Sha Yuning, Lin Naru, Zhang Guozhi, Zhang Yuan, Zhao Jingxuan, Lu Junwan, Zhu Tingting, Zhang Xueya, Li Qiaoling, Zhang Hailin, Lin Xi, Li Kewei, Bao Qiyu, Li Dong
The Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, China.
Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education, China, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China.
Front Microbiol. 2023 Aug 28;14:1224464. doi: 10.3389/fmicb.2023.1224464. eCollection 2023.
Aminoglycosides, as important clinical antimicrobials, are used as second-line drugs for treating multidrug-resistant tuberculosis or combined with β-lactam drugs for treating severe infections such as sepsis. Aminoglycoside-modifying enzyme (AME) is the most important mechanism of aminoglycoside resistance and deserves more attention.
The bacterium DW18 was isolated from the sewage of an animal farm using the conventional method. The agar dilution method was used to determine the minimum inhibitory concentrations (MICs) of antimicrobials. A novel resistance gene was cloned, and the enzyme was expressed. The kinetic parameters were measured by a SpectraMax M5 multifunctional microplate reader. Bioinformatic analysis was performed to reveal the genetic context of the gene and its phylogenetic relationship with other AMEs.
A novel aminoglycoside 3'--phosphotransferase gene designated was identified in DW18 and shared the highest amino acid identity of 77.49% with the functionally characterized aminoglycoside 3'--phosphotransferase APH(3')-Ia. The recombinant plasmid carrying the novel resistance gene (pMD19-/ DH5α) showed 1,024-, 512-, 128- and 16-fold increased MIC levels for kanamycin, ribostamycin, paromomycin and neomycin, respectively, compared with the reference strain DH5α. APH(3')-Id showed the highest catalytic efficiency for ribostamycin [ of (4.96 ± 1.63) × 10 M/s], followed by paromomycin [ of (2.18 ± 0.21) × 10 M/s], neomycin [ of (1.73 ± 0.20) × 10 M/s], and kanamycin [ of (1.10 ± 0.18) × 10 M/s]. Three conserved functional domains of the aminoglycoside phosphotransferase family and ten amino acid residues responsible for the phosphorylation of kanamycin were found in the amino acid sequence of APH(3')-Id. No mobile genetic element (MGE) was discovered surrounding the gene.
In this work, a novel aminoglycoside 3'--phosphotransferase gene designated encoded in the chromosome of the environmental isolate DW18 was identified and characterized. These findings will help clinicians select effective antimicrobials to treat infections caused by pathogens with this kind of resistance gene.
氨基糖苷类作为重要的临床抗菌药物，被用作治疗耐多药结核病的二线药物，或与β-内酰胺类药物联合用于治疗败血症等严重感染。氨基糖苷类修饰酶（AME）是氨基糖苷类耐药的最重要机制，值得更多关注。
采用常规方法从某养殖场污水中分离出DW18菌株。采用琼脂稀释法测定抗菌药物的最低抑菌浓度（MIC）。克隆了一个新的耐药基因，并表达了该酶。通过SpectraMax M5多功能微孔板读数仪测量动力学参数。进行生物信息学分析以揭示该基因的遗传背景及其与其他AMEs的系统发育关系。
在DW18中鉴定出一个新的氨基糖苷3'-磷酸转移酶基因，命名为 ，与功能已明确的氨基糖苷3'-磷酸转移酶APH(3')-Ia的氨基酸同一性最高，为77.49%。携带新耐药基因的重组质粒（pMD19- /DH5α）与参考菌株DH5α相比，对卡那霉素、核糖霉素、巴龙霉素和新霉素的MIC水平分别提高了1024倍、512倍、128倍和16倍。APH(3')-Id对核糖霉素的催化效率最高[ 为(4.96±1.63)×10 M/s]，其次是巴龙霉素[ 为(2.18±0.21)×10 M/s]、新霉素[ 为(1.73±0.20)×10 M/s]和卡那霉素[ 为(1.10±0.18)×10 M/s]。在APH(3')-Id的氨基酸序列中发现了氨基糖苷磷酸转移酶家族的三个保守功能域和负责卡那霉素磷酸化的十个氨基酸残基。在该基因周围未发现移动遗传元件（MGE）。
在本研究中，在环境分离株DW18染色体上编码的一个新的氨基糖苷3'-磷酸转移酶基因，命名为 ，被鉴定并进行了表征。这些发现将有助于临床医生选择有效的抗菌药物来治疗由携带此类耐药基因的病原体引起的感染。
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