Effects of in vitro combination therapy with polymyxin B on delaying resistance in Klebsiella pneumoniae and insights into the mechanisms of polymyxin-induced resistance.

OBJECTIVE

This study aimed to investigate the effects of polymyxin B (PMB) in combination with other antibiotics on delaying resistance in Klebsiella pneumoniae and to explore the mechanisms underlying PMB-induced resistance.

METHODS

In vitro continuous induction experiments were performed to observe changes in drug susceptibility with PMB alone vs. in combination. RNA-seq, quantitative reverse transcription PCR, and proteomic analyses were utilized to evaluate differential gene and protein expression between induced-resistant strains and those exhibiting delayed resistance. Then, gene knockout experiments were performed to validate the functional roles of relevant genes.

RESULTS

These findings indicated that PMB alone could induce resistance within 1-2 d, whereas the combination with amikacin (AMK) or tigecycline delayed the onset of resistance by 6 d. RNA-seq, quantitative reverse transcription PCR, and proteomic analyses revealed significant upregulation of nlpE, two-component systems, and AcrAB-TolC efflux pump-associated genes in PMB-induced resistant strains, whereas these genes were downregulated in the delayed resistant strains of PMB combined with AMK. Deletion and complementation experiments demonstrated that the expression levels of two-component systems and efflux pump-related genes were downregulated in nlpE knockout strains. Furthermore, PMB induction experiments revealed a significant upregulation of PmrA, PhoP, PhoQ, PagP, and AcrB proteins associated with cationic antimicrobial peptide pathways in the wild-type and nlpE complemented strains, whereas no differential change was observed in the nlpE knockout strain.

CONCLUSIONS

nlpE contributes to PMB resistance by modulating the AcrAB-TolC efflux pump and the PhoP/Q and PmrA/B two-component systems. The combined use of PMB with AMK effectively delays the development of resistance in K. pneumoniae through the regulation of nlpE and its associated signalling pathways.