Our previous research indicated the excellent in vitro antibacterial activity of Eravacycline (Erava) and its heteroresistance frequency against clinical Staphylococcus aureus isolates. In this study, we further aimed to investigate the mechanisms of Erava resistance and heteroresistance in S. aureus. Eight parental S. aureus isolates were induced under Erava pressure in vitro and the Erava-resistant isolates were selected and identified. Then, the genetic mutations of 30S ribosomal subunits were analyzed by PCR and sequence alignment. RT-qPCR analysis were performed to compare the relative expression of eight candidate genes impacting the susceptibility of tetracycline (Tet) between the resistant or heteroresistant and parental isolates. Furthermore, the in vitro overexpression vectors of three selected candidate genes were constructed to test their impact on the heteroresistance and resistance of Erava in S. aureus. The MICs elevation in Erava-induced resistant S. aureus isolates were identified and the increasing MICs values of another two Tet class antibiotics, including both omadacycline (Omada) and tigecycline (Tige) were also tested. Genetic mutations in 30S ribosomal protein S10 were found frequently in Erava-derived resistant isolates. RT-qPCR analysis and the in vitro overexpression experiments indicated that USA300HOU_RS00550 (an Na/Pi cotransporter family protein) and USA300HOU_RS01625 (a branched-chain amino acid transport system II carrier protein) contributed to Erava heteroresistance in S. aureus. Genetic mutation of 30S ribosome subunits contributed to Erava resistance, and the transcriptional overexpression of USA300HOU_RS01625 and USA300HOU_RS00550 also participated in the occurrence of Erava heteroresistance in S. aureus.

中文翻译：

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金黄色葡萄球菌中30S核糖体亚基的遗传突变以及支链氨基酸转运系统II载体蛋白，Na / Pi共同转运蛋白家族蛋白对Eravacycline的敏感性产生影响。

我们先前的研究表明，Eravacycline（Erava）的优异体外抗菌活性及其对临床金黄色葡萄球菌分离株的异抗性频率。在这项研究中，我们进一步旨在研究金黄色葡萄球菌的抗Erava和异抗性的机制。体外在Erava压力下诱导了8个亲本金黄色葡萄球菌分离株，选择并鉴定了抗Erava的分离株。然后，通过PCR和序列比对分析30S核糖体亚基的遗传突变。进行RT-qPCR分析，以比较影响抗性或异抗性和亲本分离株之间影响四环素（Tet）敏感性的八个候选基因的相对表达。此外，构建了三个候选候选基因的体外过表达载体，以测试它们对金黄色葡萄球菌Erava的异抗性和抗性的影响。确定了Erava诱导的抗性金黄色葡萄球菌分离株的MICs升高，还测试了另外两种Tet类抗生素，包括奥马达环素（Omada）和tigecycline（Tige）的MICs升高。30S核糖体蛋白S10的遗传突变经常在源自Erava的耐药菌株中发现。RT-qPCR分析和体外过表达实验表明，USA300HOU_RS00550（Na / Pi共转运蛋白家族蛋白）和USA300HOU_RS01625（支链氨基酸转运系统II载体蛋白）有助于金黄色葡萄球菌的Erava异抗性。30S核糖体亚基的遗传突变促成Erava抗性，