Exploring the additive antibacterial potential of Cinnamomum cassia volatile oil and imipenem against Acinetobacter baumannii: a multi-omics investigation

Abstract

Introduction: Acinetobacter baumannii 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 A. baumannii. However, the antibacterial activity and mechanism of the volatile oil derived from the dried bark of Cinnamomum cassia (CBV), as well as its additive effect when combined with imipenem (IPM) against A. baumannii, remain unclear.

Methods: 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 A. baumannii 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 A. baumannii, 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 A. baumannii. 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 A. baumannii. Additionally, the antibacterial mechanism was elucidated through a combination of transcriptomic and proteomic analyses.

Results: An additive effect (FICI = 0.53) was observed when CBV and IPM were combined against A. baumannii, 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 A. baumannii. 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 A. baumannii. CBV and IPM also induced significant changes at both the transcriptomic and proteomic levels in A. baumannii. 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.

Conclusion: This study demonstrated that the combination of CBV and IPM exhibits promising antimicrobial activity against A. baumannii, 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 in vivo investigations are crucial to validate the antibacterial efficacy and safety profile of CBV/IPM prior to clinical application.

Keywords: Acinetobacter baumannii; antibiotic agents; antimicrobial activity; imipenem; volatile oil of Cinnamomum cassia.

Figure 1

(A) Time-kill curves showing the activity of CBV (0.125 μL/mL) and IPM (4 μg/mL), tested alone and in combination, against A. baumannii in vitro. Compared to the control, ^***p < 0.001. (B) Crystal violet staining assay evaluation of the anti-biofilm activity of CBV (0.125 μL/mL) and IPM (4 μg/mL), individually and in combination, against A. baumannii. ^***p < 0.001; ns means p > 0.05. (C) Changes in intracellular protein content of A. baumannii following treatment with CBV (0.125 μL/mL), IPM (4 μg/mL), or their combination. Compared to the control, ^***p < 0.001, ^*p < 0.05.

Figure 2

CBV, IPM, and their combination exert antibacterial effects by disrupting membrane integrity and permeability. Live/dead staining images of A. baumannii were obtained using a fluorescent microscope (200 × magnification). (SYTO 9) SYTO 9 stains all bacteria green, indicating high membrane permeability. (PI) PI only penetrates damaged membranes, staining bacteria with compromised membranes red. (PI/SYTO 9) Merged images of live/dead fluorescent staining. (SEM) Cell morphology of A. baumannii was observed using SEM at 10000x magnification.

Figure 3

Transcriptomic profiling of A. baumannii treated with CBV, IPM, or their combination. Control1-3: control groups (no exposure to CBV and IPM); IPM1-3: IPM group (exposed to 4 μg/mL IPM); CBV1-3: CBV group (exposed to 0.125 μL/mL CBV); Combined1-3: combination group (exposed to 4 μg/mL IPM and 0.125 μL/mL CBV). (A) The correlation heatmap demonstrated high reproducibility among biological replicates. (B) Principal component analysis (PCA) of transcriptomic profiles resulted in a clear separation of the three groups. The CBV-treated group and the combination-treated group clustered together. (C) Hierarchical clustering based on differentially expressed genes (DEGs) resulted in three clusters, which is consistent with the PCA results. (D) The Venn diagram displayed shared and group-specific DEGs among the treatment groups and the control.

Figure 4

Analysis of DEGs. (A) Volcano plot of DEGs in A. baumannii following CBV (0.125 μL/mL) treatment. (B) KEGG pathways enriched by DEGs following CBV treatment in A. baumannii. (C) Volcano plot of DEGs in A. baumannii following IPM (4 μg/mL) treatment. (D) KEGG pathways enriched by DEGs following IPM treatment in A. baumannii. (E) Volcano plot of DEGs in A. baumannii treated with CBV (0.125 μL/mL) and IPM (4 μg/mL). (F) KEGG pathways enriched by DEGs following CBV and IPM treatments in A. baumannii.

Figure 5

Proteomic profiling of A. baumannii treated with CBV (0.125 μL/mL), IPM (4 μg/mL), or their combination. (A) The coefficient of variation analysis demonstrated high consistency across biological replicates. (B) PCA of proteomic profiles showed clear separation among the three groups, with the control group and IPM-treated group clustering together. (C) GO enrichment analysis of the identified proteome following treatment with CBV, IPM, or their combination reveals the top 10 most enriched terms. (D) Hierarchical clustering based on differentially expressed proteins (DEPs) revealed a clear distinction among the groups.

Figure 6

Analysis of DEPs. Dots highlighted in red (FC > 1.2) and green (FC < 0.83) indicate proteins whose expression was significantly altered (p < 0.05). (A) Volcano plot of DEPs in A. baumannii following CBV treatment. (B) GO enrichment analysis of DEPs following CBV treatment. (C) KEGG pathways enriched in downregulated DEPs after CBV treatment. (D) Volcano plot of DEPs in A. baumannii following IPM treatment. (E) GO enrichment analysis of DEPs following IPM treatment. (F) KEGG pathways enriched in downregulated DEPs after IPM treatment. (G) Volcano plot of DEPs in A. baumannii following combination treatment. (H) GO enrichment analysis of DEPs following combination treatment. (I) KEGG pathways enriched in downregulated DEPs after combination treatment.