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Article

Lipidome Disturbances of Vibrio alginolyticus Associated with Citral Exposure

by
Yanni Zhao
1,2,
Zi Wang
1,
Jie Han
1,
Yi Wang
1,
Jiamin Ren
1,
Ting Shao
1,
Hua Li
3,* and
Huan Liu
1,2,*
1
School of Food Science and Engineering, Shaanxi University of Science & Technology, Xi’an 710021, China
2
Shaanxi Research Institute of Agricultural Products Processing Technology, Xi’an 710021, China
3
SUSTech Core Research Facilities, Southern University of Science and Technology, Shenzhen 518055, China
*
Authors to whom correspondence should be addressed.
Microorganisms 2026, 14(2), 509; https://doi.org/10.3390/microorganisms14020509
Submission received: 27 January 2026 / Revised: 12 February 2026 / Accepted: 20 February 2026 / Published: 22 February 2026
(This article belongs to the Section Biofilm)
Browse Figures
Versions Notes

Abstract

Vibrio alginolyticus is an important antibiotic-resistant pathogen in aquaculture that can cause mortality in a wide range of aquatic animals and infect humans. It is urgently necessary to discover and develop effective antibiotic alternatives. Citral, a key antibacterial component of lemongrass oil, can be used as a food flavoring and additive. Although the antimicrobial activity and antibiofilm effect of citral against V. alginolyticus have been noted in our previous study, the potential lipidome influence of citral remains unclear. Accordingly, a non-targeted lipidomics approach was employed to investigate citral-induced lipidome disturbances and reveal potential regulated targets of citral against V. alginolyticus. We found that the citral exposure triggered substantial lipidome alterations (i.e., composition, contents, and structure) in V. alginolyticus. Specifically, the content of most phospholipids (e.g., phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylserines (PSs), phosphatidylinositols (PIs), and phosphatidylglycerols (PGs)) decreased with the increase in citral concentration, while ceramides (Cers) and lysophospholipids (LPLs) (e.g., lyso-PAs, lyso-PCs, lyso-PEs, and lyso-PGs) showed concentration-dependent accumulation under citral treatment. Notably, the critical lipid remodeling in response to citral exposure mainly involved the phospholipid and sphingolipid metabolic pathways. Collectively, our study reveals the bacterial lipidome response to citral exposure and highlights pivotal metabolic pathways, potentially offering a novel perspective for future investigations into lipid-centric antibacterial targets.

1. Introduction

Vibrio alginolyticus, as a facultatively anaerobic, Gram-negative marine bacterium, is extensively found in coastal regions, rivers, and aquatic environments globally [1]. The characteristic virulence factors of V. alginolyticus mainly include motility, biofilm, extracellular products (e.g., lipopolysaccharides, hemolysin and extracellular alkaline serine proteases), adhesins, the iron uptake system, and the quorum-sensing system (QS), among others [2,3]. Fish [4], shellfish [5], crabs [6], prawns [7] and other mariculture species are susceptible to infection by V. alginolyticus, leading to substantial economic losses for the mariculture industry. Furthermore, V. alginolyticus, as a zoonotic pathogen, can cause human disease, otitis media, traumatic infection, septicemia, and other symptoms through the consumption of contaminated seafood or direct exposure to seawater [8,9,10].
Antibiotics are extensively utilized in aquatic animals to prevent and cure infections caused by V. alginolyticus. However, the widespread use of antibiotics in aquaculture and the environment has caused serious ecological and environmental pollution, resulting in the emergence of antibiotic-resistant bacteria [11]. Previous investigations have shown that many species of Vibrios with antibiotic resistance genes have been detected in marine aquaculture animals and the surrounding environments [12]. Currently, the European Union, China and the United States of America have successively banned the abuse of antibiotics in aquaculture. There is an enormous demand to develop novel and effective bacteriostatic substances to replace conventional antibiotics [13]. Bioactive substances identified as probiotics, enzyme preparations, and plant extracts have been regarded as possible antibiotic alternatives due to their various benefits [14]. Plant-derived antimicrobial agents, as a natural and safe type of compound, possess broad-spectrum antibacterial and fungicidal activity, and have become a research focus across multiple fields, including food, medicine, and cosmetics, among others [15,16].
Citral, as an acyclic monoterpenoid aldehyde with a lemon flavor in aromatic plant essential oils (EOs), exhibits notable antibacterial, antioxidant, and antiviral functions [17], which are attributed to its chemical structure of citral, which allow its accumulation in the cell membranes and walls in microorganisms, thereby disrupting the membrane integrity and inhibiting microbial growth [18,19,20]. Furthermore, the α- and β-unsaturated aldehyde moieties of citral show high electrophilic reactivity, which allows citral to participate in nucleophilic addition reactions with the electron-rich residues on the surface of membrane proteins (such as cysteine residues), leading to conformational changes or potential deactivation of the membrane proteins [21,22]. Our previous research has demonstrated that citral at minimum inhibitory concentration (MIC) can disrupt cell membranes and cell walls, leading to the leakage of nucleic acids and proteins within the cells of V. alginolyticus [23] and V. parahaemolyticus [24]. In addition, citral can diminish the pathogenicity of V. alginolyticus by suppressing the expression of virulence factors (e.g., motility, biofilm formation, and alkaline serine protease production) [23]. Although citral showed potential as a novel natural bacteriostatic agent and an antibiotic alternative for pathogen prevention and control, the mechanism by which citral impaired the cell membrane integrity of V. alginolyticus remains inadequately elucidated.
Lipids, as a crucial constituent of cell membranes, serve a multitude of functions in membrane structural scaffolding and various cellular communication processes [25]. Substantial research has demonstrated that bacteriostatic agents can induce alterations in lipid metabolism of bacterial cells [26,27]. The contents of glycerophospholipids, glycolipids, and sphingomyelin, which were significantly correlated with membrane stability, underwent notable alterations in methicillin-resistant Staphylococcus aureus following treatment with menthol [28]. Cinnamaldehyde could significantly reduce the synthesis pathway of glycerophospholipids within S. aureus and Escherichia coli, leading to substantial damage to the cell membrane’s integrity [29]. Lipidomics, as a potent analytical approach in systems biology, enables comprehensive qualitative and quantitative profilings of lipids within cellular or tissue samples to elucidate the relationship between signal transduction pathways mediated by lipid metabolism and pathophysiological alterations [30]. To our knowledge, research on the inhibitory impacts of citral on V. alginolyticus at the lipid pattern level has not been reported. Consequently, our study employed a non-targeted lipidomics approach based on ultra-high-performance liquid chromatography–mass spectrometry (UHPLC-MS) to determine the alterations of lipid metabolism in V. alginolyticus under citral exposure and to reveal prospective targets for prevention and treatment to mitigate infections caused by V. alginolyticus in aquaculture.

2. Materials and Methods

2.1. Chemicals and Reagents

Citral (CAS 5392-40-5) was purchased from Shanghai Yuanye Biotechnology Co., LTD (Shanghai, China). Ammonium acetate (AmAc) was provided by Adamas (Shanghai, China). HPLC-grade methanol (MeOH), methyl tert-butyl ether (MTBE), acetonitrile (ACN) and isopropyl alcohol (IPA) were obtained from Merck (Darnstadt, Germany) or Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water was from Milli-Q water purification system (Millipore, MA, USA).

2.2. Bacterial Culture and Collection

V. alginolyticus EPGS (CCTCC No. AB209306) was provided by East China University of Science and Technology. The overnight V. alginolyticus cultures were collected, then inoculated to the fresh LBS (Luria–Bertani (LB) supplemented with 3% (w/v) NaCl) medium with 100 μg/mL ampicillin (Amp), and then agitated at 200 rpm at 37 °C for 9 h. According to our previous study, the cultures were subsequently supplemented with citral at different concentrations (Control (CK), 1/2 MIC (0.0625 mg/mL) and MIC (0.125 mg/mL)) and incubated for 3 h with shaking at 200 rpm. Following centrifugation at 7000 rpm and 4 °C for 3 min to remove the supernatant, bacterial samples were collected and promptly frozen in liquid nitrogen to inhibit enzyme activity and terminate metabolic processes. Subsequently, the bacterial samples underwent two rinses with chilled physiological saline and were then subjected to vortexing for 5 min to eliminate residual media. After centrifugation at 14,000 rpm for 10 min at 4 °C, the resultant pellet was placed in a Simon Freeze-Dry concentrator (Los Angeles, CA, USA) to obtain uniform bacteria powder for lipidomic analysis.

2.3. Lipidome Extraction

Bacterial lipids were extracted according to the previous report with minor modifications [31]. Briefly, 20 mg of bacterial powder was weighed in a 2 mL Eppendorf tube and immersed in 300 μL MeOH and 1 mL of MTBE. Following 1 h of vortexing, 300 μL of milli-Q water was introduced to generate two distinct phases. The upper layer, specifically the MTBE layer, which contained residual hydrophobic metabolites, was gathered after centrifugation and then freeze-dried. The lipid extracts obtained from the V. alginolyticus were stored at −80 °C. Six independent biological replicates were obtained from the CK group and the citral treatment groups. Quality control samples (QC) were obtained by uniform mixing of equal-quality bacterial powder of each sample.

2.4. LC-MS-Based Lipidomic Data Acquisition

A UHPLC-quadrupole orbitrap high-resolution mass spectrometer (UHPLC-QE-Orbitrap MS) (Thermo Fisher Scientific, Waltham, MA, USA) was used to examine the lipidome. The specific analysis conditions of LC were as follows: Waters ACQUITY UPLC C8 column (1.7 µm, 2.1 × 100 mm, Waters, Milford, MA, USA) was employed for separation in both positive and negative ion modes. The column temperature was kept at 55 °C with a flow rate of 0.26 mL/min. Mobile phase A consisted of ACN and water in a 6:4 ratio (containing 10 mM AmAc), while mobile phase B comprised IPA and ACN in a 9:1 ratio (containing 10 mM AmAc). The elution gradient was as follows: starting with 32% B and maintaining for 1.5 min, followed by a linear increase to 85% B over 14 min, then reaching 97% B within 0.1 min and maintaining it for 2.4 min, before returning to the initial mobile phase equilibrium for 1.9 min.
The mass spectrometer utilized an electrospray ionization (ESI) ion source to deionize the lipids. In the positive ionization mode, the spray voltage was set at 3.5 kV, with a scanning range of 300 to 2000 m/z. The temperature of the ion transport tube was maintained at 300 °C, while the sheath gas flow rate was 45 L/min and the auxiliary gas flow rate was 10 L/min. Conversely, in the negative ionization mode, the spray voltage was increased to 3.8 kV, with a scanning range of 150 to 1500 m/z. The ion transport tube temperature was elevated to 320 °C, the sheath gas flow rate was adjusted to 30 L/min, and the auxiliary gas flow rate remained at 10 L/min. Quality control samples were systematically introduced into the analytical process to assess the instrument’s stability and the experimental methods’ reliability.
The lipid abbreviations used in this study are as follows: (lyso-)phosphatidic acid, LPA/PA; (lyso-)phosphatidylcholine, LPC/PC; (lyso-)phosphatidylethanolamine, LPE/PE; (lyso-)phosphatidylglycerol, LPG/PG; (lyso-)phosphatidylinositol, LPI/PI; phosphatidylserine, PS; diacylglycerol, DG; triacylglycerol, TG; sphingomyelin, SM; ceramide, Cer; glucosylceramides, CerG1; coenzyme Q, CoQ.

2.5. Data Processing and Statistics

Lipid Search (Thermo Fisher Science, Waltham, MA, USA) and Lipid Maps databases (http://www.lipidmaps.org/) (accessed on 25 March 2025) were used to identify lipid species. The exact mass measurement, retention behavior and specific fragment ions of lipids were utilized to validate the identification. Quantitative levels of lipid species were assessed via ion feature and retention time (RT) using the X-Calibur software (version 4.0). The m/z tolerance was ± 0.5 ppm, and the retention time extraction window was ± 0.5 min.
Prior to statistical analysis, the peak area of all lipid features was normalized to the total peak area to eliminate system variance. The normalized data set was imported into SIMCA-P (version 14.0, Umeå, Sweden) for principal component analysis (PCA) and S-plot diagramming. The non-parametric test (Mann–Whitney U) between the three strains was used to compare lipid differences and screen different lipids with significant differences (p < 0.05). Hierarchical cluster analysis (HCA) was performed by Multi Experiment Viewer (version 4.7.4, Boston, MA, USA). ChiPlot cloud platform (https://www.chiplot.online/) (accessed on 28 November 2025) was applied to evaluate the volcano map. Lipid metabolic pathway analysis was carried out utilizing the Lipid Maps data.

3. Results

3.1. Global Profiling of Lipidome Changes

The separation of lipids was achieved by gradient elution, and the total ion current (TIC) diagrams were generated in positive and negative ion modes (Figure 1A,B). The major lipids identified in the positive ion mode were glycerides, sphingolipids, and glycerophospholipids. LPLs were the predominant lipids identified in the negative ion mode. The large-scale lipid profiling of the QC sample identified over 900 lipids. Among them, a total of 593 lipids were identified, involving 17 subtypes (Figure 1C). There were 465 glycerophospholipids (112 PGs, 104 PEs, 98 PCs, 80 PAs, 26 PIs, 25 PSs, 10 LPEs, 5 LPGs, 3 LPCs, 1 LPA, 1 LPI), 62 glycerolipids (52 TGs, 10 DGs), 63 sphingolipids (31 SMs, 30 Cers, 2 CerG1s), and 3 ubiquinones (3 CoQs) (Figure 1C). According to quantitative results, V. alginolyticus had the highest number of glycerophospholipids, making up 78.41% of all the lipid molecules. Among them, the primary glycerophospholipid species were PEs (17.54%), PGs (18.89%), PCs (16.53%), and PAs (13.49%). The PCA model was used to evaluate the quality of the lipid mass spectrum. The QCs were tightly clustered and significantly separated from the test samples, indicating that the instrument was stable and the data obtained in this experiment were reliable (Figure S1).
The lipidome changes in V. alginolyticus following citral treatment were subsequently displayed on a PCA score plot. The results indicated a distinct separation tendency among CK, 1/2 MIC, and MIC on the first principal component. Furthermore, the divergence between the samples escalates with rising citric acid content (Figure 1D), which was corroborated by the Euclidean-distance map of PCA (Figure 1E). Univariate statistical analysis was used to analyze the differential lipids of V. alginolyticus caused by citral. The results showed that a total of 347 differential lipid molecules were obtained between the citral treatment group (1/2 MIC and MIC) and the CK group (p < 0.05). Among them, 193 and 313 different lipid molecules were found at 1/2 MIC vs. CK and MIC vs. CK, respectively, of which 159 lipids displayed common changes between the two groups (1/2 MIC vs. CK and MIC vs. CK) (Figure 2A). The alterations of differential lipids were further visualized using a volcano plot. The results showed that 69 and 124 lipids were significantly down-regulated and up-regulated at 1/2 MIC compared to CK (p < 0.05) (Figure 2B). Compared to CK, 105 and 208 lipids exhibited substantially decreased and increased levels at MIC, respectively (Figure 2C). The above results indicated that the quantity of differential lipids increased in a concentration-dependent manner with the rise in citral concentration. Moreover, a substantial overlap of different lipids was observed between the two groups (1/2 MIC vs. CK and MIC vs. CK). Specifically, 56 of the 69 lipids that were significantly down-regulated at 1/2 MIC were also present in the down-regulated list at MIC. Of the 124 lipids up-regulated at 1/2 MIC, 97 were also included in the up-regulated list at MIC.

3.2. Citral Exposure Disturbs Lipidome Homeostasis

Differential lipidome analysis showed that citral changes the lipid metabolism of V. alginolyticus. The sum responses of each lipid class were hierarchically clustered to visualize the overall lipid perturbation pattern and discover co-regulated lipid responses among distinct groups. According to the Pearson correlation coefficient between lipid abundances, these lipids were mainly clustered into two categories (group I and group II) (Figure 3). Compared with the CK group, the abundances of most of the glycerophospholipids (PGs, PEs and PSs) displayed decreased abundance at 1/2 MIC and MIC (group I). On the contrary, group II, including glycerophospholipids (e.g., LPAs, LPEs, and LPGs) and sphingolipids (CerG1s), showed increased abundance in citral-treated groups (MIC and 1/2 MIC) compared to the CK. Furthermore, glycerides (DGs and TGs) and Cers were significantly more abundant in the MIC group than in the CK (Figure 3). Specific quantities of individual lipids were provided in Supplementary Materials (Table S1). The above results indicated that citral seriously disturbed the lipid compositions of V. alginolyticus, especially the glycerophospholipids.
Subsequently, we further studied the structural changes in glycerophospholipids with affected abundances, and the results indicated that the acyl chain carbon numbers of differential glycerophospholipids primarily ranged between 20 and 50 (Figure 4A,C). In addition, in comparison to CK, the number of double bonds of differential glycerophospholipids was from 0 to 5, and the fold variations of these differential lipids between citral-treated groups and the CK displayed an upward trend across the increase in citral concentration (Figure 4B,D).
To identify the alterations to characteristics in glycerophospholipids under different citral exposure levels, we delineated the metabolic pathway of glycerophospholipids through Lipid Maps (Figure 5A). An increase in LPL was noticed alongside a reduction in the corresponding diacyl phospholipids. Specifically, compared with the CK, the concentrations of LPGs, LPEs, LPAs and LPCs were increased at 1/2 MIC and MIC, whereas the levels of PGs, PEs, PIs and PSs were decreased at 1/2 MIC and MIC (Figure 5A). The total LPLs at 1/2 MIC and MIC increased to 1.63 and 2.43 times that of the CK. Conversely, the relative concentration of the sum of phospholipids at 1/2 MIC and MIC decreased to 0.84 and 0.70 times that of the CK (Figure 5B).
To identify candidate markers closely related to citral treatment, we ulilized SIMCA-P software to plot the S-plot and identify key differential lipid molecules based on the absolute values of their projection points from the origin p [1] (representing the contribution of each variable to the discriminant component) and p (corr) [1] (indicating the statistical reliability of that contribution). The findings indicated that compared with the CK, a total of 10 critical differential lipids (i.e., | p [1] | > 0.1, | p (corr) [1] | > 0.8) were screened between 1/2 MIC and CK. Among them, PA (20:0/22:6), PG (20:0/16:1), PE (20:0/18:2), PE (20:1/18:1), PC (18:1p/16:1), PC (21:0/14:3), PE (17:0/18:2), PE (17:1/18:1), PE (19:1/16:1) were significantly down-regulated, whereas only LPE (18:1) was significantly up-regulated at 1/2 MIC compared to CK (Figure 6A). Moreover, 15 lipids have been chosen as potential candidate markers (i.e., | p [1] | > 0.1, | p (corr) [1] | > 0.8) between MIC and CK. Among them, six lipids (i.e., LPE (18:1), LPE (16:0), LPG (18:1), PC (11:0/14:0), PC (12:0/13:0), LPE (18:0)) and eight lipids (i.e., PC (18:1p/16:1), PA (20:0/22:6), PG (20:0/16:1), PE (17:0/18:2), PC (21:0/14:3), PG (18:1/20:4), PG (18:2/23:0), PI (16:1/10:2)) exhibited significantly up-regulation and down-regulation at MIC than those in the CK, respectively (Figure 6B).
Finally, six lipid molecules, namely, (PC (18:1p/16:1), PE (17:0/18:2), PE (19:1/16:1), LPG (18:1), LPE (16:0) and LPE (18:1)) were further filtered based on significant difference threshold value (p < 0.01, FC > ± 1.5, | p [1] | > 0.1, | p (corr) [1] | > 0.8). Compared to CK, three phospholipid molecules (PC (18:1p/16:1), PE (17:0/18:2) and PE (19:1/16:1)) and three LPL molecules (LPG (18:1), LPE (16:0) and LPE (18:1)) exhibited significantly downward and upward trends with the increase in citral concentration, respectively, which may serve as potential lipid-centric antibacterial targets for the control of V. alginolyticus (Figure 6C).

4. Discussion

V. alginolyticus not only induces diseases and mortality in multiple mariculture species but also poses a risk to human health [32]. Our previous study proved that citral has an antibacterial effect on V. alginolyticus through the determination of physiological indicators [23]. Lipids are regarded as the primary barrier to external signals and chemicals [33], and are also a key target of citral’s physiological effects on live cells [23]. In this study, we investigate the changes in lipid metabolism of V. alginolyticus under citral stress, providing a mechanistic foundation for its prevention and control. Our results showed that citral-induced lipid metabolism alterations in V. alginolyticus intensified along with increasing citral concentration (Figure 1D,E and Figure 2B,C), thus resulting in cell membrane instability and adversely impacting bacterial growth. The biomarker candidates were involved in glycerophospholipid metabolism, which is intricately linked to cell membrane integrity.

4.1. Glycerophospholipid Metabolism

Phospholipids are the major components of prokaryotic cell membranes and play an important role in maintaining cell membrane integrity, biosynthesis, signaling and other functional properties [34]. LPL is produced as a metabolic intermediate during phospholipid synthesis or membrane degradation [35]. Our study revealed that as citral concentration increased, phospholipid content diminished while LPL content increased, indicating that citral addition may facilitate phospholipid decomposition of the cell membrane, thereby impacting its integrity (Figure 5). Furthermore, the accumulation of LPE can trigger membrane stress events that threaten outer-membrane integrity [35], including heat shock [36], T4 phage-induced lysis [37] and antimicrobial lipopeptide exposure [38]. The cone-shaped LPEs can diminish the stored curvature stress, subsequently leading to the relaxation of the “frustrated” bilayer due to its inverted-cone-like geometrical shape and compromising the stability of the membrane structure [35]. This likely pertains to the pores in the cell membrane of V. alginolyticus that we previously identified, together with wrinkles and potential partial rupture [23]. The addition of LPCs modulated the activity of both the OmpF-like porin of Y. pseudotuberculosis (YOmpF) [39] and the mechanosensitive channel (MscL) of Escherichia coli [40]. In both cases, the mechanisms were suggested to entail intrinsic membrane curvature stress and the corresponding physical distortion of the lipid bilayer caused by inclusion of the LPL [35]. Our findings indicated that LPEs, LPCs and LPGs in V. alginolyticus accumulated with rising citral concentrations, potentially impacting membrane fluidity, permeability and cell shape. This result is consistent with our previous study [23], which illuminated citral-induced membrane invaginations and damages, resulting in the increase in conductivity and extracellular malondialdehyde levels of V. alginolyticus.
PC serves as a specific recognition molecule and can affect the physicochemical properties of bacterial membranes [41]. The absence of PC in the bacterial membrane phospholipids impaired the secretion of alkaline phosphatase (PhoA) via the Xcp type II secretion system (T2SS) [42] and facilitated the translocation of Sec-dependent β-lactamase (AmpC) from the cytoplasm to the periplasm in Pseudomonas [43]. In this study, PCs decreased in the 1/2 MIC group, potentially influencing the composition of membrane phospholipids and consequently impacting the output of proteins and membrane stability.
PG is a principal phospholipid constituent of the cell membrane in most Gram-negative bacteria, participating in membrane formation, signal transduction, and the regulation of various metabolic processes [44]. Kóbori et al. [45] investigated the cell division and metabolism of the Synechococcus wild strain, the PG-deficient strain, and the PG-complementing strain. They observed that the cell morphology of the PG-deficient strain significantly altered, while the PG-complementing strain reinstated the original cell structure and normal cell size, demonstrating that PGs were associated with bacterial metabolism and division. Our study demonstrated that citral reduces the levels of PGs in V. alginolyticus, suggesting that citral may disrupt the associated pathways of PGs and impair cell membrane formation and cell division, ultimately inhibiting the morphology of V. alginolyticus.
PE, as the predominant phospholipid in numerous prokaryotic cell membranes, plays important roles in various membrane functions [46]. Studies have shown that an E. coli PE-deficient mutant exhibited filamentous growth [47], and PE was essential for the virulence and sustained host interaction of Brucella abortus [48]. Our study demonstrated a substantial reduction in PE with increasing citral concentrations in V. alginolyticus, implying that citral supplementation could damage cell membrane integrity and bacterial pathogenicity, which was consistent with our previous study [23]. In addition, a substantial increase in the levels of LPEs and decrease in PEs were observed in the MIC strain (Figure 5), which probably implied high-rate catabolism of phospholipids to lysophosphatide.
PSs are negatively charged phospholipids present in relatively low content in the cell membrane [49]. Our study revealed that the PS content of V.alginolyticus significantly decreased with the increase in citral treatment concentration, indicating that citral may interfere with the synthesis or decomposition of PSs to cause the imbalance of membrane potential and affect the membrane structure. In addition, PSs serve as a key precursor for the synthesis of PEs [49]. Upon exposure to citral, V. alginolyticus may accelerate the conversion of PSs to PEs to compensate for the reduction in PE levels caused by decomposition into LPEs.

4.2. Sphingolipid Metabolism

Sphingolipids are amphiphilic molecules with both hydrophilic and hydrophobic characteristics, allowing them to engage in various intricate metabolic pathways in vivo. Sphingolipids, as integral components of cell and organelle membranes, not only preserve membrane stability and fluidity, but also significantly contribute to various biological processes, such as cell signal transduction, proliferation, oxidative stress management, and cell wall remodeling [50,51]. Specifically, Cer is a bioactive sphingolipid that modulates cell growth, senescence, adhesion, and migration [52]. This investigation indicated that Cer levels in the bacteria escalated with rising citral concentration, a result perhaps linked to sphingomyelinase activation, which hydrolyzes the phosphodiester bonds of sphingomyelin to produce Cers [52], hence regulating bacterial growth and metabolic processes.

5. Conclusions

A non-targeted lipidomic method was utilized to investigate the lipid remodeling in V. alginolyticus triggered by citral. Specifically, citral treatment promoted the conversion of phospholipids (e.g., PCs, Pes and PGs) to LPLs (e.g., LPCs, LPEs and LPGs) and stimulated the accumulation of Cers. These alterations are likely to influence membrane architecture and function, potentially serving as the key mechanism of citral antibacterial efficacy. In general, these findings provided a mechanistic basis at the lipidomic level for the antibacterial activity of citral and highlight its potential as a natural antibacterial agent.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms14020509/s1, Table S1: Detailed information on the differential lipids in positive and negative ion modes. Figure S1: PCA score plots for all samples. Red dots and gray triangles represent QCs and other test samples, respectively.

Author Contributions

Conceptualization, Y.Z.; methodology, J.H., H.L. (Hua Li) and J.R.; software, Z.W., Y.W. and T.S.; validation, J.H.; formal analysis, Y.Z., Y.W. and T.S.; investigation, Y.Z. and J.R.; resources, H.L. (Huan Liu); data curation, Y.Z., Z.W. and H.L. (Hua Li); writing—original draft preparation, Z.W. and J.H.; writing—review and editing, Y.Z. and H.L. (Huan Liu); visualization, Y.Z., Z.W. and J.R.; supervision, Y.W. and H.L. (Huan Liu); project administration, H.L. (Huan Liu); funding acquisition, Y.Z. and H.L. (Huan Liu). All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the grants from Shaanxi Provincial Science and Technology Department (No. 2023-ZDLNY-40, 2023-YBNY-171 and 2024NC-ZDCYL-04-30), Xi’an Science and Technology Plan Project (No. 23NYGG0058 and 22NYYF036), Shaanxi Provincial Department of Education Project (No. 24JC012).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data in this study are included in this published article and its supplementary information files. Further inquiries can be directed to the corresponding author.

Acknowledgments

We appreciate the support of the fund providers and the contributions of the participants to this article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Microorganisms 14 00509 g001
Figure 1. Representative total ion chromatograms in positive (A) and negative (B) ion modes. Lipid categories and quantities identified in V. alginolyticus (C). The PCA of the lipid profile (D). Euclidean distance between citral exposure group and CK (E).
Figure 1. Representative total ion chromatograms in positive (A) and negative (B) ion modes. Lipid categories and quantities identified in V. alginolyticus (C). The PCA of the lipid profile (D). Euclidean distance between citral exposure group and CK (E).
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Figure 2. The Venn diagram of significantly different lipids related to citral treatment (A). The volcano plots for 1/2 MIC/CK (B) and MIC/CK (C). Red and blue dots represent the differential lipids with higher and lower levels in the citral-treated group than those in the CK, respectively. Fold-change was abbreviated as FC.
Figure 2. The Venn diagram of significantly different lipids related to citral treatment (A). The volcano plots for 1/2 MIC/CK (B) and MIC/CK (C). Red and blue dots represent the differential lipids with higher and lower levels in the citral-treated group than those in the CK, respectively. Fold-change was abbreviated as FC.
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Figure 3. Heat map of the total lipid subclass abundance patterns. The pink and blue colors show higher and lower intensity levels, respectively, compared to the average level of all the samples. The summed amount of each lipid subclass was calculated by calculating the intensities of individual lipid molecules within each lipid subclass. Six replicates are displayed for each condition, and each column represents one biological replicate.
Figure 3. Heat map of the total lipid subclass abundance patterns. The pink and blue colors show higher and lower intensity levels, respectively, compared to the average level of all the samples. The summed amount of each lipid subclass was calculated by calculating the intensities of individual lipid molecules within each lipid subclass. Six replicates are displayed for each condition, and each column represents one biological replicate.
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Figure 4. The geometric mean ratio of glycerophospholipid levels at 1/2 MIC/CK (A,B) and MIC/CK (C,B). The FC values at MIC/CK and 1/2 MIC/CK were plotted on the y-axis. The carbon numbers (A,C) and double bonds (B,D) of the acyl chains in glycerophospholipids were plotted on the x-axis. The red line indicates the ordinate value FC = 1.
Figure 4. The geometric mean ratio of glycerophospholipid levels at 1/2 MIC/CK (A,B) and MIC/CK (C,B). The FC values at MIC/CK and 1/2 MIC/CK were plotted on the y-axis. The carbon numbers (A,C) and double bonds (B,D) of the acyl chains in glycerophospholipids were plotted on the x-axis. The red line indicates the ordinate value FC = 1.
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Figure 5. Glycerophospholipid metabolic pathway associated with citral exposure (A). Proportional levels of total LPLs and phospholipids linked to citral exposure (B). Green, pink, and blue bars represent the CK group, 1/2 MIC group, and MIC group, respectively. The values are shown as the means ± SE (n = 6). Non-parametric tests were used for statistical analysis. *: 0.01 ≤ p < 0.05; **: p < 0.01.
Figure 5. Glycerophospholipid metabolic pathway associated with citral exposure (A). Proportional levels of total LPLs and phospholipids linked to citral exposure (B). Green, pink, and blue bars represent the CK group, 1/2 MIC group, and MIC group, respectively. The values are shown as the means ± SE (n = 6). Non-parametric tests were used for statistical analysis. *: 0.01 ≤ p < 0.05; **: p < 0.01.
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Figure 6. S-plots generated from OPLS-DA models of MIC vs. CK (A) and 1/2 MIC vs. CK (B), where the gray dots denote all lipid ions involved in the model, whereas the red symbols highlight the ions showing the most significant changes and higher contributions to the classification pattern. Histogram of relative levels of potential biomarkers (C). Potential biomarkers were screened by p < 0.01, FC > ± 1.5, | p [1] | > 0.1 and | p (corr) [1] | > 0.8. The values are shown as the means ± SE (n = 6). Non-parametric tests were used for statistical analysis. *: 0.01 ≤ p < 0.05; **: p < 0.01.
Figure 6. S-plots generated from OPLS-DA models of MIC vs. CK (A) and 1/2 MIC vs. CK (B), where the gray dots denote all lipid ions involved in the model, whereas the red symbols highlight the ions showing the most significant changes and higher contributions to the classification pattern. Histogram of relative levels of potential biomarkers (C). Potential biomarkers were screened by p < 0.01, FC > ± 1.5, | p [1] | > 0.1 and | p (corr) [1] | > 0.8. The values are shown as the means ± SE (n = 6). Non-parametric tests were used for statistical analysis. *: 0.01 ≤ p < 0.05; **: p < 0.01.
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MDPI and ACS Style

Zhao, Y.; Wang, Z.; Han, J.; Wang, Y.; Ren, J.; Shao, T.; Li, H.; Liu, H. Lipidome Disturbances of Vibrio alginolyticus Associated with Citral Exposure. Microorganisms 2026, 14, 509. https://doi.org/10.3390/microorganisms14020509

AMA Style

Zhao Y, Wang Z, Han J, Wang Y, Ren J, Shao T, Li H, Liu H. Lipidome Disturbances of Vibrio alginolyticus Associated with Citral Exposure. Microorganisms. 2026; 14(2):509. https://doi.org/10.3390/microorganisms14020509

Chicago/Turabian Style

Zhao, Yanni, Zi Wang, Jie Han, Yi Wang, Jiamin Ren, Ting Shao, Hua Li, and Huan Liu. 2026. "Lipidome Disturbances of Vibrio alginolyticus Associated with Citral Exposure" Microorganisms 14, no. 2: 509. https://doi.org/10.3390/microorganisms14020509

APA Style

Zhao, Y., Wang, Z., Han, J., Wang, Y., Ren, J., Shao, T., Li, H., & Liu, H. (2026). Lipidome Disturbances of Vibrio alginolyticus Associated with Citral Exposure. Microorganisms, 14(2), 509. https://doi.org/10.3390/microorganisms14020509

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