Meekes Lisa M, Heikema Astrid P, Tompa Manuela, Astorga Alsina Ana L, Hiltemann Saskia D, Stubbs Andrew P, Dekker Lennard J M, Foudraine Dimard E, Strepis Nikolaos, Pitout Johann D D, Peirano Gisele, Klaassen C H W, Goessens W H F
Department of Medical Microbiology & Infectious Diseases, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands.
Central Laboratory, Regional Institute of Gastroenterology and Hepatology "Prof. Dr. Octavian Fodor", Cluj-Napoca, Romania.
Antimicrob Agents Chemother. 2025 Jul 2;69(7):e0010725. doi: 10.1128/aac.00107-25. Epub 2025 Jun 13.
Antimicrobial resistance arises from complex genetic and regulatory changes, including single mutations, gene acquisitions, or cumulative effects. Advancements in genomics and proteomics facilitate a more comprehensive understanding of the mechanisms behind antimicrobial resistance. In this study, 74 clinically obtained isolates with increased meropenem and/or imipenem MICs were characterized by broth microdilution and PCR to check for the presence of carbapenemase genes. Subsequently, a representative subset of 15 isolates was selected for whole-genome sequencing (WGS) by Illumina and Nanopore sequencing, and proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the mechanisms underlying the differences in carbapenem susceptibility of isolates. Identical techniques were applied to characterize four mutants obtained after sequential meropenem exposure. We demonstrated that in clinically obtained isolates, increased copy numbers of -containing plasmids, combined with OmpK36 loss, contributed to high carbapenem MICs without the involvement of OmpK35 or other porins or efflux systems. In the meropenem-exposed mutants, increased copy numbers of or -containing plasmids, combined with OmpK36 loss, were demonstrated. The OmpK36 loss resulted from the insertion of IS1 transposable elements or partial deletion of the gene. Additionally, we identified two mutations, C59A and C58A, in the DNA coding the copA antisense RNA of IncFII plasmids and multiple mutations of an IncR plasmid, associated with increased plasmid copy numbers. This study demonstrates that by combining WGS and LC-MS/MS, the effect of genomic changes on protein expression related to antibiotic resistance and the mechanisms behind antibiotic resistance can be elucidated.
抗菌药物耐药性源于复杂的基因和调控变化，包括单基因突变、基因获得或累积效应。基因组学和蛋白质组学的进展有助于更全面地了解抗菌药物耐药性背后的机制。在本研究中，通过肉汤微量稀释法和PCR对74株临床分离的美罗培南和/或亚胺培南最低抑菌浓度（MIC）升高的菌株进行了鉴定，以检测碳青霉烯酶基因的存在。随后，选择15株具有代表性的菌株进行全基因组测序（WGS），采用Illumina和纳米孔测序技术，并通过液相色谱-串联质谱（LC-MS/MS）进行蛋白质组学分析，以研究菌株对碳青霉烯类药物敏感性差异背后的机制。采用相同的技术对经连续美罗培南暴露后获得的4个突变体进行了鉴定。我们证明，在临床分离菌株中，含 质粒的拷贝数增加，与外膜孔蛋白K36（OmpK36）缺失共同导致了高碳青霉烯MIC，而未涉及外膜孔蛋白K35或其他孔蛋白或外排系统。在美罗培南暴露的突变体中，证明了含 或 质粒的拷贝数增加，与OmpK36缺失共同存在。OmpK36缺失是由IS1转座元件的插入或 基因的部分缺失导致的。此外，我们在编码IncFII质粒copA反义RNA的DNA中鉴定出两个突变，即C59A和C58A，以及IncR质粒的多个突变，这些突变与质粒拷贝数增加有关。本研究表明，通过结合WGS和LC-MS/MS，可以阐明基因组变化对抗生素耐药性相关蛋白质表达的影响以及抗生素耐药性背后的机制。
