一株致病性溶藻弧菌的多重耐药与毒力基因分子分析

摘要

A pathogenic Vibrio alginolyticus strain,named VA-76,was isolated from ceca contents of a diseased Litopenaeus vannamei collected from Fuqing,Fujian Province.Antimicrobial susceptibilty tests for 13 antimicrobials and infection assay for Litopenaeus vannamei were performed to determine the phenotype of antimicrobial resistance and pathogenicity of VA-76,respectivly.To explore the genotype of antimicrobial resistance and pathogenicity,the isolate was also analyzed by PCR detection for virulence genes,integron-related genes and antimircobial resistance genes.In artificial infection assay,death occurred in all 4 groups of Litopenaeus vannamei infected with 3.5 ×107cfu,3.5 ×106cfu,3.5 ×105cfu,and 3.5 ×104cfu,and the death rates were 100%,100%,80%,and 65%,respectively.The virulence-related genes such as tdh,tlh, toxS,collagenase,flaA,ompW,aspA,and fur were identified in VA-76.The results of antimicrobial susceptibility tests showed that this bacteria was resistant to multiple antimicribals,including florfenicol (32μg· mL-1 ),doxycycline (32μg · mL-1 ),ciprofloxacin (>64μg · mL-1 ),chloramphenicol (128μg·mL-1 ),streptomycin (>5 12μg·mL-1 ),erythromycin (256μg·mL-1 ),sulfamethoxazole (>2048μg·mL-1),TMP/SMZ (>128/2432μg·mL-1),and rifampin (>64μg·mL-1).Especially,VA-76 exhibited high-level resistance to the latter 7 antimicrobials,and had relatively high MIC for neomycine (>32μg·mL-1 ).Furthermore,integron 1 and SXT was also detected in this isolate,and the gene cassete arrangement arr2-dfrA27 was identified inside this class 1 integron.Besides,insertion sequence common region1 (ISCR1 ),containing a chloramphenicol acetyltransferase gene cat2,was also detected.Several other antimicrobial resistance genes,cluding strA,strB,floR and qnrVC,were found in VA-76.However,no mutation was identified in gyrA and ParC genes of VA-76,which were usually involved in fluroquinolones resistance.This study revealed that Vibrio alginolyticus VA-76 could cause the death of Litopenaeus vannamei, exhibit resistance to multiple antimicrobials,and contain multiple virulence and antimicrobial resistance genes,indicating that prudent use of antimicrobial agents in aquaculture should be emphasized to control the development and distribution of antimicrobial resistant Vibrio alginolyticus.%从发病的凡纳滨对虾中分离并经鉴定得到一株溶藻弧菌（命名为：VA-76），通过对虾回感实验和药物敏感性实验分别对VA-76菌株的耐药表型和致病能力进行了研究，并运用PCR方法检测了该菌的毒力相关基因、整合子和耐药基因的携带情况。对虾感染实验结果表明，溶藻弧菌VA-76能够导致凡纳滨对虾发病死亡。经PCR检测发现，该菌携带8种毒力相关基因：tdh、tlh、toxS、collagenase、flaA、ompW、aspA和fur。药敏结果显示，VA-76对环丙沙星、氯霉素、链霉素、红霉素、磺胺甲唑、复方新诺明和利福平7种药物表现出较高的耐药水平，且该菌包含1类整合子、4类超级整合子SXT和1型插入序列共同区（ISCR1）。此外，该菌携带arr-2、dfrA27、strA、strB、floR、cat和qnrvC耐药相关基因。从上述研究结果可知，菌株VA-76既具有一定的感染能力，同时对多种抗菌药物表现出较高的耐药水平，提示应对养殖源致病性弧菌的耐药性现状予以更多的关注。