Polymyxin B combined with amikacin delays the resistance of Klebsiella pneumoniae to polymyxin B by modulating the expression of NlpE

Objectives: This study investigated the mechanisms underlying polymyxin B (PMB)-induced resistance and examined the role of PMB in combination with amikacin (AMK) in delaying the development of resistance.

Methods: In vitro continuous induction of three Klebsiella pneumoniae strains was performed using PMB alone or in combination with AMK, and variations in the minimum inhibitory concentration (MIC) were determined via the microdilution method. The Kp81 strain, which presented the most significant delay in resistance development, was selected for analysis of the expression of relevant genes via qRT-PCR. Homologous recombination techniques were employed to construct nlpE and cpxR gene knockout and complemented Kp81 strains, and the expression levels of these genes, as well as changes in the MIC following continuous induction, were assessed. Additionally, the biofilm-forming abilities of the strains were analyzed via crystal violet staining and confocal laser scanning microscopy.

Results: The results indicate that the combination of PMB and AMK can delay the development of resistance to PMB in K. pneumoniae. qRT-PCR analysis demonstrated a significant increase in the expression of phoP, nlpE, cpxR, and acrA in the resistant strain Kp81·R, whereas these genes were significantly downregulated in the resistant delay strain Kp81·DR. Notably, after 20 h of PMB treatment, the expression of nlpE was markedly elevated in the nlpE knockout strain. cpxR exhibited synchronized dynamic changes with nlpE in both the nlpE knockout and complemented strains, whereas acrA showed a similar expression pattern in the cpxR knockout and complemented strains. Additionally, the absence of nlpE or cpxR was found to delay PMB resistance. Furthermore, biofilm formation was significantly increased in both the resistant strains and those treated with PMB, while the combination of PMB with AMK inhibited biofilm formation.

Conclusions: The combination of PMB and AMK can delay the development of resistance in K. pneumoniae to PMB. The nlpE gene, as a key regulatory factor, can influence PMB resistance by modulating the CpxA/R two-component system and biofilm formation.

Amikacin; Combination therapy; Delayed resistance; Klebsiella pneumoniae; Polymyxin B; Resistance mechanism.