随着消毒剂的广泛滥用,肺炎克雷伯菌(K.肺炎)对氯己定(CHX)逐渐减少,给临床消毒和感染控制带来重大挑战。肺炎克雷伯菌利用外排泵的过表达和厚生物膜的形成来逃避CHX的致死作用。plumbagin(PLU)是一种天然植物提取物,可增强膜渗透性并降低质子动力。在这项研究中,我们阐明了PLU与CHX联合使用的协同抗菌活性,有效降低CHX对肺炎克雷伯菌的MIC至1µg/mL及以下。至关重要的是,通过结晶紫染色和共聚焦激光扫描显微镜活/死染色,我们发现PLU显著增强CHX的抗生物膜能力。机械上,涉及膜渗透性的实验,碱性磷酸酶渗漏,活性氧,和RT-qPCR表明,PLU和CHX的组合提高了细菌内膜和外膜的通透性,促进细菌氧化应激,并抑制oqxA/B外排泵表达。此外,我们对医疗器械进行了表面消毒实验,以模拟临床环境,证明该组合有效减少细菌负荷超过3log10CFU/mL。此外,抗性突变频率实验的结果表明,联合处理减少了细菌种群中抗性突变体的产生。总之,PLU可以作为佐剂,增强CHX的抗生物膜能力,减少抗性突变的发生,从而延长CHX的寿命。重要物质消毒剂在世界范围内被广泛和过度使用,临床病原体正在逐步获得对这些物质的耐药性。然而,高浓度的消毒剂会导致对抗生素的交叉耐药性,同时使用不同的消毒剂可以促进细菌抗性突变,促进抗性基因的水平转移,这对临床治疗提出了重大挑战。与研制新型消毒剂的漫长过程相比,用天然植物提取物提高现有消毒剂的有效性是重要和有意义的。与其他消毒剂相比,CHX特别常见且使用广泛。同时,肺炎克雷伯菌,作为一种临床上重要的病原体,表现出很高的抗性和致病性。以前的研究和我们的数据表明临床肺炎克雷伯菌对CHX的敏感性显着降低,强调迫切需要新的策略来解决这个问题。鉴于此,我们的研究是有意义的。
With the widespread misuse of disinfectants, the clinical susceptibility of Klebsiella pneumoniae (K. pneumoniae) to chlorhexidine (CHX) has gradually diminished, posing significant challenges to clinical disinfection and infection control. K. pneumoniae employs overexpression of efflux pumps and the formation of thick biofilms to evade the lethal effects of CHX. Plumbagin (PLU) is a natural plant extract that enhances membrane permeability and reduces proton motive force. In this study, we elucidated the synergistic antimicrobial activity of PLU in combination with CHX, effectively reducing the MIC of CHX against K. pneumoniae to 1 µg/mL and below. Crucially, through crystal violet staining and confocal laser scanning microscopy live/dead staining, we discovered that PLU significantly enhances the anti-biofilm capability of CHX. Mechanistically, experiments involving membrane permeability, alkaline phosphatase leakage, reactive oxygen species, and RT-qPCR suggest that the combination of PLU and CHX improves the permeability of bacterial inner and outer membranes, promotes bacterial oxidative stress, and inhibits oqxA/B efflux pump expression. Furthermore, we conducted surface disinfection experiments on medical instruments to simulate clinical environments, demonstrating that the combination effectively reduces bacterial loads by more than 3 log10 CFU/mL. Additionally, results from resistance mutation frequency experiments indicate that combined treatment reduces the generation of resistant mutants within the bacterial population. In summary, PLU can serve as an adjuvant, enhancing the anti-biofilm capability of CHX and reducing the occurrence of resistance mutations, thereby extending the lifespan of CHX.IMPORTANCEAs disinfectants are extensively and excessively utilized worldwide, clinical pathogens are progressively acquiring resistance against these substances. However, high concentrations of disinfectants can lead to cross-resistance to antibiotics, and concurrent use of different disinfectants can promote bacterial resistance mutations and facilitate the horizontal transfer of resistance genes, which poses significant challenges for clinical treatment. Compared with the lengthy process of developing new disinfectants, enhancing the effectiveness of existing disinfectants with natural plant extracts is important and meaningful. CHX is particularly common and widely used compared with other disinfectants. Meanwhile, Klebsiella pneumoniae, as a clinically significant pathogen, exhibits high rates of resistance and pathogenicity. Previous studies and our data indicate a significant decrease in the sensitivity of clinical K. pneumoniae to CHX, highlighting the urgent need for novel strategies to address this issue. In light of this, our research is meaningful.