Abstract
BACKGROUND: 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 bla (KPC-2) and bla (NDM-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 bla (NDM-5) (+2.58-fold, p<0.05) and bla (KPC-2) (+1.49-fold, p=0.156), which may be associated with upregulation of the repA gene on the bla (KPC-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: bla (KPC-2) and bla (NDM-5) co-producing K. pneumoniae employ a two-pronged resistance strategy: bla (NDM-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.