To investigate the prevalence, serotype, antibiotic resistance, and genetic relationship of Vibrio parahaemolyticus (Vp) in the retail freshwater fish in Wanzhou, 141 samples of retail freshwater fish were collected from 2022 to 2023.The Vp was isolated and quantified according to the method of GB 4789.7—2013, and then PCR was used to amplify the thermolabile hemolysin gene (tlh), heat-resistant direct hemolysin gene (tdh), heat-resistant-related hemolysin gene (trh), transmembrane transcriptional regulator gene (toxR), Photorhabdus insect-related toxin gene (pirA), type Ш secretion system 1 gene (T3SS1), and type Ш secretion system 2 genes (T3SS2α and T3SS2β) that were associated with acute hepatopancreas necrosis.Multiplex PCR was used to detect serotypes.The Kirby-Bauer and enterobacterial repeat intergenic consensus sequence PCR (ERIC-PCR) were used to detect antimicrobial susceptibility and genetic relationship, respectively.Results showed that 54 samples were Vp positive out of 141 samples (38.3%), When the quantitative range of positive samples was in the range of 1.5 MPN/g to 1 100 MPN/g, the pollution of Aristichys nobilis was the highest (66.7%).Temperature significantly affected the abundance of Vp, and the seasonal differences in sample pollution were significant, with the highest pollution rate of 57.1% in summer.All 54 Vp isolates carried tlh, toxR, and T3SS1 genes, one isolate carried both tdh and T3SS2α genes, and trh, pirA, and T3SS2β genes were not found.The serotypes distribution was more dispersed and divided into nine O groups, with an O1 dominant cluster (33.3%).The resistance rates of 54 Vp strains to benzoxacillin, penicillin, and ampicillin were 100%, 98.1%, and 90.7%, respectively.54 Vp strains were 100% susceptible to ceftazidime, ceftriaxone, gentamicin, tetracycline, doxycycline, and minocycline.ERIC-PCR showed that the isolate carrying pathogenic genes was genetically similar to clinical isolates.The results of this study might provide a theoretical basis for freshwater fish farming, consumption and clinical treatment of foodborne diseases caused by Vp.
[1] ZHOU H B, LIU X M, HU W Y, et al.Prevalence, antimicrobial resistance and genetic characterization of Vibrio parahaemolyticus isolated from retail aquatic products in Nanjing, China[J].Food Research International, 2022, 162:112026.
[2] RYU A R, MOK J S, LEE D E, et al.Occurrence, virulence, and antimicrobial resistance of Vibrio parahaemolyticus isolated from bivalve shellfish farms along the southern coast of Korea[J].Environmental Science and Pollution Research International, 2019, 26(20):21 034-21 043.
[3] 毛雪丹, 胡俊峰, 刘秀梅.用文献综述法估计我国食源性副溶血性弧菌病发病率[J].中华疾病控制杂志, 2013, 17(3):265-267.
MAO X D, HU J F, LIU X M.Study on incidence of foodborne disease caused by Vibrio parahaemolyticus by literature review method[J].Chinese Journal of Disease Control and Prevention, 2013, 17(3):265-267.
[4] 卢俊, 袁冬梅.一起致病性弧菌引起的食物中毒的调查[J].现代预防医学, 2013, 40(7):1216-1217.
LU J, YUAN D M.Investigation and analysis of a food poisoning caused by pathogenic Vibrio[J].Modern Preventive Medicine, 2013, 40(7):1216-1217.
[5] 孔令汉, 贺良, 唐敏, 等.2021年重庆市万州区两起食物中毒事件副溶血性弧菌溯源和耐药性分析[J].中国预防医学杂志, 2023, 24(7):643-647.
KONG L H, HE L, TANG M, et al.The origins and antibiotic resistance of Vibrio parahaemolyticus isolated from two food poisoning incidents in Wanzhou District of Chongqing[J].Chinese Preventive Medicine, 2023, 24(7):643-647.
[6] BEJ A K, PATTERSON D P, BRASHER C W, et al.Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh[J].Journal of Microbiological Methods, 1999, 36(3):215-225.
[7] KIM Y B, OKUDA J, MATSUMOTO C, et al.Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxR gene[J].Journal of Clinical Microbiology, 1999, 37(4):1173-1177.
[8] 郭书林, 陈信忠, 尤颖哲, 等.福建省南美白对虾幼体急性肝胰腺坏死病病原分离与PCR检测[J].中国动物检疫, 2019, 36(6):96-99.
GUO S L, CHEN X Z, YOU Y Z, et al.Pathogen isolation and PCR detection of AHPND in Penaeus vannamei larvae in Fujian Province[J].China Animal Health Inspection, 2019, 36(6):96-99.
[9] MORIEA N F 3rd, JOHNSON C N, GRIFFITT K J, et al.Distribution of type III secretion systems in Vibrio parahaemolyticus from the northern Gulf of Mexico[J].Journal of Applied Microbiology, 2010, 109(3):953-962.
[10] CABURLOTTO G, GENNARI M, GHIDINI V, et al.Presence of T3SS2 and other virulence-related genes in tdh-negative Vibrio parahaemolyticus environmental strains isolated from marine samples in the area of the Venetian Lagoon, Italy[J].FEMS Microbiology Ecology, 2009, 70(3):506-514.
[11] CHEN M, GUO D, WONG H C, et al.Development of O-serogroup specific PCR assay for detection and identification of Vibrio parahaemolyticus[J].International Journal of Food Microbiology, 2012, 159(2):122-129.
[12] Clinical and Laboratory Standards Institute.M45 Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria;approved guideline[S].Wayne:CLSI, 2016.
[13] ALI S, HOSSAIN M, AZAD A B, et al.Diversity of Vibrio parahaemolyticus in marine fishes of Bangladesh[J].Journal of Applied Microbiology, 2021, 131(5):2539-2551.
[14] WARD L N, BEJ A K.Detection of Vibrio parahaemolyticus in shellfish by use of multiplexed real-time PCR with TaqMan fluorescent probes[J].Applied and Environmental Microbiology, 2006, 72(3):2031-2042.
[15] 徐秋琼, 彭明益, 单桂花, 等.网售生鲜海产品中副溶血性弧菌定量污染状况调查分析[J].热带医学杂志, 2021, 21(3):385-388.
XU Q Q, PENG M Y, SHAN G H, et al.Investigation and analysis on quantitative contamination of Vibrio parahaemolyticus in fresh seafood sold online[J].Journal of Tropical Medicine, 2021, 21(3):385-388.
[16] LEI T, JIANG F F, HE M, et al.Prevalence, virulence, antimicrobial resistance, and molecular characterization of fluoroquinolone resistance of Vibrio parahaemolyticus from different types of food samples in China[J].International Journal of Food Microbiology, 2020, 317:108461.
[17] LI M Z, XU H Y, TIAN Y Q, et al.Comparative genomic analysis reveals the potential transmission of Vibrio parahaemolyticus from freshwater food to humans[J].Food Microbiology, 2023, 113:104277.
[18] 吴斌, 胡元玮, 方琼楼, 等.金华市淡水产品中副溶血性弧菌污染状况及PFGE分型研究[J].中国卫生检验杂志, 2022, 32(15):1829-1831.
WU B, HU Y W, FANG Q L, et al.A study on the pollution and pulse field gel electrophoresis typing of Vibrio parahaemolyticus in fresh water products in Jinhua city[J].Chinese Journal of Health Laboratory Technology, 2022, 32(15):1829-1831.
[19] CHEN H H, DONG S L, YAN Y, et al.Prevalence and population analysis of Vibrio parahaemolyticus isolated from freshwater fish in Zhejiang province, China[J].Foodborne Pathogens and Disease, 2021, 18(2):139-146.
[20] ALABOUDI A R, ABABNEH M, OSAILI T M, et al.Detection, identification, and prevalence of pathogenic Vibrio parahaemolyticus in fish and coastal environment in Jordan[J].Journal of Food Science, 2016, 81(1):M130-M134.
[21] HAM H, ORTH K.The role of type III secretion system 2 in Vibrio parahaemolyticus pathogenicity[J].Journal of Microbiology (Seoul, Korea), 2012, 50(5):719-725.
[22] STRATEV D, FASULKOVA R, KRUMOVA-VALCHEVA G.Incidence, virulence genes and antimicrobial resistance of Vibrio parahaemolyticus isolated from seafood[J].Microbial Pathogenesis, 2023, 177:106050.
[23] 周倩, 向婧姝, 黄靖宇, 等.贵阳市淡水产品及养殖环境致病性弧菌监测结果分析[J].应用预防医学, 2022, 28(4):352-354;396.
ZHOU Q, XIANG J S, HUANG J Y, et al.Analysis of monitoring results of pathogenic Vibrio in freshwater products and culture environment in Guiyang City[J].Applied Preventive Medicine, 2022, 28(4):352-354;396.
[24] 王小龙, 张梦寒, 朱莉勤, 等.2016—2019苏州市副溶血性弧菌的毒力基因和耐药性及分子分型研究[J].现代预防医学, 2020, 47(21):3975-3980.
WANG X L, ZHANG M H, ZHU L Q, et al.Virulence gene, drug resistance,molecular typing of Vibrio parahaemolyticus in Suzhou, 2016—2019[J].Modern Preventive Medicine, 2020, 47(21):3975-3980.
[25] 贺悦, 齐小娟, 王绩凯, 等.浙江省2021年食源性疾病监测结果分析[J].中国卫生检验杂志, 2023, 33(4):504-508.
HE Y, QI X J, WANG J K, et al.Analysis of surveillance results of foodborne diseases in Zhejiang Province in 2021[J].Chinese Journal of Health Laboratory Technology, 2023, 33(4):504-508.
[26] CHIOU J C, LI R C, CHEN S.CARB-17 family of β-lactamases mediates intrinsic resistance to penicillins in Vibrio parahaemolyticus[J].Antimicrobial Agents and Chemotherapy, 2015, 59(6):3593-3595.
[27] 强鑫华, 蔡云祥.食源性腹泻患者分离副溶血性弧菌毒力基因及药敏分析[J].中国卫生检验杂志, 2021, 31(2):155-158.
QIANG X H, CAI Y X.Virulence and antimicrobial susceptibility of Vibrio Parahaemolyticus in patients with foodborne diarrhea[J].Chinese Journal of Health Laboratory Technology, 2021, 31(2):155-158.
[28] VAN BELKUM A, TASSIOS P T, DIJKSHOORN L, et al.Guidelines for the validation and application of typing methods for use in bacterial epidemiology[J].Clinical Microbiology and Infection, 2007, 13:1-46.
