浙江省养鳖场中细菌耐药性及blaNDM传播特性

Antibiotic resistance and blaNDM transmission characteristics of bacteria from turtle farms in Zhejiang Province

摘要:

【目的】 调查浙江省不同地区6家养鳖场[浙江萧山(Xiaoshan, XS)和兰溪(Lanxi, LX)两地各3家]的细菌耐药情况，对养殖场细菌的耐药性进行监测并分析两地养鳖场细菌耐药特征和所携耐药基因的差异；对养鳖场内bla_NDM的传播特性进行分析。【方法】 通过16s rRNA基因进行菌株鉴定，使用Kirby-Bauer (K-B)药敏试验测定分离菌的药物敏感性，使用PCR技术检测分离菌所携带的耐药基因，通过接合转移实验获得携bla_NDM质粒接合子并根据接合转移频率评价各供体菌的接合转移能力，通过改良Carba NP法产酶实验、质粒复制子分型和最小抑菌浓度测定进一步验证携bla_NDM质粒在细菌间传播的能力。【结果】 共分离得到244株细菌，其中两地养殖场的菌群分布相似，均以肠杆菌科细菌为主。此外，对整合子的PCR扩增结果表明存在多种耐药基因，其中包括氨基糖苷类和甲氧苄胺嘧啶类耐药基因。实验还发现大量未形成完整整合子结构的耐药菌，具有整合更多外源耐药基因的潜力。所有分离菌对至少一种抗生素耐药，而且部分耐药菌对碳青霉烯类抗生素也表现出耐药性，其对应的碳青霉烯类耐药基因已在部分养殖场内传播。本研究证明bla_NDM存在于质粒上并可在细菌间传播，LX携带bla_NDM质粒的接合移频率显著高于XS。【结论】 水产养殖环境中的细菌耐药性问题日益严峻，特别是碳青霉烯类耐药基因bla_NDM在养鳖场中呈现出扩散趋势。

Abstract:

[Objective] To investigate the antibiotic resistance profiles, resistance genes, and bla_NDM transmission characteristics of bacterial isolates from six turtle farms in two regions (three in Lanxi (LX) and three in Xiaoshan (XS)) of Zhejiang Province. [Methods] Bacterial strains were identified by 16S rRNA gene sequencing. The Kirby-Bauer method was employed to examine the antibiotic susceptibility of isolated bacteria. PCR was employed to detect resistance genes carried by the isolated bacteria. Conjugation transfer experiments were conducted to obtain bla_NDM-carrying plasmids, and the conjugation frequency was analyzed to assess the transfer capabilities of bacterial donors. Additionally, the modified Carba NP test, plasmid replicon typing, and minimum inhibitory concentration determination were carried out to evaluate the transmission of bla_NDM-carrying plasmids among bacteria. [Results] A total of 244 bacterial strains were isolated, with similar distribution between the two regions and Enterobacteriaceae being dominant. PCR amplification of integrons revealed various resistance genes, including those conferring resistance to aminoglycosides and trimethoprim. We identified numerous resistant bacteria capable of integrating additional exogenous resistance genes. All the isolated strains exhibited resistance to at least one antibiotic, with some displaying resistance to carbapenems. PCR detection of resistance genes indicated the dissemination of carbapenem resistance genes within some farms. This study confirmed the presence of bla_NDM on plasmids capable of inter-bacterial transmission. Moreover, the farms in LX exhibited significantly higher conjugation frequency of bla_NDM-carrying plasmids than the farms in XS. [Conclusion] Antibiotic resistance of bacteria in aquaculture environments is becoming increasingly serious. The carbapenem resistance gene bla_NDM has spread within turtle farms.