噬菌体降低铜绿假单胞菌所致腐蛋的效果

doi:10.11843/j.issn.0366-6964.2020.07.028

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (7): 1756-1763.doi: 10.11843/j.issn.0366-6964.2020.07.028

噬菌体降低铜绿假单胞菌所致腐蛋的效果

刘晓文^1,2, 张庆^2*, 宋新慧², 骆延波², 胡明², 李璐璐², 齐静^1,2, 张印², 赵效南², 刘玉庆^1,2*

1. 山东师范大学生命科学学院, 济南 250014;
2. 山东省农业科学院畜牧兽医研究所, 济南 250100

收稿日期:2019-11-12 出版日期:2020-07-25 发布日期:2020-07-22
通讯作者: 刘玉庆,主要从事细菌抗药性分析与噬菌体治疗研究,E-mail:liuiuqing@163.com;张庆,主要从事噬菌体治疗研究,E-mail:zhangqingchina@163.com
作者简介:刘晓文(1995-),女,山东枣庄人,硕士,主要从事噬菌体生物学性质和应用研究,E-mail:Juneliu22@163.com
基金资助:
国家重点研发计划（2018YFD0500500；2016YFD0501300）；山东省重大科技创新工程（2019JZZY010719）；山东省农业科学院农业科技创新工程（GXGC2017A01；CXG2018E10）；国家科技重大专项（2018ZX10733402-013）；传染病预防控制国家重点实验室开放课题（2019SKLID317）

Effect of Phage on the Reduction of Rotten Eggs Caused by Pseudomonas aeruginosa

LIU Xiaowen^1,2, ZHANG Qing^2*, SONG Xinhui², LUO Yanbo², HU Ming², LI Lulu², QI Jing^1,2, ZHANG Yin², ZHAO Xiaonan², LIU Yuqing^1,2*

1. School of Life Sciences, Shandong Normal University, Jinan 250014, China;
2. Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan 250100, China

Received:2019-11-12 Online:2020-07-25 Published:2020-07-22

摘要/Abstract

摘要： 铜绿假单胞菌是孵化过程中造成腐蛋的主要病原菌，拟尝试用裂解性噬菌体防治。选用腐蛋中筛选的铜绿假单胞菌PA-MH12，以此为宿主从河水中筛选到1株裂解性噬菌体MH12-Q，对其进行了生物学特性研究，体外进行了对铜绿假单胞菌所致腐蛋的防控效果试验。结果显示：噬菌体MH12-Q属于肌尾噬菌体科（Myoviridae），效价可达到10¹³pfu·mL^-1，完全裂解效价（CLC）为10¹⁰pfu·mL^-1，最适生长温度为37℃，pH稳定性好，最适pH为6~8，且其在pH4~10的效价都能维持在较高的水平。最佳感染复数（OMOI）为0.001，感染宿主的潜伏期为30 min，爆发期为70 min。噬菌体MH12-Q可使高浓度铜绿假单胞菌的感染率从70%降低到40%。铜绿假单胞菌噬菌体MH12-Q的生物学性质适合生产应用，对孵化场腐蛋具有较好的防控作用，能够提高感染铜绿假单胞菌的鸡胚成活率。

关键词: 铜绿假单胞菌, 腐蛋, 裂解性噬菌体, 生物学性质, 防治

Abstract: Pseudomonas aeruginosa is the main pathogen causing rotten eggs during incubation, we attempted to control it with lytic phage. In this work, a strain of virulent phage MH12-Q was sifted from the river water sample by using Pseudomonas aeruginosa PA-MH12 isolated from the rotten eggs. The biological characteristics of the phage were studied and its prevention and control effect on Pseudomonas aeruginosa induced rotten egg were tested. The phage MH12-Q was sequenced and identified as Myoviridae. The titer of phage MH12-Q can reach 10¹³pfu·mL^-1, the complete lysis concentration (CLC) of phage MH12-Q is 10¹⁰pfu·mL^-1, and the optimal growth temperature is 37℃. The optimum pH is 6-8 for the stability of the phage and its titer can maintain a high level at pH 4-10. Its optimal multiplicity of infection (OMOI) is 0.001, the incubation period of infected hosts is 30 min, and the outbreak period is 70 min. The phage with the biological properties above can reduce the infection rate of high concentration Pseudomonas aeruginosa from 70% to 40%. The biological properties of Pseudomonas aeruginosa phage MH12-Q are suitable for production and application, which has a good prevention and control effect on rotten eggs in hatchery and can improve the survival rate of chicken embryos infected with Pseudomonas aeruginosa.

Key words: Pseudomonas aeruginosa, rotten eggs, lytic phage, biological property, prevention and control

中图分类号:

刘晓文, 张庆, 宋新慧, 骆延波, 胡明, 李璐璐, 齐静, 张印, 赵效南, 刘玉庆. 噬菌体降低铜绿假单胞菌所致腐蛋的效果[J]. 畜牧兽医学报, 2020, 51(7): 1756-1763.

LIU Xiaowen, ZHANG Qing, SONG Xinhui, LUO Yanbo, HU Ming, LI Lulu, QI Jing, ZHANG Yin, ZHAO Xiaonan, LIU Yuqing. Effect of Phage on the Reduction of Rotten Eggs Caused by Pseudomonas aeruginosa[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7): 1756-1763.

参考文献 34

[1]	刘凤荣. 孵化中臭蛋的产生及处理[J]. 中国禽业导刊, 1998, 14(2):11.LIU F R. Production and treatment of rotten eggs during incubation[J]. China Poultry Industry Guide, 1998, 14(2):11. (in Chinese)
[2]	鲍文龙, 李德福. 臭蛋在孵化室中的危害及预防方法[J]. 养禽与禽病防治, 1997(12):15.BAO W L, LI D F. The harm of rotten eggs in hatching chamber and prevention methods[J]. Poultry Husbandry and Disease Control, 1997(12):15. (in Chinese)
[3]	杨景满. 种蛋孵化过程中臭蛋产生的原因及防制[J]. 中国家禽, 2000, 22(7):42.YANG J M. Causes and control of rotten eggs during hatching of breeding eggs[J]. China Poultry, 2000, 22(7):42. (in Chinese)
[4]	HARPER D R, ENRIGHT M C. Bacteriophages for the treatment of Pseudomonas aeruginosa infections[J]. J Appl Microbiol, 2011, 111(1):1-7.
[5]	魏光. 铜绿假单胞菌耐药性分析及碳青霉烯类耐药铜绿假单胞菌外排泵MexAB-OprM的相关研究[D]. 合肥:安徽医科大学, 2014.WEI G. Analysis of antibiotics resistance among Pseudomonas aeruginosa and correlated study on efflux pump MexAB-OprM in carbapenem-resistant Pseudomonas aeruginosa[D]. Hefei:Anhui Medical University, 2014. (in Chinese)
[6]	周小青. 铜绿假单胞菌的耐药情况及基因同源性分析[D]. 南宁:广西医科大学, 2014.ZHOU X Q. Analysis of drug resistance and gene homology of Pseudomonas aeruginosa[D]. Nanning:Guangxi Medical University, 2014. (in Chinese)
[7]	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]. Chinese Journal of Health Laboratory Technology, 2016, 26(10):1409-1411. (in Chinese)
[8]	OFIR G, SOREK R. Contemporary phage biology:from classic models to new insights[J]. Cell, 2018, 172(6):1260-1270.
[9]	ABEDON S T, THOMAS-ABEDON C. Phage therapy pharmacology[J]. Curr Pharm Biotechnol, 2010, 11(1):28-47.
[10]	毛普加, 曾韦锟, 洪愉, 等. 宿主菌抗噬菌体机制的研究进展[J]. 病毒学报, 2015, 31(4):474-479.MAO P J, ZENG W K, HONG Y, et al. Advances of researches on anti-phage mechanisms of host[J]. Chinese Journal of Virology, 2015, 31(4):474-479. (in Chinese)
[11]	CHAN B K, ABEDON S T. Phage therapy pharmacology:phage cocktails[J]. Adv Appl Microbiol, 2012, 78:1-23.
[12]	ABEDON S T. Phage therapy pharmacology:calculating phage dosing[J]. Adv Appl Microbiol, 2011, 77:1-40.
[13]	NILSSON A S. Phage therapy-constraints and possibilities[J]. Upsala J Med Sci, 2014, 119(2):192-198.
[14]	CHATURONGAKUL S, OUNJAI P. Phage-host interplay:examples from tailed phages and Gram-negative bacterial pathogens[J]. Front Microbiol, 2014, 5:442.
[15]	REHMAN S, ALI Z, KHAN M, et al. The dawn of phage therapy[J]. Rev Med Virol, 2019, 29(4):e2041.
[16]	JOŃCZYK-MATYSIAK E, ŁODEJ N, KULA D, et al. Factors determining phage stability/activity:challenges in practical phage application[J]. Exp Rev Anti-infective Ther, 2019, 17(8):583-606.
[17]	RYAN E M, GORMAN S P, DONNELLY R F, et al. Recent advances in bacteriophage therapy:how delivery routes, formulation, concentration and timing influence the success of phage therapy[J]. J Pharm Pharmacol, 2011, 63(10):1253-1264.
[18]	KORTRIGHT K E, CHAN B K, KOFF J L, et al. Phage therapy:a renewed approach to combat antibiotic-resistant bacteria[J]. Cell Host Microbe, 2019, 25(2):219-232.
[19]	RYLOV V N. Bacteriophages of Pseudomonas aeruginosa:long-term prospects for use in phage therapy[J]. Adv Virus Res, 2014, 88:227-278.
[20]	VIERTEL T M, RITTER K, HORZ H P. Viruses versus bacteria-novel approaches to phage therapy as a tool against multidrug-resistant pathogens[J]. J Antimicrob Chemother, 2014, 69(9):2326-2336.
[21]	REINA J, REINA N. Phage therapy, an alternative to antibiotic therapy?[J]. Rev Esp Quimioter, 2018, 31(2):101-104.
[22]	JAMALLUDEEN N, JOHNSON R P, FRIENDSHIP R, et al. Isolation and characterization of nine bacteriophages that lyse O149 enterotoxigenic Escherichia coli[J]. Vet Microbiol, 2007, 124(1-2):47-57.
[23]	王杰, 李璐璐, 张庆. 噬菌体RZⅢ-3的生物学特性分析及对鸡大肠杆菌的疗效[J]. 畜牧兽医学报, 2017, 48(11):2166-2172.WANG J, LI L L, ZHANG Q. Biological characteristics analysis of phage RZⅢ-3 to Escherichia coli and the therapeutic effect on chicken[J]. Acta Veterinaria et Zootechnica Sinica, 2017, 48(11):2166-2172. (in Chinese)
[24]	商延. 抗药性沙门氏菌及其噬菌体的相互作用关系[D]. 济南:山东大学, 2018.SHANG Y. Interaction relationship between antimicrobial-resistant Salmonella and their phages[D]. Ji'nan:Shandong University, 2018. (in Chinese)
[25]	JIANG Y H, LIU J Q, ZHAO C Y, et al. Isolation and genome sequencing of a novel Pseudomonas aeruginosa phage PA-YS35[J]. Curr Microbiol, 2020, 77(1):123-128, doi:10. 1007/s00284-019-01792-8.
[26]	YU S, HUANG H L, HAO Y C, et al. Complete genome sequence of the Myoviral bacteriophage YS35, which causes the lysis of a multidrug-resistant Pseudomonas aeruginosa strain[J]. Genome Announc, 2018, 6(6):e01395-17.
[27]	BORYSOWSKI J, MIEDZYBRODZKI R, GÓRSKI A. 噬菌体治疗-当前的研究和应用[M]. 王冉, 刘玉庆, 译. 北京:化学工业出版社, 2019.BORYSOWSKI J, MIEDZYBRODZKI R, GORSKI A. Phage therapy-current research and applications[M]. WANG R, LIU Y Q, trans. Beijing:Chemical Industry Press, 2019. (in Chinese)
[28]	GOLKAR Z, BAGASRA O, JAMIL N. Experimental phage therapy on multiple drug resistant Pseudomonas aeruginosa infection in mice[J/OL]. J Antivir Antiretrovir, 2013, S10(6):S10-005.[2020-04-30] . https://www.longdom.org/open-access/experimental-phage-therapy-on-multiple-drug-resistant-pseudomonas-aeruginosa-infection-in-mice-jaa.S10-005.pdf.
[29]	MCVAY C S, VELÁSQUEZ M, FRALICK J A. Phage therapy of Pseudomonas aeruginosa infection in a mouse burn wound model[J]. Antimicrob Agents Chemother, 2007, 51(6):1934-1938.
[30]	KHAIRNAR K, RAUT M P, CHANDEKAR R H, et al. Novel bacteriophage therapy for controlling metallo-beta-lactamase producing Pseudomonas aeruginosa infection in catfish[J]. BMC Vet Res, 2013, 9(1):264.
[31]	MARZA J A S, SOOTHILL J S, BOYDELL P, et al. Multiplication of therapeutically administered bacteriophages in Pseudomonas aeruginosa infected patients[J]. Burns, 2006, 32(5):644-646.
[32]	WRIGHT A, HAWKINS C H, ÄNGGÅRD E E, et al. A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic-resistant; a preliminary report of efficacy[J]. Clin Otolaryngol, 2009, 34(4):349-357.
[33]	XIE H J, ZHUANG X L, KONG J, et al. Bacteriophage Esc-A is an efficient therapy for Escherichia COLI 3-1 caused diarrhea in chickens[J]. Gen Appl Microbiol, 2005, 51(3):159-163.
[34]	HUFF W E, HUFF G R, RATH N C, et al. Therapeutic efficacy of bacteriophage and Baytril (enrofloxacin) individually and in combination to treat colibacillosis in broilers[J]. Poult Sci, 2004, 83(12):1944-1947.

噬菌体降低铜绿假单胞菌所致腐蛋的效果

Effect of Phage on the Reduction of Rotten Eggs Caused by Pseudomonas aeruginosa

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