Isolation, identification and characteristic analysis of three new pathogenic bacteria of Apostichopus japonicus

doi:10.14088/j.cnki.issn0439-8114.2023.04.021

Abstract

Abstract: Three dominant pathogenic bacteria WY-1, WY-8 and LY-2 were isolated from the skin canker tissue of Apostichopus japonicus infected with severe skin rot. The isolated strains were identified by morphological observation, biochemical identification, 16S rRNA molecular identification, drug sensitivity analysis, growth characteristics analysis and other methods. WY-1, WY-8 and LY-2 were arranged in a grape-like shape on 2216E solid medium with the color of milky white, and Gram staining was positive. The artificial infection test showed that the three strains of bacteria had strong pathogenicity to pseudoacanthus japonicus, and could cause the skin ulcer and other symptoms, which were the same as natural infection symptoms. Through 16S rRNA molecular sequencing and phylogenetic tree construction, it was confirmed that the strains WY-8, WY-1 and LY-2 had the highest homology with Staphylococcus haematolyticus, Staphylococcus saprophytius and Staphylococcus epidermidis, respectively, and clustered into one branch in the phylogenetic tree. Combined with morphological and biochemical identification, WY-8, WY-1 and LY-2 were Staphylococcus haematolyticus,Staphylococcus saprophytius and Staphylococcus epidermidis, respectively. The drug sensitivity test showed that tetracycline and oxytetracycline, commonly used antibiotics in marine aquatic products, had inhibitory effects on three strains of pathogenic Staphylococcus. The growth characteristics test showed that the three strains of pathogenic Staphylococcus had the growth characteristics of adapting to high temperature and low salt.

Key words: Apostichopus japonicus, Staphylococcus, skin ulceration syndrome, 16S rRNA, growth characteristics

CLC Number:

S947.9

XU Xiao-ran, GE Chang-zi, ZHANG Yu-qun, LIU Hong-zhan. Isolation, identification and characteristic analysis of three new pathogenic bacteria of Apostichopus japonicus[J]. HUBEI AGRICULTURAL SCIENCES, 2023, 62(4): 118-122.

References

[1] XU C, ZHANG R, WEN Z Y.Bioactive compounds and biological functions of sea cucumbers as potential functional foods[J]. Journal of functional foods, 2018, 49: 73-84.
[2] PANGESTUTI R, ARIFIN Z.Medicinal and health benefit effects of functional sea cucumbers[J]. Journal of traditional and complementary medicine, 2018, 8(3):341-351.
[3] HUO D, SUN L N, ZHANG L B, et al.Time course analysis of immunity-related gene expression in the sea cucumber Apostichopus japonicus during exposure to thermal and hypoxic stress[J]. Fish&shellfish immunology, 2019, 95:383-390.
[4] YU Z H, ZHOU Y, YANG H S, et al.Bottom culture of the sea cucumber Apostichopus japonicus, Selenka (Echinodermata: Holothuroidea) in a fish farm, southern China[J]. Aquacul ture resarch, 2014, 45(9): 1434-1441.
[5] 李翠苹, 翟倩倩, 王想,等. 对虾养殖池副溶血弧菌的分离鉴定及其耐药特征、毒力基因分析[J]. 渔业科学进展, 2020(6): 174-180.
[6] 兰欣, 李杰, 李贵阳, 等. 发病鲆鲽类分离菌株的16S rRNA基因测序分析[J]. 渔业科学进展, 2020(3): 142-150.
[7] 王凯, 王印庚, 姜勇, 等. 一株感染深水网箱养殖许氏平鲉的病原菌分离与鉴定[J]. 渔业科学进展,2019,40(1):119-126.
[8] 王印庚, 郭伟丽, 荣小军, 等. 养殖刺参“化板症”病原菌的分离与鉴定[J]. 渔业科学进展, 2012, 33(6): 81-86.
[9] 张春云, 王印庚, 荣小军. 养殖刺参腐皮综合征病原菌的分离与鉴定[J]. 水产学报, 2006(1): 118-123.
[10] 张春云,陈国福,徐仲,等. 养殖刺参附着期“化板症”病原菌的分离鉴定及来源分析[J]. 微生物学报,2009,49(5):632-638.
[11] LI H, QIAO G, GU J Q, et al.Phenotypic and genetic characterization of bacteria isolated from diseased cultured sea cucumber Apostichopus japonicus in northeastern China[J]. Diseases of aquatic organisms, 2010, 91(3): 223-235.
[12] LIU H Z,ZHENG F Z,SUN X Q, et al.Identification of the pathogens associated with skin ulceration and peristome tumescence in cultured sea cucumbers Apostichopus japonicus (Selenka)[J]. Journal of invertebrate pathology, 2010, 105(3): 236-242.
[13] LI H, QIAO G, LI Q, et al.Biological characteristics and pathogenicity of a highly pathogenic Shewanella marisflavi infecting sea cucumber, Apostichopus japonicus[J]. Journal of fish diseases, 2010, 33(11):865-877.
[14] ZHANG Z,XING R L, LYU Z M, et al.Analysis of gut microbiota revealed Lactococcus garviaeae could be an indicative of skin ulceration syndrome in farmed sea cucumber Apostichopus japonicus[J]. Fish & shellfish immunology, 2018, 80: 148-154.
[15] ALONSO B, LUCIO J, PÉREZ-GRANDA M J, et al. Does biomass production correlate with metabolic activity in Staphylococcus aureus[J]. Journal of microbiological method,2016,131:110-112.
[16] SUGITA H, SHIBUYA K, SHIMOOKA H, et al.Antibacterial abilities of intestinal bacteria in freshwater cultured fish[J]. Aquaculture, 1996, 145(1-4):195-203.
[17] ATYAH M A S, ZAMRI-SAAD M, SITI-ZAHRAH A. First report of methicillin-resistant Staphylococcus aureus from cage-cultured tilapia(Oreochromis niloticus)[J]. Veterinary microbiology, 2010,144(3-4): 502-504.
[18] DIVYA S, THINESH T, KIRAN G S, et al.Emergence of a multi host biofilm forming opportunistic pathogen Staphylococcus sciuri D26 in coral Favites abdita[J]. Microbial pathogenesis, 2018, 120: 204-212.
[19] LU A J, HU X C, WANG Y, et al.Immune response in the skin of zebrafish, Danio rerio (Hamilton), against Staphylococcus chromogenes[J]. Journal of fish diseases, 2012, 35(9):699-704.
[20] 张云鹏, 朱立颖, 肖朝耿, 等. 南美白对虾体表与肠道细菌菌群结构的DGGE分析[J]. 中国食品学报,2016,16(8):218-224.
[21] 刘青, 赵铭武, 曹鑫磊, 等. 血鹦鹉鱼木糖葡萄球菌的分离鉴定和耐药性研究[J]. 水产科学, 2016, 35(2): 174-178.
[22] 葛丽婷, 刘青, 王一帆, 等. 鱼源葡萄球菌对金鱼和斑马鱼致病性的研究[J]. 黑龙江农业科学, 2014(8): 69-71.
[23] YE M Q, HAN J R, WANG C, et al.Alteromonas sediminis sp. nov., isolated from sediment in a sea cucumber culture pond[J]. International journal of systematic and evolutionary microbiology, 2019, 69(6):1579-1584.
[24] 黄梦诗, 杨倩倩, 张艳, 等. 响应面法研究温度、盐度、pH及磷酸盐、硝氮、氨氮对副溶血弧菌生长的影响[J]. 渔业科学进展,2019,40(2):132-140.
[25] BARGHINI P, PASQUALETTI M, GORRASI S, et al.Bacteria from the “Saline di Tarquinia” marine salterns reveal very atypical growth profiles with regards to salinity and temperature[J]. Mediterranean marine science, 2018, 19(3):513-525.
[26] 王倩,蓝蔚青,张墨言,等. 冷链与断链流通对冰藏大黄鱼品质与微生物多样性的影响[J]. 中国食品学报,2019,19(9):221-229.
[27] ABDELFATAH E N, MAHBOUB H H H. Studies on the effect of Lactococcus garvieae of dairy origin on both cheese and Nile tilapia (O. niloticus)[J]. International journal of veterinary science and medicine, 2018,6(2):201-207.
[28] MA Y X, LI L Y, LI M, et al.Effects of dietary probiotic yeast on growth parameters in juvenile sea cucumber, Apostichopus japonicus[J]. Aquaculture, 2019, 499: 203-211.
[29] HU X, YANG H L, YAN Y Y, et al.Effects of fructooligosaccharide on growth, immunity and intestinal microbiota of shrimp(Litopenaeus vannamei) fed diets with fish meal partially replaced by soybean meal[J]. Aquaculture nutrition, 2019, 25(1): 194-204.