产金属β-内酰胺酶猪源ST201型多杀性巴氏杆菌的耐药性和致病性分析

doi:10.11843/j.issn.0366-6964.2024.12.032

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (12): 5692-5705.doi: 10.11843/j.issn.0366-6964.2024.12.032

产金属β-内酰胺酶猪源ST201型多杀性巴氏杆菌的耐药性和致病性分析

杨荣荣¹(), 张婷¹, 唐萍萍², 何爽方³, 赵苗苗¹, 雷连成¹^,⁴, 张付贤¹^,*()

1. 长江大学动物科学技术学院，荆州 434023
2. 湖北省潜江市农业农村局，潜江 433199
3. 湖北省潜江市张金镇畜牧兽医技术服务中心，潜江 433140
4. 吉林大学动物医学学院，长春 130062

收稿日期:2024-01-26 出版日期:2024-12-23 发布日期:2024-12-27
通讯作者: 张付贤 E-mail:yangrr0702@163.com;zhangfuxian99@163.com
作者简介:杨荣荣(2001-)，女，陕西宝鸡人，硕士生，主要从事多杀性巴氏杆菌相关研究, E-mail: yangrr0702@163.com
基金资助:
国家科技部“十四五”重点研发项目(2021YFD1800405)；国家自然科学基金面上项目(32072823)

Analysis of Drug Resistance and Pathogenicity of Metallo-β-Lactamase-producing Porcine ST201 Pasteurella multocida

YANG Rongrong¹(), ZHANG Ting¹, TANG Pingping², HE Shuangfang³, ZHAO Miaomiao¹, LEI Liancheng¹^,⁴, ZHANG Fuxian¹^,*()

1. College of Animal Science and Technology, Yangtze University, Jingzhou 434023, China
2. Qianjiang Bureau of Agriculture and Rural Affairs, Qianjiang 433199, China
3. Zhangjin Town for Animal Husbandry and Veterinary Technical Service, Qianjiang 433140, China
4. College of Veterinary Medicine, Jilin University, Changchun 130062, China

Received:2024-01-26 Online:2024-12-23 Published:2024-12-27
Contact: ZHANG Fuxian E-mail:yangrr0702@163.com;zhangfuxian99@163.com

摘要/Abstract

摘要：

为探究湖北荆州某生猪养殖企业育肥猪呼吸道疾病的致病原，采集病猪肺组织进行病原的筛查和分离鉴定。本研究对分离菌株进行形态学观察、生理生化、16S rDNA基因序列测定、血清型分型、多位点序列分析、药物敏感性试验、耐药基因测序分析以及动物致病性试验。结果显示，分离菌株Pm0525为革兰阴性短杆菌；生化试验、PCR和16S rDNA测序鉴定为多杀性巴氏杆菌。该菌为荚膜血清型A型，分子分型为ST201型，与ST1615型聚为一支；分离菌株对庆大霉素、卡那霉素、麦迪霉素等13种抗生素敏感，对氯霉素、克林霉素、头孢呋辛、头孢他啶和四环素耐药；分离菌株具有中等生物被膜形成能力，携带SulⅠ、SulⅡ、tetA和bla-TEM四种耐药基因，可通过产金属β-内酰胺酶(MBL)方式发挥抗β-内酰胺类抗生素的作用。中药药敏试验结果显示，该分离菌株对中药黄连呈现明显的敏感性。该分离株携带18种毒力基因，并对小鼠具有较强的致病性。本研究系统性分析了猪源多杀性巴氏杆菌的血清型、分子分型、药物敏感性和致病性，为进一步研究多杀性巴氏杆菌感染的致病机制、防控和精准施治提供科学依据和数据参考。

关键词: 多杀性巴氏杆菌, 金属β-内酰胺酶, MLST分析, 耐药性, 致病性

Abstract:

The study was design to investigate the pathogenic agents of respiratory tract diseases of fattening pigs in a pig breeding enterprise in Jingzhou, Hubei Province. The pathogen was screened and isolated from the diseased pig lung tissue. Morphological observation, physiological and biochemical analysis, 16S rDNA gene sequence, serotype typing, multilocus sequence typing, drug sensitivity test, drug resistance gene sequencing analysis and animal pathogenicity test were performed for the isolated strains. The results showed that the isolated strain Pm0525 was Gram-negative brevibacterium. It was identified as Pasteurella multocida by biochemical assay, PCR and 16S rDNA sequence analysis. The isolated strain was identified as Pasteurella multocida type A. Multilocus sequence typing analysis confirmed that the isolated strain is type ST201 and merged with type ST1615. The isolated strain was sensitive 13 kinds of antibiotics, such as gentamicin, kanamycin and medemycin, and resistant to chloramphenicol, clindamycin, cefuroxime, ceftazidime and tetracycline. The isolated strain showed moderate biofilm formation ability, carrying out four resistance genes, SulⅠ, SulⅡ, tetA and bla-TEM. The isolated strain may play the role of anti-β-lactam antibiotics by producing metal-beta-lactamase (MBL). In addition, traditional Chinese medicine Coptis had obvious inhibitory effect on the isolated strain in vitro. The isolated strain carried 18 virulence genes, and showed strong pathogenicity to mice. This study systematically analyzed the serotype, molecular typing, drug sensitivity and pathogenicity of swine Pasteurella multocida, providing scientific basis and data reference for further research on the transmission, prevention and precise treatment of Pasteurella infection.

Key words: Pasteurella multocida, metallo-beta-lactamase, MLST analysis, drug resistance, pathogenicity

中图分类号:

S852.61

杨荣荣, 张婷, 唐萍萍, 何爽方, 赵苗苗, 雷连成, 张付贤. 产金属β-内酰胺酶猪源ST201型多杀性巴氏杆菌的耐药性和致病性分析[J]. 畜牧兽医学报, 2024, 55(12): 5692-5705.

YANG Rongrong, ZHANG Ting, TANG Pingping, HE Shuangfang, ZHAO Miaomiao, LEI Liancheng, ZHANG Fuxian. Analysis of Drug Resistance and Pathogenicity of Metallo-β-Lactamase-producing Porcine ST201 Pasteurella multocida[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5692-5705.

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血清型 Serotype	基因 Gene	引物序列(5′→3′) Primer sequences	片段大小/bp Fragment size
A	hyaD-hyaC	F: TGCCAAAATCGCAGTCAG	1 044
A	hyaD-hyaC	R: TTGCCATCATTGTCAGTG	1 044
B	bcbD	F: CATTTATCCAAGCTCCACC	760
B	bcbD	R: GCCCGAGAGTTTCAATCC	760
D	dcbF	F: TTACAAAAGAAAGACTAGGAGCCC	657
D	dcbF	R: CATCTACCCACTCAACCATATCAG	657
E	ecbJ	F: TCCGCAGAAAATTATTGACTC	511
E	ecbJ	R: GCTTGCTGCTTGATTTTGTC	511
F	fcbD	F: AATCGGAGAACGCAGAAATCAG	851
F	fcbD	R: TTCCGCCGTCAATTACTCTG	851

分离株 Isolate	地区 Region	时间 Year	宿主 Host	来源 Sample	MLST
分离株 Isolate	地区 Region	时间 Year	宿主 Host	来源 Sample	mhadk	mharoA	mhdeoD	mhgdhA	mhg6pd	mhmdh	mhpgi	ST
XJ-PM99	中国	2020	牛	鼻拭子	1	1	1	53	1	1	1	201
XJ-PM100	中国	2020	牛	鼻拭子	1	1	1	53	1	1	1	201
PM0525	中国	2022	猪	鼻拭子	1	1	1	53	1	1	1	201

耐药类型 Types of drug resistance	耐药谱 Antibiotic resistance
耐药	氯霉素Chloramphenicol，克林霉素Clindamycin，头孢呋辛Cefuroxime，头孢他啶Ceftazidime，四环素Tetracycline
中介	红霉素Erythromycin，羧苄西林Carbenicillin，氨苄西林Ampicillin，呋喃唑酮Furazolidone
敏感	庆大霉素Gentamicin，卡那霉素Kanamycin，环丙沙星Ciprofloxacin，麦迪霉素Midecamycin，新霉素Neomycin，氧氟沙星Ofloxacin，诺氟沙星Norfloxacin，哌拉西林Piperacillin，丁胺卡那Amikacin，头孢氨苄Cephalexin，头孢咪唑Cefpimizole，头孢拉定Cefradine，头孢哌酮Cefoperazone

中药 Traditional Chinese medicine	抑菌圈直径/mm IZD/mm	敏感性 Sensitivity	中药 Traditional Chinese medicine	抑菌圈直径/mm IZD/mm	敏感性 Sensitivity
连翘 Forsythia suspensa	7	+	乌梅 Dark plum	8	+
黄连 Coptis chinensis	19	++	秦皮 Ash bark	7	+
五倍子 Gallnut	12	++	荆芥 Schizonepeta	10	++
肉桂 Cinnamomum cassia	7	+	五味子 Schisandra chinensis	0	-
款冬花 Coltsfoot flower	2	-	蒲公英 Dandelion	2	-
金银花 Honeysuckle	0	-	枳壳 Fructus Aurantii	0	-
炒僵蚕 Fried Silkworm	0	-	板蓝根 Radix isatidis	0	-
金蝎 Golden Scorpion	3	-	羌活 Notopterygium root	0	-
桑皮 Mulberry bark	0	-	炙白附子 Roasted White Aconite	4	-
薄荷 Mint	0	-	鱼腥草 Houttuynia cordata	0	-
苏子 Perillaseed	0	-	贝川母 Beichuan mother	0	-
桑白皮 Morus alba	0	-	胖大海 scaphium scaphigerum	0	-
黄芩 Scutellaria baicalensis	0	-	首乌 Polygonum multiflorum	1	-
茯苓 Poria cocos	0	-	罗汉果 Siraitia grosvenorii	0	-

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产金属β-内酰胺酶猪源ST201型多杀性巴氏杆菌的耐药性和致病性分析

Analysis of Drug Resistance and Pathogenicity of Metallo-β-Lactamase-producing Porcine ST201 Pasteurella multocida

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