Shropshire William C, Song Xinhao, Bremer Jordan, Seo Seokju, Rodriguez Susana, Anand Selvalakshmi Selvaraj, Dinh An Q, Bhatti Micah M, Konovalova Anna, Arias Cesar A, Kalia Awdhesh, Shamoo Yousif, Shelburne Samuel A
bioRxiv. 2024 Jul 31:2024.07.31.606066. doi: 10.1101/2024.07.31.606066.
It remains unclear how high-risk lineages, like sequence type (ST) 131, initially adapt to carbapenem exposure in their progression to becoming carbapenem resistant.
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/ 30Rx isolate, MB1860, under prolonged, increasing carbapenem exposure was performed using two distinct experimental evolutionary platforms to measure fast vs. slow adaptation.
All thirteen ESBL positive ST131 strains selected from a diverse (n=184) ST131 bacteremia cohort had detectable ertapenem (ETP) mutational frequencies with a statistically positive correlation between initial ESBL gene copy number and mutation frequency (r = 0.87, -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) encoding 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 copy numbers via modular, insertion sequence (IS ) mediated pseudocompound transposons (PCTns). Transposase activity driven by PCTn upregulation 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.
ESBL gene amplification is a conserved response to initial carbapenem exposure, especially within the high-risk ST131 C2/ 30Rx subclade. Targeting such amplification could assist with mitigating carbapenem resistance development.
目前尚不清楚像序列类型（ST）131这样的高风险谱系在其发展为碳青霉烯耐药的过程中最初是如何适应碳青霉烯暴露的。
使用波动试验测量超广谱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突变频率，随后进行全基因组测序（WGS）表征。使用两个不同的实验进化平台对ST131 C2/30Rx分离株MB1860在长时间增加碳青霉烯暴露的情况下进行基因组、转录组和孔蛋白分析，以测量快速适应与缓慢适应。
从一个多样化的（n = 184）ST131菌血症队列中选出的所有13株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之间存在统计学上的正相关（r = 0.87，P值<1e - 5）。对突变体的WGS分析表明，对ETP暴露的初始反应导致ESBL基因拷贝数显著增加或外膜孔蛋白（Omp）编码基因发生突变，且不存在ESBL基因扩增，并具有亚分支特异性关联。在两个实验进化平台中，MB1860对初始ETP暴露的反应都是通过模块化的插入序列（IS）介导的假复合转座子（PCTn）增加基因拷贝数。由PCTn上调驱动的转座酶活性是两个实验进化平台中的保守表达信号。仅在长时间增加碳青霉烯暴露后才检测到Omp编码基因的稳定突变，这与临床观察结果一致。
ESBL基因扩增是对初始碳青霉烯暴露的保守反应，尤其是在高风险的ST131 C2/30Rx亚分支内。针对这种扩增可能有助于减轻碳青霉烯耐药性的发展。
