西藏地区藏猪源大肠杆菌耐药性及耐药基因检测与分析

doi:10.11843/j.issn.0366-6964.2025.10.040

摘要/Abstract

摘要：

旨在探究西藏地区藏猪源大肠杆菌的耐药情况。本研究采集西藏山南、昌都和林芝地区共180份藏猪粪便样本，通过细菌分离培养、革兰染色和16S rRNA基因扩增鉴定菌株，采用K-B纸片扩散法检测耐药性，并通过PCR扩增检测毒力基因及耐药基因携带情况。结果表明：从180份藏猪粪便中分得42株致泻大肠杆菌，其中肠聚集性大肠杆菌(Enteroaggregative Escherichia coli，EAEC)和非典型肠道致病性大肠杆菌(Enteropathogenic Escherichia coli，EPEC)的总分离率分别为25.36%和5.07%。耐药性分析显示，EAEC和非典型EPEC对亚胺培南的耐药率分别为97.14%和100%，且最常见的耐药表型分别为TE+DX+MI+AM+IPM+SIZ和TE+DX+AM+IPM+SIZ。所有菌株中，β-内酰胺类bla_TEM基因检出率均为100.00%。综上，西藏地区藏猪源致泻大肠杆菌具有较高的流行率和多重耐药性，且耐药谱型多样，耐药基因均有不同程度检出，EAEC为主要流行菌株。本研究为西藏地区藏猪源致泻大肠杆菌的防控提供数据参考，有助于提升公共卫生水平和优化临床实践。

关键词: 藏猪, 致泻大肠杆菌, 耐药基因

Abstract:

The aim of this study was to investigate the antibiotic resistance of Escherichia coli isolated from Tibetan pigs in Xizang. A total of 180 fecal samples were collected from Tibetan pigs in Shannan, Changdu, and Linzhi in Xizang. Bacterial strains were isolated and identified using bacterial culture, Gram staining, and 16S rRNA gene amplification. Antibiotic resistance was assessed using the K-B disk diffusion method, while virulence genes and resistance genes carried by the strain were detected through PCR amplification. The results showed that, a total of 42 diarrheagenic E. coli strains were isolated from 180 fecal samples, with the overall isolation rates of Enteroaggregative Escherichia coli (EAEC) and atypical Enteropathogenic Escherichia coli (EPEC) being 25.36% and 5.07%, respectively. Antibiotic resistance analysis showed that EAEC and atypical EPEC exhibited imipenem resistance rates of 97.14% and 100%, respectively. The most common resistance phenotypes for EAEC and atypical EPEC were TE+DX+MI+AM+IPM+SIZ and TE+DX+AM+IPM+SIZ, respectively. All isolates carried the β-lactamase bla_TEM gene, with a detection rate of 100.00%. In summary, diarrheagenic E. coli isolated from Tibetan pigs in Xizang exhibited high prevalence and multidrug resistance, with diverse resistance phenotypes and varying levels of resistance gene detection. EAEC was the predominant strain. This study provides valuable data for the prevention and control of diarrheagenic E. coli in Tibetan pigs in Xizang, contributing to the improvement of public health and the optimization of clinical practice.

Key words: Tibetan pigs, diarrheagenic Escherichia coli, resistance genes

中图分类号:

S852.61

吴茜, 张翔峰, 李海洋, 罗侣, 韦明邦, 张辉, 叶幼荣, 商鹏. 西藏地区藏猪源大肠杆菌耐药性及耐药基因检测与分析[J]. 畜牧兽医学报, 2025, 56(10): 5212-5221.

WU Xi, ZHANG Xiangfeng, LI Haiyang, LUO Lü, WEI Mingbang, ZHANG Hui, YE Yourong, SHANG Peng. Detection and Analysis of Antibiotic Resistance and Resistance Genes in Escherichia coli from Tibetan Pigs in Xizang[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 5212-5221.

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参考文献 22

1	商鹏, 强巴央宗, 张博, 等. 藏猪选育群屠宰性能和肉质测定分析[J]. 黑龙江畜牧兽医, 2015 (2): 30- 32.
	SHANG P , CHAMBA Y Z , ZHANG B , et al. Analysis of the slaughter performance and determination of meat quality in the selective breeding population of Tibetan pigs[J]. Heilongjiang Animal Science and Veterinary Medicine, 2015 (2): 30- 32.
2	周琳, 王蜀金. 藏猪生理学研究进展[J]. 家畜生态学报, 2014, 35 (7): 7- 12.
	ZHOU L , WANG S J . Research progress on Tibetan swine physiology[J]. Acta Ecologiae Animalis Domastici, 2014, 35 (7): 7- 12.
3	王静, 张琳. 我区藏猪产业发展驶入快车道[N]. 西藏日报(汉), 2024-05-23.
	WANG J, ZHANG L. The Tibetan Pig industry in our region enters the fast lane of development[N]. Xizang Daily (Chinese Edition), 2024-05-23. (in Chinese)
4	谢筱纯, 王珊. 林芝市工布江达县: "中国藏猪之乡"的致富密码[N]. 西藏日报(汉), 2023-06-14.
	XIE X C, WANG S. Gongbo'gyamda County, Linzhi City: The Secret to Prosperity in the "Hometown of Tibetan Pigs"[N]. Xizang Daily (Chinese Edition), 2023-06-14. (in Chinese)
5	CAO Z , QI M , SHANG P , et al. Characterization, estimation of virulence and drug resistance of diarrheagenic Escherichia coli(DEC) isolated from Tibetan pigs[J]. Microb Pathog, 2023, 177, 106046. doi: 10.1016/j.micpath.2023.106046
6	ESCOBAR-PÁRAMO P , GIUDICELLI C , PARSOT C , et al. The evolutionary history of Shigella and Enteroinvasive Escherichia colirevised[J]. J Mol Evol, 2003, 57 (2): 140- 148. doi: 10.1007/s00239-003-2460-3
7	SUNTHADVANICH R , CHIEWSILP D , SERIWATANA J , et al. Nationwide surveillance program to identify diarrhea-causing Escherichia coliin children in Thailand[J]. J Clin Microbiol, 1990, 28 (3): 469- 472. doi: 10.1128/jcm.28.3.469-472.1990
8	KNUTTON S , SHAW R , PHILLIPS A D , et al. Phenotypic and genetic analysis of diarrhea-associated Escherichia coli isolated from children in the United Kingdom[J]. J Pediatr Gastroenterol Nutr, 2001, 33 (1): 32- 40.
9	朱飞舟, 陈利玉, 陈汉春. 16S rRNA基因序列分析法鉴定病原细菌[J]. 中南大学学报(医学版), 2013, 38 (10): 1035- 1041.
	ZHU F Z , CHEN L Y , CHEN H C . Identification of pathogenic microorganism by sequencing 16S rRNA gene[J]. Journal of Central South University(Medical Sciences), 2013, 38 (10): 1035- 1041.
10	刘变芳, 王涛, 王蕊, 等. 生鲜肉中产毒素大肠杆菌多重PCR检测方法构建[J]. 中国食品学报, 2018, 18 (12): 225- 231.
	LIU B F , WANG T , WANG R , et al. Construction of a multiplex pcr method for detection Enterotoxigenic Escherichia coli in fresh meat[J]. Journal of Chinese Institute of Food Science and Technology, 2018, 18 (12): 225- 231.
11	食品安全国家标准食品微生物学检验致泻大肠埃希氏菌检验[Z]. 中华人民共和国国家卫生和计划生育委员会; 国家食品药品监督管理总局. 2016: 24.
	National Food Safety Standard Microbiological Examination of Food Examination of Diarrheagenic Escherichia coli[Z]. National Health and Family Planning Commission of the People's Republic of China; China Food and Drug Administration, 2016: 24. (in Chinese)
12	任子宁, 袁孝武, 张烨淳, 等. 猪源大肠杆菌的耐药性与锌抗性相关性的研究[J]. 中国预防兽医学报, 2023, 45 (12): 1223- 1231.
	REN Z N , YUAN X W , ZHANG Y C , et al. Study on correlation between antimicrobial resistance and zinc resistance of porcine Escherichia coli[J]. Chinese Journal of Preventive Veterinary Medicine, 2023, 45 (12): 1223- 1231.
13	薛舒文. 广西某猪场猪大肠杆菌分离鉴定、耐药性分析及联合用药方案筛选[D]. 武汉: 华中农业大学, 2023.
	XUE S W. Isolation, identification, drug resistance analysis and selection of drug combination of swine Escherichia colifrom a pig farm in Guangxi[D]. Wuhan: Huazhong Agricultural University, 2023. (in Chinese)
14	车勇良, 陈秋勇, 陈如敬, 等. 福建省猪源多黏菌素耐药基因mcr-1阳性细菌的耐药特性及分布特征[J]. 西北农林科技大学学报(自然科学版), 2022, 50 (12): 1-8, 17.
	CHE Y L , CHEN Q Y , CHEN R J , et al. Resistance characteristic and distribution of polymyxin-resistant mcr-1 gene positive bacteria fron swine in Fujian[J]. Journal of Northwest A & F University(Natural Science Edition), 2022, 50 (12): 1-8, 17.
15	KAGAMBÈGA A , MARTIKAINEN O , SIITONEN A , et al. Prevalence of diarrheagenic Escherichia coli virulence genes in the feces of slaughtered cattle, chickens, and pigs in Burkina Faso[J]. Microbiologyopen, 2012, 1 (3): 276- 284. doi: 10.1002/mbo3.30
16	佟盼盼, 黄顺敏, 王芋丹, 等. 新疆地区腹泻仔猪源大肠杆菌的分群、血清型鉴定及耐药性分析[J]. 畜牧兽医学报, 2023, 54 (1): 414- 420. doi: 10.11843/j.issn.0366-6964.2023.01.038
	TONG P P , HUANG S M , WANG Y D , et al. Phylogenetic clustering, serotype and drug resistance analysis of Escherichia coli from diarrhea with piglets in Xinjiang[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54 (1): 414- 420. doi: 10.11843/j.issn.0366-6964.2023.01.038
17	BERTELLONI F , CILIA G , BOGI S , et al. Pathotypes and antimicrobial susceptibility of Escherichia coli isolated from wild boar (Sus scrofa) in Tuscany[J]. Animals (Basel), 2020, 10 (4)
18	ALONSO C A , MORA A , DÍAZ D , et al. Occurrence and characterization of stx and/or eae-positive Escherichia coli isolated from wildlife, including a typical EPEC strain from a wild boar[J]. Vet Microbiol, 2017, 207, 69- 73. doi: 10.1016/j.vetmic.2017.05.028
19	KYLLA H , DUTTA T K , ROYCHOUDHURY P , et al. Characterisation of porcine Enteropathogenic Escherichia coli isolated in Northeastern India[J]. J Vet Res, 2020, 64 (3): 391- 397. doi: 10.2478/jvetres-2020-0046
20	杨守深, 庄艺超, 华雪妃, 等. 闽西地区猪源大肠杆菌耐药基因bla_CTX-M-9G的流行性分析[J]. 中国兽医科学, 2018, 48 (5): 658- 663.
	YANG S S , ZHUANG Y C , HUA X F , et al. Investigation of bla_CTX-M-9 Gresistance gene in Escherichia coli isolated from pig origin in the west of Fujian Province[J]. Veterinary Science in China, 2018, 48 (5): 658- 663.
21	李金朋, 温文彦, 靳曼玉, 等. 河南地区猪源大肠杆菌分子分群、耐药性及生物被膜表型分析[J]. 中国预防兽医学报, 2018, 40 (03): 190- 194.
	LI J P , WEN W Y , JIN M Y , et al. Phylogenetic group, drug resistance and biofilm formation of Escherichia coli isolated from pigs in Henan regions[J]. Chinese Journal of Preventive Veterinary Medicine, 2018, 40 (3): 190- 194.
22	陈朝喜, 李宇涵, 谭敏, 等. 川西北高原牦牛和藏猪源大肠杆菌生物被膜表型、耐药基因、整合酶基因和毒力基因检测[J]. 中国农业科学, 2021, 54 (23): 5144- 5162.
	CHEN C X , LI Y H , TAN M , et al. Biofilm-forming phenotype, antibacterial resistance genes, integrase genes and virulence genes detection of Escherichia coli isolated from yaks and Tibetan pigs in Northwest Sichuan plateau[J]. Scientia Agricultura Sinica, 2021, 54 (23): 5144- 5162.

引物名称Primer name	引物序列(5′→3′) Primer sequence	退火温度/℃ Annealing temperature	产物大小/bp Product size
16S rRNA	27-F: AGAGTTTGATCMTGGCTCAG	55	1 500
	1492-R: GGTTACCTTGTTACGACTT
uidA	F: TAATGTTCTGCGACGCTCAC		321
	R: CGGCGAAATTCCATACCT

目的基因Target gene	引物序列(5′→3′) Primer sequence(5′→3′)	退火温度/℃ Annealing temperature	产物大小/bp Product size
bfpB	F: GACACCTCATTGCTGAAGTCG	56	910
	R: CCAGAACACCTCCGTTATGC
escV	F: ATTCTGGCTCTCTTCTTCTTTATGGCTG	58	544
	R: CGTCCCCTTTTACAAACTTCATCGC
stx1	F: CGATGTTACGGTTTGTTACTGTGACAGC	59	244
	R: AATGCCACGCTTCCCAGAATTG
stx2	F: GTTTTGACCATCTTCGTCTGATTATTGAG	58	324
	R: AGCGTAAGGCTTCTGCTGTGAC
iPaH	F: TTGACCGCCTTTCCGATACC	59	647
	R: ATCCGCATCACCGCTCAGAC
lt	F: GAACAGGAGGTTTCTGCGTTAGGTG	58	655
	R: CTTTCAATGGCTTTTTTTTGGGAGTC
sth	F: TGTCTTTTTCACCTTTCGCTC	54	171
	R: CGGTACAAGCAGGATTACAACAC
astA	F: TGCCATCAACACAGTATATCCG	55	102
	R: ACGGCTTTGTAGTCCTTCCAT
aggR	F: ACGCAGAGTTGCCTGATAAAG	55	400
	R: AATACAGAATCGTCAGCATCAGC
pic	F: AGCCGTTTCCGCAGAAGCC	60	1 111
	R: AAATGTCAGTGAACCGACGATTGG

类别Category	目的基因Target gene	引物序列(5′→3′) Primer sequence(5′→3′)	退火温度/℃ Annealing temperature	产物大小/bp Product size
四环素类	tetA	F: GTAATTCTGAGCACTGTCGC	54	957
Tetracyclines		R: CTGCCTGGACAACATTGCTT
	tetB	F: TTGGTTAGGGGCAAGTTTTG	54	659
		R: GTAATGGGCCAATAACACCG
β-内酰胺类	ampC	F: TTCTATCAAMACTGGCARCC	55	530
β-lactams		R: CCYTTTTATGTACCCAYGA
	bla_TEM	F: ATGAGTATTCAACATTTCCG	57	859
		R: CAATGCTTAATCAGTGAGG
	bla_NDM	F: TGACGCGGCGTAGTGCTCAGTGT	60	297
		R: GCGGCGGGGATTGCGACTTAT
磺胺类	sul1	F: GGTTCTGAAATCCATCCCTG	56	251
Sulfonamides		R: CTCTCATCGAAGAAGGAGTC
	sul2	F: CGGCATCGTCAACATAACC	57	368
		R: CCGAATGCATAACGACGAG
	sul3	F: AAACGAATCCGGAAGAGGT	57	279
		R: CAAGAGTTGGTGCTAAACGA
喹诺酮类	acc(6′)-Ib-cr	F: TTGCGATGCTCTATGAGTGGCTA	52	482
Quinolones		R: CTCGAATGCCTGGCGTGTTT
	qnrA	F: TCAGCAAGAGGATTTCTCA	55	627
		R: GGCAGCACTATTACTCCCA
	qnrB	F: GGMATHGAAATTCGCCACTG	55	264
		R: TTTGCYGYYCGCCAGTCGAA
	qnrS	F: ATGGAAACCTACAATCATAC	50	467
		R: AAAAACACCTCGACTTAAGT
氨基糖苷类	ant(3′)-Ⅰa	F: ATCTGGCTATCTTGCTGACA	49	284
Aminoglycosides		R: TATGACGGGCTGATACTGG
	aph(3′)-Ⅱa	F: TGACTGGGCACAACAGACT	50	717
		R: TCAAGAAGGCGATAGAAGGC
	aac(3′)-Ⅱ	F: TGAAACGCTGACGGAGCCTC	54	370
		R: GTCGAACAGGTAGCACTGAG
	rmtB	F: ATGAACATCAACGATGCCCTC	49	756
		R: TTATCCATTCTTTTTTATCAAGTATAT

类别Category	抗菌药物Antimicrobial agent	耐药菌株数Number of drug-resistant strains		耐药率/% Resistance rate
类别Category	抗菌药物Antimicrobial agent	EAEC	非典型EPEC Atypical EPEC	EAEC	非典型EPEC Atypical EPEC
四环素类Tetracyclines	四环素Tetracycline	24	4	68.57	57.14
	多西环素Doxycycline	14	3	40.00	42.86
	米诺环素Minocycline	9	0	25.71	0.00
β-内酰胺类β-lactams	氨苄西林Ampicillin	26	4	74.29	42.86
	头孢曲松Ceftriaxone	2	0	5.71	0.00
	头孢他啶Ceftazidime	2	0	5.71	0.00
	美罗培南Meropenem	0	0	0.00	0.00
	亚胺培南Imipenem	34	7	97.14	100.00
磺胺类Sulfonamides	磺胺异恶唑Sulfafurazole	22	4	62.86	42.86
喹诺酮类Quinolones	环丙沙星Ciprofloxacin	1	0	2.86	0.00
	氧氟沙星Ofloxacin	0	0	0.00	0.00
	诺氟沙星Norfloxacin	0	0	0.00	0.00
氨基糖苷类Aminoglycosides	庆大霉素Gentamicin	0	0	0.00	0.00
	卡那霉素Kanamycin	0	0	0.00	0.00
	阿米卡星Amikacin	2	0	5.71	0.00

耐药谱Resistance spectrum	耐药菌株数Number of drug-resistant strains		百分比/% Percentage
耐药谱Resistance spectrum	EAEC	非典型EPEC Atypical EPEC	EAEC	非典型EPEC Atypical EPEC
IPM	6	2	17.14	28.57
CIP	1	0	2.86	0.00
AM+IPM	3	0	8.57	0.00
TE+IPM	1	1	2.86	14.29
TE+AM+IPM	2	0	5.71	0.00
AM+IPM+SIZ	1	1	2.86	14.29
TE+IPM+SIZ	1	0	2.86	0.00
TE+AM+IPM+SIZ	5	0	14.29	0.00
TE+AM+IPM+SIZ+AK	1	0	2.86	0.00
TE+DX+AM+IPM+SIZ	4	3	11.43	42.86
TE+DX+MI+AM+IPM+SIZ	7	0	20.00	0.00
TE+DX+MI+AM+CTR+IPM+SIZ	1	0	2.86	0.00
TE+DX+AM+CAZ+IPM+SIZ+AK	1	0	2.86	0.00
TE+DX+MI+AM+CTR+CAZ+IPM+SIZ	1	0	2.86	0.00