Journal of global antimicrobial resistance · 2025
Effects of in vitro combination therapy with polymyxin B on delaying resistance in Klebsiella pneumoniae and insights into the mechanisms of polymyxin-induced resistance.
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Zhang Huanhuan, Feng Wei, Sun Fengjun, Xu Te, Qian Yan
Аннотация
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.
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.