Antibacterial activity and mechanism of naphthoquine phosphate against ceftazidime-resistant Acinetobacter baumannii via cell membrane disruption and ROS induction

Abstract

Introduction: Drug-resistant bacteria, particularly Acinetobacter baumannii, present a significant threat to global public health, highlighting the urgent need for novel antibacterial therapies. Drug repurposing has emerged as a promising strategy to accelerate therapeutic development by identifying new applications for existing pharmaceuticals. This study investigates the potential of naphthoquine phosphate (NQP), an antimalarial agent, as a broad-spectrum antibacterial candidate against the multidrug-resistant strain A. baumannii LAC-4.

Methods: To evaluate the antibacterial activity of NQP, we determined the minimum inhibitory concentration (MIC) against Acinetobacter baumannii LAC-4. Inhibition kinetics were analyzed to assess concentration-dependent effects. Membrane permeability assays were performed to examine NQP-induced changes in cell membrane integrity. Oxidative damage tests were conducted to investigate impacts on bacterial metabolic processes. Morphological changes in A. baumannii LAC-4 treated with NQP of MIC were observed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Additionally, transcriptome analysis was performed to identify disrupted physiological pathways associated with NQP exposure.

Results and discussion: NQP exhibited broad-spectrum antibacterial activity, with a MIC of 62.5 μg/mL against Acinetobacter baumannii LAC-4. Its inhibition kinetics curve confirmed a concentration-dependent inhibitory effect. Membrane permeability tests revealed that NQP disrupts cell membrane integrity, enhancing permeability-consistent with TEM/SEM observations showing significant structural damage in NQP-treated A. baumannii, including membrane rupture, cellular deformation, and cytoplasmic disorganization. Oxidative damage tests indicated NQP impacts bacterial metabolism, and transcriptome analysis further demonstrated that NQP disrupts multiple physiological pathways, primarily through enhanced membrane permeability and induced oxidative stress. These findings support NQP as a promising molecular scaffold for developing novel therapies against Acinetobacter baumannii infections, highlighting its potential in drug repurposing strategies for combating drug resistance.

Keywords: Acinetobacter baumannii; antibacterial activity; cell membrane permeability; naphthoquine phosphate; transcriptome analysis.

Figure 1

The molecular structure of NQP.

Figure 2

(A) Cytotoxic effect of different concentrations of NQP on A549 cells. Ns: no significance (p > 0.05), compared to the control group. (B) The hemolysis assay: the hemolysis percentage of NQP at different concentrations. (C) Visualization of tubes after incubation and centrifugation. C–: the negative control; C+: the positive control.

Figure 3

(A) Bacterial growth kinetics of NQP against Acinetobacter baumannii. (B) Time-kill test results curve for NQP against Acinetobacter baumannii LAC-4.

Figure 4

Bacterial survival fraction of Acinetobacter baumannii LAC-4 treated with different concentrations of NQP. PBS served as the negative control. Level of significance (p < 0.01) is indicated by **.

Figure 5

Detection of NQP-induced damage to the cell membranes of Acinetobacter baumannii LAC-4. (A) Cell membrane potential of Acinetobacter baumannii LAC-4 cells monitored with DiSC3(5) after incubation with NQP. (B) Intracellular ATP in bacterial cells treated with different concentrations of NQP. (C) The absorbsance at 420 nm in ONPG hydrolysis test. (D) The fluorescence intensity of PI was photographed using a fluorescence microscope and quantified using ImageJ software. Error bars show the SDs of experiments performed in triplicate. * Significantly different (p < 0.05); **** Significantly different (p < 0.0001).

Figure 6

(A) Scanning electron microscopy (SEM) and (B) transmission electron microscopy (TEM) images of Acinetobacter baumannii LAC-4 treated with 62.5 μg/mL NQP. The control group shows bacteria untreated with NQP.

Figure 7

Detection of NQP-induced oxidative damage mediated by reactive oxygen species in Acinetobacter baumannii LAC-4. (A) NADH; (B) H₂O₂; (C) ROS; (D) SOD; (E) MDA. Error bars show the SDs of experiments performed in triplicate. ns: no significance (P > 0.05); * Significantly different (p < 0.05); ** Significantly different (p < 0.01); *** Significantly different (p < 0.001); **** Significantly different (p < 0.0001).

Figure 8

Differentially expressed genes (DEGs) after NQP treatment of Acinetobacter baumannii LAC-4. (A) Expression heatmap of 498 DEGs analyzed by hierarchical clustering, where red indicates relatively high expression levels and blue indicates relatively low expression levels. (B) Volcano map of DEGs with 326 up-regulated genes (red dots) and 172 down-regulated genes (green dots). Blue dots indicate genes with no significant change in expression. MIC: Treated with a concentration of 62.5 μg/mL NQP; NT: The Control group.

Figure 9

Eight differentially expressed genes (DEGs) after NQP treatment of Acinetobacter baumannii LAC-4 identified by RNA-seq were validated using RT-qPCR. Red: RNA-seq; Blue: RT-qPCR. Error bars show the SDs of experiments performed in triplicate. 16s rRNA was used as the reference gene for normalization.

Figure 10

GO functional enrichment analysis and KEGG pathway enrichment after NQP treatment of Acinetobacter baumannii LAC-4. For genes with [|log₂FC| > 1 and p < 0.05], GO and KEGG enrichment analyses were performed. (A) Bar plots display the top 10 significantly enriched terms. (B) Bubble plots show the top 20 pathways, where bubble size represents gene count and color indicates p-value. BP: Biological Process; CC: Cellular Component; MF: Molecular Function.