Lu Youyuan, Xu Wanlin, Xue Jiahui, Xie Mingxia, Liu Haotian, Wang Ruilin, Li Gang, Wang Hanqing
College of Pharmacy, General Hospital of Ningxia Medical University, Ningxia Medical University, Yinchuan, China.
Key Laboratory of Protection, Development and Utilization of Medicinal Resources in the Liupanshan Area, Ministry of Education, Ningxia Engineering and Technology Research Center for Modernization of Regional Characteristic Traditional Chinese Medicine, Ningxia Medical University, Yinchuan, China.
Front Microbiol. 2025 Jul 2;16:1578322. doi: 10.3389/fmicb.2025.1578322. eCollection 2025.
has been identified as a critical pathogen, and new antibiotics are urgently needed. Volatile oils, which function as natural antibacterial agents, may provide an effective means of inhibiting . However, the antibacterial activity and mechanism of the volatile oil derived from the dried bark of (CBV), as well as its additive effect when combined with imipenem (IPM) against remain unclear.
CBV was extracted using the hydrodistillation method and characterized by gas chromatography-mass spectrometry (GC-MS) analysis. The minimum inhibitory concentrations (MICs) of CBV and IPM against were determined using the microdilution method. A checkerboard assay was performed to evaluate the additive effect of CBV (concentration range: 0-1 μL/mL) and IPM (concentration range: 0-256 μg/mL) against , with the fractional inhibitory concentration index (FICI) calculated. A time-kill curve analysis was performed to assess the additive effect of CBV (0.125 μL/mL) and IPM (4 μg/mL) against . Antibiofilm activity was evaluated using a crystal violet staining assay. Cell membrane integrity was assessed using SYTO 9/PI staining based on fluorescence color. Intracellular protein levels were quantified using a BCA kit according to the manufacturer's instructions. Scanning electron microscopy (SEM) was used to observe morphological changes in . Additionally, the antibacterial mechanism was elucidated through a combination of transcriptomic and proteomic analyses.
An additive effect (FICI = 0.53) was observed when CBV and IPM were combined against , reducing the MIC of IPM from 256 μg/mL to 4 μg/mL. CBV and IPM inhibited biofilm formation, damaged the cell membrane, and induced intracellular protein leakage in . Compared to CBV or IPM alone, the combination group (at the dosage showing an additive effect) caused significantly greater damage to the cell membrane of . CBV and IPM also induced significant changes at both the transcriptomic and proteomic levels in . Functional analysis revealed that the differentially expressed genes (DEGs) and proteins (DEPs) were involved in multiple pathways. Both CBV and IPM contributed to the observed antibacterial activity. CBV primarily influenced the ribosome pathway, while IPM mainly influenced oxidative phosphorylation. In the combination treatment, the simultaneous targeting of the ribosome and oxidative phosphorylation pathways was identified as the key antibacterial mechanism.
This study demonstrated that the combination of CBV and IPM exhibits promising antimicrobial activity against , suggesting that CBV could serve as a potential natural candidate for the development of novel antibiotic agents. While the current findings establish a mechanistic foundation for CBV's antimicrobial effects, further research is necessary to facilitate its clinical translation. Specifically, formulation optimization studies are necessary to enhance the therapeutic viability of the CBV/IPM combination, and comprehensive investigations are crucial to validate the antibacterial efficacy and safety profile of CBV/IPM prior to clinical application.
已被确认为一种关键病原体，迫切需要新型抗生素。挥发油作为天然抗菌剂，可能提供一种抑制该病原体的有效手段。然而，[植物名称]干燥树皮挥发油（CBV）的抗菌活性及其作用机制，以及与亚胺培南（IPM）联合使用时对该病原体的增效作用仍不清楚。
采用水蒸馏法提取CBV，并通过气相色谱 - 质谱联用（GC - MS）分析进行表征。使用微量稀释法测定CBV和IPM对该病原体的最低抑菌浓度（MIC）。进行棋盘法试验以评估CBV（浓度范围：0 - 1μL/mL）和IPM（浓度范围：0 - 256μg/mL）对该病原体的增效作用，并计算分数抑菌浓度指数（FICI）。进行时间 - 杀菌曲线分析以评估CBV（0.125μL/mL）和IPM（4μg/mL）对该病原体的增效作用。使用结晶紫染色试验评估抗生物膜活性。基于荧光颜色，使用SYTO 9/PI染色评估细胞膜完整性。根据制造商说明，使用BCA试剂盒定量细胞内蛋白质水平。使用扫描电子显微镜（SEM）观察该病原体的形态变化。此外，通过转录组学和蛋白质组学分析相结合阐明抗菌机制。
CBV与IPM联合使用时对该病原体表现出增效作用（FICI = 0.53），将IPM的MIC从256μg/mL降至4μg/mL。CBV和IPM抑制生物膜形成，破坏细胞膜，并诱导该病原体细胞内蛋白质泄漏。与单独使用CBV或IPM相比，联合组（在显示增效作用的剂量下）对该病原体的细胞膜造成的损伤明显更大。CBV和IPM在该病原体的转录组学和蛋白质组学水平上也诱导了显著变化。功能分析表明，差异表达基因（DEG）和蛋白质（DEP）参与多种途径。CBV和IPM均对观察到的抗菌活性有贡献。CBV主要影响核糖体途径，而IPM主要影响氧化磷酸化。在联合治疗中，同时靶向核糖体和氧化磷酸化途径被确定为关键抗菌机制。
本研究表明，CBV与IPM联合使用对该病原体具有良好的抗菌活性，表明CBV可作为开发新型抗生素药物的潜在天然候选物。虽然目前的研究结果为CBV的抗菌作用建立了机制基础，但仍需要进一步研究以促进其临床转化。具体而言，需要进行制剂优化研究以提高CBV/IPM联合制剂的治疗可行性，并且在临床应用前进行全面的安全性研究对于验证CBV/IPM的抗菌疗效和安全性至关重要。
