牛源荚膜A型、D型多杀性巴氏杆菌的全基因组测序及基因组进化分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0371

摘要/Abstract

摘要：

【目的】对乌拉盖管理区分离的牛源巴氏杆菌进行荚膜血清分型，对不同血清型菌株进行全基因组测序及基因组进化分析。【方法】对肉牛肺脏进行病原菌分离纯化、16S rRNA鉴定、荚膜血清型分型、药敏试验和小鼠致病性实验；对不同血清型巴氏杆菌进行全基因组测序及基因组进化分析。【结果】成功分离得到两株牛源巴氏杆菌，命名为Pm-YQ与Pm-SM，其中Pm-YQ为荚膜血清A型，Pm-SM为荚膜血清D型；两株分离菌均具有较强毒力，但耐药性较弱。分离菌全基因组长度分别为2 274 102 bp与2 244 957 bp，分别编码2 070与2 007个基因。菌株Pm-YQ为ST 179型，菌株Pm-SM为ST 1型。通过构建全基因组进化树分析，Pm-YQ与德国、丹麦、美国在GenBank中登录的3株巴氏杆菌亲缘关系较近；Pm-SM株与国内重庆所分离的一株荚膜F型巴氏杆菌在同一进化分支。【结论】完成了两株不同血清型巴氏杆菌的分离鉴定及全基因组测序，Pm-YQ菌株与德国分离株亲缘关系较近，Pm-SM菌株与目前登录的牛源巴氏杆菌亲缘关系均较远。

关键词: 牛, 多杀性巴氏杆菌, 荚膜血清型, 耐药基因, 毒力基因, 全基因组测序

Abstract:

【Objective】The capsular serotype of the Pasteurella multocida isolated from bovine in the Wulagai management area was identified. Whole genome sequencing and genome evolution analysis were performed for the strains with different serotypes. 【Method】Taking the lung tissue samples from bovine for pathogen isolation and purification, the 16S rRNA identification, capsular serotype typing, drug sensitivity experiments, and mouse pathogenicity experiments were performed. Whole-genome sequencing and genomic evolution analysis were performed for the strains with different serotypes. 【Result】Two strains of bovine Pasteurella multocida were successfully isolated, designated as Pm-YQ and Pm-SM. The Pm-YQ strain was capsular serotype A and the Pm-SM strain was capsular serotype D. Both strains presented relatively high virulence but low resistance to antibiotics. The whole genome lengths of the isolates were 2 274 102 bp and 2 244 957 bp, respectively, encoding 2 070 and 2 007 genes. The Pm-YQ strain belonged to sequence type ST 179, while Pm-SM strain belonged to ST 1. Through the construction of whole-genome phylogenetic tree, Pm-YQ strain was found to be closely related to three strains registered in GenBank from Germany, Denmark, and the United States, Pm-SM strain was found in the same evolutionary branch with a capsular serotype F of P. multocida strain isolated from Chongqing, China. 【Conclusion】The isolation, identification and whole genome sequencing of two strains with different capsular serotypes of bovine P. multocida were completed. The Pm-YQ strain was closely related to the German isolate, and the Pm-SM strain was relatively far related to the currently registered bovine P. multocida strains.

Key words: bovine, Pasteurella multocida, capsular serotype, drug-resistant gene, virulence gene, whole genome sequencing

王梓, 石金川, 王永强, 孙淼, 孟令浩, 耿超, 刘锴. 牛源荚膜A型、D型多杀性巴氏杆菌的全基因组测序及基因组进化分析[J]. 生物技术通报, 2024, 40(12): 282-290.

WANG Zi, SHI Jin-chuan, WANG Yong-qiang, SUN Miao, MENG Ling-hao, GENG Chao, LIU Kai. Whole Genome Sequencing and Genome Evolution Analysis of Capsular Serotype A and D Pasteurella multocida of Bovine[J]. Biotechnology Bulletin, 2024, 40(12): 282-290.

图/表 11

参考文献 37

[1]	Harper M, Boyce JD, Adler B. Pasteurella multocida pathogenesis: 125 years after Pasteur[J]. FEMS Microbiol Lett, 2006, 265(1): 1-10. doi: 10.1111/j.1574-6968.2006.00442.x pmid: 17107417
[2]	Piorunek M, Brajer-Luftmann B, Walkowiak J. Pasteurella multocida infection in humans[J]. Pathogens, 2023, 12(10): 1210.
[3]	Jiang JF, Zhao YS, Chen AH, et al. Efficient markerless genetic manipulation of Pasteurella multocida using lacZ and pheS m as selection markers[J]. Appl Environ Microbiol, 2024, 90(4): e0204323.
[4]	Kasivalu JK, Omwenga GI, Aboge GO. Molecular detection and characterization of Pasteurella multocida infecting camels in marsabit and turkana counties, Kenya[J]. Int J Microbiol, 2022, 2022: 9349303.
[5]	Peng Z, Liu JY, 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.
[6]	马文戈, 于力. 牛源荚膜血清A型多杀性巴氏杆菌的分离鉴定[J]. 中国预防兽医学报, 2008, 30(10): 747-750, 754.
	Ma WG, Yu L. Isolation and identification of bovine capsular serotype A Pasteurella mutocida[J]. Chin J Prev Vet Med, 2008, 30(10): 747-750, 754.
[7]	刘朋, 陈萌, 程子龙, 等. 奶牛荚膜血清A型巴氏杆菌的分离鉴定及致病性[J]. 中国兽医学报, 2018, 38(8): 1548-1552.
	Liu P, Chen M, Cheng ZL, et al. Isolation, identification and pathogenicity of the serotype a Pasteurella multocida in cow[J]. Chin J Vet Sci, 2018, 38(8): 1548-1552.
[8]	孔令聪. 牛A型多杀性巴氏杆菌耐药性分析及对部分常用药物耐药机制研究[D]. 长春: 吉林农业大学, 2016.
	Kong LC. Analysis of drug resistance of Pasteurella multocida type A in cattle and study on drug resistance mechanism of some commonly used drugs[D]. Changchun: Jilin Agricultural University, 2016.
[9]	贾开文, 操义恒, 王子杰, 等. 新疆牛源溶血性曼氏杆菌和多杀性巴氏杆菌的分离鉴定及耐药性和毒力分析[J]. 中国预防兽医学报, 2023, 45(2): 201-206.
	Jia KW, Cao YH, Wang ZJ, et al. Isolation, identification, drug resistance and virulence analysis of Mannheimia haemolytica and Pasteurella multocida from a cattle farm in Xinjiang[J]. Chin J Prev Vet Med, 2023, 45(2): 201-206.
[10]	范正波. 牛源A型多杀性巴氏杆菌毒力相关基因和药物敏感性分析[D]. 重庆: 西南大学, 2020.
	Fan ZB. Analysis of virulence-related genes and drug sensitivity of Pasteurella multocida type A from cattle[D]. Chongqing: Southwest University, 2020.
[11]	Snyder E, Credille B. Mannheimia haemolytica and Pasteurella multocida in bovine respiratory disease: how are they changing in response to efforts to control them?[J]. Vet Clin North Am Food Anim Pract, 2020, 36(2): 253-268.
[12]	Richardson NI, Ravenscroft N, Kuttel MM. Conformational comparisons of Pasteurella multocida types B and E and structurally related capsular polysaccharides[J]. Glycobiology, 2023, 33(9): 745-754.
[13]	Wang HJ, Xin LX, Wu Y, et al. Construction of a one-step multiplex real-time PCR assay for the detection of serogroups A, B, and E of Pasteurella multocida associated with bovine pasteurellosis[J]. Front Vet Sci, 2023, 10: 1193162.
[14]	Calderón Bernal JM, Fernández A, Arnal JL, et al. Molecular epidemiology of Pasteurella multocida associated with bovine respiratory disease outbreaks[J]. Animals, 2022, 13(1): 75.
[15]	李偲, 郭双, 王永强, 等. 一株牛源都柏林沙门氏菌的全基因组测序及毒力与耐药性分析[J]. 微生物学通报, 2023, 50(6): 2569-2581.
	Li C, Guo S, Wang YQ, et al. Whole-genome sequencing, virulence, and drug resistance of a bovine-derived strain of Salmonella enterica subsp.enterica serovar Dublin[J]. Microbiol China, 2023, 50(6): 2569-2581.
[16]	王永强, 李偲, 耿超, 等. 通辽地区犊牛腹泻大肠杆菌耐药性检测及一株多重耐药菌全基因组测序分析[J]. 微生物学通报, 2022, 49(12): 4964-4977.
	Wang YQ, Li C, Geng C, et al. Drug resistance of Escherichia coli strains causing calf diarrhea in Tongliao and whole-genome sequencing of a multi-drug resistant strain[J]. Microbiol China, 2022, 49(12): 4964-4977.
[17]	Townsend KM, Boyce JD, Chung JY, 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. doi: 10.1128/JCM.39.3.924-929.2001 pmid: 11230405
[18]	Humphries R, Bobenchik AM, Hindler JA, et al. Overview of changes to the clinical and laboratory standards institute Performance standards for antimicrobial susceptibility testing, M100, 31st edition[J]. J Clin Microbiol, 2021, 59(12): e0021321.
[19]	Koren S, Walenz BP, Berlin K, et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation[J]. Genome Res, 2017, 27(5): 722-736.
[20]	Hyatt D, Chen GL, Locascio PF, et al. Prodigal: prokaryotic gene recognition and translation initiation site identification[J]. BMC Bioinformatics, 2010, 11: 119. doi: 10.1186/1471-2105-11-119 pmid: 20211023
[21]	Bertelli C, Brinkman FSL, Valencia A. Improved genomic island predictions with IslandPath-DIMOB[J]. Bioinformatics, 2018, 34(13): 2161-2167. doi: 10.1093/bioinformatics/bty095 pmid: 29905770
[22]	Fu LM, Niu BF, Zhu ZW, et al. CD-HIT: accelerated for clustering the next-generation sequencing data[J]. Bioinformatics, 2012, 28(23): 3150-3152. doi: 10.1093/bioinformatics/bts565 pmid: 23060610
[23]	Ashburner M, Ball CA, Blake JA, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium[J]. Nat Genet, 2000, 25(1): 25-29. doi: 10.1038/75556 pmid: 10802651
[24]	Kanehisa M, Goto S, Kawashima S, et al. The KEGG resource for deciphering the genome[J]. Nucleic Acids Res, 2004, 32(Database issue): D277-D280.
[25]	Jia BF, Raphenya AR, Alcock B, et al. CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database[J]. Nucleic Acids Res, 2017, 45(D1): D566-D573.
[26]	罗婷, 徐业芬, 韩著, 等. 西藏牦牛源牛支原体T10株全基因组测序及其序列分析[J]. 畜牧兽医学报, 2024, 55(5): 2154-2167. doi: 10.11843/j.issn.0366-6964.2024.05.033
	Luo T, Han Z, Xu YF, et al. Whole genome sequencing and sequence analysis of Mycoplasma bovis T10 strain from Tibetan yak[J]. Annals of Animal Husbandry and Veterinary Medicine, 2024, 55(5): 2154-2167.
[27]	Chen CJ, Wu Y, Li JW, et al. TBtools-II: a “one for all, all for one” bioinformatics platform for biological big-data mining[J]. Mol Plant, 2023, 16(11): 1733-1742.
[28]	李贵琴. 牛巴氏杆菌的分离鉴定及黄芩苷对巴氏杆菌肺炎的治疗作用[D]. 杨凌: 西北农林科技大学, 2022.
	Li GQ. Isolation and identification of Pasteurella bovis and therapeutic effect of baicalin on Pasteurella pneumonia[D]. Yangling: Northwest A & F University, 2022.
[29]	Alhamami T, Roy Chowdhury P, Venter H, et al. Genomic profiling of Pasteurella multocida isolated from feedlot cases of bovine respiratory disease[J]. Vet Microbiol, 2023, 283: 109773.
[30]	李轩宇. 牛支原体、A型巴氏杆菌二联灭活疫苗模型动物免疫保护性评价[D]. 长春: 吉林农业大学, 2023.
	Li XY. Evaluation of animal immune protection of combined inactivated vaccine model of Mycoplasma bovis and Pasteurella A[D]. Changchun: Jilin Agricultural University, 2023.
[31]	王镭. 猪源多杀性巴氏杆菌A型、D型分离株病原学特性比较研究[D]. 合肥: 安徽农业大学, 2022.
	Wang L. Comparative study on pathogenic characteristics of Pasteurella multocida isolates A and D from pigs[D]. Hefei: Anhui Agricultural University, 2022.
[32]	魏诗谣, 王丽扬, 张信军, 等. 3株羊源D型多杀性巴氏杆菌的生物学特性研究[J]. 扬州大学学报: 农业与生命科学版, 2021, 42(4): 12-17.
	Wei SY, Wang LY, Zhang XJ, et al. Study on biological characteristics of three strains of Pasteurella multocida serotype D from goat[J]. J Yangzhou Univ Agric Life Sci Ed, 2021, 42(4): 12-17.
[33]	Gharib Mombeni E, Gharibi D, Ghorbanpoor M, et al. Toxigenic and non-toxigenic Pasteurella multocida genotypes, based on capsular, LPS, and virulence profile typing, associated with pneumonic pasteurellosis in Iran[J]. Vet Microbiol, 2021, 257: 109077.
[34]	Kim J, Kim JW, Oh SI, et al. Characterisation of Pasteurella multocida isolates from pigs with pneumonia in Korea[J]. BMC Vet Res, 2019, 15(1): 119.
[35]	Smith E, Miller E, Aguayo JM, et al. Genomic diversity and molecular epidemiology of Pasteurella multocida[J]. PLoS One, 2021, 16(4): e0249138.
[36]	Peng Z, Wang XR, Zhou R, et al. Pasteurella multocida: genotypes and genomics[J]. Microbiol Mol Biol Rev, 2019, 83(4): e00014-e00019.
[37]	Wang Z, Kong LC, Jia BY, et al. Aminoglycoside susceptibility of Pasteurella multocida isolates from bovine respiratory infections in China and mutations in ribosomal protein S5 associated with high-level induced spectinomycin resistance[J]. J Vet Med Sci, 2017, 79(10): 1678-1681. doi: 10.1292/jvms.17-0219 pmid: 28867688

目的基因 Target gene	引物序列 Primer sequence（5'-3'）	扩增长度Amplification length/bp
16S rRNA	F: AGAGTTTGATCCTGGCTCAG R: GGTTACCTTGTTACGACTT	1 369
KMT1	F:ATCCGCTATTTACCCAGTGG R:GCTGTAAACGAACTCGCCAC	460
hyaD-hyaC	F:TGCCAAAATCGCAGTCAG R: TTGCCATCATTGTCAGTG	1 044
bcbD	F: CATTTATCCAAGCTCCACC R: GCCCGAGAGTTTCAATCC	760
dcbF	F:TTACAAAAGAAAGACTAGGAGCCC R: CATCTACCCACTCAACCATATCAG	657
ecbJ	F: TCCGCAGAAAATTATTGACTC R: GCTTGCTGCTTGATTTTGTC	511
fcbD	F:AATCGGAGAACGCAGAAATCAG R: TTCCGCCGTCAATTACTCTG	851

目的基因 Target gene	引物序列 Primer sequence（5'-3'）	扩增长度Amplification length/bp
16S rRNA	F: AGAGTTTGATCCTGGCTCAG R: GGTTACCTTGTTACGACTT	1 369
KMT1	F:ATCCGCTATTTACCCAGTGG R:GCTGTAAACGAACTCGCCAC	460
hyaD-hyaC	F:TGCCAAAATCGCAGTCAG R: TTGCCATCATTGTCAGTG	1 044
bcbD	F: CATTTATCCAAGCTCCACC R: GCCCGAGAGTTTCAATCC	760
dcbF	F:TTACAAAAGAAAGACTAGGAGCCC R: CATCTACCCACTCAACCATATCAG	657
ecbJ	F: TCCGCAGAAAATTATTGACTC R: GCTTGCTGCTTGATTTTGTC	511
fcbD	F:AATCGGAGAACGCAGAAATCAG R: TTCCGCCGTCAATTACTCTG	851

抗生素 Antibiotic	菌株编号Strain No.
抗生素 Antibiotic	Pm-YQ	Pm-SM
强力霉素	S（17）	I（10）
诺氟沙星	R（9）	S（21）
头孢噻肟	S（15）	S（20）
氟苯尼考	S（26）	S（21）
阿米卡星	I（10）	S（17）
庆大霉素	R（9）	S（16）
恩诺沙星	R（7）	S（25）
多粘菌素B	S（18）	S（13）
环丙沙星	S（22）	S（23）
青霉素	S（34）	S（34）
氧氟沙星	I（14）	S（21）
卡那霉素	R（0）	R（0）

抗生素 Antibiotic	菌株编号Strain No.
抗生素 Antibiotic	Pm-YQ	Pm-SM
强力霉素	S（17）	I（10）
诺氟沙星	R（9）	S（21）
头孢噻肟	S（15）	S（20）
氟苯尼考	S（26）	S（21）
阿米卡星	I（10）	S（17）
庆大霉素	R（9）	S（16）
恩诺沙星	R（7）	S（25）
多粘菌素B	S（18）	S（13）
环丙沙星	S（22）	S（23）
青霉素	S（34）	S（34）
氧氟沙星	I（14）	S（21）
卡那霉素	R（0）	R（0）

菌株名称Name of strain	基因岛Genomic island	位置Location	基因位点Position	长度Length/bp
Pm-YQ	Genomic-island-1	染色体Chromosome	845109-852871	7763
	Genomic-island-2	染色体Chromosome	1105564-1116467	10904
	Genomic-island-3	染色体Chromosome	1602281-1618085	15805
	Genomic-island-4	染色体Chromosome	1876215-1889636	13422
Pm-SM	Genomic-island-1	染色体Chromosome	160470-193937	33468
	Genomic-island-2	染色体Chromosome	953950-964232	10283
	Genomic-island-3	染色体Chromosome	1143374-1159357	15984