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

doi:10.3969/j.issn.1004-1524.2023.06.07

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (6): 1297-1306.DOI: 10.3969/j.issn.1004-1524.2023.06.07

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S947.2

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.

Figures/Tables 11

References 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

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

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