罗非鱼不同养殖阶段喹诺酮类耐药菌群数量与组成分析

Quantity and composition of quinolone-resistant microbiota at different breeding stages of tilapia

【目的】评估不同养殖阶段罗非鱼肠道及养殖环境中喹诺酮类耐药菌产生的风险,筛选出水产养殖耐药性监测指示菌,为建立科学的水产品耐药防控技术提供参考依据。【方法】选取萘啶酸(第一代)和恩诺沙星(第三代)2种喹诺酮类药物,通过平板计数法统计不同养殖阶段(苗种阶段、中间阶段及上市阶段)罗非鱼肠道及养殖环境(池塘水和底泥)中喹诺酮类耐药菌的数量及耐药率,然后采用高通量测序分析不同养殖阶段喹诺酮类耐药菌群的结构特征及组成变化。【结果】在不同养殖阶段罗非鱼肠道及其养殖环境中的细菌总数相对稳定,罗非鱼肠道的细菌总数维持在4.6×10^(5)~1.6×10^(6)CFU/g,池塘水的细菌总数维持在5.2×10^(3)~2.1×10^(4)CFU/g,池塘底泥的细菌总数维持在4.8×10^(3)~3.2×10^(4)CFU/g。苗种阶段罗非鱼肠道菌群对萘啶酸和恩诺沙星的耐药率均高于中间阶段和上市阶段,分别为34.7%和19.0%。各养殖阶段的罗非鱼肠道及养殖环境优势菌群均以气单胞菌属、埃希菌—志贺氏菌属、鲸杆菌属、邻单胞菌属、芽孢杆菌属和肠杆菌属细菌为主。随着养殖阶段的推移,罗非鱼肠道中的气单胞菌属相对丰度呈下降趋势,而鲸杆菌属呈上升趋势;而养殖环境中的菌群结构无明显规律性变化。在罗非鱼肠道及养殖环境喹诺酮类耐药菌群中,埃希菌—志贺氏菌属和气单胞菌属的相对丰度较高,且随着养殖阶段的推移呈不同程度上升趋势。【结论】罗非鱼养殖的耐药风险主要集中在养殖前期阶段,埃希菌属和气单胞菌属是喹诺酮类耐药的主要菌群,应加强不同养殖阶段的饲养管理,减少病害发生以减轻抗菌药物使用带来的风险。此外,可选用埃希菌属和气单胞菌属作为水产养殖细菌耐药性监测的指示菌,实时监控喹诺酮类药物耐药性的产生与传播。

【Objective】In order to evaluate the bacterial resistance risk to quinolones in tilapia intestine and its culture envrionment,and find out the bacterial indicator of antimicrobial resistance surveillance,which could provide reference for the establishment of scientific prevention and control technology of aquatic product drug resistance.【Method】Two quinolones of nalidixic acid(first generation)and enrofloxacin(third generation)were selected.The number and resistance rate of quinolones resistant bacteria in intestinal tract and culture environment(pond water and sediment)of tilapia were calculated by plate counting method,then high throughput sequencing was used to analyze the structural characteristics and composition of quinolones resistant flora in different breeding stages.【Result】The results showed that total viable counts of bacteria in the tilapia intestine and the culture environment was comparatively stable at different breeding stages.The total number of bacteria in tilapia intestine,pond water and sediment maintained at 4.6×10^(5)-1.6×10^(6)CFU/g,5.2×10^(3)-2.1×10^(4)CFU/g and 4.8×10^(3)-3.2×10^(4)CFU/g,respectively.The resistance rates of fish intestinal bacteria to nalidixic acid and enrofloxacin at the fingerling stage were higher than other two stages,reaching 34.7%and 19.0%,respectively.The dominant flora of tilapia intestines and culture environment at each breeding stage were Aeromonas,Escherichia-Shigella,Cetobacterium,Plesiomonas,Bacillus and Enterobacter.As breeding time progressed,the flora of tilapia intestine was observed that the relative abundance of Aeromonas was decreased,while the relative abundance of Cetobacterium was increased.There was no obvious regular change in the structure of the bacterial community in the culture environment.Among the quinolone-resistant flora of tilapia intestines and culture environment,the relative abundance of Escherichia-Shigella and Aeromonas was relatively high,and increased in varying degrees with the pass of the breeding stage.【Conclusion】In conclusion,high risk of quinolones resistance is observed in tilapia intestine and aquatic environment at the fingerlings stage.Escherichia and Aeromonas are the dominant flora of quinolone resistance.It is necessary to strengthen the feeding management of different breeding stages and reduce the occurrence of diseases to reduce the risk of antimicrobial use.Escherichia and Aeromonas should be as indicator bacteria of antimiorcobial resistance in aquaculture,to monitor drug resistance occurrence and transmission of quinolones.