Background It remains unclear how high-risk Escherichia coli lineages, like sequence type (ST) 131, initially adapt to carbapenem exposure in their progression to carbapenem resistance. Methods Carbapenem mutation frequency was measured in multiple subclades of extended-spectrum β-lactamase (ESBL) positive ST131 clinical isolates using a fluctuation assay followed by whole genome sequencing (WGS) characterization. Genomic, transcriptomic, and porin analyses of ST131 C2/H30Rx isolate, MB1860, under prolonged, increasing carbapenem exposure was performed using two experimental evolutionary platforms to measure fast vs. slow adaptation. Results All thirteen ESBL positive ST131 strains selected from a diverse (n=184) ST131 bacteremia cohort had detectable ertapenem (ETP) mutational frequencies with a positive correlation between initial ESBL gene copy number and mutation frequency (r = 0.87, P-value <1e-5). WGS analysis of mutants showed initial response to ETP exposure resulted in significant increases in ESBL gene copy numbers or mutations in outer membrane porin (Omp) genes in the absence of ESBL gene amplification with subclade specific associations. In both experimental evolutionary platforms, MB1860 responded to initial ETP exposure by increasing blaCTX-M-15 copy numbers via modular, insertion sequence 26 (IS26) mediated pseudocompound transposons (PCTns). Increased transcript levels of genes present within the PCTn was a conserved expression signal in both experimental evolutionary platforms. Stable mutations in Omp encoding genes were detected only after prolonged increasing carbapenem exposure consistent with clinical observations. Conclusions ESBL gene amplification is a conserved response to initial carbapenem exposure, especially within the high-risk ST131 C2/H30Rx subclade. Targeting such amplification could assist with mitigating carbapenem resistance development.

中文翻译：

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ESBL 阳性 ST131 大肠杆菌对碳青霉烯类暴露初始适应的综合评估


背景 目前尚不清楚高危大肠杆菌谱系，如序列类型 （ST） 131，在发展为碳青霉烯类耐药的过程中最初如何适应碳青霉烯类暴露。方法 使用波动测定法测量广谱 β-内酰胺酶 （ESBL） 阳性 ST131 临床分离株的多个亚分支中的碳青霉烯类突变频率，然后进行全基因组测序 （WGS） 表征。使用两个实验进化平台对长期、增加的碳青霉烯类暴露的 ST131 C2/H30Rx 分离株 MB1860 进行基因组、转录组和孔蛋白分析，以测量快速适应与慢适应。结果 从不同的 （n=184） ST131 菌血症队列中选择的所有 13 个 ESBL 阳性 ST131 菌株均具有可检测到的厄他培南 （ETP） 突变频率，初始 ESBL 基因拷贝数与突变频率呈正相关 （r = 0.87，P 值 <1e-5）。突变体的 WGS 分析显示，在没有具有亚分支特异性关联的 ESBL 基因扩增的情况下，对 ETP 暴露的初始反应导致 ESBL 基因拷贝数或外膜孔蛋白 （Omp） 基因突变显着增加。在这两个实验进化平台中，MB1860 通过模块化插入序列 26 （IS26） 介导的假化合物转座子 （PCTn） 增加 blaCTX-M-15 拷贝数来响应初始 ETP 暴露。PCTn 中存在的基因转录水平的增加在两个实验进化平台中都是一个保守的表达信号。只有在与临床观察一致的碳青霉烯类药物暴露时间延长后，才能检测到 Omp 编码基因的稳定突变。 结论 ESBL 基因扩增是对初始碳青霉烯类暴露的保守反应，尤其是在高危 ST131 C2/H30Rx 亚分支内。靶向这种扩增可能有助于减轻碳青霉烯类耐药性的发展。