一株克氏原螯虾雷氏普罗威登斯菌的分离鉴定与联合药敏试验

doi:10.3969/j.issn.1004-1524.2022.01.04

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (1): 24-32.DOI: 10.3969/j.issn.1004-1524.2022.01.04

一株克氏原螯虾雷氏普罗威登斯菌的分离鉴定与联合药敏试验

董靖¹(), 张露珊¹, 张国栋¹, 刘永涛¹, 刘绍春², 杨移斌¹, 艾晓辉¹^,^*()

1.中国水产科学研究院长江水产研究所,湖北省水产品质量安全工程技术研究中心,湖北武汉 430223
2.岳阳渔美康生物科技有限公司,湖南岳阳 414100

收稿日期:2020-09-16 出版日期:2022-01-25 发布日期:2022-02-05
通讯作者: 艾晓辉
作者简介:* 艾晓辉,E-mail: aixh@yfi.ac.cn
董靖(1985—),男,山东烟台人,博士,助理研究员,研究方向为水产药理学。 E-mail: dongjing@yfi.ac.cn
基金资助:
湖北省技术创新专项重大项目(2019ABA077);国家自然科学基金(31702368)

Identification and synergistic susceptibility of pathogenic Providencia rettgeri isolated from diseased Procambarus clarkii

DONG Jing¹(), ZHANG Lushan¹, ZAHNG Guodong¹, LIU Yongtao¹, LIU Shaochun², YANG Yibin¹, AI Xiaohui¹^,^*()

1. Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Hubei Province Engineering and Technology Research Center of Aquatic Product Quality and Safety, Wuhan 430223, China
2. Yueyang Yumeikang Biological Technology Co., Ltd., Yueyang 414100, Hunan, China

Received:2020-09-16 Online:2022-01-25 Published:2022-02-05
Contact: AI Xiaohui

摘要/Abstract

摘要：

为了探明克氏原螯虾(Procambarus clarkii)5—6月死亡的病因并制定相应的治疗方案,本试验从患病克氏原螯虾肝胰腺中分离到1株病原菌(L20190509),通过生理生化特征、16S rRNA序列分析和进化树进行鉴定,通过琼脂扩散法测定了该菌株的药物敏感性,进一步研究了抗菌药物与天然化合物的联合抑菌作用。结果发现,该菌株对克氏原螯虾的半数致死浓度为9.27×10⁴ CFU·mL^-1,其生理生化特征与雷氏普罗威登斯菌(Providencia rettgeri)一致,16S rRNA测序和进化树分析发现,分离株与雷氏普罗威登斯菌的亲缘关系最近,综合以上结果将该菌株鉴定为雷氏普罗威登斯菌。进一步通过药敏试验发现该菌株对20种受试药物中的亚胺培南、头孢噻肟、恩诺沙星3种药物敏感,对氟苯尼考和多西环素2种药物中度敏感,对其他受试药物均不敏感。此外,联合抑菌试验发现氟苯尼考与厚朴酚联用具有协同抑菌作用。以上研究发现,分离株L20190509是一株能导致克氏原螯虾发病的病原菌,具有多重耐药性,研究结果有助于进一步了解克氏原螯虾“五月瘟”的病因,并制定科学有效的治疗方案。

关键词: 克氏原螯虾, 雷氏普罗威登斯菌, 耐药性, 厚朴酚

Abstract:

To clarify the cause of death of Procambarus clarkii (P. clarkii) during May to June and bring out treatment protocols, a bacterial pathogen (L20190509) was isolated from P. clarkia with typical symptom. The isolated pathogen was characterized by physiological and biochemical assay, 16S rRNA sequencing and phylogenetic analysis. Then the sensitivity of the isolated pathogen was tested by agar diffusion method, the synergistic effect of antibiotics and natural compounds were determined by checkerboard assays. The results showed that the 50% lethal concentration of the isolate to healthy P. clarkii was 9.27×10⁴ CFU·mL^-1, which exhibited similar physiological and biochemical characteristics to Providencia rettgeri (P. rettgeri) and shared over 99% homologies with P. rettgeri. Thus, the isolate was determined to be a P. rettgeri strain. The sensitivity study showed that the isolate was susceptible to imipenem, cefotaxime and enrofloxacin; intermediate to florfenicol and doxycycline; resistant to other tested antibiotics. Moreover, florfenicol combined with magnolol had synergistic effect against L20190509. Taken together, L20190509 is a bacterial pathogen of farmed P. clarkii which exhibited multi-resistance to antibiotics, these findings help to further acknowledge the pathogens of P. clarkii in May and formulate scientific and effective treatments.

Key words: Procambarus clarkii, Providencia rettgeri, resistance, magnolol

中图分类号:

S945.1⁺2

董靖, 张露珊, 张国栋, 刘永涛, 刘绍春, 杨移斌, 艾晓辉. 一株克氏原螯虾雷氏普罗威登斯菌的分离鉴定与联合药敏试验[J]. 浙江农业学报, 2022, 34(1): 24-32.

DONG Jing, ZHANG Lushan, ZAHNG Guodong, LIU Yongtao, LIU Shaochun, YANG Yibin, AI Xiaohui. Identification and synergistic susceptibility of pathogenic Providencia rettgeri isolated from diseased Procambarus clarkii[J]. Acta Agriculturae Zhejiangensis, 2022, 34(1): 24-32.

图/表 5

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组别 Group	感染浓度 Concentration/ (CFU·mL^-1)	感染后不同时间(d)死亡数量 Deaths after infection								死亡率 Mortality/%
组别 Group	感染浓度 Concentration/ (CFU·mL^-1)	1	2	3	4	5	6	7	8	死亡率 Mortality/%
阴性对照	0	0	0	0	0	0	0	0	0	0
Negative control
1	1.5×10⁸	9	13	8	0	0	0	0	0	100
2	1.5×10⁷	10	12	8	0	0	0	0	0	100
3	1.5×10⁶	5	6	2	3	2	1	2	0	70
4	1.5×10⁵	6	3	2	2	1	1	0	0	50
5	1.5×10⁴	4	2	2	1	1	1	0	0	36.67

组别 Group	感染浓度 Concentration/ (CFU·mL^-1)	感染后不同时间(d)死亡数量 Deaths after infection								死亡率 Mortality/%
组别 Group	感染浓度 Concentration/ (CFU·mL^-1)	1	2	3	4	5	6	7	8	死亡率 Mortality/%
阴性对照	0	0	0	0	0	0	0	0	0	0
Negative control
1	1.5×10⁸	9	13	8	0	0	0	0	0	100
2	1.5×10⁷	10	12	8	0	0	0	0	0	100
3	1.5×10⁶	5	6	2	3	2	1	2	0	70
4	1.5×10⁵	6	3	2	2	1	1	0	0	50
5	1.5×10⁴	4	2	2	1	1	1	0	0	36.67

测定项目Tested parameters	反应结果Reactions	测定项目Tested parameters	反应结果Reactions
β-半乳糖苷 β-galactosidase	-	肌醇 Inositol	+
精氨酸双水解酶 Arginine dihydrolase	-	山梨醇 Sorbierite	-
赖氨酸脱羧酶 Lysine decarboxylase	-	鼠李糖 Rhamnose	+
鸟氨酸脱羧酶 Ornithine decarboxylase	-	蔗糖 Sucrose	-
柠檬酸利用 Citrate	-	蜜二糖 Meilibiose	-
硫化氢产生 H₂S	-	苦杏仁苷 Amygdalin	-
尿素酶 Urease	+	阿拉伯糖 Arabinose	-
色氨酸脱氨酶 Tryptophan deaminase	+	氧化酶 Oxidase	-
吲哚产生 Indole	+	运动性 Mobility	+
丙酮酸盐 V-P	-	硝酸盐还原 Nitrate	+
明胶酶 Gelatinase	-	麦康凯平板 McC growth	+
葡萄糖 Glucose	+	氧化型 Oxidation	+
甘露醇 Mannitol	-	发酵型 Fermentation	+

测定项目Tested parameters	反应结果Reactions	测定项目Tested parameters	反应结果Reactions
β-半乳糖苷 β-galactosidase	-	肌醇 Inositol	+
精氨酸双水解酶 Arginine dihydrolase	-	山梨醇 Sorbierite	-
赖氨酸脱羧酶 Lysine decarboxylase	-	鼠李糖 Rhamnose	+
鸟氨酸脱羧酶 Ornithine decarboxylase	-	蔗糖 Sucrose	-
柠檬酸利用 Citrate	-	蜜二糖 Meilibiose	-
硫化氢产生 H₂S	-	苦杏仁苷 Amygdalin	-
尿素酶 Urease	+	阿拉伯糖 Arabinose	-
色氨酸脱氨酶 Tryptophan deaminase	+	氧化酶 Oxidase	-
吲哚产生 Indole	+	运动性 Mobility	+
丙酮酸盐 V-P	-	硝酸盐还原 Nitrate	+
明胶酶 Gelatinase	-	麦康凯平板 McC growth	+
葡萄糖 Glucose	+	氧化型 Oxidation	+
甘露醇 Mannitol	-	发酵型 Fermentation	+

药物名称 Antibiotics	含量 Content/ (μg·disc^-1)	抑菌圈直径 Inhibition zone/mm	敏感度 Sensitivity
庆大霉素Gentamicin	10	5.22±0.93	R
妥布霉素Tobramycin	10	8.39±1.26	R
新霉素Neomycin	30	8.58±2.19	R
链霉素Streptomycin	10	0	R
卡那霉素Kanamycin	30	0	R
亚胺培南Imipenem	10	19.25±3.17	S
头孢唑啉Cefazolin	30	0	R
头孢噻肟Cefotaxime	30	25.61±2.84	S
阿莫西林Amoxicillin	20	0	R
氨苄西林Ampicillin	10	0	R
苯唑西林Oxacillin	1	0	R
氟苯尼考Florfenicol	30	15.23±2.59	I
多黏菌素B	300	9.17±3.11	R
Polymyxin B
红霉素Erythromycin	15	0	R
阿奇霉素Azithromycin	15	8.25±0.84	R
恩诺沙星Enrofloxacin	10	19.36±2.32	S
多西环素Doxycycline	30	12.58±1.84	I
四环素Tetracycline	30	12.03±1.95	R
阿米卡星Amikacin	30	11.24±1.56	R
头孢拉定Cefradine	30	0	R

一株克氏原螯虾雷氏普罗威登斯菌的分离鉴定与联合药敏试验

Identification and synergistic susceptibility of pathogenic Providencia rettgeri isolated from diseased Procambarus clarkii

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天然化合物 Natural products	单独使用 MIC MIC of agents alone/(μg·mL^-1)		联合使用 MIC MIC in combination/(μg·mL^-1)		结果 Results
天然化合物 Natural products	天然化合物 Natural products	氟苯尼考 Florfenicol	天然化合物 Natural products	氟苯尼考 Florfenicol	联合抑菌指数 FICI	作用效应 Interpretation
血根碱 Sanguinarine	32		16	16	1.5	IND
漆黄素 Fisetin	512		256	4	0.75	IND
厚朴酚 Magnolol	64		16	2	0.375	SYN
麝香草酚 Thymol	128	16	64	16	1.5	IND
槲皮苷 Quercetin	256		256	8	1.5	IND
甘草素 Liquiritigenin	128		32	16	1.25	IND
山奈酚 Kaempferol	512		256	4	0.75	IND
桑色素 Morin	256		64	8	0.75	IND
木犀草素 Luteolin	128		128	16	2	IND
没食子酸 Gallic acid	256		64	16	1.25	IND

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