Servicio de Microbiología, Hospital Universitario Son Espases, IdISBa, CIBERINFEC, Palma de Mallorca, Spain.
Servicio de Medicina Intensiva, Hospital Universitario Son Espases, IdISBa, CIBERINFEC, Palma de Mallorca, Spain.
EBioMedicine. 2024 Oct;108:105367. doi: 10.1016/j.ebiom.2024.105367. Epub 2024 Sep 26.
Pseudomonas aeruginosa is a major cause of hospital-acquired and chronic infections, characterised by an extraordinary capacity to develop antimicrobial resistance through the selection of chromosomal mutations, leading to treatment failure. Here, we designed and tested a hybridisation-based capture system for the enrichment of genes of interest before sequencing to monitor resistant populations genomics directly from clinical samples.
A panel for enrichment before sequencing of close to 200 genes related to P. aeruginosa antimicrobial resistance, multilocus sequence typing, mutability or virulence was designed, synthesised (KAPA HyperCap, Roche) and initially validated in vitro using a multidrug-resistant ST175 isolate and representative isolates from major P. aeruginosa clades. In vivo testing included ventilator associated pneumonia by MDR P. aeruginosa in ICU (3-10 sequential samples from 3 patients) and chronic respiratory infection by hypermutable P. aeruginosa in cystic fibrosis (8 sequential samples from a single patient covering a 4-year period). Results from direct sequencing with the enrichment panel were compared with those of whole genome sequencing (WGS) and phenotypic profiling of 10 isolated colonies per sample.
In vitro assays confirmed the selectivity of the enrichment panel and the correct identification of the vast mutational resistome of ST175, including specific mutations even when introduced in a 1:100 proportion. In vivo performance was at least equivalent to sequencing 10 colonies per sample, including the accurate identification of the sequence types and the basal and acquired mutational resistome. To note, specific resistance mutations, such as those in ampC leading to resistance to novel β-lactams, could be traced even at frequencies of 1%. Moreover, the coselection of mutator populations and antibiotic resistance mutations, predicted in theoretical and in vitro studies, was evidenced in vivo.
This proof-of-concept study demonstrates that resistance genomics of P. aeruginosa can be analysed directly from clinical samples, determining not only a considerable reduction in turnaround time and cost from a diagnostics perspective, but also an unprecedented potency for accurate monitoring of in vivo population dynamics in bacterial infections.
Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea-NextGenerationEU.
铜绿假单胞菌是医院获得性和慢性感染的主要原因，其特点是通过选择染色体突变来产生抗微生物药物耐药性的能力非凡，导致治疗失败。在这里，我们设计并测试了一种基于杂交的捕获系统，用于在测序前富集感兴趣的基因，以便直接从临床样本中监测耐药人群的基因组学。
设计了一个接近 200 个与铜绿假单胞菌抗微生物药物耐药性、多位点序列分型、可变性或毒力相关的基因的测序前富集面板，并用 KAPA HyperCap（罗氏）合成，并在体外使用多药耐药 ST175 分离株和主要铜绿假单胞菌谱系的代表性分离株进行了初步验证。体内测试包括 ICU 中由 MDR 铜绿假单胞菌引起的呼吸机相关性肺炎（来自 3 名患者的 3-10 个连续样本）和囊性纤维化中由高突变性铜绿假单胞菌引起的慢性呼吸道感染（来自单个患者的 8 个连续样本，涵盖 4 年时间）。用富集面板进行直接测序的结果与全基因组测序（WGS）和每个样本 10 个分离菌落的表型分析结果进行了比较。
体外试验证实了富集面板的选择性和 ST175 广泛的突变耐药组的正确鉴定，包括即使以 1:100 的比例引入的特定突变。体内性能至少与每个样本测序 10 个菌落相当，包括序列类型的准确鉴定以及基础和获得性突变耐药组。值得注意的是，即使在频率为 1%的情况下，也可以追踪到 ampC 导致对新型β-内酰胺类药物耐药的特定耐药突变。此外，在理论和体外研究中预测的突变体种群和抗生素耐药突变的共选择在体内得到了证实。
这项概念验证研究表明，可以直接从临床样本中分析铜绿假单胞菌的耐药基因组学，不仅从诊断的角度大大缩短了周转时间和成本，而且还具有对细菌感染中体内群体动态进行准确监测的前所未有的能力。
西班牙卡洛斯三世健康研究所、西班牙科学与创新部和欧盟下一代欧盟。
