三十四株猪源多杀性巴氏杆菌的分型及耐药性分析

doi:10.11843/j.issn.0366-6964.2025.08.044

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (8): 4074-4085.doi: 10.11843/j.issn.0366-6964.2025.08.044

三十四株猪源多杀性巴氏杆菌的分型及耐药性分析

马彩萍¹^,²(), 祝瑶²(), 刘红道³, 周光斌⁴, 许秋², 林龙华², 柴霁芸², 侯杰², 孙宏飞²^,⁵, 杜素素¹^,², 范翠龙¹^,², 夏利宁¹^,*(), 张万江²^,*()

1. 新疆农业大学动物医学学院，乌鲁木齐 830052
2. 中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室/动物细菌病创新团队，哈尔滨 150069
3. 吉安市农业农村产业发展服务中心，吉安 343000
4. 丰城市现代农业技术服务中心，丰城 331100
5. 东北林业大学野生动物与自然保护地学院，哈尔滨 150040

收稿日期:2024-07-29 出版日期:2025-08-23 发布日期:2025-08-28
通讯作者: 夏利宁,张万江 E-mail:1757841410@qq.com;yaozhu922@163.com;xln@edu.xjau.cn;zhangwanjiang@caas.cn
作者简介:马彩萍(1995-)，女，甘肃定西人，硕士，主要从事动物源细菌耐药性研究，E-mail: 1757841410@qq.com
祝瑶(1990-), 男，河北石家庄人，助理研究员，主要从事动物源细菌耐药性研究, E-mail: yaozhu922@163.com
第一联系人：
马彩萍与祝瑶为同等贡献作者
基金资助:
国家重点研发计划(2021YFD1800401)；中央级公益性科研院所基本科研业务费专项(1610302022001；1610302024001)

Typing and Antimicrobial Resistance Analysis of Thirty-four Pasteurella multocida Strains from Pigs

MA Caiping¹^,²(), ZHU Yao²(), LIU Hongdao³, ZHOU Guangbin⁴, XU Qiu², LIN Longhua², CHAI Jiyun², HOU Jie², SUN Hongfei²^,⁵, DU Susu¹^,², FAN Cuilong¹^,², XIA Lining¹^,*(), ZHANG Wanjiang²^,*()

1. College of Animal Medicine, Xinjiang Agricultural University, Urumqi 830052, China
2. State Key Laboratory for Animal Control and Prevention/Innovation Team of Animal Bacterial Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China
3. Ji'an Agricultural and Rural Industry Development Service Center, Ji'an 343000, China
4. Fengcheng Modern Agricultural Technology Service Center, Fengcheng 331100, China
5. College of Wildlife and Natural Reserves, Northeast Forestry University, Harbin 150040, China

Received:2024-07-29 Online:2025-08-23 Published:2025-08-28
Contact: XIA Lining, ZHANG Wanjiang E-mail:1757841410@qq.com;yaozhu922@163.com;xln@edu.xjau.cn;zhangwanjiang@caas.cn

摘要/Abstract

摘要：

旨在了解我国猪源多杀性巴氏杆菌(Pm)的流行特征和耐药情况。本研究对来自黑龙江、辽宁、吉林、内蒙古、陕西、宁夏、河南的病死猪脏器进行细菌分离培养，经16S rRNA测序和kmt基因鉴定，利用荚膜分型、脂多糖基因型分型、多位点序列分型(MLST)、脉冲场凝胶电泳(PFGE)等方法对Pm进行分型；通过微量肉汤稀释法分析Pm对8种抗生素的敏感性；通过Illumina PE 150对细菌全基因组测序，采用ResFinder数据库分析细菌的耐药基因，最后通过小鼠致病性试验确定分离菌株的毒力。结果显示，分离菌在血平板上形成表面光滑、湿润、中央微凸、半透明的菌落；革兰染色可见呈红色、球状或短杆状的细菌；共获得34株Pm，荚膜A型19株(占55.88%)，荚膜D型14株(占41.18%)和荚膜F型1株(2.94%)；脂多糖基因型L3有8株，脂多糖基因型L6有26株。MLST分析得到8个MLST型，ST11为优势基因型，占比为35.29%。可将Pm分为21个PFGE谱型，菌株间具有明显的多样性。耐药性结果显示，34株Pm对多黏菌素B、四环素、氟苯尼考、氯霉素、头孢噻呋、环丙沙星、卡那霉素的耐药率分别为38.23%、35.29%、23.53%、14.71%、11.76%、8.82%、2.94%，对庆大霉素不耐药；耐药基因检测结果显示，18株Pm携带9种耐药基因，分别为tet(B)、tet(C)、tet(L)、floR、aadA14、sul2、aph(6)-Id、aph(3″)-Ib和aph(3′)-Ia。Pm荚膜A型菌株毒力强于荚膜D型菌株，经腹腔注射31.5 CFU·mL^-1 PmA1菌株后的试验组小鼠死亡率为100%。综上表明，34株猪源Pm中流行血清型为荚膜A型和D型，MLST型为ST11，荚膜A型菌株毒力较荚膜D型菌株强，且PFGE分型表明菌株间存在多样性，本研究为多杀性巴氏杆菌病的疫苗研发及防治提供了理论依据。

关键词: 多杀性巴氏杆菌, 荚膜分型, MLST分型, PFGE, 耐药性

Abstract:

In order to understand the epidemiological characteristics and antibiotic resistance of Pasteurella multocida (Pm) from pigs in China, isolation and culture of bacteria recovered from diseased and dead porcine organs from Heilongjaing, Liaoning, Jilin, Inner Mongolia, Shaanxi, Ningxia and Henan provinces were carried out. The bacteria were identified by 16S rRNA sequencing and kmt gene, and typed using capsular typing, lipopolysaccharide genotype typing, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). The sensitivity of Pm to 8 antibiotics was analyzed by the broth microdilution method. The whole genome sequencing of the bacteria was performed using Illumina PE 150, and the antibiotic resistance genes were analyzed using the ResFinder database. Finally, the virulence of the isolates was determined through the pathogenicity experiment in mice. The results showed that the isolates formed smooth, moist colonies with a slightly convex and surface on blood agar plates, Gram staining revealed that it was red, coccus or short rod-shaped bacteria. A total of 34 isolates of Pm were obtained, including 19 isolates of capsule serotype A (55.88%), 14 isolates of capsule serotype D (41.18%), and 1 isolate of capsule serotype F. There were 8 isolates of LPS genotype L3 and 26 isolates of LPS genotype L6. MLST analysis identified 8 MLST types, with ST11 being the predominant genotype, accounting for 35.29%. Pm can be divided into 21 PFGE profiles, with obvious diversity among isolates. The antibiotic resistance results showed that the resistance rates of the 34 isolates of Pm to polymyxin B, tetracycline, florfenicol, chloramphenicol, ceftiofur, ciprofloxacin, and kanamycin were 38.23%, 35.29%, 23.53%, 14.71%, 11.76%, 8.82%, and 2.94%, respectively, and none was resistant to gentamicin. The antibiotic resistance genes detection showed that 18 isolates of Pm carried 9 types of antibiotic resistance genes, namely tet(B), tet(C), tet(L), floR, aadA14, sul2, aph(6)-Id, aph(3″)-Ib, and aph(3′)-Ia. The capsule serotype A isolates of Pm were more virulent than capsule serotype D isolates. The experimental group of mice exhibited an 100% mortality rate following intraperitoneal injection of the PmA1 strain at a concentration of 31.5 CFU·mL^-1.In conclusion, it was shown that the prevalent serotypes of the 34 porcine-origin Pm isolates were capsule serotype A and capsule serotype D, and the MLST type was ST11. The virulence of Pm capsular serotype A isolates was higher than that of capsular serotype D isolates. Moreover, PFGE revealed that there is diversity among the isolates. The study provided a theoretical basis for the development of vaccines and prevention strategies against Pm infections.

Key words: Pasteurella multocida, capsular serotypes, MLST types, PFGE, antimicrobial resistance

中图分类号:

S852.612

马彩萍, 祝瑶, 刘红道, 周光斌, 许秋, 林龙华, 柴霁芸, 侯杰, 孙宏飞, 杜素素, 范翠龙, 夏利宁, 张万江. 三十四株猪源多杀性巴氏杆菌的分型及耐药性分析[J]. 畜牧兽医学报, 2025, 56(8): 4074-4085.

MA Caiping, ZHU Yao, LIU Hongdao, ZHOU Guangbin, XU Qiu, LIN Longhua, CHAI Jiyun, HOU Jie, SUN Hongfei, DU Susu, FAN Cuilong, XIA Lining, ZHANG Wanjiang. Typing and Antimicrobial Resistance Analysis of Thirty-four Pasteurella multocida Strains from Pigs[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 4074-4085.

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引物用途 Primer usage	目的基因 Target genes	引物序列(5′→3′) Primer sequences (5′→3′)	产物大小/bp Size of products
鉴定Pm Identify Pm	kmt	ATCCGCTATTTACCCAGTGG GCTGTAAACGAACTCGCCAC	460
鉴定A型荚膜 Identify serotypes A	hyaD-hyaC	TGCCAAAATCGCAGTCAG TTGCCATCATTGTCAGTG	1 044
鉴定B型荚膜 Identify serotypes B	bcbD	CATTTATCCAAGCTCCACC GCCCGAGAGTTTCAATCC	760
鉴定D型荚膜 Identify serotypes D	dcbF	TTACAAAAGAAAGACTAGGAGCCC CATCTACCCACTCAACCATATCAG	657
鉴定E型荚膜 Identify serotypes E	ecbJ	TCCGCAGAAAATTATTGACTC GCTTGCTGCTTGATTTTGTC	511
鉴定F型荚膜 Identify serotypes F	fcbD	AATCGGAGAACGCAGAAATCAG TTCCGCCGTCAATTACTCTG	851
鉴定L1型脂多糖基因型 Identify L1 LPS genotypes	-	ACATTCCAGATAATACACCCG ATTGGAGCACCTAGTACCC	1 307
鉴定L2型脂多糖基因型 Identify L2 LPS genotypes	-	CTTAAAGTAACACTCGCTATTGC TTTGATTTCCCTTGGGATAGC	810
鉴定L3型脂多糖基因型 Identify L3 LPS genotypes	-	TGCAGGCGAGAGTTGATAAACCATC CAAAGATTGGTTCCAAATCTGAATGGA	4 747
鉴定L4型脂多糖基因型 Identify L4 LPS genotypes	-	TTTCCATAGATTAGCAATGCCG CTTTATTTGGTCTTTATATATACC	550
鉴定L5型脂多糖基因型 Identify L5 LPS genotypes	-	AGATTGCATGGCGAAATGGC CAATCCTCGTAAGACCCCC	1 175
鉴定L6型脂多糖基因型 Identify L6 LPS genotypes	-	TCTTTATAATTATACTCTCCCAAGG AATGAAGGTTTAAAAGAGATAGCTGGAG	668
鉴定L7型脂多糖基因型 Identify L7 LPS genotypes	-	CCTATATTTATATCTCCTCCCC CTAATATATAAACCATCCAACGC	931
鉴定L8型脂多糖基因型 Identify L8 LPS genotypes	-	GAGAGTTACAAAAATGATCGGC TCCTGGTTCATATATAGGTAGG	255
16S rRNA	-	AGAGTTTGATCCTGGCTCAG CTACGGCTACCTTGTTACG	1 500

耐药基因 Resistance genes	分类Class	数量 Numbers	菌株 Strains
tet(B)	四环素 Tetracycline	6	Pm50、Pm226、Pm241、Pm271、Pm277、PmD2
tet(C)	四环素 Tetracycline	3	Pm279、PmD1、Pm291
tet(L)	四环素 Tetracycline	1	Pm282
floR	酰胺醇 Amphenicol	7	Pm2、Pm-LN5、Pm267、PmD2、PmA3、PmA4、Pm-JL
sul2	磺胺类 Sulfonamides	2	Pm226、Pm245
aph(6)-Id	氨基糖苷类 Aminoglycoside	1	Pm226
aph(3′)-Ia	氨基糖苷类 Aminoglycoside	1	Pm226
aph(3″)-Ib	氨基糖苷类 Aminoglycoside	1	Pm226
aadA14	氨基糖苷类 Aminoglycoside	1	Pm254

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三十四株猪源多杀性巴氏杆菌的分型及耐药性分析

Typing and Antimicrobial Resistance Analysis of Thirty-four Pasteurella multocida Strains from Pigs

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