Kasai Ayako, Ohta Ayaka, Maeda Yuina, Yamada Kageto, Tao Kazuyuki, Saito Ryoichi
Department of Molecular Microbiology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan,
Department of Clinical Laboratory, Toho University Medical Center Omori Hospital, Ota-ku, Tokyo 143-8541, Japan.
Infect Drug Resist. 2018 Nov 1;11:2137-2140. doi: 10.2147/IDR.S181714. eCollection 2018.
High-level macrolide-resistant strains have been isolated; however, the underlying mechanism has not been well elucidated. We investigated the role of mutations in the 23S rRNA gene and the L4 and L22 ribosomal proteins using spontaneous erythromycin-resistant mutants and transformants.
The erythromycin-susceptible ATCC25238 and clinical isolate Mc19 were used as parental strains. To obtain spontaneous erythromycin-resistant mutants, in vitro stepwise selection was performed using brain-heart infusion agar plates containing various concentrations of erythromycin. The role of the mutations identified in the spontaneous mutants was validated using transformation experiments.
We obtained two spontaneous mutants with high-level resistance to erythromycin, S25-32-af10 and S19-256-af10, from ATCC25238 and Mc19, respectively. S25-32-af10 exhibited mutations of Q61R in L4 and Insertion98SRADRIS in L22. S19-256-af10 exhibited three C2611T-mutated alleles in the 23S rRNA gene and G65A in L4. Transformants with single mutations identified in S25-32-af10 or S19-256-af10 showed higher erythromycin and azithromycin minimum inhibitory concentrations (MICs) than those of each parental strain. However, transformants with multiple mutations identified in S25-32-af10 or S19-256-af10 showed macrolide MICs similar to those of each parental strain.
Our results provide the first evidence suggesting that Q61R in L4 and Insertion98SRADRIS in L22 are involved in the synergistic acquisition of high-level resistance to both 14- and 15-member macrolides, and that C2611T in the 23S rRNA gene and G65A in L4 also synergistically contribute toward conferring high-level 14-member macrolide resistance to .
已分离出对大环内酯类药物具有高水平耐药性的菌株；然而，其潜在机制尚未完全阐明。我们使用自发的红霉素耐药突变体和转化体，研究了23S rRNA基因以及L4和L22核糖体蛋白中突变的作用。
将对红霉素敏感的ATCC25238和临床分离株Mc19用作亲本菌株。为获得自发的红霉素耐药突变体，使用含有不同浓度红霉素的脑心浸液琼脂平板进行体外逐步筛选。通过转化实验验证自发突变体中鉴定出的突变的作用。
我们分别从ATCC25238和Mc19中获得了两个对红霉素具有高水平耐药性的自发突变体，即S25 - 32 - af10和S19 - 256 - af10。S25 - 32 - af10在L4中表现出Q61R突变，在L22中表现出Insertion98SRADRIS突变。S19 - 256 - af10在23S rRNA基因中表现出三个C2611T突变等位基因，在L4中表现出G65A突变。在S25 - 32 - af10或S19 - 256 - af10中鉴定出的具有单个突变的转化体，其红霉素和阿奇霉素的最低抑菌浓度（MIC）高于各自的亲本菌株。然而，在S25 - 32 - af10或S19 - 256 - af10中鉴定出的具有多个突变的转化体，其大环内酯类药物的MIC与各自的亲本菌株相似。
我们的结果提供了首个证据，表明L4中的Q61R和L22中的Insertion98SRADRIS参与了对14元和15元大环内酯类药物高水平耐药性的协同获得，并且23S rRNA基因中的C2611T和L4中的G65A也协同作用，赋予对14元大环内酯类药物的高水平耐药性。
