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

doi:10.3969/j.issn.1004-1524.2022.01.04

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (1): 24-32.DOI: 10.3969/j.issn.1004-1524.2022.01.04

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S945.1⁺2

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.

Figures/Tables 5

References 34

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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

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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