A:L1 ST128型鸭源多杀性巴氏杆菌的耐药性及毒力分析

doi:10.11843/j.issn.0366-6964.2021.010.016

摘要/Abstract

摘要： 旨在阐明贵州某规模化鸭场临床疑似鸭霍乱的病原菌耐药性及毒力特征，通过细菌的分离鉴定、多位点序列分型（MLST）、ERIC-PCR同源性分析、药敏试验、动物回归试验、细菌全基因组测序以及基因组局部比较分析对分离株进行系统研究。结果显示，分离得到5株鸭源多杀性巴氏杆菌（PmCW1~5），均为A：L1 ST128型且同源性较高；分离株均对氨苄西林、阿莫西林、左氧氟沙星和林可霉素4种药物耐药，其中PmCW1还对环丙沙星低水平耐药；对强毒株PmCW1的全基因组数据分析显示，PmCW1中存在β-内酰胺类、喹诺酮类、四环素类、大环内酯类和多肽类等耐药基因，同时存在多药外排泵及多药耐药蛋白基因，耐药表型与耐药基因检测结果基本相符；PmCW1中存在的毒力基因总数为201个，主要是脂寡糖/脂多糖（LOS/LPS）、荚膜、黏附因子等编码基因；此外，还检测到IV型菌毛基因（ptfA、comE、hofB、hofC、vfr）、铁摄取相关蛋白基因（ccmABCEF、hgbBC、fur、hscB等）以及部分外膜蛋白基因（ompP5等）等；PmCW1具有典型的A：L1型多杀性巴氏杆菌的特征。综上表明，本研究分离得到的ST128型鸭源多杀性巴氏杆菌目前鲜有报道，对其耐药性及毒力的研究可为鸭霍乱的临床用药及疫苗研发提供理论依据。

关键词: 鸭源多杀性巴氏杆菌, 分离鉴定, MLST, 耐药性与毒力, 基因组局部比较分析

Abstract: The aim of this study was to characterize the drug resistance and virulence of pathogens from ducks suspected of cholera in a farm in Guizhou. The pathogenic bacteria were cultured and confirmed by PCR. Capsule genotyping and lipopolysaccharide genotyping, multilocus sequence typing (MLST), ERIC-PCR, animal regression test, drug susceptibility test, whole genome sequencing(WGS) and comparative analysis of local genomes were conducted on those isolated strains. Five Pasteurella multocida isolates (PmCW1-5) were identified as A:L1 ST128 and shared high homology. All of them were resistant to ampicillin, amoxicillin, levofloxacin, and lincomycin. PmCW1 also showed low level resistance to ciprofloxacin. WGS data showed PmCW1 which is highly virulent also harbored genes mediating β-lactams, quinolones, tetracyclines, macrolides and peptides resistance and encoding multi-drug efflux pumps and multi-drug resistance proteins. Meanwhile, PmCW1 carried 201 different virulence-mediating genes including those encoding lipooligosaccharide/lipopolysaccharide (LOS/LPS), capsule, adhesion factor, and etc. In addition, type IV fimbriae genes (ptfA, comE, hofB, hofC, vfr), iron uptake related protein genes (ccmABCEF, hgbBC, fur, hscB), outer membrane protein genes (ompP5) were also detected. PmCW1 has the characteristics of typical A:L1 P. multocida. P. multocida ST128 isolated from ducks in this study has been rarely reported. The drug resistance and virulence analysis on those isolates will provide theoretical basis for clinical medication and vaccine development of duck cholera.

Key words: Pasteurella multocida from ducks, isolation and identification, MLST, drug resistance and virulence, comparative analysis of local genomes

中图分类号:

S852.61

张亚楠, 李亚菲, 陈汝佳, 余波, 彭珊, 李婷, 蒲龄, 徐景峨. A:L1 ST128型鸭源多杀性巴氏杆菌的耐药性及毒力分析[J]. 畜牧兽医学报, 2021, 52(10): 2852-2863.

ZHANG Yanan, LI Yafei, CHEN Rujia, YU Bo, PENG Shan, LI Ting, PU Ling, XU Jinge. Resistance and Virulence Analysis of Type A: L1 ST128 Pasteurella multocida from Ducks[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(10): 2852-2863.

参考文献 44

[1]	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. Berlin:Springer, 2012:1-22.
[2]	WILSON B A, HO M. Pasteurella multocida:from zoonosis to cellular microbiology[J]. Clin Microbiol Rev, 2013, 26(3):631-655.
[3]	PENG Z, LIANG W, WANG F, et al. Genetic and phylogenetic characteristics of Pasteurella multocida isolates from different host species[J]. Front Microbiol, 2018, 9:1408.
[4]	HONNORAT E, SENG P, SAVINI H, et al. Prosthetic joint infection caused by Pasteurella multocida:a case series and review of literature[J]. BMC Infect Dis, 2016, 16(1):435.
[5]	RYAN J M, FEDER JR H M. Dog licks baby. Baby gets Pasteurella multocida meningitis[J]. Lancet, 2019, 393:E41.
[6]	LIU Q, HU Y L, LI P, et al. Identification of fur in Pasteurella multocida and the potential of its mutant as an attenuated live vaccine[J]. Front Vet Sci, 2019, 6:5.
[7]	HOLSCHBACH C L, AULIK N, POULSEN K, et al. Prevalence and temporal trends in antimicrobial resistance of bovine respiratory disease pathogen isolates submitted to the Wisconsin Veterinary Diagnostic Laboratory:2008-2017[J]. J Dairy Sci, 2020, 103(10):9464-9472.
[8]	刘海燕, 杨有文, 王远微, 等. 6株山羊源多杀性巴氏杆菌血清型、耐药性及致病性分析[J]. 黑龙江畜牧兽医, 2020, (16):65-68.LIU H Y, YANG Y W, WANG W Y, et al. Analysis of serotype, drug resistance and pathogenicity of 6 strains of Pasteurella multocida from goats[J]. Heilongjiang Animal Science and Veterinary Medicine, 2020, (16):65-68. (in Chinese)
[9]	PRAJAPATI A, CHANDA M M, DHAYALAN A, et al. Variability in in vitro biofilm production and antimicrobial sensitivity pattern among Pasteurella multocida strains[J]. Biofouling, 2020, 36(8):938-950.
[10]	DABO S M, TAYLOR J D, CONFER A W. Pasteurella multocida and bovine respiratory disease[J]. Anim Health Res Rev, 2007, 8(2):129-150.
[11]	HARPER M, BOYCE J D, ADLER B. Pasteurella multocida pathogenesis:125 years after pasteur[J]. FEMS Microbiol Lett, 2006, 265(1):1-10.
[12]	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.
[13]	PENG Z, LIANG W, LIU W J, et al. Genomic characterization of Pasteurella multocida HB01, a serotype A bovine isolate from China[J]. Gene, 2016, 581(1):85-93.
[14]	MARZA A D, JESSE ABDULLAH F F, 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.
[15]	PERIASAMY S, PRAVEENA P E, SINGH N. Effects of Pasteurella multocida lipopolysaccharides on bovine leukocytes[J]. Microb Pathog, 2018, 119:225-232.
[16]	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.
[17]	PENG Z, WANG X R, ZHOU R, et al. Pasteurella multocida:genotypes and genomics[J]. Microbiol Mol Biol Rev, 2019, 83(4):e00014-19.
[18]	Clinical Laboratory Standards Institute (CLSI). Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals;Approved standard-fourth edition. CLSI document VET01-A4[R]. Wayne, PA, USA:CLSI, 2013.
[19]	WICK R R, JUDD L M, GORRIE C L, et al. Unicycler:resolving bacterial genome assemblies from short and long sequencing reads[J]. PLoS Comput Biol, 2017, 13(6):e1005595.
[20]	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 Res, 2001, 29(12):2607-2618.
[21]	BUCHFINK B, XIE C, HUSON D H. Fast and sensitive protein alignment using DIAMOND[J]. Nat Methods, 2015, 12(1):59-60.
[22]	SULLIVAN M J, PETTY N K, BEATSON S A. Easyfig:a genome comparison visualizer[J]. Bioinformatics, 2011, 27(7):1009-1010.
[23]	刘玉庆, 李璐璐, 骆延波, 等. EUCAST 2017欧盟药敏试验标准[M]. 北京:中国标准出版社, 2016.LIU Y Q, LI L L, LUO Y B, et al. European committee on antimicrobial susceptibility testing[M]. Beijing:Standards Press of China, 2016. (in Chinese)
[24]	王林柏, 孙久鹤, 郭东春, 等. 国内部分地区多杀性巴氏杆菌荚膜血清型和基因型的研究[J]. 中国预防兽医学报, 2016, 38(2):116-119.WANG L B, SUN J H, GUO D C, et al. Identification of capsule serotype and genotype of Pasteurella multocida in some areas of China[J]. Chinese Journal of Preventive Veterinary Medicine, 2016, 38(2):116-119. (in Chinese)
[25]	LI Z C, CHENG F J, LAN S M, et al. Investigation of genetic diversity and epidemiological characteristics of Pasteurella multocida isolates from poultry in southwest China by population structure, multi-locus sequence typing and virulence-associated gene profile analysis[J]. J Vet Med Sci, 2018, 80(6):921-929.
[26]	UJVÁRI B, MAKRAI L, MAGYAR T. Virulence gene profiling and ompA sequence analysis of Pasteurella multocida and their correlation with host species[J]. Vet Microbiol, 2019, 233:190-195.
[27]	EWERS C, LUBKE-BECKER A, BETHE A, et al. Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status[J]. Vet Microbiol, 2006, 114(3-4):304-317.
[28]	TURNI C, SINGH R, BLACKALL P J. Genotypic diversity of Pasteurella multocida isolates from pigs and poultry in Australia[J]. Aust Vet J, 2018, 96(10):390-394.
[29]	UJVÁRI B, WEICZNER R, DEIM Z, et al. Characterization of Pasteurella multocida strains isolated from human infections[J]. Comp Immunol, Microbiol Infect Dis, 2019, 63:37-43.
[30]	PETERSEN A, BISGAARD M, TOWNSEND K, et al. MLST typing of Pasteurella multocida associated with haemorrhagic septicaemia and development of a real-time PCR specific for haemorrhagic septicaemia associated isolates[J]. Vet Microbiol, 2014, 170(3-4):335-341.
[31]	WANG Y H, ZHU J, LU C P, et al. Evidence of circulation of an epidemic strain of Pasteurella multocida in Jiangsu, China by multi-locus sequence typing (MLST)[J]. Infect, Genet Evol, 2013, 20:34-38.
[32]	李伟杰, 田野, 岂晓鑫, 等. 禽源多杀性巴氏杆菌多位点序列分型研究[J]. 中国兽药杂志, 2017, 51(2):1-6.LI W J, TIAN Y, QI X X, et al. Multi-locus sequence typing of Pasteurella multocida isolated from Avian[J]. Chinese Journal of Veterinary Drug, 2017, 51(2):1-6. (in Chinese)
[33]	陈红梅, 程龙飞, 邓辉, 等. 鸭源多杀性巴氏杆菌对磺胺类药物的耐药性检测及耐药基因分析[J]. 中国畜牧兽医, 2019, 46(1):223-230.CHEN H M, CHENG L F, DENG H, et al. Detection of sulfonamides sensitivity and analysis of resistant genes of Pasteurella multocida isolated from ducks[J]. China Animal Husbandry & Veterinary Medicine, 2019, 46(1):223-230. (in Chinese)
[34]	陈国权, 张旭, 阎朝华, 等. 鸭源多杀性巴氏杆菌的分离鉴定及耐药性分析[J]. 中国畜牧兽医, 2020, 47(2):620-628.CHEN G Q, ZHANG X, YAN C H, et al. Isolation, identification and drug resistance analysis of Pasteurella multocida from ducks[J]. China Animal Husbandry & Veterinary Medicine, 2020, 47(2):620-628. (in Chinese)
[35]	HOOPER D C, JACOBY G A. Mechanisms of drug resistance:quinolone resistance[J]. Ann N Y Acad Sci, 2015, 1354(1):12-31.
[36]	KONG L C, GAO D, GAO Y H, et al. Fluoroquinolone resistance mechanism of clinical isolates and selected mutants of Pasteurella multocida from bovine respiratory disease in China[J]. J Vet Med Sci, 2014, 76(12):1655-1657.
[37]	HAN J, SAHIN O, BARTON Y W, et al. Key role of Mfd in the development of Fluoroquinolone resistance in Campylobacter jejuni[J]. PLoS Pathog, 2008, 4(6):e1000083.
[38]	RUFFOLO C G, TENNENT J M, MICHALSKI W P, et al. Identification, purification, and characterization of the type 4 fimbriae of Pasteurella multocida[J]. Infect Immun, 1997, 65(1):339-343.
[39]	HATFALUDI T, AL-HASANI K, BOYCE J D, et al. Outer membrane proteins of Pasteurella multocida[J]. Vet Microbiol, 2010, 144(1-2):1-17.
[40]	ALLEN J W A, LEACH N, FERGUSON S J. The histidine of the c-type cytochrome CXXCH haem-binding motif is essential for haem attachment by the Escherichia coli cytochrome c maturation (Ccm) apparatus[J]. Biochem J, 2005, 389:587-592.
[41]	JACOBSEN I, GERSTENBERGER J, GRUBER A D, et al. Deletion of the ferric uptake regulator Fur impairs the in vitro growth and virulence of Actinobacillus pleuropneumoniae[J]. Infect Immun, 2005, 73(6):3740-3744.
[42]	HARPER M, COX A D, ADLER B, et al. Pasteurella multocida lipopolysaccharide:the long and the short of it[J]. Vet Microbiol, 2011, 153(1-2):109-115.
[43]	PETRUZZI B, BRIGGS R E, SWORDS W E, et al. Capsular polysaccharide interferes with biofilm formation by Pasteurella multocida Serogroup A[J]. mBio, 2017, 8(6):e01843-17.
[44]	TOMICH M, PLANET P J, FIGURSKI D H. The tad locus:Postcards from the widespread colonization island[J]. Nat Rev Microbiol, 2007, 5(5):363-375.