患白内障病黑斑蛙米尔伊丽莎白菌的分离鉴定与PNGase基因克隆

doi:10.3969/j.issn.1004-1524.2023.06.07

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (6): 1297-1306.DOI: 10.3969/j.issn.1004-1524.2023.06.07

患白内障病黑斑蛙米尔伊丽莎白菌的分离鉴定与PNGase基因克隆

龚保荣(), 吴红军, 李本镇, 徐大洋, 邹文腾, 曲君艺, 鲍传和, 朱若林^*()

安徽农业大学动物科技学院,安徽合肥 230036

收稿日期:2022-04-28 出版日期:2023-06-25 发布日期:2023-07-04
通讯作者: *朱若林,E-mail: jollinz@163.com
作者简介:龚保荣(1995—),男,安徽六安人,硕士研究生,主要从事水产动物疾病与免疫研究。E-mail: 768615015@qq.com
基金资助:
安徽省高校自然科学基金(KJ2019A0164);安徽省大学生创新创业训练计划项目(201910364056)

Isolation, identification of Elizabethkingia miricola of Pelophylax nigromaculatus with cataract and cloning of PNGase gene

GONG Baorong(), WU Hongjun, LI Benzhen, XU Dayang, ZOU Wenteng, QU Junyi, BAO Chuanhe, ZHU Ruolin^*()

College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China

Received:2022-04-28 Online:2023-06-25 Published:2023-07-04

摘要/Abstract

摘要：

为给安徽舒城地区患白内障病黑斑蛙(Pelophylax nigromaculatus)提供治疗方案,对病蛙进行了细菌分离、鉴定和药敏检测。从典型患病黑斑蛙的肝脏、眼睛、脑和脊柱中分离出4株优势菌株,经16S rRNA和gyrB基因序列分析,结合生理生化鉴定,结果表明,4株菌株均为米尔伊丽莎白菌(Elizabethkingia miricola)。对分离菌的N-糖苷酶(PNGase)基因进行了克隆和分析,结果显示,其包含PNGase F和PNGase F-Ⅱ两种类型,进化分析结果同样显示,其与米尔伊丽莎白菌聚为一支,表明PNGase基因序列可作为伊丽莎白菌属内部菌分类的参考依据。药敏试验结果显示,4株分离株仅对酰胺醇类中的氟苯尼考敏感,对香豆素类中的新生霉素、大环内酯类中的红霉素和利福霉素类中的利福平等3种抗生素中介,而对测试的其他14种抗生素耐药。实验结果为伊丽莎白菌属细菌的分类鉴定提供了参考方法,且为黑斑蛙细菌病防治提供参考依据。

关键词: 黑斑蛙, 米尔伊丽莎白菌, 16S rRNA, gyrB, N-糖苷酶, 药敏试验

Abstract:

In order to provide treatment protocols for the cataract disease of black-spotted pond frog (Pelophylax nigromaculatus) in Shucheng area, Anhui Province, four dominant bacterial strains were isolated from the liver, eyes, spine and brain, respectively. The 16S rRNA gene and gyrB gene sequences of the isolated strains were analyzed, and the results showed that the four strains were Elizabethkingia miricola, which was confirmed by physiological and biochemical identification. N-glycosidase (PNGase) genes of the isolated strain was cloned and analysed, and the results showed it contained two types, PNGase F and PNGase F-Ⅱ. The phylogenetic analysis of PNGase F and PNGase F-Ⅱ both showed that the isolated strain was clustered with Elizabethkingia miricola in Elizabethkingia genus, which indicated that they can be used as reference genes for the classification of Elizabetha genus. Antimicrobial susceptibility test showed that the four isolates were only sensitive to florfenicol in amidols, intermediary to neosporin in coumarin, erythromycin in macrolide and rifampin in rifamycin, and resistant to other 14 antibiotics. The experiment results will provide a reference for the prevention and treatment of the bacterial diseases of black-spotted pond frog.

Key words: Pelophylax nigromaculatus, Elizabethkingia miricola, 16S rRNA, gyrB, N-glycosidase, antimicrobial susceptibility test

中图分类号:

S947.2

龚保荣, 吴红军, 李本镇, 徐大洋, 邹文腾, 曲君艺, 鲍传和, 朱若林. 患白内障病黑斑蛙米尔伊丽莎白菌的分离鉴定与PNGase基因克隆[J]. 浙江农业学报, 2023, 35(6): 1297-1306.

GONG Baorong, WU Hongjun, LI Benzhen, XU Dayang, ZOU Wenteng, QU Junyi, BAO Chuanhe, ZHU Ruolin. Isolation, identification of Elizabethkingia miricola of Pelophylax nigromaculatus with cataract and cloning of PNGase gene[J]. Acta Agriculturae Zhejiangensis, 2023, 35(6): 1297-1306.

图/表 11

参考文献 33

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基因 Gene	引物名称 Primer name	引物序列 Primer sequence(5'→3')
16S rRNA	27F	AGAGTTTGATCCTGGCTCAG
	1492R	GGTTACCTTGTTACGACTT
gyrB	gyrB-F	GTTTTGTAAGGTTTAGTGCC
	gyrB-R	ACATCGGTGATGTCGGA
PNGase F	PNGase F-F	AATCCTGTGGTGCTTTTTGTCC
	PNGase F-R	AGCTAAACTTGCTCAGCTTCTCG
PNGase F-Ⅱ	PNGase F-Ⅱ-F	TTGGTAAATAGTCATCTTCAGGC
	PNGase F-Ⅱ-R	CAATAGGTGAATAAGTGAATGGG

基因 Gene	引物名称 Primer name	引物序列 Primer sequence(5'→3')
16S rRNA	27F	AGAGTTTGATCCTGGCTCAG
	1492R	GGTTACCTTGTTACGACTT
gyrB	gyrB-F	GTTTTGTAAGGTTTAGTGCC
	gyrB-R	ACATCGGTGATGTCGGA
PNGase F	PNGase F-F	AATCCTGTGGTGCTTTTTGTCC
	PNGase F-R	AGCTAAACTTGCTCAGCTTCTCG
PNGase F-Ⅱ	PNGase F-Ⅱ-F	TTGGTAAATAGTCATCTTCAGGC
	PNGase F-Ⅱ-R	CAATAGGTGAATAAGTGAATGGG

测定项目 Measurement items	分离株Isolated strain				参考株Reference strain^*
测定项目 Measurement items	pn1907-1	pn1907-2	pn1907-3	pn1907-4	脑膜炎败血伊丽莎白菌 E. meningoseptica	米尔伊丽莎白菌 E. miricola
淀粉水解Starch hydrolysis	-	-	-	-	-	-
D-木糖D-Xylose	-	-	-	-	-	-
L-阿拉伯糖L-Arabinose	-	-	-	-	-	-
硝酸盐还原Nitrate reduction	-	-	-	-	-	-
乳糖Lactose	+	+	+	+	+	+
麦芽糖Maltose	+	+	+	+	+	+
甘油Glycerol	-	-	-	-	+	-
硫化氢H₂S	+	+	+	+	-	+
卵磷脂吐温-80培养基	+	+	+	+	-	+
Lecithin Tween-80 medium

测定项目 Measurement items	分离株Isolated strain				参考株Reference strain^*
测定项目 Measurement items	pn1907-1	pn1907-2	pn1907-3	pn1907-4	脑膜炎败血伊丽莎白菌 E. meningoseptica	米尔伊丽莎白菌 E. miricola
淀粉水解Starch hydrolysis	-	-	-	-	-	-
D-木糖D-Xylose	-	-	-	-	-	-
L-阿拉伯糖L-Arabinose	-	-	-	-	-	-
硝酸盐还原Nitrate reduction	-	-	-	-	-	-
乳糖Lactose	+	+	+	+	+	+
麦芽糖Maltose	+	+	+	+	+	+
甘油Glycerol	-	-	-	-	+	-
硫化氢H₂S	+	+	+	+	-	+
卵磷脂吐温-80培养基	+	+	+	+	-	+
Lecithin Tween-80 medium

药物种类 Drug Category	抗菌药物 Antibiotic	抑菌圈直径Antibacterial circle diameter/mm				敏感度 Sensibility
药物种类 Drug Category	抗菌药物 Antibiotic	pn1907-1	pn1907-2	pn1907-3	pn1907-4	敏感度 Sensibility
酰胺醇类Amphenicols	氟苯尼考Florfenicol	21	27	21	20	S
香豆素类Coumarin	新生霉素Novobiocin	16	16	13	13	I
大环内酯类Macrolides	红霉素Erythromycin	16	16	17	18	I
利福霉素类Rifamycins	利福平Rifampicin	17	18	17	18	I
林可酰胺类Lincosamides	林可霉素Lincomycin	0	0	0	0	R
青霉素类Penicillins	氨苄西林Ampicillin	0	0	0	0	R
头孢菌素类Cephalosporins	头孢氨苄Cephalexin	0	0	0	0	R
四环素类Tetracyclines	四环素Tetracycline	0	0	0	0	R
多肽类Polypeptides	多粘菌素B Polymyxin B	0	0	0	0	R
磺胺类Sulfonamides	甲氧苄氨嘧啶Trimethoprim	0	9	0	0	R
	复方新诺明Compound Sulfamethoxazole	0	9	0	0	R
氟喹诺酮类Quinolones	恩诺沙星Enrofloxacin	11	10	10	12	R
	氧氟沙星Ofloxacin	10	10	10	9	R
	诺氟沙星Norfloxacin	0	0	0	0	R
氨基糖苷类Aminoglycosides	卡那霉素Kanamycin	0	0	0	0	R
	新霉素Neomycin	0	9	0	8	R
	庆大霉素Gentamicin	0	9	0	0	R
	丁胺卡那Amikacin	0	9	0	9	R

患白内障病黑斑蛙米尔伊丽莎白菌的分离鉴定与PNGase基因克隆

Isolation, identification of Elizabethkingia miricola of Pelophylax nigromaculatus with cataract and cloning of PNGase gene

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