引用本文:	金晶磊,周前进,邵鑫斌,王瑶华,陈炯,闫茂仓,苗亮,唐标,张严峻.东海温州水域4类常见耐药基因的流行情况研究.海洋与湖沼,2021,52(4):947-959.

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东海温州水域4类常见耐药基因的流行情况研究
金晶磊^1,2, 周前进^1,2, 邵鑫斌³, 王瑶华³, 陈炯^1,2, 闫茂仓³, 苗亮², 唐标⁴, 张严峻⁵
1.宁波大学农产品质量安全危害因子与风险防控国家重点实验室宁波 315211;2.宁波大学海洋学院宁波 315832;3.浙江省海洋水产养殖研究所浙江省近岸水域生物资源开发与保护重点实验室温州 325005;4.浙江省农业科学院农产品质量标准研究所杭州 310021;5.浙江省疾病预防控制中心杭州 310051

摘要:

磺胺类、四环素、喹诺酮类和多粘菌素抗生素是医学与养殖业中的常用抗生素，其中多粘菌素在畜牧养殖业中常作为促长剂添加。调查分析了这4类抗生素的常见耐药基因在东海温州近海水域的分布情况。利用抗性平板筛选温州近海水域对磺胺类、四环素类及喹诺酮类抗生素耐药的可培养细菌分离株，对耐药分离株通过PCR方法进行目标耐药基因检测；对目标耐药基因阳性的耐药分离株进行多粘菌素耐药基因MCR检测以及采用微量二倍稀释法测定对上述4类抗生素的耐药性。结果表明，利用抗性平板筛选获得可培养耐药分离株1 605株，从中筛选到目标耐药基因阳性的51株，检出率为3.18%，包括含磺胺类耐药基因35株、含四环素耐药基因36株、含喹诺酮耐药基因17株、含多粘菌素耐药基因2株；51株耐药菌归于7个菌属，其中气单胞菌属（Aeromonas sp.）43株，占比84.31%，枸橼酸杆菌属（Citrobacter sp.）和希瓦氏菌属（Shewanella sp.）各2株，埃希氏菌属（Escherichia sp.）、类芽孢杆菌属（Paenibacillus sp.）、Thalassobius sp.及Amphritea sp.各1株；上述2株非常见分离株不统计在内，常见的49株均对四环素耐药，41株对多粘菌素耐药，对磺胺类和喹诺酮类耐药的分离株均为34株；同时，发现23株对4类抗生素均产生耐药，16株对多达3种抗生素耐药，多重耐药现象普遍存在。同时，鉴定出MCR-3阳性的条件致病菌维氏气单胞菌和豚鼠气单胞菌各1株。综上，温州市近海水域中含有常见耐药基因的耐药分离株检出率较低，但同一耐药基因的细菌分布多样，对磺胺类、四环素类、喹诺酮类及多粘菌素类等4类抗生素表现出较强的耐药性，多重耐药现象明显。研究结果将为评估温州近海水域的细菌耐药性风险，规范抗生素的使用提供科学依据。

关键词: 东海温州水域耐药基因多重耐药 MCR-3

DOI：10.11693/hyhz20210200040

分类号:X172;Q789;S931.3

基金项目:浙江省重点研发计划，2021C02059号；农产品质量安全危害因子与风险防控国家重点实验室自主设计课题，2010DS700124-ZZ2008号；农产品质量安全危害因子与风险防控国家重点实验室2020开放基金项目，KF20200105号；浙江省近岸水域生物资源开发与保护重点实验室开放基金，J2018004号。

附件

INVESTIGATION ON RESISTANCE GENES AGAINST FOUR TYPES OF COMMON ANTIBIOTICS IN WENZHOU WATERS OF THE EAST CHINA SEA

JIN Jing-Lei^1,2, ZHOU Qian-Jin^1,2, SHAO Xin-Bin³, WANG Yao-Hua³, CHEN Jiong^1,2, YAN Mao-Cang³, MIAO Liang², TANG Biao⁴, ZHANG Yan-Jun⁵

1.State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, China;2.School of Marine Science, Ningbo University, Ningbo 315832, China;3.Zhejiang Key Laboratory of Exploitation and Preservation of Coastal Bio-resource, Zhejiang Mariculture Research Institute, Wenzhou 325005, China;4.Institute of Quality and Standard for Agro-Products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China;5.Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China

Abstract:

Sulfonamide, tetracyclines, quinolone, and polymyxin are antibiotics commonly used in medicine and animal breeding, of which polymyxin is often used as addictive to promote animal growth. The prevalence of the common resistance genes against the four types of antibiotics in coastal water of Wenzhou, Zhejiang, East China was investigated. Firstly, culturable bacterial strains resistant to sulfonamides, tetracyclines, and quinolones were isolated from the coastal waters of Wenzhou using the resistance plate method, in which the common resistance genes against sulfonamides, tetracyclines, and quinolones were identified using polymerase chain reaction. Secondly, polymyxin resistance-associated MCR genes were detected from the bacterial isolates carrying the resistance genes against sulfonamides, tetracyclines, and quinolones. Finally, the antimicrobial resistance of all the isolates carrying the four types of the resistance gene were determined using microdilution broth method. The results show that 1 605 culturable bacterial isolates were resistant to sulfonamides, tetracyclines, and quinolones, of which 51 were proven to carry different resistance genes against sulfonamides (35 bacterial isolates), tetracyclines (36 bacterial isolates), quinolones (17 bacterial isolates), and polymyxin (2 bacterial isolates), with a detection rate of 3.18%. These bacterial isolates could be classified into 7 genera, i.e., Aeromonas sp. (43 isolates, 84.31%), Citrobacter sp., Shewanella sp., Escherichia sp., Paenibacillus sp., Thalassobius sp., and Amphritea sp. Except for Thalassobius sp. and Amphritea sp., the remaining 49 isolates were all resistant to sulfonamides, of which 41 were resistant to tetracyclines, 34 to quinolones and polymyxin, 23 to all the four antibiotics, and 16 to three antibiotics, indicating a ubiquitous multi-drug resistance in the coastal waters in Wenzhou. In addition, 2 bacterial isolates carrying resistance gene MCR-3 were identified, i.e., Aeromonas veronica and Aeromonas caviae. Therefore, a small amount of antibiotic resistant isolates carrying common resistant genes against sulfonamides, tetracyclines, quinolones, and polymyxin were identified in the coastal waters, which characterized a diverse multi-drug resistance and strong resistance tolerance. These findings provide a reference for evaluating the risk of bacterial resistance and standardizing the use of antibiotics in Wenzhou coastal waters.

Key words: Wenzhou waters of the East China Sea resistance genes multi-drug resistance MCR-3