MLST typing of Vibrio parahaemolyticus from diseased shrimp ponds in Zhanjiang area and isolation and identification of novel drug-resistant strains
WU Junmin, WANG Yi, HAO Jingwei, WANG Qingyao, ZHANG Jin, FU Songzhe*, LIU Ying
1.Key Laboratory of Environment Controlled Aquaculture(Dalian Ocean University), Ministry of Education, Dalian 116023,China; 2.College of Marine Science and Environment, Dalian Ocean University, Dalian 116023,China; 3.College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023,China; 4.College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China
Abstract: In order to evaluate the genetic diversity of Vibrioparahaemolyticus in shrimp ponds in Zhanjiang area, seven housekeeping genes dnaE, gyrB, recA, dtdS, pntA, pyrC and tnaA of pathogenic V.parahaemolyticus were selected in the experiment, and the isolated strains were subjected to MLST typing for genetic evolution analysis by MEGA 6.0 software.Also, the virulence genes of tdh, trh, tlh, mam7, vcrD1, vcrD2 and pirAB were detected by a PCR method. The drug susceptibility test was carried out on all strains by disc diffusion method, and one multi-drug resistant strain was selected using the Illumina Hiseq 2500 platform. Subsequently, the spliced sequences were detected by IslandPath-DIOMB software to analyze the origin of drug resistance gene islands. The 58 strains of V.parahaemolyticus were divided into 34 ST types by MLST, all of which contained virulence genes tlh, mam7 and vcrD1, without tdh, trh and vcrD2, 8 strains containing the virulence gene pirAB causing AHPND. It was found that the 58 strains were 100% resistant to penicillin and high resistance rates to ampicillin, clarithromycin and gentamicin. Four strains of ST1740 were found to be multi-drug resistant. One of the 4 ST1740 strains (ZJ20-3/2020) was selected for whole genome sequencing, and contained 8 drug resistance genes, namely blaCARB-41, aadA16, aph(6)-Id, aph(3″)-Ib, tet(A), sul1, dfrA27 and ARR-3, and were resistant to penicillin, kanamycin, tetracycline and rifampicin. Four gene islands were found in ST1740 strain by IslandPath-DIOMB software, with 99% DNA sequence similarity with the corresponding sequences in Shewanella NCTC12093, indicating that the V.parahaemolyticus in the shrimp culture area in Zhanjiang area had rich genetic diversity. MLST typing identified 2 new pirA-containing genotypes, and subsequent genomic analysis identified a novel drug-resistant strain. A new ST strain (ST1740) carrying pirAB was identified, which carried 4 gene islands from Shiwanella and were multidrug-resistant strains. The horizontal gene transfer from Shewanella in V.parahaemolyticus was the first reported and the findings provided important information for understanding of the population dynamics and pathogenic characteristics of V.parahaemolyticus in shrimp ponds in Zhanjiang area, and also provided a basis for the prevention and control of AHPND outbreaks.
吴俊敏, 王艺, 郝婧薇, 王青瑶, 张晋, 傅松哲, 刘鹰. 湛江地区患病对虾池塘中副溶血性弧菌的MLST分型和新型耐药毒株的分离鉴定[J]. 大连海洋大学学报, 2023, 38(1): 59-67.
WU Junmin, WANG Yi, HAO Jingwei, WANG Qingyao, ZHANG Jin, FU Songzhe, LIU Ying. MLST typing of Vibrio parahaemolyticus from diseased shrimp ponds in Zhanjiang area and isolation and identification of novel drug-resistant strains. Journal of Dalian Ocean University, 2023, 38(1): 59-67.