Abstract：This study aims to determine and characterize the pathogen of the diseased turbot Scophthalmus maximus in a farm in Yantai, Shandong Province. The suspicious pathogenic bacteria were isolated from the diseased turbot and identified combined with morphological characteristics, physiological and biochemical tests, and 16S rRNA sequence. The artificial infection tests, drug sensitivity tests, and whole-genome sequencing were then carried out for the isolates. The results showed the strains of gram-negative brevibacterium was isolated from the liver, kidney, and spleen tissues of 5 diseased turbot with symptoms of abdominal distension and inflamed anus, which was motile, negative for VP reaction and methyl red reaction, and positive for nitrate reduction reaction and oxidase reaction. It was identified as Pseudomonas putida through sequencing comparison and named as D2. The strains had median lethal concentration(LD50) of 8.13×107 cfu/mL. D2 were sensitive to twelve antibiotics (such as quinolones, and aminoglycosides) and resistant to seven antibiotics (such as penicillin, and ampicillin). The full-length genome of strain D2 is 6.32 Mb, with G+C content accounting for 62.0%, and a total of 5878 genes were predicted. In previous studies, 947 proteins related to cell adhesion, iron absorption, and secretion functions had been identified. Previous studies had shown that the D2 strain carried resistance to various antibiotics, including quinolones and polymyxins. Based on the preliminary work, this project plans to further analyze the pathogenic mechanism of Pseudomonas putida on farmed turbot, predict its pathogenic factors and resistance genes, and provide scientific basis for the rational use of doxycycline hydrochloride in aquaculture. This study has important practical significance for the prevention and control of this disease.
[1] 雷霁霖,刘新富.大菱鲆Scophthalmus maximus L.引进养殖的初步研究[J].现代渔业信息,1995(11):1-3. [2] QIN L, XU J, WANG Y G. Edwardsiellosis in farmed turbot, Scophthalmus maximus (L. ), associated with an unusual variant of Edwardsiella tarda:a clinical, aetiological and histopathological study[J]. Journal of Fish Diseases,2014,37(2):103-111. [3] SOUTO S, OLVEIRA J G, DOPAZO C P, et al. Reassortant betanodavirus infection in turbot (Scophthalmus maximus)[J]. Journal of Fish Diseases,2016,39(11):1347-1356. [4] COSCELLI G A, BERMÚDEZ R, SANCHO SILVA A R, et al. Granulomatous dermatitis in turbot (Scophthalmus maximus L. ) associated with natural Aeromonas salmonicida subsp. salmonicida infection[J]. Aquaculture,2014,428:111-116. [5] 王鹤,王文豪,黄华,等.一株大菱鲆源杀鲑气单胞菌非典型株的分离鉴定及毒力基因型[J].大连海洋大学学报,2022,37(4):558-567. [6] 方振华,代小梅,丁利,等.中华草龟恶臭假单胞菌的分离鉴定及药物敏感性分析[J].中国畜牧兽医,2019,46(10):3115-3123. [7] 杨敏,吴青,沙莎.养殖大鲵恶臭假单胞菌的分离鉴定及药物敏感性分析[J].中国兽医杂志,2018,54(1):87-90. [8] 白明焕,耿毅,邓龙君,等.长薄鳅恶臭假单胞菌的分离鉴定及其感染的病理损伤[J].浙江农业学报,2019,31(2):207-215. [9] 樊海平.恶臭假单胞菌引起的欧洲鳗鲡烂鳃病[J].水产学报,2001,25(2):147-150. [10] 闫茂仓,单乐州,马爱敏,等.条石鲷烂尾白浊症病原菌的分离鉴定及系统发育分析[J].上海海洋大学学报,2010,19(1):50-55. [11] 毛芝娟,王美珍,陈吉刚,等.黑鲷肠炎病原恶臭假单胞菌的分离和鉴定[J].渔业科学进展,2010,31(3):23-28. [12] 杨圆圆,杨移斌,曹海鹏,等.杂交鲟源恶臭假单胞菌的分离鉴定及药敏特性研究[J].浙江农业学报,2017,29(12):1978-1985. [13] 段亚佼,黄小丽,詹瑰然,等.杂交鲇恶臭假单胞菌的分离鉴定及其病理损伤研究[J].水生生物学报,2017,41(2):371-378. [14] 许斌福,程海华,池洪树,等.大黄鱼内脏白点病的病原分析与鉴定[J].福建农业学报,2015,30(7):631-635. [15] 郭玲玲.微生物菌种保藏方法及关键技术[J].微生物学杂志,2019,39(3):105-108. [16] 何宏港,吴悠,刘乃瑜,等.鳖源铜绿假单胞菌的分离鉴定及多位点序列与全基因组分析[J].微生物学报,2022,62(7):2751-2767. [17] 王鹤,李战军,黄华,等.大菱鲆源副乳房链球菌的分离鉴定及其毒力基因型[J].大连海洋大学学报,2021,36(4):563-572. [18] 刁菁,李乐,王晓璐,等.虹鳟致病性杀鲑气单胞菌的分离鉴定及其毒力因子的检测[J].大连海洋大学学报,2018,33(4):435-443. [19] 马越,李景云,金少鸿.美国临床实验室标准委员会推荐药敏试验操作方法和判断标准(2005年修订版)[J].中华医学杂志,2005,85(17):1182-1184. [20] 东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:166-167. [21] 朱鹏飞.大菱鲆养殖环境细菌多样性分析及水产病原菌基因芯片检测体系的构建[D].青岛:国家海洋局第一海洋研究所,2015. [22] WANG H R, HU Y H, ZHANG W W, et al. Construction of an attenuated Pseudomonas fluorescens strain and evaluation of its potential as a cross-protective vaccine[J]. Vaccine,2009,27(30):4047-4055. [23] BERTHE F, MICHEL C, BERNARDET J F. Identification of Pseudomonas anguilliseptica isolated from several fish species in France[J]. Diseases of Aquatic Organisms, 1995,21:151-155. [24] OCHMAN H, DAVALOS L M. The nature and dynamics of bacterial genomes[J]. Science,2006,311(5768):1730-1733. [25] RANEA J A G, BUCHAN D W A, THORNTON J M, et al. Evolution of protein superfamilies and bacterial genome size[J]. Journal of Molecular Biology,2004,336(4):871-887. [26] 方源,谢柏盛,黄婷,等.34株假单胞菌的泛基因组分析[J].应用与环境生物学报,2021,27(4):1031-1038. [27] POOLE K, MCKAY G A. Iron acquisition and its control in Pseudomonas aeruginosa:many roads lead to Rome[J]. Frontiers in Bioscience,2003,8:d661-d686. [28] RAVEL J, CORNELIS P. Genomics of pyoverdine-mediated iron uptake in pseudomonads[J]. Trends in Microbiology,2003,11(5):195-200. [29] DAHYOT S, OXARAN V, NIEPCERON M, et al. Role of the LytSR two-component regulatory system in Staphylococcus lugdunensis biofilm formation and pathogenesis[J]. Frontiers in Microbiology,2020,11:39. [30] SHON A S, BAJWA R P S, RUSSO T A. Hypervirulent (hypermucoviscous) Klebsiella pneumoniae:a new and dangerous breed[J]. Virulence,2013,4(2):107-118. [31] CORNELIS P. Iron uptake and metabolism in pseudomonads[J]. Applied Microbiology and Biotechnology,2010,86(6):1637-1645. [32] DASGUPTA N, WOLFGANG M C, GOODMAN A L, et al. A four-tiered transcriptional regulatory circuit controls flagellar biogenesis in Pseudomonas aeruginosa[J]. Molecular Microbiology,2003,50(3):809-824. [33] SONG Z J, WU H, CIOFU O, et al. Pseudomonas aeruginosa alginate is refractory to Th1 immune response and impedes host immune clearance in a mouse model of acute lung infection[J]. Journal of Medical Microbiology,2003,52(Pt 9):731-740. [34] GROHMANN E, CHRISTIE P J, WAKSMAN G, et al. Type IV secretion in Gram-negative and Gram-positive bacteria[J]. Molecular Microbiology,2018,107(4):455-471. [35] CHUNG J M, SHEEDLO M J, CAMPBELL A M, et al. Structure of the Helicobacter pylori cag type Ⅳ secretion system[J]. Biophysical Journal,2020,118(3):295a. [36] JUHAS M, CROOK D W, HOOD D W. Type Ⅳ secretion systems:tools of bacterial horizontal gene transfer and virulence[J]. Cellular Microbiology,2008,10(12):2377-2386. [37] 杨雨齐.猪源大肠杆菌对达氟沙星敏感性临界值及其耐药机制研究[D].哈尔滨:东北农业大学,2019. [38] GUNN J S, RICHARDS S M. Recognition and integration of multiple environmental signals by the bacterial sensor kinase PhoQ[J]. Cell Host & Microbe,2007,1(3):163-165. [39] SUN J J, DENG Z Q, YAN A X. Bacterial multidrug efflux pumps:mechanisms, physiology and pharmacological exploitations[J]. Biochemical and Biophysical Research Communications,2014,453(2):254-267. [40] MASUDA N, SAKAGAWA E, OHYA S, et al. Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-oprM efflux pumps in Pseudomonas aeruginosa[J]. Antimicrobial Agents and Chemotherapy,2000,44(12):3322-3327. [41] HOCQUET D, VOGNE C, EL GARCH F, et al. MexXY-OprM efflux pump is necessary for a adaptive resistance of Pseudomonas aeruginosa to aminoglycosides[J]. Antimicrobial Agents and Chemotherapy,2003,47(4):1371-1375. [42] GUÉNARD S, MULLER C, MONLEZUN L, et al. Multiple mutations lead to MexXY-OprM-dependent aminoglycoside resistance in clinical strains of Pseudomonas aeruginosa[J]. Antimicrobial Agents and Chemotherapy,2014,58(1):221-228. [43] 崔羽茜,丹雨晴,武慧敏,等.7株羊源大肠埃希氏菌的分离鉴定及相关耐药基因的研究[J].动物医学进展,2022,43(10):27-34.
2025, Vol. 44 
