摘要
为研究水产食品源副溶血性弧菌的多重耐药情况以及质粒介导其多重耐药的传播机制,本研究采购太平洋鲭鱼、凡纳滨对虾和太平洋牡蛎等水产食品样品共计300份,经传统培养法、PCR法、革兰氏染色法、生化鉴定、16S rRNA基因的PCR扩增与测序分析,分离并鉴定分离的副溶血性弧菌,利用PCR法分析分离菌株携带的耐热直接溶血素基因(tdh)和耐热直接溶血毒素相关基因(trh),采用K-B药敏纸片扩散法检测分离菌株对9类共19种药物的耐药性并分析其的多重耐药性,通过SDS法质粒消除试验、质粒提取及分型试验和质粒接合转移试验研究质粒介导的多重耐药副溶血性弧菌耐药性的传播情况。结果显示:300份水产食品中共分离鉴定到95株副溶血性弧菌,分离率为31.7%(95/300);未检出tdh和trh毒力基因;药敏试验结果显示,有30.5%(29/95)的分离菌株具有多重耐药性且分离菌株主要对氨苄青霉素和利福平耐药。对29株多重耐药分离菌株进行质粒消除试验后,发现有15株发生全部或部分耐药表型消失现象;对该15株分离菌株提取质粒并分型,其中13株分离菌株携带IncF型质粒;对10株质粒携带氨苄青霉素抗性的分离菌株进行接合转移试验,并采用K-B法检测结合子的耐药性及耐药性的稳定性。结果显示10株分离菌株均携带具有接合转移能力的质粒且其对氨苄青霉素和利福平的耐药性均稳定。综上所述,本研究从水产食品中分离到的副溶血性弧菌且均不含tdh和trh毒力基因,因此分离菌株的致病性低,但多重耐药率较高,其中多重耐药副溶血性弧菌中耐药质粒的携带率为51.7%(15/29),其以耐药质粒为载体的水平传播可能由IncF型质粒主导。本研究为规范水产养殖用药、阻断水产食品源耐药微生物的传播扩散奠定一定的实验基础。
To investigate the situation of multi-drug resistant(MDR)of V.parahaemolyticus from aquatic food sources and the transmission mechanism of MDR mediated by plasmids,in this study,a total of 300 aquatic food samples including Pneumatophorus japonicus,Litopenaeus vannamei and Crassostrea gigas were collected,and V.parahaemolyticus was isolated and identified by traditional culture method,PCR,Gram staining,biochemical identification and sequencing verification.PCR was used to analyze the two virulence genes of thermostable direct hemolysin(tdh)and TDH related hemolysin(trh)carried by the V.parahaemolyticus isolates,and Kirby-Bauer disk diffusion method was employed to detect the isolates to 19 types of drugs in 9 categories and analyze the multidrug resistance of the isolates.The plasmid elimination test,plasmid extraction and typing test and plasmid conjugation transfer test were fulfilled to examine the plasmid-mediated transmission of antimicrobial resistance(AMR)of MDR V.parahaemolyticus isolates.The results showed that 95 strains of V.parahaemolyticus were isolated and identified from 300 aquatic food samples,the isolation rate was 31.7%(95/300),and the virulence genes of tdh and trh were not detected.The antimicrobial susceptibility testing revealed that the MDR rate of V.parahaemolyticus isolates was 30.5%(29/95)and the most isolates resistance to ampicillin and rifampicin.Plasmid elimination test of 29 MDR isolates showed that antimicrobial resistance phenotype of 15 isolates disappeared completely or partially.Plasmids of the 15 isolates were extracted and typed,of which 13 isolates carried IncF type of plasmid.Conjugation transfer test of 10 isolates carrying plasmids with ampicillin resistance showed that all plasmids had ability of conjugation transfer and the antimicrobial resistance was stable.In conclusion,the V.parahaemolyticus isolated from aquatic food in this study were all devoid of the tdh and trh virulence genes,hence exhibiting low pathogenicity.However,the rate of multi-drug resistance was rel
作者
董羽织
徐曼如
王靖雯
焦雪
方结红
蒋晗
DONG Yu-zhi;XU Man-ru;WANG Jing-wen;JIAO Xue;FANG Jie-hong;JIANG Han(Key Laboratory of Specialty Agri-products Quality and Hazard Controlling Technology of Zhejiang Province,College of Life Sciences,China Jiliang University,Hangzhou 310018,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2024年第6期576-583,共8页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(31901792、31801655)
浙江省基础公益研究计划项目(LGN22C200013)
宁波市公益类科技计划项目(2022S014)。
关键词
副溶血性弧菌
多重耐药
水产食品源
质粒
传播
Vibrio parahaemolyticus
multi-drug resistance
aquatic food
plasmid
dissemination
