Isolation and biological characteristics of a pathogenic ST-251 Aeromonas hydrophila from Procypris Rabaudi (rock carp)

doi:10.3969/j.issn.1004-1524.2021.12.08

Acta Agriculturae Zhejiangensis ›› 2021, Vol. 33 ›› Issue (12): 2286-2294.DOI: 10.3969/j.issn.1004-1524.2021.12.08

• Animal Science • Previous Articles Next Articles

Isolation and biological characteristics of a pathogenic ST-251 Aeromonas hydrophila from Procypris Rabaudi (rock carp)

CHEN Mengzhu¹(), KANG Zhenya¹, GUO Xianghui¹, GENG Yi¹^,^*(), BAI Minghuan², OUYANG Ping¹, CHEN Defang³, HUANG Xiaoli³, LAI Weimin¹

1. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2. Zigong Center for Disease Control & Prevention, Zigong 643000, China
3. College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China

Received:2021-01-22 Online:2021-12-25 Published:2022-01-10
Contact: GENG Yi

Abstract

Abstract:

To explore the causes of epidemic diseases characterized by body surface bleeding in a Procypris rabaudi farm in Yibin, the pathogen and its biological characteristics were identified by physiological and biochemical characteristics,research on the pathogenesis,16S rDNA, multilocus sequence typing (MLST) and Kirby-Bauer (K-B). A pathogenic bacterium (YYL) was isolated from liver and kidney of diseased fish, and identified as Aeromonas hydrophila.When the water temperature was (24±1) ℃, its median lethal dose (LD₅₀) was 5.8 × 10 ⁴ CFU·g^-1 for rock carp weight of (12.6±0.8) g. Drug sensitivity test showed that the YYL was sensitive to many drugs, such as ceftazidime, gentamicin, doxycycline, enrofloxacin, florfenicol, but resistant to imipenem, roxithromycin, compound sulfamethoxazole, clindamycin. The isolated A. hydrophila belonged to ST-251 type by multilocus sequence typing (MLST), and carried virulence genes aerA, hlyA, aphA, act, ast. In this study, ST-251 A. hydrophila was isolated from the diseased rock carp characterized by body surface hemorrhage. This strain carried a variety of virulence factors and had strong pathogenicity. Therefore, it was necessary to strengthen the prevention and control of this pathogen infection in the cultivation of rock carp.

Key words: Aeromonas hydrophila, isolation and identification, multilocus sequence typing, virulence factor, drug resistance, Procypris rabaudi

CLC Number:

S917.1

CHEN Mengzhu, KANG Zhenya, GUO Xianghui, GENG Yi, BAI Minghuan, OUYANG Ping, CHEN Defang, HUANG Xiaoli, LAI Weimin. Isolation and biological characteristics of a pathogenic ST-251 Aeromonas hydrophila from Procypris Rabaudi (rock carp)[J]. Acta Agriculturae Zhejiangensis, 2021, 33(12): 2286-2294.

Figures/Tables 11

References 32

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引物 Primer	上游引物 Forward primer (5'-3')	下游引物 Reverse primer(5'-3')	长度 Length/bp	退火温度 Annealing temperature/℃
gayrB	GGGGTCTACTGCTTCACCAA	CTTGTCCGGGTTGTACTCGT	477	59
groL	CAAGGAAGTTGCTTCCAAGG	CATCGATGATGGTGGTGTTC	510	56
metG	TGGCAACTGATCCTCGTACA	TCTTGTTGGCCATCTCTTCC	504	58
ppsA	AGTCCAACGAGTACGCCAAC	TCGGCCAGATAGAGCCAGGT	537	57
recA	AGAACAAACAGAAGGCACTGG	AACTTGAGCGCGTTACCAC	561	60
gltA	TTCCGTCTGCTCTCCAAGAT	GAAGATCACGGTGAACATGG	495	57

引物 Primer	上游引物 Forward primer (5'-3')	下游引物 Reverse primer(5'-3')	长度 Length/bp	退火温度 Annealing temperature/℃
gayrB	GGGGTCTACTGCTTCACCAA	CTTGTCCGGGTTGTACTCGT	477	59
groL	CAAGGAAGTTGCTTCCAAGG	CATCGATGATGGTGGTGTTC	510	56
metG	TGGCAACTGATCCTCGTACA	TCTTGTTGGCCATCTCTTCC	504	58
ppsA	AGTCCAACGAGTACGCCAAC	TCGGCCAGATAGAGCCAGGT	537	57
recA	AGAACAAACAGAAGGCACTGG	AACTTGAGCGCGTTACCAC	561	60
gltA	TTCCGTCTGCTCTCCAAGAT	GAAGATCACGGTGAACATGG	495	57

毒力基因 Virulence gene	上游引物 Forward primer (5'-3')	下游引物 Reverse primer(5'-3')	长度 Length/ bp	退火温度 Annealing temperature/℃
Aerolysin (aerA)	CCTATGGCCTGAGCGAGAAG	CCAGTTCCAGTCCCACCACT	431	55.5
Hemolysin (hlyA)	CAAGGAAGTTGCTTCCAAGG	CATCGATGATGGTGGTGTTC	592	62.0
Cytotoxic enterotoxin (act)	GAGAAGGTGACCACCAAGAACA	AACTGACATCGGCCTTGAACTC	232	56.0
Cytotonic heat-stable	TCTCCATGCTTCCCTTCCACT	GTGTAGGGATTGAAGAAGCCG	331	55.0
enterotoxin (ast)
Type III secretion system inner	AGAACAAACAGAAGGCACTGG	AACTTGAGCGCGTTACCAC	577	60.0
membrane component(ascV)
Serine protease (ahp)	ATTGGATCCCTGCCTATCGCTTCAGTTCA	GCTAAGCTTGCATCCGTGCCGTATTCC	911	55.0

毒力基因 Virulence gene	上游引物 Forward primer (5'-3')	下游引物 Reverse primer(5'-3')	长度 Length/ bp	退火温度 Annealing temperature/℃
Aerolysin (aerA)	CCTATGGCCTGAGCGAGAAG	CCAGTTCCAGTCCCACCACT	431	55.5
Hemolysin (hlyA)	CAAGGAAGTTGCTTCCAAGG	CATCGATGATGGTGGTGTTC	592	62.0
Cytotoxic enterotoxin (act)	GAGAAGGTGACCACCAAGAACA	AACTGACATCGGCCTTGAACTC	232	56.0
Cytotonic heat-stable	TCTCCATGCTTCCCTTCCACT	GTGTAGGGATTGAAGAAGCCG	331	55.0
enterotoxin (ast)
Type III secretion system inner	AGAACAAACAGAAGGCACTGG	AACTTGAGCGCGTTACCAC	577	60.0
membrane component(ascV)
Serine protease (ahp)	ATTGGATCCCTGCCTATCGCTTCAGTTCA	GCTAAGCTTGCATCCGTGCCGTATTCC	911	55.0

鉴定项目 Test items	结果 Result	鉴定项目 Test items	结果 Result
丙氨酸-苯丙氨酸-脯氨酸芳胺酶Ala-Phe-Pro arylamidase	+	蔗糖Saccharose	+
侧金盏花醇Adonitol	-	D-塔格糖D-tagatose	-
吡咯烷基芳胺酶L-prroldonyi arylamidase	-	D-海藻糖D-trehalose	+
L-阿拉伯醇L-arabitol	-	柠檬酸盐Citrate	-
D-纤维二糖D-cellose	-	丙二酸盐Malonate	-
β-半乳糖苷酶β-galactosidase	+	5-酮-葡萄糖苷5-Keto-d-gluconate	-
H₂S	+	乳酸盐产碱L-lactate alkalinisation	+
β-N-乙酰葡萄糖苷酶β-N-acetyl glucosaminidase	+	α-葡萄糖α-glucosidase	-
谷氨酰芳胺酶Glutamyl arylamidase pNA	-	琥珀酸盐产碱Succinate alkalinisation	+
D-葡萄糖D-glucose	+	N-乙酰-β-半乳糖氨酶β-N-acetyl-galactosaminidase	-
γ-谷氨酰转移酶γ-glutamyl transferase	-	α-半乳糖苷酶β-galactosidase	-
葡萄糖发酵Glucose fermentation	+	磷酸酶Phosphatase	-
β-葡萄糖苷酶β-glucosidase	-	氨基乙酸芳胺酶Glycine arylamidase	+
D-麦芽糖D-maltose	+	鸟氨酸脱羧酶Ornithine decarboxylase	-
D-甘露醇(dMAN)D-mannitol	+	赖氨酸脱羧酶Lysine decarboxylase	-
D-甘露糖D-mannose	+	组氨酸同化L-Histidine assimilation	-
β-木糖苷酶β-xylosidase	-	COURMARATE (CMT)	+
β-丙氨酸芳胺酶β-alanine arylamidase pNA	-	β-葡萄糖苷酸酶β-glucoronidase	-
L-脯氨酸芳胺酶L-proline arylamidase	-	0/129耐受0/129 Resistance	+
脂酶Lipase	+	谷氨酸-甘氨酸-精氨酸芳胺酶Glu-Gly-Arg arylamidase	-
酪氨酸芳胺酶Tyrosine arylamidase	-	L-苹果酸盐同化L-malate assimilation	-
古老糖Palatinose	+	ELLMAN (ELLM)	+
尿酸酶Urease	-	L-乳酸盐同化L-lactate assimilation	-
D-山梨醇D-sorbitol	-

Isolation and biological characteristics of a pathogenic ST-251 Aeromonas hydrophila from Procypris Rabaudi (rock carp)

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注射浓度 Concentration/ (CFU·mL^-1)	试验鱼数量 Fish number	死亡总数量 Number of death	死亡率 Mortality rate/%
5.0×10⁸	12	12	100.0
5.0×10⁷	12	8	66.7
5.0×10⁶	12	6	50.0
5.0×10⁵	12	2	16.7
5.0×10⁴	12	0	0
0	12	0	0

抗生素 Antibiotics	药物 Drugs	抑菌圈直径 Diameter/mm	判断标准Assessment criteria			敏感性 Sensitivity
抗生素 Antibiotics	药物 Drugs	抑菌圈直径 Diameter/mm	R	I	S	敏感性 Sensitivity
β-内酰胺类 β-lactams	亚胺培南Imipenem	10	≤13	14-15	≥16	R
	头孢他定Ceftazidime	27	≤14	15-17	≥18	S
	头孢西丁Cefoxitin	19.5	≤14	15-17	≥18	S
氨基糖苷类Aminoglycosides	庆大霉素Gentamicin	17.5	≤12	13-14	≥15	S
	妥布霉素Tobramycin	13.5	≤12	13-14	≥15	I
	萘啶酸Nalidixic acid	25	≤13	14-18	≥19	S
	链霉素Streptomycin	16.5	≤11	13-14	≥15	S
	卡那霉素Kanamycin	20	≤13	14-22	≥18	S
	大观霉素Spectinomycin	29	≤14	13-17	≥18	S
四环素类Tetracycline	多西环素Doxycycline	19	≤12	13-15	≥16	S
	四环素Tetracycline	15	≤14	15-18	≥19	I
大环内酯类macrolides	罗红霉素Roxithromycin	19	≤21	22-30	≥31	R
喹诺酮类Quinolones	恩诺沙星Enrofloxacin	31	≤20	21-27	≥28	S
	左氧氟沙星Levofloxacin	30	≤12	13-15	≥16	S
	环丙沙星Ciprofloxacin	32	≤15	16-20	≥21	S
磺胺类Sulfonamides	复方新诺Sulfamethoxazole	20	≤24	25-31	≥32	R
	磺胺异恶唑Bacteresulf	13	≤15	16-21	≥21	R
酰胺醇类Amide alcohols	氟苯尼考Florfenicol	31	≤12	13-17	≥18	S
林可胺类Lincosamides	克林霉素Clindamycin	9	≤14	15-20	≥21	R
多肽类Polypeptide	多黏菌素BPolymyxin B	11.5	≤8	9-11	≥12	I

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