黑龙江省地区犊牛腹泻源致病性大肠杆菌流行情况调查、毒力基因及耐药性分析

doi:10.11843/j.issn.0366-6964.2025.07.031

摘要/Abstract

摘要：

大肠杆菌是引起新生犊牛腹泻和死亡的主要病原之一，本研究旨在探明黑龙江省犊牛腹泻源大肠杆菌致病性、携带毒力基因和耐药的流行情况。对2020年1月至2023年7月收集的黑龙江省8个城市35个牛群252份犊牛腹泻样品，进行细菌分离、革兰染色、鉴别培养基鉴定、生化试验、16S rRNA鉴定、小鼠致病性试验和药敏试验，通过PCR方法检测15种毒力基因，试验数据应用SPSS 22.0软件卡方检验方法对致病性大肠杆菌的地区流行情况、不同年限和不同季节因素进行分析。结果显示：共分离鉴定出153株致病性大肠杆菌，其中49.02%(75/153)携带毒力基因，其中Irp-2阳性率最高为37.25%(57/153)，FyuA、K99、F41、F17、STa、HlyA、Stx1、Stx2和Eaea阳性率依次为19.61%(30/153)、4.58%(7/153)、4.58%(7/153)、3.92%(6/153)、2.61%(4/153)、3.27%(5/153)、0.65%(1/153)、0.65%(1/153)和0.65%(1/153)。药敏试验结果显示153株分离菌对磷霉素敏感率最高56.21%(86/153)，而对磺胺异噁唑、阿莫西林等耐药严重，所有菌株都耐2重及以上药物，部分菌株最高达到11重耐药。通过统计分析发现2020年致病性大肠杆菌分离率77.59%(45/58)显著高于2021年与2022年(P < 0.001)。在一年四季中夏季致病性大肠杆菌分离率最低(P < 0.05)，各地区之间致病性大肠杆菌分离率有统计学差异，其中鸡西市最高，齐齐哈尔市最低(P < 0.001)。以上结果表明黑龙江省犊牛腹泻源牛致病性大肠杆菌携带毒力基因种类具有多样性，普遍多重耐药，致病性大肠杆菌的流行与地区、年限和季节均有一定的相关性。本研究为黑龙江地区牛大肠杆菌感染的防治提供了理论依据。

关键词: 犊牛腹泻, 大肠杆菌, 毒力基因, 耐药性, 流行病学

Abstract:

Escherichia coli is one of the main pathogens causing diarrhea and death in newborn calves. The pathogenicity, virulence gene carriage, and antibiotic resistance prevalence of Escherichia coli causing diarrhea in calves in Heilongjiang Province were investigated. Two hundred and fifty-two diarrhea samples of calf were collected from 35 herds in 8 cities from January 2020 to July 2023. Bacterial isolation, Gram staining, identification on differential media, biochemical tests, 16S rRNA identification, mouse pathogenicity tests, and drug sensitivity tests were conducted. The presence of 15 virulence genes was detected using PCR. Experimental data were analyzed using the chi-square test method of SPSS 22.0 software to assess the regional prevalence of pathogenic E. coli, as well as factors related to different years and seasons. Results showed that a total of 153 strains of pathogenic E. coli were isolated and identified, of which 49.02% (75/153) carried virulence genes. The highest positive rate was for Irp-2 at 37.25% (57/153), followed by FyuA, K99、F41, F17, STa, HlyA, Stx1, Stx2 and EaeA at 19.61% (30/153), 4.58% (7/153), 4.58% (7/153), 3.92% (6/153), 2.61% (4/153), 3.27% (5/153), 0.65% (1/153), 0.65% (1/153), and 0.65% (1/153), respectively. Drug sensitivity tests indicated that the highest sensitivity rate of the 153 isolated strains was to fosfomycin at 56.21% (86/153), while there was severe resistance to sulfisoxazole, amoxicillin, etc. All strains were resistant to two or more drugs, with some reaching resistance to as many as 11 drugs. Statistical analysis revealed that the isolation rate of pathogenic E. coli in 2020 was significantly higher at 77.59% (45/58) compared to 2021 and 2022 (P < 0.001). Among the four seasons, the summer had the lowest isolation rate of pathogenic E. coli (P < 0.05). And the rate of pathogenic E. coli was statistically different among regions, with the highest in Jixi and the lowest in Qiqihar (P < 0.001). These results indicate that the pathogenic E. coli strains causing calf diarrhea in the Heilongjiang region carry a diverse range of virulence genes and are generally resistant to multiple drugs. The prevalence of pathogenic E. coli is correlated with region, year, and season. This study provides a theoretical basis for the prevention and control of E. coli infections in cattle in the Heilongjiang region.

Key words: calf diarrhea, Escherichia coli, virulence genes, drug resistance, epidemiology

中图分类号:

S852.612

孔祥鹤, 苏欣雨, 钟一铭, 卢赵梓暄, 廖献茂, 张哲, 张旭, 高萌萌, 周玉龙, 樊祜卿. 黑龙江省地区犊牛腹泻源致病性大肠杆菌流行情况调查、毒力基因及耐药性分析[J]. 畜牧兽医学报, 2025, 56(7): 3390-3398.

KONG Xianghe, SU Xinyu, ZHONG Yiming, LUZHAO Zixuan, LIAO Xianmao, ZHANG Zhe, ZHANG Xu, GAO Mengmeng, ZHOU Yulong, FAN Huqing. To Investigate the Prevalence, Virulence Genes and Drug Resistance of Pathogenic Escherichia coli Isolated from Calves with Diarrhea in Heilongjiang Province[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3390-3398.

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参考文献 22

1	PAITAN Y. Current trends in antimicrobial resistance of escherichia coli[M]//FRANKE G, RON E. Escherichia coli, a versatile pathogen. Springer, Cham, 2018, 416: 181-211.
2	马忠. 河西走廊地区犊牛腹泻致病性大肠杆菌分离鉴定、毒力因子及耐药性检测[J]. 中国牛业科学, 2023, 49 (2): 29- 33. doi: 10.3969/j.issn.1001-9111.2023.02.006
	MA Z . Isolation, identification virulence factors and drug resistance test of pathogenic E. coli from calf diarrhea in Hexi Corridor Region[J]. China Cattle Science, 2023, 49 (2): 29- 33. doi: 10.3969/j.issn.1001-9111.2023.02.006
3	DA SILVA G J , MENDONÇA N . Association between antimicrobial resistance and virulence in escherichia coli[J]. Virulence, 2012, 3 (1): 18- 28.
4	VU KHAC H , HOLODA E , PILIPCINEC E , et al. Serotypes, virulence genes, and PFGE profiles of escherichia coli isolated from pigs with postweaning diarrhoea in Slovakia[J]. BMC Vet Res, 2006, 2, 10.
5	诸明欣, 张焕容, 汤承, 等. 四川阿坝州牦牛源溶血性大肠杆菌血清型鉴定及毒力相关基因检测[J]. 中国预防兽医学报, 2018, 40 (1): 9- 13.
	ZHU M X , ZHANG H R , TANG C , et al. Serotype identification and virulence-related genes detection of hemolytic E.coli isolated from yak in Sichuan Aba state[J]. Chinese Journal of Preventive Veterinary Medicine, 2018, 40 (1): 9- 13.
6	贾垌, 张迪, 张兆天, 等. 京津冀部分地区致犊牛腹泻大肠杆菌毒力基因、耐药基因检测及药物敏感性分析[J]. 中国畜牧兽医, 2022, 49 (12): 4820- 4831.
	JIA D , ZHANG D , ZHANG Z T , et al. Detection of virulence and resistance genes and drug sensitivity analysis of Escherichia coli derived from diarrheic calves in Beijing-Tianjin-Hebei Partial Regions[J]. China Animal Husbandry & Veterinary Medicine, 2022, 49 (12): 4820- 4831.
7	张培东. 大肠杆菌的分离及生化鉴定[J]. 中国畜牧兽医文摘, 2017, 33 (9): 68.
	ZHANG P D . Isolation and biochemical identification of Escherichia coli[J]. China Animal Husbandry and Veterinary Abstracts, 2017, 33 (9): 68.
8	孙月, 王琪, 毛伟, 等. 内蒙古地区犊牛腹泻大肠杆菌耐药性分析及毒力基因检测[J]. 中国畜牧兽医, 2023, 50 (9): 3811- 3822.
	SUN Y , WANG Q , MAO W , et al. Drug resistance analysis and virulence gene detection of Escherichia coli causing calf diarrhea in Inner Mongolia[J]. China Animal Husbandry & Veterinary Medicine, 2023, 50 (9): 3811- 3822.
9	PAKBIN B , BRVCK W M , ROSSEN J W . Virulence factors of enteric pathogenic escherichia coli: A review[J]. Int J Mol Sci, 2021, 22 (18): 9922.
10	王玮, 王景松, 郭亚男, 等. 宁夏部分地区犊牛腹泻样本大肠杆菌分离菌株的毒力因子检测与分析[J]. 畜牧兽医学报, 2024, 55 (7): 3261- 3266. doi: 10.11843/j.issn.0366-6964.2024.07.044
	WANG W , WANG J S , GUO Y N , et al. Toxicity factor detection and analysis of Escherichia coli isolated strains from calf diarrhea samples in some areas of Ningxia[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55 (7): 3261- 3266. doi: 10.11843/j.issn.0366-6964.2024.07.044
11	高海慧, 黎玉琼, 高小斐, 等. 犊牛腹泻源大肠杆菌耐药情况及HPI相关基因的检测[J]. 中国畜牧兽医, 2020, 47 (6): 1942- 1951.
	GAO H H , LI Y Q , GAO X F , et al. Drug resistance and detection of HPI related genes in E. coli from calves with diarrhea[J]. China Animal Husbandry & Veterinary Medicine, 2020, 47 (6): 1942- 1951.
12	张玥, 张学迪, 徐楷, 等. 河北省部分地区腹泻犊牛源大肠埃希氏菌毒力基因检测与耐药性分析[J]. 动物医学进展, 2024, 45 (10): 40- 47.
	ZHANG Y , ZHANG X D , XU K , et al. Detection of Escherichia coli virulence genes and analysis of drug resistance in diarrhoeal calf sources in some areas of Hebei Province[J]. Progress in Veterinary Medicine, 2024, 45 (10): 40- 47.
13	DUBREUIL J D , ISAACSON R E , SCHIFFERLI D M . Animal enterotoxigenic Escherichia coli[J]. EcoSal Plus, 2016, 7 (1)
14	关一凡, 李妍, 高亚桃, 等. 河北省部分地区犊牛腹泻大肠杆菌优势血清型筛选及兼性菌毛株的致病性[J]. 中国兽医学报, 2021, 41 (7): 1282- 1289.
	GUAN Y F , LI Y , GAO Y T , et al. Sereening of predominant serotypes of Escherichia coli from calf diarrhea and isolation and identification of facultative fimbriae strains in some areas of HebeiProvince[J]. Chin J Vet Sci Jul, 2021, 41 (7): 1282- 1289.
15	宋康, 王静梅, 蒋建军, 等. 新疆部分地区致犊牛腹泻大肠杆菌的分离鉴定及药物敏感性测定[J]. 畜牧与兽医, 2016, 48 (7): 89- 92.
	SONG K , WANG J M , JIANG J J , et al. Isolation, identification and drug sensitivity of Escherichia coli causing calf diarthea in parts of Xinjiang[J]. Animal Husbandry & Veterinary Medicine, 2016, 48 (7): 89- 92.
16	赵宇骐, 刘滢芳, 林一凡, 等. 《中华人民共和国生物安全法》实施背景下我国生物技术和人类遗传资源的伦理问题[J]. 中国公共卫生, 2024, 40 (3): 389- 392.
	ZHAO Y Q , LIU Y F , LIN Y F , et al. Ethical issues of biotechnology and human genetic resources in China under the background of the implementation of the Biosecurity Law of the People 's Republic of China[J]. Chinese Journal of Public Health, 2024, 40 (3): 389- 392.
17	RAMABHADRAN T V , JAGGER J . Mechanism of growth delay induced in Escherichia coli by near ultraviolet radiation[J]. Proc Natl Acad Sci U S A, 1976, 73 (1): 59- 63.
18	张玉杰, 王晓旭, 徐锋, 等. 牛源大肠杆菌耐药性研究进展[J]. 中国奶牛, 2023 (10): 29- 36.
	ZHANG Y J , WANG X X , XU F , et al. Progress on the antibiotics resistance of Escheichia coli from bovine[J]. China Dairy Cattle, 2023 (10): 29- 36.
19	左扬, 李田, 胡秀花, 等. 奶牛牧场养殖环境中产ESBL耐药菌的流行特征[J]. 畜牧兽医学报, 2022, 53 (11): 4027- 4034. doi: 10.11843/j.issn.0366-6964.2022.11.028
	ZUO Y , LI T , HU X H , et al. Epidemiological characteristics of ESBL-producing resistant bacteria in dairy farming environment[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53 (11): 4027- 4034. doi: 10.11843/j.issn.0366-6964.2022.11.028
20	MESELE F , LETA S , AMENU K , ABUNNA F . Occurrence of Escherichia coli O157:H7 in lactating cows and dairy farm environment and the antimicrobial susceptibility pattern at Adami Tulu Jido Kombolcha District, Ethiopia[J]. BMC Vet Res, 2023, 19 (1): 6.
21	刘佳惠, 吴开开, 王磊, 等. 黄芪多糖、皂苷及益生菌复合物对感染大肠杆菌肉鸡肠道的保护作用[J]. 畜牧兽医学报, 2024, 55 (5): 2241- 2252. doi: 10.11843/j.issn.0366-6964.2024.05.041
	LIU J H , WU K K , WANG L , et al. Protective effeets of Astragalus Polysaccharides, saponins and probiotie compounds on intestinal tract of broilers infected with E. coli[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55 (5): 2241- 2252. doi: 10.11843/j.issn.0366-6964.2024.05.041
22	林焱, 罗润波, 刘燕坤, 等. 1株感染多种致病性大肠杆菌噬菌体的生物学特性研究[J]. 畜牧兽医学报, 2022, 53 (1): 282- 289. doi: 10.11843/j.issn.0366-6964.2022.01.028
	LIN Y , LUO R B , LIU Y K , et al. Comparison of infection characteristies of an Escherichia coli phage C6 on different pathogenic Escherichia coli strains[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53 (1): 282- 289. doi: 10.11843/j.issn.0366-6964.2022.01.028

药物类别Drug category	抗菌药物Antibacterials	菌株数Number of strains			耐药情况Drug resistance situation
药物类别Drug category	抗菌药物Antibacterials	耐药Resistance	中介Intermediary	敏感Sensitivity	耐药率/%Rate of resistance	敏感率/%Rate of sensitivity
β-内酰胺类β-lactam class	头孢唑林Cefazolin	86	45	22	56.21	14.37
	阿莫西林Amoxicillin	103	16	34	67.32	22.22
	头孢他啶Ceftazidime	93	27	33	60.78	21.57
喹诺酮类Quinolones	左氧氟沙星Levofloxacin	44	43	66	28.75	43.13
	恩诺沙星Enrofloxacin	54	37	62	35.29	40.52
	环丙沙星Ciprofloxacin	72	33	48	47.06	31.35
氨基糖苷类Aminoglycoside	庆大霉素Gentamicin	66	48	39	43.13	25.49
	卡那霉素Kanamycin	71	43	39	46.41	25.49
	链霉素Streptomycin	69	67	17	45.10	11.11
酰胺醇类Amidochols	氯霉素Chloramphenicol	53	98	2	34.64	1.31
磺胺类Sulfanilamide	磺胺异噁唑Sulfisoxazole	115	37	1	75.16	0.65
四环素类Tetracyclines	四环素Tetracycline	107	40	6	69.93	3.92
大环内酯类Macrolides	阿奇霉素Azithromycin	41	41	71	26.80	46.41
多磷类Polyphosphates	磷霉素Fosfomycin	56	11	86	36.60	56.21

统计指标Statistical indicators	样本来源Sample source								X²	P
统计指标Statistical indicators	大庆市Daqing city	哈尔滨市Harbin city	黑河市Heihe city	齐齐哈尔市Qiqihar city	鸡西市Jixi city	绥化市Suihua city	牡丹江市Mudanjiang city	佳木斯市Jiamusi city	X²	P
阳性率(阳性数/总数)Positive rate(positive number/Total)	70.09(75/105)	35.29(12/34)	61.36(27/44)	16.60(2/12)	100.00(6/6)	66.67(4/6)	80.00(4/5)	36.80(7/19)	139.957	< 0.001
阴性率(阴性数/总数)Negative rate(negative number/Total)	29.91(30/105)	64.71(22/34)	38.64(17/44)	83.40(10/12)	0	33.34(2/6)	20.00(1/5)	63.20(12/19)

统计指标Statistical indicators	样本来源(年) Sample source(Year)			X²	P
统计指标Statistical indicators	2020	2021	2022	X²	P
阳性率(阳性数/总数)Positive rate(positive number/Total)	77.59(45/58)	59.46(44/74)	52.38(33/63)	15.530	< 0.001
阴性率(阴性数/总数)Negative rate(negative number/Total)	22.41(13/58)	40.54(30/74)	47.62(30/63)

统计指标Statistical indicators	样本来源Sample source				X²	P
统计指标Statistical indicators	春季Spring	夏季Summer	秋季Autumn	冬季Winter	X²	P
阳性率(阳性数/总数)Positive rate(positive number/Total)	67.5(54/80)	48.81(41/84)	66.00(33/50)	65.79(25/38)	9.756	0.021
阴性率(阴性数/总数)Negative rate(negative number/Total)	32.5(26/80)	51.19(43/84)	34.00(17/50)	34.21(13/38)

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