多重耐药胸膜肺炎放线杆菌GD2107株全基因组测序及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2022.10.035

中国畜牧兽医 ›› 2022, Vol. 49 ›› Issue (10): 4019-4031.doi: 10.16431/j.cnki.1671-7236.2022.10.035

多重耐药胸膜肺炎放线杆菌GD2107株全基因组测序及生物信息学分析

徐民生^1,2, 柯海意^1,2, 杨冬霞², 施科达^1,2, 孙泽仪², 牛佳伟², 常鑫^1,2, 翟少伦², 臧莹安¹, 李春玲²

1. 仲恺农业工程学院动物科技学院, 广州 510305;
2. 广东省农业科学院动物卫生研究所, 广东省畜禽疫病防治研究重点实验室, 农业农村部兽用药物与诊断技术广东科学观测实验站, 广州 510640

收稿日期:2022-06-10 出版日期:2022-10-05 发布日期:2022-09-30
通讯作者: 臧莹安,E-mail:1134580900@qq.com;李春玲,E-mail:lclclare@163.com
作者简介:徐民生,E-mail:379852566@qq.com;柯海意,E-mail:1056124851@qq.com。
基金资助:
广东省现代农业产业技术体系创新团队项目(2021KJ119);广州市重点研发项目(202206010192);广东省动物疫病野外科学观测研究站项目(2021B1212050021);广州市科技特派员项目(20212100027、20212100010);广东省农业科学院学科建设新兴团队项目(202122TD)

Whole Genome Sequencing and Biological Characteristics Analysis of Multidrug-resistant Actinobacillus pleuropneumoniae GD2107 Strain

XU Minsheng^1,2, KE Haiyi^1,2, YANG Dongxia², SHI Keda^1,2, SUN Zeyi², NIU Jiawei², CHANG Xin^1,2, ZHAI Shaolun², ZANG Yingan¹, LI Chunling²

1. College of Animal Science and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510305, China;
2. Guangdong Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Technology, Ministry of Agriculture Rural Affairs, Guangdong Key Laboratory of Livestock Disease Prevention, Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China

Received:2022-06-10 Online:2022-10-05 Published:2022-09-30

摘要/Abstract

摘要： 【目的】通过对多重耐药胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)GD2107株进行全基因组测序及生物信息学分析,丰富APP基因组数据库信息;构建基于ApxⅣ基因的系统进化树,分析该菌株的进化关系,为探索APP致病机制和临床防控猪传染性胸膜肺炎(porcine contagious pleuropneumia,PCP)提供参考。【方法】通过药敏试验测定分离菌株的耐药谱;采用全基因组测序技术(whole genome sequencing,WGS)对细菌DNA进行全基因组测序,分别利用Illumina NovaSeq、PacBio SequeI测序平台对全基因组测序结果进行基因功能注释及生物信息学分析(包括基因组基本信息、功能元件分析及亚系统分析等);基于ApxⅣ基因构建系统进化树。【结果】药敏试验结果显示,GD2107菌株对青霉素、头孢拉定(先锋Ⅵ)、卡那霉素等14种抗菌药均耐药。对GD2107株全基因组测序得到1条大小为2 271 987 bp的环状染色体(GC含量为41.21%)和2个大小分别为5 027和3 497 bp的环状质粒,共预测到2 290个编码基因,包含19个rRNA (7个5S rRNA、6个16S rRNA、6个23S rRNA)、21个tRNA基因、20个ncRNA;23个基因岛、4个原噬菌体和2组CRISPR相关序列;分别有2 112、1 549和1 866个基因在COG、KEGG和GO数据库中得到注释,且相关蛋白集中分布于APP的代谢过程;另外在毒力因子(VFDB)和耐药因子(CARD)数据库中还注释到48个毒力基因和22个耐药基因(仅floR基因位于质粒上)。绘制该菌株的全基因组圈图,将基因组信息提交至NCBI,获得染色体GenBank登录号为CP097377,质粒登录号分别为CP097378和CP097379。系统进化树分析发现,该菌株与来自中国的APP菌株(CP063424.1)进化关系最近。【结论】本研究完成了对多重耐药菌株GD2107的全基因组测序与生物信息学分析,全面认识了该菌株基因组的结构和功能并探究了耐药和致病机制中的相关基因,进化关系显示该菌株具有一定的地域流行性,为预防PCP的流行和探索APP的致病机制提供了参考。

关键词: 胸膜肺炎放线杆菌(APP); 全基因组测序; 生物信息学分析; 耐药基因; 系统进化分析

Abstract: 【Objective】 Through genome sequencing and bioinformatics analysis of GD2107 strain of multi-drug resistant Actinobacillus pleuropneumoniae (APP),the genome database information of APP was enriched,the phylogenetic tree based on ApxⅣ gene was constructed,and the evolutionary relationship of the strain was analyzed,so as to provide reference for exploring the pathogenic mechanism of APP and clinical prevention and control of porcine infectious porcine contagious pleuropneumia (PCP).【Method】 The drug resistance spectrum of the isolated strains was determined by drug sensitivity test. The whole genome of bacterial DNA was sequenced by whole genome sequencing (WGS) method.Based on Illumina NovaSeq and PacBio SequeI sequencing platforms,the whole genome sequencing results were analyzed by gene function annotation and bioinformatics analysis (including basic genome information,functional element analysis and subsystem analysis,etc.). Phylogenetic tree was constructed based on ApxⅣ gene.【Result】 The results of drug sensitivity test of GD2107 strain showed that it was resistant to 14 kinds of antimicrobials including penicillin,cefradine (pioneer Ⅵ),kanamycin and so on.The whole genome of GD2107 strain was sequenced to obtain a circular chromosome of 2 271 987 bp (GC content was 41.21%) and two circular plasmids of 5 027 and 3 497 bp,respectively.A total of 2 290 coding genes were predicted,including 19 rRNA (7 5S rRNA,6 16S rRNA,6 23S rRNA),21 tRNA genes,20 ncRNA;23 gene islands,4 prophages and 2 groups of CRISPR related sequences.2 112,1 549 and 1 866 genes were annotated in the databases of COG,KEGG and GO,respectively,and the related proteins were mainly distributed in the metabolic process of APP.In addition,48 virulence genes and 22 drug resistance genes were annotated in VFDB and CARD databases (only floR gene was located on the plasmid).The whole genome circle map of the strain was drawn and the genome information was submitted to NCBI.The chromosome GenBank accession No. was CP097377 and the plasmids accession No. were CP097378 and CP097379, respectively. Phylogenetic tree analysis showed that the evolutionary relationship between this strain and APP strain from China (CP063424.1) was the closest.【Conclusion】 In this study, we completed the whole genome sequencing and bioinformatics analysis of the multi-drug resistant strain GD2107, fully understood the structure and function of the genome of the strain, and explored the related genes in drug resistance and pathogenic mechanism. The evolutionary relationship showed that the strain had a certain regional epidemic, which provided a reference for preventing the epidemic of PCP and exploring the pathogenic mechanism of APP.

Key words: Actinobacillus pleuropneumoniae (APP); whole genome sequencing; bioinformatics analysis; resistance genes; phylogenetic analysis

中图分类号:

S852.61^＋9

徐民生, 柯海意, 杨冬霞, 施科达, 孙泽仪, 牛佳伟, 常鑫, 翟少伦, 臧莹安, 李春玲. 多重耐药胸膜肺炎放线杆菌GD2107株全基因组测序及生物信息学分析[J]. 中国畜牧兽医, 2022, 49(10): 4019-4031.

XU Minsheng, KE Haiyi, YANG Dongxia, SHI Keda, SUN Zeyi, NIU Jiawei, CHANG Xin, ZHAI Shaolun, ZANG Yingan, LI Chunling. Whole Genome Sequencing and Biological Characteristics Analysis of Multidrug-resistant Actinobacillus pleuropneumoniae GD2107 Strain[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 4019-4031.

参考文献

[1] ZHANG L,WU X,HUANG Z,et al.Pharmacokinetic/pharmacodynamic assessment of cefquinome against Actinobacillus pleuropneumoniae in a piglet tissue cage infection model[J].Veterinary Microbiology,2018,219(6):100-106.
[2] SCHERRER S,PETERHANS S,NEUPERT C,et al.Development of a novel high resolution melting assay for identification and differentiation of all known 19 serovars of Actinobacillus pleuropneumoniae[J].MicrobiologyOpen,2022,11(2):e1272
[3] SHIN M,KANG M L,CHA S B,et al.An immunosorbent assay based on the recombinant ApxⅠa,ApxⅡa,and ApxⅢa toxins of Actinobacillus pleuropneumoniae and its application to field sera[J].Journal of Veterinary Diagnostic Investigation,2011,23(4):736-742.
[4] STRINGER O W,LI Y,BOSS J T,et al.JMM Profile:Actinobacillus pleuropneumoniae:A major cause of lung disease in pigs but difficult to control and eradicate[J].Journal of Medical Microbiology,2022,71(3):1483.
[5] STRINGER O W,LI Y,BOSS J T,et al.Rapid detection of Actinobacillus pleuropneumoniae from clinical samples using recombinase polymerase amplification[J].Frontiers in Veterinary Science,2022,9:805382.
[6] 祝希辉,庞喆羽,王志伟,等.致病性大肠杆菌携带原噬菌体的预测及耐药性与毒力研究[J].中国畜牧兽医,2022,49(3):1126-1134. ZHU X H,PANG Z Y,WANG Z W,et al.Prediction of prophage carrying by pathogenic Escherichia coli and its drug resistance and virulence[J].China Animal Husbandry & Veterinary Medicine,2022,49(3):1126-1134.(in Chinese)
[7] RHOADS A,AU K F.PacBio sequencing and its applications[J].Genomics, Proteomics & Bioinformatics,2015,13(5):278-289.
[8] LI S C,HUANG J F,HUNG Y T,et al.In silico capsule locus typing for serovar prediction of Actinobacillus pleuropneumoniae[J].Microbial Genomics,2022,8(4).doi:10.1099/mgen.0.000780.
[9] 徐卓菲.胸膜肺炎放线杆菌和猪瘟病毒的基因组测序与比较基因组学研究[D].武汉:华中农业大学,2008. XU Z F.Genome sequencing and comparative genomics of Actinobacillus pleuropneumoniae and Classical swine fever virus[D].Wuhan:Huazhong Agricultural University,2008.(in Chinese)
[10] ZHANG B Z,KU X G,YU X X,et al.Prevalence and antimicrobial susceptibilities of bacterial pathogens in Chinese pig farms from 2013 to 2017[J].Scientific Reports,2019,9(1):9908.
[11] PATEL R K,JAIN M.NGS QC Toolkit:A toolkit for quality control of next generation sequencing data[J].PLoS One,2012,7(2):e30619.
[12] SCHUBERT M,LINDGREEN S,ORLANDO L.AdapterRemoval v2:Rapid adapter trimming,identification,and read merging[J].BMC Research Notes,2016,12(9):88.
[13] LUO R B,LIU B H,XIE Y L,et,al.SOAPdenovo2:An empirically improved memory-efficient short-read de novo assembler[J]. Gigascience,2012,1(1):18.
[14] CHIN C,PELUSO P,SEDLAZECK F J,et al.Phased diploid genome assembly with single-molecule Real-time sequencing[J].Nature Methods,2016,13(12):1050-1054.
[15] KOREN S,WALENZ B P,BERLIN K,et al.Canu:Scalable and accurate long-read assembly via adaptivek k-mer weighting and repeat separation[J].Genome Research,2017,27(5):722-736.
[16] WALKER B J,ABEEL T,SHEA T,et al.Pilon:An integrated tool for comprehensive microbial variant detection and genome assembly improvement[J].PLoS One,2014,9(11):e112963.
[17] STOTHARD P,WISHART D S.Circular genome visualization and exploration using CGView[J].Bioinformatics,2005,21(4):537-539.
[18] BESEMER J,LOMSADZE A,BORODOVSKY M.GeneMarkS:A self-training method for prediction of gene starts in microbial genomes.Implications for finding sequence motifs in regulatory regions[J].Nucleic Acids Research,2001,29(12):2607-2618.
[19] LOWE T M,EDDY S R.tRNAscan-SE:A program for improved detection of transfer RNA genes in genomic sequence[J].Nucleic Acids Research,1997,25(5):955-964.
[20] KALVARI I,ARGASINSKA J,QUINONES-OLVERA N,et al.Rfam 13.0:Shifting to a genome-centric resource for non-coding RNA families[J].Nucleic Acids Research,2018,46(D1):D335-D342.
[21] GRISSA I,VERGNAUD G,POURCEL C.CRISPRFinder:A web tool to identify clustered regularly interspaced short palindromic repeats[J].Nucleic Acids Research,2007,35(Web Server):W52-W57.
[22] ARNDT D,GRANT J R,MARCU A,et al.PHASTER:A better,faster version of the PHAST phage search tool[J].Nucleic Acids Research,2016,44(W1):W16-W21.
[23] BERTELLI C,LAIRD M R,WILLIAMS K P,et al.IslandViewer 4:Expanded prediction of genomic islands for larger-scale datasets[J].Nucleic Acids Research,2017,45(W1):W30-W35.
[24] BUCHFINK B,XIE C,HUSON D H.Fast and sensitive protein alignment using DIAMOND[J].Nature Methods,2015,12(1):59-60.
[25] KROGH A,BROWN M,MIAN I S,et al.Hidden Markov models in computational biology.Applications to protein modeling[J].Journal of Molecular Biology,1994,235(5):1501-1531.
[26] CHEN L,ZHENG D,LIU B,et al.VFDB 2016:Hierarchical and refined dataset for big data analysis——10 years on[J].Nucleic Acids Research,2016,44(D1):D694-D697.
[27] MCARTHUR A G,WAGLECHNER N,NIZAM F,et al.The comprehensive antibiotic resistance database[J].Antimicrobial Agents and Chemotherapy,2013,57(7):3348-3357.
[28] ZHANG L F,XIE H B,WANG H J,et al.Kill rate and evaluation of ex vivo PK/PD integration of cefquinome against Actinobacillus pleuropneumoniae[J].Frontiers in Veterinary Science,2021,13(8):751957.
[29] 王丹丹,陈国强,张常印,等.猪胸膜肺炎放线杆菌血清8型菌株的分离鉴定及耐药性研究[J].中国畜牧兽医,2019,46(10):3124-3130. WANG D D,CHEN G Q,ZHANG C Y,et al.Isolation,identification and drug sensitive tests of Actinobacillus pleuropneumoniae serotype 8[J].China Animal Husbandry & Veterinary Medicine,2019,46(10):3124-3130.(in Chinese)
[30] 杨德智,侯冠彧,施力光,等.全基因组测序在畜禽中应用的研究进展[J].中国畜牧兽医,2021,48(9):3403-3414. YANG D Z,HOU G Y,SHI L G,et al.Research progress on whole genome sequencing in livestock and poultry[J].China Animal Husbandry & Veterinary Medicine,2021,49(9):3403-3414.(in Chinese)
[31] BOYCE J D,ADLER B.How does Pasteurella multocida respond to the host environment?[J].Current Opinion Microbiology,2006,9(1):117-122.
[32] COSTA T R,CATARINA F R,MEIR A,et al.Secretion systems in Gram-negative bacteria:Structural and mechanistic insights[J].Nature Reviews Microbiology,2015,6(13):343-359.
[33] BOSS J T,LI Y W,ROGERS J,et al.Whole genome sequencing for surveillance of antimicrobial resistance in Actinobacillus pleuropneumoniae[J].Frontiers in Microbiology,2017,6(8):311.
[34] KONG C,NEOH H,NATHAN S.Targeting Staphylococcus aureus toxins:A potential form of anti-virulence therapy[J].Toxins,2016,8(3):72.
[35] PETRUZZI B,BRIGGS R E,TATUM F M,et al.Capsular polysaccharide interferes with biofilm formation by Pasteurella multocida serogroup A[J].mBio,2017,6(8):e01843-17.
[36] LIU B,ZHENG D D,ZHOU S Y,et al.VFDB 2022:A general classification scheme for bacterial virulence factors[J].Nucleic Acids Research,2022,50(D1):D912-D917.
[37] LIU Z C,DENG D,LU H J,et al.Evaluation of machine learning models for predicting antimicrobial resistance of Actinobacillus pleuropneumoniae from whole genome sequences[J].Frontiers in Microbiology,2020,11:48.
[38] WISSING A,NICOLET J,BOERLIN P.The current antimicrobial resistance situation in Swiss veterinary medicine[J].Schweizer Archiv fur Tierheikunde,2001,143(10):503-510.
[39] CHANG Y F,SHI J,SHIN S J.Sequence analysis of the ROB-1 beta-lactamase gene from Actinobacillus pleuropneumoniae[J].Veterinary Microbiology,1992,32(3-4):319-325.
[40] KWONG J C,MCCALLUM N,SINTCHENKO V,et al.Whole genome sequencing in clinical and public health microbiology[J].Pathology,2015,47(3):199-210.
[41] LIN H,FENG C,ZHU T,et al.Molecular mechanism of the β-lactamase mediated β-lactam antibiotic resistance of Pseudomonas aeruginosa isolated from a Chinese teaching hospital[J].Frontiers in Microbiology,2022,13:855961.
[42] DON V,RAMETTE A,PERRETEN V.Comparative genomics of 26 complete circular genomes of 18 different serotypes of Actinobacillus pleuropneumoniae[J].Microbial Genomics,2022,8(2):000776.

多重耐药胸膜肺炎放线杆菌GD2107株全基因组测序及生物信息学分析

Whole Genome Sequencing and Biological Characteristics Analysis of Multidrug-resistant Actinobacillus pleuropneumoniae GD2107 Strain

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