一株牛源福氏志贺菌的分离鉴定及全基因组测序分析

doi:10.11843/j.issn.0366-6964.2026.01.033

摘要/Abstract

摘要：

近年来，牛易患腹泻病在牛场中进行传播，并有着较高的致死率，造成经济损失。本试验旨在对引起北京某牛场牛腹泻的福氏志贺菌进行分离鉴定，明确病原菌的血清型、耐药性和致病性等生物学特性及基因组特征。本研究采集腹泻牛粪便样本，进行细菌分离纯化、16S rRNA扩增子测序分析、毒力基因检测和分离株全基因组测序。结果显示，经细菌分离纯化、PCR鉴定发现一株福氏志贺菌Fc；基因组测序获得福氏志贺菌株Fc株全基因组大小为4.98 Mp，GC含量为50.76%；含有91个tRNA基因，7个23S rRNA，7个16S rRNA，8个5S rRNA，283个misc_RNA，97个碳水化合物活性酶基因；TVBD注释分析发现1 363个转运蛋白基因；CARD注释分析发现80个抗性基因；VFDB和PHI-base注释分析发现1 410个潜在的毒力因子相关基因及2 052个表型突变基因等。系统进化树分析发现福氏志贺菌与福氏志贺菌29903属于同一分支，进化关系最近。本研究完成了一株牛源福氏志贺菌的分离鉴定和全基因组测序分析，为志贺氏菌病的防控研究提供了理论依据。

关键词: 福氏志贺菌, 全基因组测序, 耐药性, 毒力

Abstract:

In recent years， diarrhea in cattle has been spreading in cattle farms with a high mortality rate， causing economic losses. This experiment aims to isolate and identify Shigella flexneri， which causes diarrhea in a Beijing cattle farm， and to clarify the biological characteristics and genomic features of the pathogen’s serotype， drug resistance， and pathogenicity. In this study， fecal samples from diarrheal cattle were collected for bacterial isolation and purification， 16S rRNA amplicon sequencing analysis， virulence gene detection， and whole-genome sequencing of isolated strains. The results showed that one strain of Shigella flexneri Fc was identified through bacterial isolation and purification， and PCR identification. Whole-genome sequencing revealed that the Shigella flexneri Fc strain has a genome size of 4.98 Mb and a GC content of 50.76%. It contains 91 tRNA genes， 7 23S rRNA， 7 16S rRNA， 8 5S rRNA， 283 misc_RNA， and 97 carbohydrate-active enzyme genes. TVBD annotation analysis identified 1 363 transporter genes； CARD annotation analysis identified 80 resistance genes； VFDB and PHI-base annotation analysis identified 1 410 potential virulence factor-related genes and 2 052 phenotypic mutation genes. Phylogenetic tree analysis revealed that Shigella flexneri is closely related to Shigella flexneri 29903， belonging to the same branch. This study completed the isolation and identification of a bovine-origin Shigella flexneri strain and whole-genome sequencing analysis， providing a theoretical basis for the prevention and control research of shigellosis.

Key words: Shigella flexneri, whole-genome sequencing, drug resistance, virulence

中图分类号:

S852.61

张晶, 王亚平, 樊曦雯, 王真. 一株牛源福氏志贺菌的分离鉴定及全基因组测序分析[J]. 畜牧兽医学报, 2026, 57(1): 378-391.

ZHANG Jing, WANG Yaping, FAN Xiwen, WANG Zhen. Isolation， Identification and Whole Genome Sequencing Analysis of a Bovine-derived Shigella flexneri Strain[J]. Acta Veterinaria et Zootechnica Sinica, 2026, 57(1): 378-391.

图/表 13

表1

图1

图2

图3

表1

图4

图5

图6

图7

表2

表3

表4

图8

参考文献 28

[1]	JENNISON A V，VERMA N K.Shigella flexneri infection：pathogenesis and vaccine development［J］.Fems Microbiol Rev，2004，28（1）：43-58.
[2]	RANJBAR R，BEHNOOD V，MEMARIANI H，et al.Molecular characterisation of quinolone-resistant Shigella strains isolated in Tehran，Iran［J］.J Glob Antimicrob Resist，2016，5：26-30.
[3]	MILES L S，HOLT E K，MOSTOWY S.Recent advances in modelling Shigella infection［J］.Trends Microbiol，2024，32（9）：917-924.
[4]	KHALIL A I，TROEGER C，BLACKER F B，et al.Morbidity and mortality due to Shigella and enterotoxigenic Escherichia coli diarrhoea：The global burden of disease study 1990-2016［J］.Lancet Infect Dis，2018，18（11）：1229-1240.
[5]	ZHU Z，SHI Y，ZHOU X，et al.Molecular characterization of fluoroquinolone and/or cephalosporin resistance in Shigella sonnei isolates from yaks［J］.BMC Vet Res，2018，14（1）：1-9.
[6]	MOHD A，MITESH P，SUMUKH D，et al.Effect of Adiantum philippense extract on biofilm formation，adhesion with its antibacterial activities against foodborne pathogens，and characterization of bioactive metabolites：An in vitro-in silico approach［J］.Front Microbiol，2020，11：823.
[7]	PAKBIN B，AMANI Z，ALLAHYARI S，et al.Genetic diversity and antibiotic resistance of Shigella spp.isolates from food products［J］.Food Sci Nutr，2021，9：6362-6371.
[8]	PAKBIN B，BRÜCK W M，BRÜCK T B.Molecular mechanisms of Shigella pathogenesis； recent advances［J］.Int J Mol Sci，2023，24（3）：2448.
[9]	KELLY M，JANARDHANAN J，WAGH C，et al.Development of a Shigella conjugate vaccine targeting Shigella flexneri 6 that is immunogenic and provides protection against virulent challenge［J］.Vaccine，2024，42（24）：126263.
[10]	WIEGAND I，HILPERT K，HANCOCK R E.Agar and broth dilution methods to determine the minimal inhibitory concentration （MIC） of antimicrobial substances［J］.Nat Protoc，2008，3（2）：163-175.
[11]	LI H.Minimap and miniasm：fast mapping and de novo assembly for noisy long sequences［J］.Bioinformatics，2016，32（14）：2103-2110.
[12]	LI H，HANDSAKER B，WYSOKER A，et al.The sequence alignment/map format and SAMtools［J］.Bioinformatics，2009，25（16）：2078-2079.
[13]	TORSTEN S.Prokka：rapid prokaryotic genome annotation［J］.Bioinformatics （Oxford，England），2014，30（14）：2068-2069.
[14]	APWEILER R，BAIROCH A，WU C H，et al.UniProt：The universal protein knowledgebase［J］.Nucleic Acids Res，2004，32：D115- D119.
[15]	MINORU K，SUSUMU G，SHUICHI K，et al.The KEGG resource for deciphering the genome［J］.Nucleic Acids Res，2004，32：D277-D280.
[16]	ASHBURNER M，BALL A C，BLAKE A J，et al.Gene Ontology：Tool for the unification of biology［J］.Nat Genet，2000，25（1）：25-29.
[17]	SARA E，JAINA M，ALEX B，et al.The Pfam protein families database in 2019［J］.Nucleic Acids Res，2019，47（D1）：D427-D432.
[18]	TATUSOV R L，GALPERIN M Y，NATALE D A，et al.The COG database：A tool for genome-scale analysis of protein functions and evolution［J］.Nucleic Acids Res，2000，28（1）：33-36.
[19]	LIU B，POP M.ARDB—Antibiotic resistance genes database［J］.Nucleic Acids Res，2009，37：D443- D447.
[20]	ALCOCK B P，RAPHENYA A R，LAU T T Y，et al.CARD 2020：antibiotic resistome surveillance with the comprehensive antibiotic resistance database［J］.Nucleic Acids Res，2020，48（D1）：D517- D525.
[21]	KOTLOFF K L，RIDDLE M S，PLATTS-MILLS J A，et al.Shigellosis［J］.Lancet，2018，391（10122）：801-812.
[22]	HOSANGADI D，SMITH P G，KASLOW D C，et al.WHO consultation on ETEC and Shigella burden of disease，Geneva，6-7th April 2017：Meeting report［J］.Vaccine，2019，37（50）：7381-7390.
[23]	廖驰真，曾竣.志贺菌流行耐药现状及溯源研究进展［J］.中国卫生检验杂志，2023，33（16）：2046-2048.
	LIAO C Z，ZENG J.Current status of antimicrobial resistance and traceability research on Shigella ［J］.Chinese Journal of Health Laboratory Technology，2023，33（16）：2046-2048.
[24]	中华人民共和国国家卫生健康委员会.细菌性痢疾诊疗方案（2023年版）［EB/OL］.（2023-12-14）.https：//www.nhc.gov.cn/cms-search/downFiles/58e9d15c81b945708f2afc8b64d6c698.pdf
	National Health Commission of the People's Republic of China.Diagnosis and treatment protocol for bacillary dysentery （2023 Edition）［EB/OL］.（2023-12-14）.https：//www.nhc.gov.cn/cms-search/downFiles/58e9d15c81b945708f2afc8b64d6c698.pdf
[25]	彭杰夫，姜仁杰，董晨，等.一起耐多药福氏1a志贺菌感染暴发疫情的调查和分子特征［J］.江苏预防医学，2024，35（4）：464-468.
	PENG J F，JIANG R J，DONG C，et al.Investigation and molecular characteristics of an outbreak caused by multidrug resistant Shigella flexneri subtype 1a infection［J］.Jiangsu Journal of Preventive Medicine，2024，35（4）：464-468.
[26]	吴自豪，王雪，刘鹏，等.新疆塔额垦区牛羊粪源志贺菌耐药性、毒力基因检测及其致病性分析［J］.中国草食动物科学，2024，44（5）：37-42.
	WU Z H，WANG X，LIU P，et al.Analysis of antimicrobial susceptibility testing，virulence genes，and pathogenicity of shigella spp.isolated from cattle and sheep feces in Taer Reclamation Area，Xinjiang［J］.China Herbivore Science，2024，44（5）：37-42.
[27]	GOMEZ D E，ARROYO L G，COSTA M C，et al.Characterization of the fecal bacterial microbiota of healthy and diarrheic dairy calves［J］.J Vet Intern Med，2017，31（3）：928-939.
[28]	孙睿雪，李玉铎，赵建军.牛新发病毒性传染病及其诊断技术研究进展［J］.动物医学进展，2024，45（5）：90-96.
	SUN R X，LI Y Z，ZHAO J J.Progress on bovine emerging viral infectious diseases and relevant diagnosis technology［J］.Progress in Veterinary Medicine，2024，45（5）：90-96.

类型 Type	数量 Number	总长度 Total length	平均长度 Average length	基因组比例/% Percentage of genome （%）
Gene	5 750	5 006 971	871	88.63
CDS	5 353	4 934 757	922	87.36
tRNA	91	7 137	78	0.13
23S rRNA	7	20 314	2 902	0.36
16S rRNA	7	10 766	1 538	0.19
5S rRNA	8	888	111	0.02
tmRNA	1	363	363	0.01
misc_RNA	283	32 746	116	0.58

分类 Class	数量 Number	说明 Descrition
糖苷水解酶GHs	42	Glycoside hydrolases
糖基转移酶GTs	36	Glycosy transferases
多糖裂合酶PLs	0	Polysaccharide lyases
碳水化合物酯酶CEs	8	Carbohydrate esterases
辅助氧化还原酶AAs	3	Auxiliary activities
碳水化合物结合模块CBMs	3	Carbohydrate-binding modules

毒力因子 Virulence factors	相关基因数 Related genes number
硝酸还原酶 Nitrate reductase	27
周毛鞭毛 Peritrichous flagella	59
荚膜 Capsule	68
寡聚糖 LOS	42
O抗原 O-antigen	23
脂多糖 LPS	27
IV型菌毛 Type IV pili	39

耐药机制 Resistance Mechanism	抗生素耐药基因数 Antibiotic resistance genes number	抗性 Resistances
抗生素外排泵 Antibiotic efflux	45	氟喹诺酮类、头孢菌素类、头霉素类、青霉烷类、大环内酯类、磷霉素、硝基咪唑类、四环素类、肽类、氨基糖苷类、核苷类、磺胺类抗生素
抗生素失活 Antibiotic inactivation	17	β-内酰胺类、林可酰胺类、氟喹诺酮类、四环素类、利福霉素类抗生素
抗生素靶标改变 Antibiotic target alteration	14	肽类、氟喹诺酮类、头孢菌素类、甘氨酰环素类、青霉烷类、四环素类、利福霉素类、苯酚类、三氯生、头霉素类、青霉烷类抗生素
抗生素靶标保护 Antibiotic target protection	2	氟喹诺酮类抗生素
抗生素靶标替换 Antibiotic target replacement	2	复合磺胺类、磺胺二甲氧嘧啶