武汉大学,2013.
[2]李静,王配军,李文春.猪胆管作为人胆管移植替代材料的生物力学评价[J].湖北医药学院学报,2016,33(5):425-432.
[3]李红玉.现代化生猪养殖环境测控技术现状与发展趋势[J].国外畜牧学,2017,23(4):78-79.
[4]刘燕.规模化猪场疾病防控存在的问题及对策[J].湖北畜牧兽医,2017,26(7):43.
[5]杨发.宁夏生物环保养猪技术研究与示范[D].陕西:西北农林大学,2015.
[6]何燕柳.猪舍结构及日粮类型对猪舍内环境、猪生长性能及血液各指标的影响[D].广西:广西大学,2013.
[7]WATKINS J,SARTORY D P.Evaluation of a membrane filtration method for the rapid enumeration of confirmed Clostridium perfringens from water[J].Lett Appl Microbiol,2015,60(4):367-371.
[8]KIM O S,CHO Y J,et al.Introducing Ez Taxon-e:a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species[J].Int J Syst Evol Microbiol,2016,12(62):716-721.
[9]ASNANI M R,QUIMBY K R.Interventions for patients and caregivers to improve knowledge of sickle cell disease and recognition of its related complications[J].Nucleic Acids Res,2016,10(6):73-80.
[10]LI X J,MA T B,WANG P.Molecular epidemiology of Cryptosporidium infection in infants with diarrhea in Wuhan City[J].Zhongguo Xue Xi Chong Bing Za Zhi,2017,29(2):188-191.
[11]卢舒娴.养猪发酵床垫料微生物及其猪细菌性病原群落动态的研究[J].农业资源与环境学报,2016,5(33):425-432.
[12]朱双红.猪生物发酵床垫料中细菌群落结构动态变化研究[D].武汉:华中农业大学,2012.
[13]LEE K,LEE S S.Aneurinibacillus humi sp.nov.,isolated from soil collected in ukraine[J].Int J Syst Evol Microbiol,2016,72(2):139-144.
[14]NQUYEN T M,KIM J.A rapid and simple method for identifying bacterial polar lipid components in wet biomass[J].J Microbiol,2017,55(8):635-639.
[15]ZHANG G X,ZENG G Q,CAI X W,et al.Brachybacterium zhongshanense sp.nov.,a cellulose-decomposing bacterium from sediment along the Qijiang River,Zhongshan City,China[J].Int J Syst Evol Microbiol,2013(57):2519-2524.
[16]WU H G,LIU W S.Research and analysis of 74 bloodstream infection cases of acinetobacter baumannii and drug resistance[J].Eur Rev Med Pharmacol Sci,2018,22(6):1782-1786.
[17]GHAZNAVI H,AALI E.Association study of the ATPbinding cassette transporter A1(ABCA1)Rs2230806 genetic variation with lipid profile and coronary artery disease risk in an iranian population[J].Open Access Maced J Med Sci,2018,6(2):274-279.
[18]LINHARES P,VIANA P,FERREIRA M.Genetic variants of vascular endothelial growth factor predict risk and survival of gliomas[J].Tumour Biol,2018,40(3):50-56.
[19]LI L,PAN Y,DAI L.Association of genetic polymorphisms on vascular endothelial growth factor and its receptor genes with susceptibility to coronary heart disease[J].Med Sci Monit,2016,4(22):31-40.
[20]ORIA-JENSEN,S.Penicillin-binding proteins in Actinobacteria[J].J Antibiot,2015,68(4):223-245.
[21]ASAF S,KHAN A L,KHAN M A,et al.Qsmoprotective functions conferred to soybean plants via inoculation with Sphingomonas sp.LK11 and exogenous trehalose[J].Microbiol Res,2017,12(205):135-145.
[22]BRENNAN N M,BROWN R,GOODFELLOW M,et al.Microbacterium gubbeenense sp.nov.,from the surface of a smear-ripened cheese[J].Int J Syst Bacteriol,2016,3(51):1969-1976.
[23]KAMPFER P,SCHAFER J,LODDERS N,et al.Microbacterium murale sp.nov.,isolated from an indoor wall[J].Int J Syst Bacteriol,2012,7(62):2669-2673.
[24]YANG Z W,SALAM N,MOHANY M,et al.Microbacterium album sp.nov.and microbacterium deserti sp.nov.,two halotolerant actinobacteria isolated from desert soil[J].Int J Syst Bacteriol,2017,1(68):217-222.
[2]李静,王配军,李文春.猪胆管作为人胆管移植替代材料的生物力学评价[J].湖北医药学院学报,2016,33(5):425-432.
[3]李红玉.现代化生猪养殖环境测控技术现状与发展趋势[J].国外畜牧学,2017,23(4):78-79.
[4]刘燕.规模化猪场疾病防控存在的问题及对策[J].湖北畜牧兽医,2017,26(7):43.
[5]杨发.宁夏生物环保养猪技术研究与示范[D].陕西:西北农林大学,2015.
[6]何燕柳.猪舍结构及日粮类型对猪舍内环境、猪生长性能及血液各指标的影响[D].广西:广西大学,2013.
[7]WATKINS J,SARTORY D P.Evaluation of a membrane filtration method for the rapid enumeration of confirmed Clostridium perfringens from water[J].Lett Appl Microbiol,2015,60(4):367-371.
[8]KIM O S,CHO Y J,et al.Introducing Ez Taxon-e:a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species[J].Int J Syst Evol Microbiol,2016,12(62):716-721.
[9]ASNANI M R,QUIMBY K R.Interventions for patients and caregivers to improve knowledge of sickle cell disease and recognition of its related complications[J].Nucleic Acids Res,2016,10(6):73-80.
[10]LI X J,MA T B,WANG P.Molecular epidemiology of Cryptosporidium infection in infants with diarrhea in Wuhan City[J].Zhongguo Xue Xi Chong Bing Za Zhi,2017,29(2):188-191.
[11]卢舒娴.养猪发酵床垫料微生物及其猪细菌性病原群落动态的研究[J].农业资源与环境学报,2016,5(33):425-432.
[12]朱双红.猪生物发酵床垫料中细菌群落结构动态变化研究[D].武汉:华中农业大学,2012.
[13]LEE K,LEE S S.Aneurinibacillus humi sp.nov.,isolated from soil collected in ukraine[J].Int J Syst Evol Microbiol,2016,72(2):139-144.
[14]NQUYEN T M,KIM J.A rapid and simple method for identifying bacterial polar lipid components in wet biomass[J].J Microbiol,2017,55(8):635-639.
[15]ZHANG G X,ZENG G Q,CAI X W,et al.Brachybacterium zhongshanense sp.nov.,a cellulose-decomposing bacterium from sediment along the Qijiang River,Zhongshan City,China[J].Int J Syst Evol Microbiol,2013(57):2519-2524.
[16]WU H G,LIU W S.Research and analysis of 74 bloodstream infection cases of acinetobacter baumannii and drug resistance[J].Eur Rev Med Pharmacol Sci,2018,22(6):1782-1786.
[17]GHAZNAVI H,AALI E.Association study of the ATPbinding cassette transporter A1(ABCA1)Rs2230806 genetic variation with lipid profile and coronary artery disease risk in an iranian population[J].Open Access Maced J Med Sci,2018,6(2):274-279.
[18]LINHARES P,VIANA P,FERREIRA M.Genetic variants of vascular endothelial growth factor predict risk and survival of gliomas[J].Tumour Biol,2018,40(3):50-56.
[19]LI L,PAN Y,DAI L.Association of genetic polymorphisms on vascular endothelial growth factor and its receptor genes with susceptibility to coronary heart disease[J].Med Sci Monit,2016,4(22):31-40.
[20]ORIA-JENSEN,S.Penicillin-binding proteins in Actinobacteria[J].J Antibiot,2015,68(4):223-245.
[21]ASAF S,KHAN A L,KHAN M A,et al.Qsmoprotective functions conferred to soybean plants via inoculation with Sphingomonas sp.LK11 and exogenous trehalose[J].Microbiol Res,2017,12(205):135-145.
[22]BRENNAN N M,BROWN R,GOODFELLOW M,et al.Microbacterium gubbeenense sp.nov.,from the surface of a smear-ripened cheese[J].Int J Syst Bacteriol,2016,3(51):1969-1976.
[23]KAMPFER P,SCHAFER J,LODDERS N,et al.Microbacterium murale sp.nov.,isolated from an indoor wall[J].Int J Syst Bacteriol,2012,7(62):2669-2673.
[24]YANG Z W,SALAM N,MOHANY M,et al.Microbacterium album sp.nov.and microbacterium deserti sp.nov.,two halotolerant actinobacteria isolated from desert soil[J].Int J Syst Bacteriol,2017,1(68):217-222.