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

BACKGROUND

The emergence of 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 and co-producing 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 (+2.58-fold, <0.05) and (+1.49-fold, =0.156), which may be associated with upregulation of the gene on the -located plasmid and the gene on the chromosome. Meropenem exposure enhanced biofilm formation by 42% (<0.01), driven by the upregulation (tryptophan synthesis, 4.2-fold), (exopolysaccharide production, 2.1-fold), and (glycine metabolism, 2.05-fold).

CONCLUSION

and co-producing employ a two-pronged resistance strategy: 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.