铜绿假单胞菌噬菌体PA-27-1的分离及其生物学特性
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摘要
目的从医院污水中分离筛选铜绿假单胞菌噬菌体,检测其裂解谱和生物学特性,探讨其应用价值。方法以铜绿假单胞菌标准菌株ATCC 27853和临床分离菌株为宿主,从未处理的医院污水中分离筛选铜绿假单胞菌噬菌体,检测其裂解谱,观测裂解效率最高的噬菌体的形态及生物学特性等。结果从污水中分离纯化到6株铜绿假单胞菌噬菌体,对162株不同药敏试验结果的临床菌株(包括63株多重耐药菌)裂解效率最高(79. 63%)的噬菌体命名为PA-27-1,能裂解80. 95%(51/63)的耐药菌株。噬菌体PA-27-1在双层琼脂平板上形成直径2～3 mm的圆形透明且边界清晰的噬菌斑;头部呈多面立体对称结构,尾短;最佳感染复数为0. 000 1,与宿主菌液混合后即有80%的噬菌体吸附,孵育4 min后有98%的噬菌体吸附;潜伏期为0～20 min,以MOI=10试验时,裂解期为20～60 min,60 min后为平台期,裂解量约为248;在pH 6～10和≤50℃的环境中稳定性良好,对氯仿具有较高的耐受性,核酸类型为dsDNA。结论分离筛选到的铜绿假单胞菌噬菌体PA-27-1具有较宽的宿主谱,对多重耐药铜绿假单胞菌具有较强的裂解能力,吸附力强,裂解量大,酸碱和热稳定性好,可作为储备噬菌体,并为临床治疗铜绿假单胞菌感染提供基础和依据。
Objective To isolate and screen Pseudomonas aeruginosa( P. aeruginosa) phages from hospital sewage,identify their lysis spectra and biological characteristics,and explore their application value.Methods P. aeruginosa standard strain ATCC 27853 and the clinical isolates in our department were used as hosts to isolate and screen P. aeruginosa phages from the untreated hospital sewage of our hospital. Then the lysis spectra of the obtained phages were examined. The morphological and biological characteristics of the phage of highest lysis efficiency was studied. Results Six P. aeruginosa phages were isolated and purified from the sewage. The phage with the highest lysis efficiency( 79. 63%) to 162 clinical isolates with different drug susceptibilities( including 63 multi-drug resistant strains) was named PA-27-1,and the phage lysed80. 95%( 51/63) resistant strains. The plaque of the phage PA-27-1 was circular,transparent,and about2 ～ 3 mm in diameter with clear borders,on a double-layer agar plate. Its head was multi-faceted and symmetrical,its tail was short,and the optimal multiplicity of infection was 0. 000 1. Eighty percent of the phage was adhered to the host after being mixed with the host bacterial solution,and the percentage was increased to 98% after incubation for 4 min. One-step growth curve of phage PA-27-1 showed that its latent period were 0 to 20 min,the burst period was 20 to 60 min,and the platform period was subsequently,and the average burst size was about 248( tested on MOI = 10). It was stable in the environment of pH 6 ～ pH 10and≤50 ℃,and had high tolerance to chloroform. Its nucleic acid type was ds DNA. Conclusion P. aeruginosa phage PA-27-1 has a broad host spectrum,strong lysis ability to multi-drug resistant strains,large burst size,good acid-base and thermal stability. It could be used as a reserve phage and provide a scientific basis for the clinical treatment of P. aeruginosa infection.
Objective To isolate and screen Pseudomonas aeruginosa( P. aeruginosa) phages from hospital sewage,identify their lysis spectra and biological characteristics,and explore their application value.Methods P. aeruginosa standard strain ATCC 27853 and the clinical isolates in our department were used as hosts to isolate and screen P. aeruginosa phages from the untreated hospital sewage of our hospital. Then the lysis spectra of the obtained phages were examined. The morphological and biological characteristics of the phage of highest lysis efficiency was studied. Results Six P. aeruginosa phages were isolated and purified from the sewage. The phage with the highest lysis efficiency( 79. 63%) to 162 clinical isolates with different drug susceptibilities( including 63 multi-drug resistant strains) was named PA-27-1,and the phage lysed80. 95%( 51/63) resistant strains. The plaque of the phage PA-27-1 was circular,transparent,and about2 ～ 3 mm in diameter with clear borders,on a double-layer agar plate. Its head was multi-faceted and symmetrical,its tail was short,and the optimal multiplicity of infection was 0. 000 1. Eighty percent of the phage was adhered to the host after being mixed with the host bacterial solution,and the percentage was increased to 98% after incubation for 4 min. One-step growth curve of phage PA-27-1 showed that its latent period were 0 to 20 min,the burst period was 20 to 60 min,and the platform period was subsequently,and the average burst size was about 248( tested on MOI = 10). It was stable in the environment of pH 6 ～ pH 10and≤50 ℃,and had high tolerance to chloroform. Its nucleic acid type was ds DNA. Conclusion P. aeruginosa phage PA-27-1 has a broad host spectrum,strong lysis ability to multi-drug resistant strains,large burst size,good acid-base and thermal stability. It could be used as a reserve phage and provide a scientific basis for the clinical treatment of P. aeruginosa infection.
引文
[1]李明远,宝福凯.医学微生物学[M].北京:科学出版社,2016.LI M Y,BAO F K. Medical Microbiology[M]. Beijing:Science Press,2016.
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[3]郭棵棵,杨雨卉,沈伟,等.一株感染铜绿假单胞菌的双链RNA噬菌体Pa P6的分离和鉴定[J].微生物学通报,2016,43(5):948-954.GUO K K,YANG Y H,SHEN W,et al. Isolation and characterization of Pa P6,a newly isolated Pseudomonas aeruginosa bacteriophage containing three ds RNA genomic segments[J].Microbiol China,2016,43(5):948-954.
[4] BURROWS L L. The Therapeutic Pipeline for Pseudomonas aeruginosa Infections[J]. ACS Infect Dis,2018,4(7):1041-1047. DOI:10. 1021/acsinfecdis. 8b00112.
[5]李亚辉,王小亭,靳静,等.铜绿假单胞菌宽谱噬菌体PL39生物学特性研究[J].中国病原生物学杂志,2016(2):144-146.LI Y H,WANG X T,JIN J,et al. Characterization of a bacteriophage,PL39,that infects a broad range of Pseudomonas aeruginosa[J]. J Pathogen Biol,2016(2):144-146.
[6]付丽娜,王丽丽,李晓宇,等.噬菌体对三大耐药菌的防控作用研究进展[J].中国抗生素杂志,2017,42(10):836-841.FU L N,WANG L L,LI X Y,et al. Advances in phage control of three drug-resistant strains[J]. Chin J Antibiot,2017,42(10):836-841.
[7]王文杰.宽嗜性铜绿假单胞菌噬菌体的生物学特性及对临床菌株的嗜性覆盖研究[J].医学临床研究,2014,31(7):1259-1262. DOI:10. 3969/j. issn. 1671-7171. 2014.07. 004.WANG W J. Study on biological characteristics and coverage on clinical strains of wide tropism pseudomonas aeruginosa phages[J]. J Clin Res,2014,31(7):1259-1262. DOI:10. 3969/j. issn. 1671-7171. 2014. 07. 004.
[8]杨梅,杜崇涛,顾敬敏,等.铜绿假单胞菌噬菌体裂解酶Lys YH6的表达与活性[J].吉林农业大学学报,2015,37(3):357-362.YANG M,DU C T,GU J M,et al. Expression and antibacterial activity of endolysin lys YH6 derived from a novel Pseudomonas aeruginosa phage YH6[J]. J Jilin Agricult Univ,2015,37(3):357-362.
[9]曹振辉,张建城,金礼吉,等.噬菌体控制绿脓杆菌感染的研究进展[J].黑龙江畜牧兽医,2014(11):58-61.CAO Z H,ZHANG J C,JIN L J,et al. Research process on the use of bacteriophages to control the infection of Pseudomonas aeruginosa[J]. Heilongjiang Animal Sci Veterin Med,2014(11):58-61.
[10] AL-WRAFY F,BRZOZOWSKA E,GRSKA S,et al.Pathogenic factors of Pseudomonas aeruginosa—the role of biofilm in pathogenicity and as a target for phage therapy[J]. Postepy Hig Med Dosw(Online),2017,71(0):78-91. DOI:10. 5604/01. 3001. 0010. 3792.
[11]徐彬,高晶,郭晓奎,等.铜绿假单胞菌噬菌体D204的生物学特性和基因组学研究[J].上海交通大学学报(医学版),2016,36(1):1-5. DOI:10. 3969/j. issn. 1674-8115. 2016. 01. 001.XU B,GAO J,GUO X K,et al. Study on the biological and genomic characteristics of Pseudomonas aeruginosa phage D204[J]. J Shanghai Jiaotong Univ(Med Sci),2016,36(1):1-5. DOI:10. 3969/j. issn. 1674-8115.2016. 01. 001.
[12]罗娟,张然,吴柳,等.鲍曼不动杆菌噬菌体SWH-Ab-1分离鉴定及其重要功能基因的生物信息学分析[J].第三军医大学学报,2018,40(1):23-30. DOI:10.16016/j. 1000-5404. 201708010.LUO J,ZHANG R,WU L,et al. Isolation and identification of phage SWH-Ab-1 against Acinetobacter baumannii and bioinformatic analysis of its major functional genes[J].J Third Mil Med Univ,2018,40(1):23-30. DOI:10.16016/j. 1000-5404. 201708010.
[13]陈龙,胡佳萍,徐文刚,等.噬菌体治疗铜绿假单胞菌感染的初步应用研究[J].中国卫生检验杂志,2016,26(10):1409-1411.CHEN L,HU J P,XU W G,et al. Preliminary application of bacteriophages against Pseudomonas aeruginosa infection[J]. Chin J Health Lab Tec,2016,26(10):1409-1411.
[14]李宁.宽嗜性铜绿假单胞菌噬菌体YAPa及其用途:中国,201410120067. 9[P]. 2014-03-02.LI N. Application of wide tropism pseudomonas aeruginosa phages YAPa:China,201410120067. 9[P]. 2014-03-02.
[15]张然,刘智勇,吴柳,等. 2012-2017年某综合教学医院临床分离病原菌的变迁及耐药趋势分析[J].中华医院感染学杂志,2018,28(16):2463-2469. DOI:10.11816/cn. ni. 2018-181279.ZHANG R,LIU Z Y,WU L,et al. Distribution and variation of antimicrobial resistance of pathogenic bacteria isolated from clinical specimens in a teaching hospital in 2012-2017[J]. Chin J Nosocomiol,2018,28(16):2463-2469.DOI:10. 11816/cn. ni. 2018-181279.
[16] ROHWER F,SEGALL A M. In retrospect:A century of phage lessons[J]. Nature,2015,528(7580):46-48.DOI:10. 1038/528046a.
[17]代芳芳,赵秀英,于艳华,等.医院内宽嗜性铜绿假单胞菌噬菌体的分离筛选[J].首都医科大学学报,2013,34(5):727-732.DAI F F,ZHAO X Y,YU Y H,et al. Isolation and identification of pseudomonas aeruginosa phages with broad host range in hospital[J]. J Capit Med Univ,2013,34(5):727-732.
[18]张凡,陈昊,柳鹏程,等. 6株铜绿假单胞菌噬菌体的分离及其生物学特性[J].兰州大学学报(医学版),2017,43(6):28-34. DOI:10. 13885/j. issn. 1000-2812.2017. 06. 007.ZHANG F,CHEN H,LIU P C,et al. Identification and biological characteristics of six Pseudomonas aeruginosa phage strains[J]. J Lanzhou Univ(Med Sci),2017,43(6):28-34. DOI:10. 13885/j. issn. 1000-2812. 2017.06. 007.
[19]马兴铭.一株可裂解多重耐药铜绿假单胞菌的噬菌体及其治疗感染的应用:201310164780. 9[P]. 2014-11-12.MA X M. Phage capable of lysing multi-drug resistant Pseudomonas aeruginosa and application thereof for treating infection:201310164780. 9[P]. 2014-11-12.
[20]孙策,赵龙,魏云林,等.一株裂解性低温假单胞菌噬菌体的分离及特性研究[J].生命科学研究,2014,18(3):222-226.SUN C,ZHAO L,WEI Y L,et al. Isolation and characterization of a lytic cold-active bacteriophage of pseudomonas fluorescens[J]. Life Sci Res,2014,18(3):222-226.
[21]崔尹赡,易萍,许忠福,等.一株假单胞菌属低温噬菌体的分离及其特性研究[J].中国微生态学杂志,2013,25(3):249-253.CUI Y S,YI P,XU Z F,et al. Isolation and characterization of a Pseudomonas-specific cold-active bacteriophage[J]. Chin J Microecol,2013,25(3):249-253.
[22]范华昊.耐药性嗜麦芽窄食单胞菌和绿脓杆菌噬菌体初步应用研究[D].长沙:中南大学,2013.FAN H H,et al. Preliminary application of bacteriophages against antibiotics-resistant Stenotrophomonas maltophilia and Pseudomonas aeruginosa[D]. Changsha:Central South University,2013.
[23]朱丹,祝思路,付玉荣,等.噬菌体裂解酶作用机制及用于细菌感染治疗的研究进展[J].基础医学与临床,2018(2):241-245.ZHU D,ZHU S L,FU Y R,et al. Mechanism and progress of the research on the treatment of bacterial infection with bacteriophage lysin[J]. Basic Clin Med,2018(2):241-245.
[24] KIM E S,BAE H W,CHO Y H. A pilin region affecting host range of the Pseudomonas aeruginosa RNA phage,PP7[J]. Front Microbiol,2018,9:247. DOI:10. 3389/fmicb. 2018. 00247.
[25] ZHAO X,SHEN M,JIANG X,et al. Transcriptomic and metabolomics profiling of phage-host interactions between phage Pa P1 and Pseudomonas aeruginosa[J]. Front Microbiol,2017,8:548. DOI:10. 3389/fmicb. 2017. 00548.
[2]杨雨卉,乐率.铜绿假单胞菌噬菌体治疗的研究进展[J].中国抗生素杂志,2017,42(10):814-820.YANG Y H,LE S. Progress on Pseudomonas aeruginosa bacteriophage therapy[J]. Chin J Antibiot,2017,42(10):814-820.
[3]郭棵棵,杨雨卉,沈伟,等.一株感染铜绿假单胞菌的双链RNA噬菌体Pa P6的分离和鉴定[J].微生物学通报,2016,43(5):948-954.GUO K K,YANG Y H,SHEN W,et al. Isolation and characterization of Pa P6,a newly isolated Pseudomonas aeruginosa bacteriophage containing three ds RNA genomic segments[J].Microbiol China,2016,43(5):948-954.
[4] BURROWS L L. The Therapeutic Pipeline for Pseudomonas aeruginosa Infections[J]. ACS Infect Dis,2018,4(7):1041-1047. DOI:10. 1021/acsinfecdis. 8b00112.
[5]李亚辉,王小亭,靳静,等.铜绿假单胞菌宽谱噬菌体PL39生物学特性研究[J].中国病原生物学杂志,2016(2):144-146.LI Y H,WANG X T,JIN J,et al. Characterization of a bacteriophage,PL39,that infects a broad range of Pseudomonas aeruginosa[J]. J Pathogen Biol,2016(2):144-146.
[6]付丽娜,王丽丽,李晓宇,等.噬菌体对三大耐药菌的防控作用研究进展[J].中国抗生素杂志,2017,42(10):836-841.FU L N,WANG L L,LI X Y,et al. Advances in phage control of three drug-resistant strains[J]. Chin J Antibiot,2017,42(10):836-841.
[7]王文杰.宽嗜性铜绿假单胞菌噬菌体的生物学特性及对临床菌株的嗜性覆盖研究[J].医学临床研究,2014,31(7):1259-1262. DOI:10. 3969/j. issn. 1671-7171. 2014.07. 004.WANG W J. Study on biological characteristics and coverage on clinical strains of wide tropism pseudomonas aeruginosa phages[J]. J Clin Res,2014,31(7):1259-1262. DOI:10. 3969/j. issn. 1671-7171. 2014. 07. 004.
[8]杨梅,杜崇涛,顾敬敏,等.铜绿假单胞菌噬菌体裂解酶Lys YH6的表达与活性[J].吉林农业大学学报,2015,37(3):357-362.YANG M,DU C T,GU J M,et al. Expression and antibacterial activity of endolysin lys YH6 derived from a novel Pseudomonas aeruginosa phage YH6[J]. J Jilin Agricult Univ,2015,37(3):357-362.
[9]曹振辉,张建城,金礼吉,等.噬菌体控制绿脓杆菌感染的研究进展[J].黑龙江畜牧兽医,2014(11):58-61.CAO Z H,ZHANG J C,JIN L J,et al. Research process on the use of bacteriophages to control the infection of Pseudomonas aeruginosa[J]. Heilongjiang Animal Sci Veterin Med,2014(11):58-61.
[10] AL-WRAFY F,BRZOZOWSKA E,GRSKA S,et al.Pathogenic factors of Pseudomonas aeruginosa—the role of biofilm in pathogenicity and as a target for phage therapy[J]. Postepy Hig Med Dosw(Online),2017,71(0):78-91. DOI:10. 5604/01. 3001. 0010. 3792.
[11]徐彬,高晶,郭晓奎,等.铜绿假单胞菌噬菌体D204的生物学特性和基因组学研究[J].上海交通大学学报(医学版),2016,36(1):1-5. DOI:10. 3969/j. issn. 1674-8115. 2016. 01. 001.XU B,GAO J,GUO X K,et al. Study on the biological and genomic characteristics of Pseudomonas aeruginosa phage D204[J]. J Shanghai Jiaotong Univ(Med Sci),2016,36(1):1-5. DOI:10. 3969/j. issn. 1674-8115.2016. 01. 001.
[12]罗娟,张然,吴柳,等.鲍曼不动杆菌噬菌体SWH-Ab-1分离鉴定及其重要功能基因的生物信息学分析[J].第三军医大学学报,2018,40(1):23-30. DOI:10.16016/j. 1000-5404. 201708010.LUO J,ZHANG R,WU L,et al. Isolation and identification of phage SWH-Ab-1 against Acinetobacter baumannii and bioinformatic analysis of its major functional genes[J].J Third Mil Med Univ,2018,40(1):23-30. DOI:10.16016/j. 1000-5404. 201708010.
[13]陈龙,胡佳萍,徐文刚,等.噬菌体治疗铜绿假单胞菌感染的初步应用研究[J].中国卫生检验杂志,2016,26(10):1409-1411.CHEN L,HU J P,XU W G,et al. Preliminary application of bacteriophages against Pseudomonas aeruginosa infection[J]. Chin J Health Lab Tec,2016,26(10):1409-1411.
[14]李宁.宽嗜性铜绿假单胞菌噬菌体YAPa及其用途:中国,201410120067. 9[P]. 2014-03-02.LI N. Application of wide tropism pseudomonas aeruginosa phages YAPa:China,201410120067. 9[P]. 2014-03-02.
[15]张然,刘智勇,吴柳,等. 2012-2017年某综合教学医院临床分离病原菌的变迁及耐药趋势分析[J].中华医院感染学杂志,2018,28(16):2463-2469. DOI:10.11816/cn. ni. 2018-181279.ZHANG R,LIU Z Y,WU L,et al. Distribution and variation of antimicrobial resistance of pathogenic bacteria isolated from clinical specimens in a teaching hospital in 2012-2017[J]. Chin J Nosocomiol,2018,28(16):2463-2469.DOI:10. 11816/cn. ni. 2018-181279.
[16] ROHWER F,SEGALL A M. In retrospect:A century of phage lessons[J]. Nature,2015,528(7580):46-48.DOI:10. 1038/528046a.
[17]代芳芳,赵秀英,于艳华,等.医院内宽嗜性铜绿假单胞菌噬菌体的分离筛选[J].首都医科大学学报,2013,34(5):727-732.DAI F F,ZHAO X Y,YU Y H,et al. Isolation and identification of pseudomonas aeruginosa phages with broad host range in hospital[J]. J Capit Med Univ,2013,34(5):727-732.
[18]张凡,陈昊,柳鹏程,等. 6株铜绿假单胞菌噬菌体的分离及其生物学特性[J].兰州大学学报(医学版),2017,43(6):28-34. DOI:10. 13885/j. issn. 1000-2812.2017. 06. 007.ZHANG F,CHEN H,LIU P C,et al. Identification and biological characteristics of six Pseudomonas aeruginosa phage strains[J]. J Lanzhou Univ(Med Sci),2017,43(6):28-34. DOI:10. 13885/j. issn. 1000-2812. 2017.06. 007.
[19]马兴铭.一株可裂解多重耐药铜绿假单胞菌的噬菌体及其治疗感染的应用:201310164780. 9[P]. 2014-11-12.MA X M. Phage capable of lysing multi-drug resistant Pseudomonas aeruginosa and application thereof for treating infection:201310164780. 9[P]. 2014-11-12.
[20]孙策,赵龙,魏云林,等.一株裂解性低温假单胞菌噬菌体的分离及特性研究[J].生命科学研究,2014,18(3):222-226.SUN C,ZHAO L,WEI Y L,et al. Isolation and characterization of a lytic cold-active bacteriophage of pseudomonas fluorescens[J]. Life Sci Res,2014,18(3):222-226.
[21]崔尹赡,易萍,许忠福,等.一株假单胞菌属低温噬菌体的分离及其特性研究[J].中国微生态学杂志,2013,25(3):249-253.CUI Y S,YI P,XU Z F,et al. Isolation and characterization of a Pseudomonas-specific cold-active bacteriophage[J]. Chin J Microecol,2013,25(3):249-253.
[22]范华昊.耐药性嗜麦芽窄食单胞菌和绿脓杆菌噬菌体初步应用研究[D].长沙:中南大学,2013.FAN H H,et al. Preliminary application of bacteriophages against antibiotics-resistant Stenotrophomonas maltophilia and Pseudomonas aeruginosa[D]. Changsha:Central South University,2013.
[23]朱丹,祝思路,付玉荣,等.噬菌体裂解酶作用机制及用于细菌感染治疗的研究进展[J].基础医学与临床,2018(2):241-245.ZHU D,ZHU S L,FU Y R,et al. Mechanism and progress of the research on the treatment of bacterial infection with bacteriophage lysin[J]. Basic Clin Med,2018(2):241-245.
[24] KIM E S,BAE H W,CHO Y H. A pilin region affecting host range of the Pseudomonas aeruginosa RNA phage,PP7[J]. Front Microbiol,2018,9:247. DOI:10. 3389/fmicb. 2018. 00247.
[25] ZHAO X,SHEN M,JIANG X,et al. Transcriptomic and metabolomics profiling of phage-host interactions between phage Pa P1 and Pseudomonas aeruginosa[J]. Front Microbiol,2017,8:548. DOI:10. 3389/fmicb. 2017. 00548.