Transcriptomic Insights into Adaptive Strategies of Klebsiella pneumoniae Co-Producing KPC-2 and NDM-5 Carbapenemases Under Meropenem Stress

Abstract

Liting Dai,1,&ast; Huimin Zhang,1,&ast; Yingyi Cao,1,&ast; Jiacheng Ding,1 Jiaxin Tang,2 Zhirou Xiao,2 Zhemei Chen,2 Jiahui Ling,1 Mengxue Zou,1 Xiwu Cao,1 Lijuan Lin,1 Ziheng Xu,1 Wanting Liu,1 Dingqiang Chen,1 Peibo Yuan1 1Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, 510120, People’s Republic of China; 2Department of Clinical Laboratory, National Center for Respiratory Medicine, National Clinical Research Center for Respiratory Disease, State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, 510120, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Peibo Yuan, Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, 510120, People’s Republic of China, Tel [phone], Email [email protected] Dingqiang Chen, Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, 510120, People’s Republic of China, Tel [phone], Email [email protected]: The emergence of Klebsiella pneumoniae strains that co-produce multiple carbapenemases poses significant threats to clinical management; however, the molecular adaptations driving their resilience remain poorly characterized.Methods: This study aimed to investigate the resistance and adaptive mechanisms of the blaKPC-2 and blaNDM-5 co-producing K. pneumoniae KP4 strains. The antimicrobial susceptibility of KP4 was determined using Vitek, a carbapenemase inhibitor enhancement assay, and single-cell Raman spectroscopy. Transcriptomic profiling was used to identify key pathways involved in meropenem tolerance and key differentially expressed genes (DEGs). Transcriptomic analysis mapped stress-responsive pathways and DEGs, whereas qPCR validated carbapenemase gene expression and plasmid copy number variation. Biofilm dynamics were assessed under antibiotic pressure conditions.Results: KP4 exhibited pan-drug resistance, while retaining tigecycline susceptibility. Meropenem exposure (256 mg/L) triggered 161 DEGs primarily associated with metabolic pathways, including arginine biosynthesis, amino acid metabolism, and secondary metabolite production. Notably, qPCR quantification of bacterial DNA revealed plasmid copy number amplification of blaNDM-5 (+2.58-fold, p< 0.05) and blaKPC-2 (+1.49-fold, p=0.156), which may be associated with upregulation of the repA gene on the blaKPC-2-located plasmid and the dnaA gene on the chromosome. Meropenem exposure enhanced biofilm formation by 42% (p< 0.01), driven by the upregulation trpE (tryptophan synthesis, 4.2-fold), wecC (exopolysaccharide production, 2.1-fold), and gcvA (glycine metabolism, 2.05-fold).Conclusion: blaKPC-2 and blaNDM-5 co-producing K. pneumoniae employ a two-pronged resistance strategy: blaNDM-5 plasmid amplification ensures enzymatic overdose, while biofilm induction creates physical barriers. These findings decode the survival strategies of pan-resistant pathogens and inform novel therapeutic approaches that target plasmid stability and biofilm disruption.Keywords: carbapenem-resistant Klebsiella pneumoniae, CRKP, blaKPC-2, blaNDM-5, transcriptome, plasmid copy number, biofilm

Keywords

• carbapenem-resistant Klebsiella pneumoniae
• CRKP
• blaKPC-2，blaNDM-5
• transcriptome
• plasmid copy number
• biofilm