致脑膜炎多杀性巴氏杆菌的分离鉴定及全基因组重测序

doi:10.11843/j.issn.0366-6964.2022.12.020

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (12): 4346-4355.doi: 10.11843/j.issn.0366-6964.2022.12.020

致脑膜炎多杀性巴氏杆菌的分离鉴定及全基因组重测序

王斐^1,2, 杨洁^1,2, 吕庆杰^1,2, 王米雪^1,2, 刘鹏^1,2, 张若愚^1,2, 史聪聪^1,2, 王雪莹^1,2, 林琳^1,2, 华琳^1,2, 宋文博^1,2, 梁婉^1,2, 陈焕春^1,2, 吴斌^1,2*, 彭忠^1,2,3*

1. 华中农业大学动物医学院, 农业微生物学国家重点实验室, 武汉 430070;
2. 生猪健康养殖协同创新中心, 武汉 430070;
3. 湖北洪山实验室, 武汉 430070

收稿日期:2022-01-10 出版日期:2022-12-23 发布日期:2022-12-25
通讯作者: 彭忠，主要从事动物传染病学研究，E-mail:pengzhong@mail.hzau.edu.cn；吴斌，主要从事动物传染病学研究，E-mail:wub@mail.hzau.edu.cn
作者简介:王斐(1994-)，男，湖北黄冈人，博士生，主要从事畜禽病原微生物研究，E-mail:wf2018@webmail.hzau.edu.cn
基金资助:
国家自然科学基金(31902241)；中国博士后科学基金(2020T130232；2018M640719)；湖北省重点研发计划(2021BBA085)

Isolation and Genomic Characterization of a Meningitis Causing Pasteurella multocida

WANG Fei^1,2, YANG Jie^1,2, Lü Qingjie^1,2, WANG Mixue^1,2, LIU Peng^1,2, ZHANG Ruoyu^1,2, SHI Congcong^1,2, WANG Xueying^1,2, LIN Lin^1,2, HUA Lin^1,2, SONG Wenbo^1,2, LIANG Wan^1,2, CHEN Huanchun^1,2, WU Bin^1,2*, PENG Zhong^1,2,3*

1. State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China;
2. The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China;
3. Hubei Hongshan Laboratory, Wuhan 430070, China

Received:2022-01-10 Online:2022-12-23 Published:2022-12-25

摘要/Abstract

摘要： 旨在了解致脑膜炎多杀性巴氏杆菌的生物学特性及其引起脑部感染的生物学基础。本研究从有神经症状的病死猪脑组织中分离鉴定1株多杀性巴氏杆菌SD001，对其开展血清杀菌试验、胞内存活试验、小鼠感染试验，并利用纳米孔测序技术对其进行全基因组测序。SD001相对于猪其他器官中分离的多杀性巴氏杆菌而言表现出更强的抗血清杀菌的能力，其在小鼠巨噬细胞内存活的能力也较强。小鼠感染试验显示，相同剂量的细菌经血液感染后，SD001在小鼠血液和脑组织中的载菌量显著高于猪肺炎型多杀性巴氏杆菌(P ≤ 0.01)，并且SD001造成了小鼠脑组织发生炎症等病理损伤，提示SD001可能造成血流感染并引起小鼠脑膜炎。纳米孔测序显示，SD001的全基因组大小约为2.45 Mb，平均GC含量为40.25%，编码2 281个蛋白以及77个RNA；SD001基因组中含有2条CRISPR结构、5个基因岛、7条前噬菌体序列；毒力基因预测结果显示，SD001含有233个毒力基因；基因分型结果显示，SD001为荚膜：LPS：MLST基因型A:L6:ST10。SD001感染可以引起脑膜炎，其较强的抗血清杀菌能力以及能形成血流感染的能力可能是SD001引起脑部感染的重要原因，在兽医临床关于中枢神经系统感染的诊断中应该纳入多杀性巴氏杆菌感染的可能性。

关键词: 多杀性巴氏杆菌, 脑膜炎, 猪, 血清杀菌试验, 胞内存活试验, 小鼠感染试验, 纳米孔测序

Abstract: This study was designed to investigate the biological characteristics of a meningitis causing Pasteurella multocida and the biological basis for this bacterium causing brain infections. Serum bactericidal tests, intracellular survival tests, mouse infection tests, and nanopore sequencing were performed on a P. multocida strain SD001 which was isolated from the brain of a pig with neurological symptoms. Results were as follows: Compared to those of P. multocida isolates from the other organs of pigs, SD001 displayed a stronger anti-serum bactericidal capacity, and a stronger capacity of surviving in mouse macrophages. In mouse models, SD001 formed a higher bacterial load in both blood and brain than a pneumonic P. multocida strain at the same time points post challenge, and the challenge of SD001 led to inflammatory and other pathological damages in the experimental mice. These findings suggest that SD001 may cause bloodstream infections and meningitis animals. Results from nanopore sequencing revealed that the complete genome sequence of SD001 was 2.45 Mb in size, with an average GC content of 40.25%. The complete genome of SD001 encoded 2 281 putative proteins and 77 RNAs. It also contained two CRISPR sequences, five genomic islands, seven prophages, and 233 virulence factor encoding genes. Genotyping using the complete genome sequence showed that SD001 belonged to capsular: LPS: MLST type A: L6: ST10. SD001 infection could cause meningitis and its strong capacity of anti-serum bactericidal activity as well as inducing bloodstream infection may confer the bacterium capacity of causing brain infections. P. multocida infection should be considered for the diagnosis of brain infections in veterinary clinical settings.

Key words: Pasteurella multocida, meningitis, pig, serum bactericidal test, intracellular survival test, mouse infection test, nanopore sequencing

中图分类号:

S852.612

王斐, 杨洁, 吕庆杰, 王米雪, 刘鹏, 张若愚, 史聪聪, 王雪莹, 林琳, 华琳, 宋文博, 梁婉, 陈焕春, 吴斌, 彭忠. 致脑膜炎多杀性巴氏杆菌的分离鉴定及全基因组重测序[J]. 畜牧兽医学报, 2022, 53(12): 4346-4355.

WANG Fei, YANG Jie, Lü Qingjie, WANG Mixue, LIU Peng, ZHANG Ruoyu, SHI Congcong, WANG Xueying, LIN Lin, HUA Lin, SONG Wenbo, LIANG Wan, CHEN Huanchun, WU Bin, PENG Zhong. Isolation and Genomic Characterization of a Meningitis Causing Pasteurella multocida[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4346-4355.

参考文献 22

[1]	PENG Z, WANG X R, ZHOU R, et al. Pasteurella multocida:genotypes and genomics[J]. Microbiol Mol Biol Rev, 2019, 83(4):e00014-19.
[2]	HARPER M, BOYCE J D, ADLER B. Pasteurella multocida pathogenesis:125 years after Pasteur[J]. FEMS Microbiol Lett, 2006, 265(1):1-10.
[3]	TOWNSEND K M, BOYCE J D, CHUNG J Y, et al. Genetic organization of Pasteurella multocida cap loci and development of a multiplex capsular PCR typing system[J]. J Clin Microbiol, 2001, 39(3):924-929.
[4]	HARPER M, JOHN M, TURNI C, et al. Development of a rapid multiplex PCR assay to genotype Pasteurella multocida strains by use of the lipopolysaccharide outer core biosynthesis locus[J]. J Clin Microbiol, 2015, 53(2):477-485.
[5]	WILSON B A, HO M. Pasteurella multocida:from zoonosis to cellular microbiology[J]. Clin Microbiol Revi, 2013, 26(3):631-655.
[6]	WILKIE I W, HARPER M, BOYCE J D, et al. Pasteurella multocida: diseases and pathogenesis[M]//AKTORIES K, ORTH J H C, ADLER B. Pasteurella multocida: Molecular Biology, Toxins and Infection. Berlin: Springer, 2012:1-22.
[7]	PENG Z, LIANG W, WANG Y G, et al. Experimental pathogenicity and complete genome characterization of a pig origin Pasteurella multocida serogroup F isolate HN07[J]. Vet Microbiol, 2017, 198:23-33.
[8]	PENG Z, LIU J Y, LIANG W, et al. Development of an online tool for Pasteurella multocida genotyping and genotypes of Pasteurella multocida from different hosts[J]. Front Vet Sci, 2021, 8:771157.
[9]	SAHOO M, BALONI S, THAKOR J C, et al. Localization of Pasteurella multocida antigens in the brains of pigs naturally infected with Pasteurellosis revealing a newer aspect of pathogenesis[J]. Microb Pathog, 2020, 140:103968.
[10]	KPODÉKON M. Experimental study of the pathogenesis of meningitis and encephalitis during pasteurellosis in rabbits[J]. Ann Res Vet, 1983, 14(3):217-224.
[11]	MARZA A D, ABDULLAH F F J, AHMED I M, et al. The ability of lipopolysaccharide (LPS) of Pasteurella multocida B:2 to induce clinical and pathological lesions in the nervous system of buffalo calves following experimental inoculation[J]. Microb Pathog, 2017, 104:340-347.
[12]	DUTHEIL F, WAHL D, CHAMOUX A. Pasteurella multocida meningitis in a 93-year-old woman following a cat bite[J]. Méd Mal Infect, 2009, 39(1):61-63.
[13]	LÓPEZ C, SANCHEZ-RUBIO P, BETRÁN A, et al. Pasteurella multocida bacterial meningitis caused by contact with pigs[J]. Braz J Microbiol, 2013, 44(2):473-474.
[14]	ARMSTRONG G R, SEN R A, WILKINSON J. Pasteurella multocida meningitis in an adult:case report[J]. J Clin Pathol, 2000, 53(3):234-235.
[15]	KUMAR A, DEVLIN H R, VELLEND H. Pasteurella multocida meningitis in an adult:case report and review[J]. Rev Infect Dis, 1990, 12(3):440-448.
[16]	RODRIGUES C M C, MAIDEN M C J. A world without bacterial meningitis:how genomic epidemiology can inform vaccination strategy[J]. F1000Res, 2018, 7:401.
[17]	彭忠, 李春辉, 陈焕春, 等. 细菌性脑膜炎概述[J]. 中国感染控制杂志, 2022, 21(1):97-103.PENG Z, LI C H, CHEN H C, et al. An overview of bacterial meningitis[J]. Chinese Journal of Infection Control, 2022, 21(1):97-103. (in Chinese)
[18]	DOBRINDT U, HOCHHUT B, HENTSCHEL U, et al. Genomic islands in pathogenic and environmental microorganisms[J]. Nat Rev Microbiol, 2004, 2(5):414-424.
[19]	YANG D, WANG S, SUN E C, et al. A temperate Siphoviridae bacteriophage isolate from Siberian tiger enhances the virulence of methicillin-resistant Staphylococcus aureus through distinct mechanisms[J]. Virulence, 2022, 13(1):137-148.
[20]	GUGLIELMETTI S, TAMAGNINI I, MORA D, et al. Implication of an outer surface lipoprotein in adhesion of Bifidobacterium bifidum to Caco-2 cells[J]. Appl Environ Microbiol, 2008, 74(15):4695-702.
[21]	彭忠, 梁婉, 艾伟诚, 等. 我国猪群中多杀性巴氏杆菌的基因型分析[J]. 畜牧兽医学报, 2019, 50(5):1064-1072.PENG Z, LIANG W, AI W C, et al. Genotypical characteristics of swine Pasteurella multocida in China[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(5):1064-1072. (in Chinese)
[22]	PENG Z, WANG H N, LIANG W, et al. A capsule/lipopolysaccharide/MLST genotype D/L6/ST11 of Pasteurella multocida is likely to be strongly associated with swine respiratory disease in China[J]. Arch Microbiol, 2018, 200(1):107-118.

致脑膜炎多杀性巴氏杆菌的分离鉴定及全基因组重测序

Isolation and Genomic Characterization of a Meningitis Causing Pasteurella multocida

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 22

相关文章 15

编辑推荐

Metrics

本文评价

[1]	牛乃琪, 赵润泽, 宗文成, 刘先策, 刘海, 石国华, 井西涛, 张龙超. 北京黑猪GREB1L和MIB1基因多态性与肋骨数及胴体性状的关联分析[J]. 畜牧兽医学报, 2024, 55(1): 79-86.
[2]	祝雪丽, 张龙超, 王立贤, 蒲蕾, 刘欣. 北京黑猪AQP9和RPS10基因多态性及其与背膘厚的关联分析[J]. 畜牧兽医学报, 2024, 55(1): 87-98.
[3]	时胜洁, 王立光, 高磊, 蔡传江, 何伟先, 褚瑰燕. miR-24-3p对猪颗粒细胞雌二醇合成的作用[J]. 畜牧兽医学报, 2024, 55(1): 169-178.
[4]	陈姝宇, 朱雪蛟, 周金柱, 陶然, 张雪寒, 索朗斯珠, 贡嘎, 李彬. 鸟苷酸结合蛋白GBP1和GBP2抑制猪轮状病毒体外复制[J]. 畜牧兽医学报, 2024, 55(1): 245-257.
[5]	宋雯妍, 张瀚文, 吴澳迪, 张丽燕, 刘照, 叶桐桐, 陈创夫, 盛金良. 猪繁殖与呼吸综合征病毒GP5蛋白纳米抗体的筛选及其对病毒复制的抑制效应[J]. 畜牧兽医学报, 2024, 55(1): 258-270.
[6]	谢青云, 邢蕙萱, 于岩飞, 袁厅, 熊祺琰, 熊富强, 冯志新. 猪肺炎支原体解螺旋酶RuvA的原核表达、多克隆抗体制备及活性鉴定[J]. 畜牧兽医学报, 2024, 55(1): 271-281.
[7]	白昀, 谢青云, 欧阳伟, 甘源, 袁厅, 赵东明, 步志高, 邵国青, 冯志新. 基于黏膜sIgA抗体的非洲猪瘟病毒感染早期血清学检测方法的建立[J]. 畜牧兽医学报, 2024, 55(1): 300-310.
[8]	马亚军, 焦智慧, 刘笑凝, 陆翔羽, 刘涛, 王月, 朴晨曦, 王洪斌. 脂肪间充质干细胞对小型猪肝缺血再灌注合并肝切除组织细胞焦亡的影响[J]. 畜牧兽医学报, 2024, 55(1): 355-364.
[9]	刘传霞, 王晓, 李雪雯, 鲍苗菲, 李婷婷, 陈欣, 翁长江, 郑君. 非洲猪瘟病毒pE120R蛋白单克隆抗体的制备[J]. 畜牧兽医学报, 2024, 55(1): 388-394.
[10]	周建浩, 王东方, 刘影, 王淑娟, 马震原, 谢彩华, 赵雪丽, 杨海波, 冯桂丹, 康台生, 胡煜锋, 李博文, 闫若潜. 猪流行性腹泻病毒微滴式数字PCR定量检测方法的建立及初步应用[J]. 畜牧兽医学报, 2024, 55(1): 413-418.
[11]	袁巍, 毕欢, 张雨丹, 张依裕, 顾晓龙, 杨红文, 陈伟. 全基因组选择信号解析剑白香猪和从江香猪的遗传差异[J]. 畜牧兽医学报, 2023, 54(9): 3631-3641.
[12]	陈莹, 钟儒清, 陈亮, 张宏福. 猪饲粮纤维的利用及其对养分消化的影响[J]. 畜牧兽医学报, 2023, 54(9): 3745-3757.
[13]	张兆博, 侯黎明, 李平华, 杜陶然, 王中宇, 吴承武, 黄瑞华. 基于血浆代谢组学筛选日粮纤维影响二花脸猪肉质性状的候选代谢物[J]. 畜牧兽医学报, 2023, 54(9): 3758-3769.
[14]	郑先瑞, 卓明雪, 纪金丽, 蒋维虎, 邓在双, 张吉成, 田雅莉, 丁月云, 张晓东, 殷宗俊. 皖南黑猪不同生长阶段血清免疫指标及肠道菌群的特征分析[J]. 畜牧兽医学报, 2023, 54(9): 3770-3783.
[15]	王志远, 刘博奇, 许志颖, 徐思佳, 邢家宝, 张桂红, 王衡, 孙彦阔. 2021—2022年我国部分地区猪繁殖与呼吸综合征病毒ORF5基因变异分析[J]. 畜牧兽医学报, 2023, 54(9): 3812-3823.