鸡白痢沙门菌减毒候选疫苗株的环境应激评价及主要生物学特性测定
|
摘要
为评估鸡白痢沙门菌减毒候选疫苗株对环境应激及消毒剂的抵抗力,本研究对S6702△rfaG△csgA、S6702△rfaL△csgA、S6702△rfaG△csgA△ompR和S6702△rfaL△csgA△ompR等4种鸡白痢沙门菌双基因缺失株和三基因缺失株进行了生长曲线、生物被膜形成能力、药敏试验、环境应激试验及消毒剂的灭活效果的测定。结果显示,4株缺失株均丧失生物被膜形成能力并保留了与亲本株相似的药物敏感性。多基因缺失株对pH为4.0和8.0的培养条件较野生菌株和单基因缺失株更为敏感,呈现微耐酸和不耐碱的特性;所有菌株对54℃热处理和紫外线处理均敏感。消毒剂对实验菌株的杀灭效果显示,野生菌株和各基因缺失株均对0.01%的聚维酮碘溶液敏感,基因缺失株相比野生菌株过硫酸氢钾复合物更为敏感。因此,鸡白痢沙门菌减毒候选疫苗株对环境应激的抵抗力均有所降低,本研究为该菌后续候选疫苗株的进一步筛选奠定了基础。
To evaluate the resistance of Salmonella pullorum attenuated vaccine candidates to environmental stresses and disinfectants, four gene deletion mutants S6702△rfaG△csgA, S6702△rfaL△csgA, S6702△rfaG△csgA△ompR and S6702△rfaL△csgA△ompR were tested for growth curve, biofilm-forming ability, antibiotic susceptibility, and the sensitivity to environ-mental stresses and disinfectants. The results showed all of the four gene deletion strains lost biofilm-forming ability and retained similar drug sensitivity to parent strain. Determination of responses to environmental stresses showed multi-genes deletion mutants were significantly inactivated at pH of 4.0 and pH of 8.0 and were more sensitive than wild-type strain and single-gene deletion mutants. Besides, all strains were sensitive to 54℃ and UV light treatment. Evaluation of inactivation effect of disinfectants indicated that both wild-type and gene deletion strains were all sensitive to 0.01% povidone-iodine solution, and all gene deletion mutants were more sensitive to potassium persulfate compound powder than wild-type strain. Therefore, these four Salmonella attenuated candidate vaccine strains have lower resistance to environmental stresses and our study may aid in the further screening for attenuated vaccine candidates of S.pullorum.
To evaluate the resistance of Salmonella pullorum attenuated vaccine candidates to environmental stresses and disinfectants, four gene deletion mutants S6702△rfaG△csgA, S6702△rfaL△csgA, S6702△rfaG△csgA△ompR and S6702△rfaL△csgA△ompR were tested for growth curve, biofilm-forming ability, antibiotic susceptibility, and the sensitivity to environ-mental stresses and disinfectants. The results showed all of the four gene deletion strains lost biofilm-forming ability and retained similar drug sensitivity to parent strain. Determination of responses to environmental stresses showed multi-genes deletion mutants were significantly inactivated at pH of 4.0 and pH of 8.0 and were more sensitive than wild-type strain and single-gene deletion mutants. Besides, all strains were sensitive to 54℃ and UV light treatment. Evaluation of inactivation effect of disinfectants indicated that both wild-type and gene deletion strains were all sensitive to 0.01% povidone-iodine solution, and all gene deletion mutants were more sensitive to potassium persulfate compound powder than wild-type strain. Therefore, these four Salmonella attenuated candidate vaccine strains have lower resistance to environmental stresses and our study may aid in the further screening for attenuated vaccine candidates of S.pullorum.
引文
[1]Everest P,Wain J,Roberts M,et al.The molecular mechanisms of severe typhoid fever[J].Trends Microbiol,2001,9(7):316-320.
[2]House D,Bishop A,Parry C,et al.Typhoid fever:pathogenesis and disease[J].Curr Opin Infect Dis,2001,14(5):573-578.
[3]Wigley P,Berchieri A,J R,Page K L,et al.Salmonella enterica serovar Pullorum persists in splenic macrophages and in the reproductive tract during persistent,disease-free carriage in chickens[J].Infect Immun,2001,69(12):7873-7879.
[4]Donlan R M,Costerton J W.Biofilms:survival mechanisms of clinically relevant microorganisms[J].Clin Microbiol Rev,2002,15(2):167-193.
[5]Romling U,Bian Z,Hammar M,et al.Curli fibers are highly conserved between Salmonella typhimurium and Escherichia coli with respect to operon structure and regulation[J].J Bacteriol,1998,180(3):722-731.
[6]Quinn H J,Cameron A D,Dorman C J.Bacterial regulon evolution:distinct responses and roles for the identical OmpR proteins of Salmonella Typhimurium and Escherichia coli in the acid stress response[J].PLoS Genet,2014,10(3):e1004215.
[7]满成连.鸡白痢沙门菌LPS合成和生物被膜形成相关基因缺失株的构建及免疫效力评价[D].扬州:扬州大学,2018.
[8]Pratt L A,Kolter R.Genetic analysis of Escherichia coli biofilm formation:roles of flagella,motility,chemotaxis and type I pili[J].Mol Microbiol,1998,30(2):285-293.
[9]Han Yue,Han Xian-gan,Wang Shao-hui,et al.The waaL gene is involved in lipopolysaccharide synthesis and plays a role on the bacterial pathogenesis of avian pathogenic Escherichia coli[J].Vet Microbiol,2014,172(3-4):486-491.
[10]Hower R A,Andrews J M.BSAC standardized disc susceptibility testing method(version 11)[J].J Antimicrob Chemother,2012,67(12):2783-2784.
[11]Guo Rong-xian,Jiao Yang,Li Zhuo-yang,et al.Safety,Protective immunity,and DIVA capability of a rough mutant Salmonella Pullorum vaccine candidate in Broilers[J].Front Microbiol,2017,8:547.
[12]吴白.鸡白痢沙门菌LPS合成相关基因缺失株的构建及免疫效力评价[D].扬州:扬州大学,2016.
[13]Lianou A,Nychas G E,Koutsoumanis K P.Variability in the adaptive acid tolerance response phenotype of Salmonella enterica strains[J].Food Microbiol,2017,62:99-105.
[14]沈欣悦,程旭,戴增斌,等.禽源沙门菌的分离鉴定及耐药性分析[J].中国家禽,2014,36(14):48-51.
[15]Amass S F,Schneider J L,Gaul A M.Evaluation of current and novel protocols for disinfection of airplane passenger footwear under simulated conditions[J].Prev Vet Med,2005,71(1-2):127-134.
[16]江喜春,滑国华,熊家军,等.一种复方聚维酮碘凝胶有效碘稳定性研究[J].中国兽药杂志,2010,44(11):26-28.
[17]邱训斌,王群,伍堂洪,等.聚维酮碘预防气管插管细菌寄殖研究[J].中国消毒学杂志,2017,34(06):528-530.
[2]House D,Bishop A,Parry C,et al.Typhoid fever:pathogenesis and disease[J].Curr Opin Infect Dis,2001,14(5):573-578.
[3]Wigley P,Berchieri A,J R,Page K L,et al.Salmonella enterica serovar Pullorum persists in splenic macrophages and in the reproductive tract during persistent,disease-free carriage in chickens[J].Infect Immun,2001,69(12):7873-7879.
[4]Donlan R M,Costerton J W.Biofilms:survival mechanisms of clinically relevant microorganisms[J].Clin Microbiol Rev,2002,15(2):167-193.
[5]Romling U,Bian Z,Hammar M,et al.Curli fibers are highly conserved between Salmonella typhimurium and Escherichia coli with respect to operon structure and regulation[J].J Bacteriol,1998,180(3):722-731.
[6]Quinn H J,Cameron A D,Dorman C J.Bacterial regulon evolution:distinct responses and roles for the identical OmpR proteins of Salmonella Typhimurium and Escherichia coli in the acid stress response[J].PLoS Genet,2014,10(3):e1004215.
[7]满成连.鸡白痢沙门菌LPS合成和生物被膜形成相关基因缺失株的构建及免疫效力评价[D].扬州:扬州大学,2018.
[8]Pratt L A,Kolter R.Genetic analysis of Escherichia coli biofilm formation:roles of flagella,motility,chemotaxis and type I pili[J].Mol Microbiol,1998,30(2):285-293.
[9]Han Yue,Han Xian-gan,Wang Shao-hui,et al.The waaL gene is involved in lipopolysaccharide synthesis and plays a role on the bacterial pathogenesis of avian pathogenic Escherichia coli[J].Vet Microbiol,2014,172(3-4):486-491.
[10]Hower R A,Andrews J M.BSAC standardized disc susceptibility testing method(version 11)[J].J Antimicrob Chemother,2012,67(12):2783-2784.
[11]Guo Rong-xian,Jiao Yang,Li Zhuo-yang,et al.Safety,Protective immunity,and DIVA capability of a rough mutant Salmonella Pullorum vaccine candidate in Broilers[J].Front Microbiol,2017,8:547.
[12]吴白.鸡白痢沙门菌LPS合成相关基因缺失株的构建及免疫效力评价[D].扬州:扬州大学,2016.
[13]Lianou A,Nychas G E,Koutsoumanis K P.Variability in the adaptive acid tolerance response phenotype of Salmonella enterica strains[J].Food Microbiol,2017,62:99-105.
[14]沈欣悦,程旭,戴增斌,等.禽源沙门菌的分离鉴定及耐药性分析[J].中国家禽,2014,36(14):48-51.
[15]Amass S F,Schneider J L,Gaul A M.Evaluation of current and novel protocols for disinfection of airplane passenger footwear under simulated conditions[J].Prev Vet Med,2005,71(1-2):127-134.
[16]江喜春,滑国华,熊家军,等.一种复方聚维酮碘凝胶有效碘稳定性研究[J].中国兽药杂志,2010,44(11):26-28.
[17]邱训斌,王群,伍堂洪,等.聚维酮碘预防气管插管细菌寄殖研究[J].中国消毒学杂志,2017,34(06):528-530.