Calvo-Villamañán Alicia, Sastre-Dominguez Jorge, Barrera-Martín Álvaro, Costas Coloma, San Millan Álvaro
Department of Microbial Biotechnology, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.
Centro de Investigación Biológica en Red de Epidemiología y Salud Pública, Instituto de Salud Carlos III, Madrid, Spain.
Nat Commun. 2025 Aug 19;16(1):7700. doi: 10.1038/s41467-025-63082-1.
Conjugative plasmids are the main vehicle for the spread of antimicrobial resistance (AMR) genes in clinical bacteria. AMR plasmids allow bacteria to survive antibiotic treatments, but they also produce physiological alterations in their hosts that commonly translate into fitness costs. Despite the key role of plasmid-associated fitness effects in AMR evolution, their origin and molecular bases remain poorly understood. In this study, we introduce plasmid-wide CRISPR interference (CRISPRi) screens as a tool to dissect plasmid-associated fitness effects. We design and perform CRISPRi screens targeting the globally distributed carbapenem resistance plasmid pOXA-48 in 13 different multidrug resistant clinical Enterobacterales. Our results reveal that pOXA-48 gene-level effects are conserved across clinical strains, and expose the key role of the carbapenemase-encoding gene, bla, as the main culprit for pOXA-48 fitness costs. Moreover, our results highlight the relevance of postsegregational killing systems in pOXA-48 vertical transmission, and uncover new genes implicated in pOXA-48 stability (pri, korC, DNDJGHEP_13 and 14 and H-NS). This study sheds new light on the biology and evolution of carbapenem resistant Enterobacterales and endorses CRISPRi screens as a powerful method for studying plasmid-mediated AMR.
接合质粒是临床细菌中抗菌药物耐药性（AMR）基因传播的主要载体。AMR质粒使细菌能够在抗生素治疗中存活，但它们也会在宿主中产生生理变化，这些变化通常会转化为适应性代价。尽管质粒相关适应性效应在AMR进化中起着关键作用，但其起源和分子基础仍知之甚少。在本研究中，我们引入全质粒CRISPR干扰（CRISPRi）筛选作为剖析质粒相关适应性效应的工具。我们设计并在13种不同的多重耐药临床肠杆菌科细菌中对全球分布的碳青霉烯耐药质粒pOXA-48进行CRISPRi筛选。我们的结果表明，pOXA-48基因水平的效应在临床菌株中是保守的，并揭示了碳青霉烯酶编码基因bla作为pOXA-48适应性代价的主要罪魁祸首的关键作用。此外，我们的结果突出了后分离杀伤系统在pOXA-48垂直传播中的相关性，并发现了与pOXA-48稳定性相关的新基因（pri、korC、DNDJGHEP_13和14以及H-NS）。这项研究为耐碳青霉烯肠杆菌科细菌的生物学和进化提供了新的见解，并认可CRISPRi筛选作为研究质粒介导的AMR的有力方法。
