EOs can stimulate the production of white blood cells, which are crucial for combating infections, and activate phagocytes, the cells responsible for engulfing and eliminating pathogens. These effects indicate a wider potential benefit of EOs for preventing pathological and welfare issues in farmed fish (Mabrok and Wahdan, 2018). For example, rainbow trout fed a diet containing thyme essential oil had significantly higher levels of white blood cells and phagocytic activity compared to controls (Yousefi et al., 2022).

Dietary administration of specific essential oils, such as origanum essential oil, has been shown to enhance antioxidative status, modulate immune-related genes, and increase resistance to bacterial infections. For instance, supplementation with origanum essential oil improved antioxidative status, immune-related genes, and disease resistance in common carp challenged with Aeromonas hydrophila (Abdel-Latif et al., 2020).

These benefits of EOs indicate a potential positive impact on the overall health and disease resistance of farmed fish. The immunostimulatory properties of EOs in fish can enhance their immune responses, potentially leading to improved disease resistance and overall welfare in aquaculture. These findings underscore the potential of EOs as natural and sustainable alternatives for promoting the health and well-being of farmed fish.

The aim of the present work is to investigate the antibacterial properties of 15 EOs which are locally cultivated or easily sourced from commercial producers in the Mediterranean region, against common fish pathogens of Mediterranean aquaculture, aiming to identify EOs with potent inhibitory effects, particularly against Aeromonas, Vibrio, and Tenacibaculum. The antibacterial potential of these essential oils is largely attributed to major constituents such as thymol, carvacrol, eugenol, and cinnamaldehyde, which are known to disrupt bacterial membranes and interfere with metabolic processes (Ultee et al., 2000; Burt and Reinders, 2003; Nazzaro et al., 2013).

EOs were sourced from these MAPs: Ginger (Gingiber officinale), cutleaf geranium (Geranium dissectum), Basil (Ocimum basilicum), Tea tree (Malaleuca alternifolia), Cinnamon (Cinnamomum verum), Clove (Syzygiuma romaticum, also known as Eugenia caryophyllata), Oregano (Origanum vulgare), Patchouli (Pogostemon cablin), English Lavender (Lavandula angustifolia), Frankincense or olibanum (Boswellia carterii), Blue gum (Eucalyptus globulus), and Sweet Marjoram (Origanum majorana) were procured from a commercial company. Thyme (Thymus vulgaris), Aloe (Aloe vera), and Wormwood or Absinthe (Artemisia absinthium) EOs were extracted manually. For the extraction process, dried samples from the aerial parts (leaves, flowers, and stems) of these plants underwent a 3-hour water distillation using a Clevenger-type apparatus, yielding 2.0–2.6% v/w. The resulting EOs were subsequently dried using anhydrous sodium sulfate and stored at temperature 5°C (± 1°C).

The essential oils (EOs) selected in this study represent plants that are either widely available or already recognized for their use in food, medicine, or aquaculture-related applications. EOs of thyme and oregano are rich in thymol and carvacrol, clove contains eugenol, and cinnamon provides cinnamaldehyde, all of which are widely recognized for strong antibacterial effects through membrane disruption and enzyme inhibition (Ultee et al., 2000; Burt and Reinders, 2003). Alcohol-containing oils, including basil, lavender, and tea tree, contribute compounds such as linalool and terpinen-4-ol with similar mechanisms of action (Lee et al., 2009; Nazzaro et al., 2013). Additional oils, including ginger, eucalyptus, patchouli, frankincense, marjoram, aloe, and wormwood, were included to broaden the chemical classes evaluated and to capture compounds with antioxidant or immunomodulatory potential. With this range of tested essential oils, the study aimed to evaluate representatives of different chemical classes and plant origins, providing a first step toward identifying natural alternatives to antibiotics that can contribute to ensuring the health and sustainability of aquatic ecosystems (Dawood et al., 2021; Park et al., 2016).

Cultures of Aeromonas veronii bv. veronii and Aeromonas veronii bv. veronii were obtained from naturally infected European sea bass (Dicentrarchus labrax) collected from Chios Island and the Saronikos region (Greece), respectively. Similarly, Vibrio harveyi and Tenacibaculum maritimum were isolated from naturally infected sea bass sourced from Euboea Island (Greece). All these isolates were preserved on tryptone soy broth (TSB) slants at a temperature of 4°C. Inocula for the experiments (A. veronii bv. veronii, A. veronii bv. sobria and V. harveyi) were prepared by cultivating the isolates for 16 hours in Mueller–Hinton Broth (MHB; Oxoid) while Tenacibaculum maritimum was cultivated in marine broth (MB; Condalab), all at a temperature of 23°C.

Bacterial cultures 18 hours old were appropriately diluted using sterile physiological saline solution (0.85% w/v sodium chloride) based on the McFarland standard (bioMérieux, Marcy l’Etoile, France) to achieve an inoculum of around 10⁶ CFU ml^-1. A 5ml volume of each inoculum (A. veronii bv. veronii, A. veronii bv. sobria and V. harveyi) was applied to the surface of Mueller–Hinton agar (MHA; Oxoid) plates that had been surface-dried to remove excess moisture and allowed to make contact for 1 minute. Subsequently, sterile 6mm filter paper discs (Sigma-Aldrich) were positioned on the plates, and immediately 10 µl of each EO was administered separately. Sterile physiological saline solution was applied to separate discs as a negative control to ensure that any inhibition observed was due to the EO and not the solvent or disc material. Plates were then incubated at 23°C for 24 hours. The inhibition zones were measured to the nearest millimeter defined at complete growth inhibition according to EUCAST disk diffusion reading guide (EUCAST, 2024). Each assay was repeated ten times for every EO. These standardized reading practices improve inter-study comparability even though categorical breakpoints are not available for essential oils.

EOs demonstrating the most potent antibacterial effects in the disc diffusion assay (namely absinthe, thyme, cinnamon, and oregano), underwent further analysis to determine their bacteriostatic and bactericidal concentrations using a modified (Wang et al., 2021; Palaniyappan et al., 2023) colorimetric broth microdilution technique (Salvat et al., 2001). This evaluation involved setting up three sterile 96-well microplates with lids (VWR International). The process involved arranging 200 μl portions of 2% EO in sterile Mueller-Hinton Broth (MHB; A.veronii bv. veronii, A. veronii bv. sobria, V. harveyi) or Marine Broth (MB; Tenacibaculum maritimum) in wells of row A, while wells in rows B to H received 100 μl of sterile MHB (or MB). To enhance the stability and emulsification of the essential oil within the broths, an incorporation of soy lecithin (Oxoid) at a concentration of 0.25% (w/v) was introduced. Serial two-fold dilutions were carried out from row A to row H, with the highest dilution in row H. The inoculums, prepared from 16-hour cultures adjusted according to the McFarland standard, were further diluted with MHB (or MB) to achieve an approximate concentration of 10⁶ CFU ml^-1. Each well then received 100 μl of the inoculum and resazurin sodium salt (Sigma-Aldrich).

The microplates were incubated at temperatures of 23°C for 96 hours. A color change from blue to pink or mauve indicated bacterial growth. An extension of Salvat et al.’s method (Burt and Reinders, 2003) involved plating aliquots of 5 μl from the wells that remained blue onto Mueller-Hinton Agar (MHA; A. veronii bv. veronii, A. veronii bv. sobria, V. harveyi) or MA (Tenacibaculum maritimum), followed by a 24-hour incubation at 23°C. The experiment was conducted in six replicates for each microassay.

The bacteriostatic concentration was established as the lowest concentration at which bacteria in at least five out of the six replicates failed to grow in MHB (or MB) but were able to grow when plated on MHA (or MA). The bactericidal concentration was determined as the lowest concentration at which bacteria in at least five of the six replicates did not grow in MHB (or MB) and were not cultured after plating onto MHA (or MA). The method used for MIC and MBC determinations followed CLSI-style broth microdilution methodology commonly applied in EO studies, thereby increasing comparability with standardized antimicrobial testing. A limitation, however, is that CLSI and EUCAST clinical breakpoints are established only for antibiotics and not for essential oils, which prevents direct categorization of EO activity into susceptible, intermediate, or resistant classes (EUCAST, 2024; Xiao et al., 2020; Fokas et al., 2025).

Data were analyzed using SPSS v.1.0.0.1406. Inhibition zone diameters from the disc diffusion assays (10 replicates per each of tested essential oil) were compared using one-way ANOVA, followed by Tukey’s post hoc test to identify significant pairwise differences. MIC and MBC values obtained from broth microdilution assays (six replicates per each tested essential oil) were compared among oils using the non-parametric Kruskal–Wallis test, as distributional assumptions were not met. Correlations between inhibition zone diameters and MIC values were assessed using Pearson’s correlation coefficient to evaluate consistency of results between the two methods. Statistical significance was accepted at p<0.05. The level Significant differences are indicated in Table 1 by bold lettering and explained in the footnotes.

All MAPs and EOs were acquired from licensed commercial enterprises in adherence to regional law 3937/2011 concerning biodiversity conservation. The handling, storage, and disposal of all biological agents were carried out in accordance with Directives 2000/54/EU and 89/391/EEC. The experimental strategy employed no animals in any aspect of the experimentation taking into account article 4 of Directive 2010/63/EU. The overall experimental protocol’s principles are in accordance with the Hellenic presidential decree 56/2013(106). The bacterial isolates originated from prior isolations from naturally infected fish, and their species-level characterization was accomplished using molecular techniques.

The results of colorimetric determination of bacteriostatic and bactericidal concentrations are provided in Table 2 . The results indicate that EO of Artemisia absinthium exhibited a promising antibacterial effect across the tested pathogens. The results obtained by the disc diffusion (inhibition zones) correlated well with the MIC values (Pearson’s r = 0.84, p < 0.001), supporting consistency between the two methods.

With concentrations ranging from 312 to 625 μl/l for both bacteriostatic and bactericidal actions, A. absinthium showcases a relatively consistent performance. This suggests that it could be a versatile option for controlling various fish infections. Oregano oil exhibited notable antimicrobial effects, with concentrations varying between 312 and 1250 μl/l., notably, the absence of a specific bacteriostatic concentration for V. harveyi suggests that the lowest effective concentration was also bactericidal. Thyme oil demonstrated significant antimicrobial effects across the pathogens, displaying consistent bactericidal concentrations. Cinnamon oil showed relatively consistent performance against A. veronii bv. sobria and V. harveyi with bacteriostatic and bactericidal concentrations of 312 μl/l.

The objective of this study was to examine the antibacterial characteristics of 15 essential oils (EOs) against prevalent fish pathogens found in Mediterranean aquaculture. The primary goal was to identify EOs that exhibit strong inhibitory effects, specifically targeting Aeromonas, Vibrio, and Tenacibaculum. The escalating demand for sustainable aquaculture practices has prompted a search for effective alternatives to conventional antimicrobials in controlling fish pathogens (Nisa et al., 2023). EOs, derived from various MAPs, have gained considerable attention for their potential antimicrobial properties against a wide array of pathogens (Tiwari et al., 2009; Athanassopoulou et al., 2018; Moosavi-Nasab et al., 2019). This includes not only bacteria but also ectoparasites and endoparasites that pose significant threats to fish health and aquaculture productivity (Athanassopoulou et al., 2004; Karagouni et al., 2005; Tsantilas et al., 2005; Athanassopoulou et al., 2009; Dawood et al., 2021). EOs offer a natural and eco-friendly approach to combating these parasitic infections, reducing the reliance on synthetic chemicals and addressing concerns related to antibiotic resistance and environmental impact (Caruso, 2016; Luis et al., 2019; Okeke et al., 2022; Deekshit et al., 2023). By harnessing the inherent antimicrobial properties of essential oils, researchers and aquaculture practitioners are exploring innovative strategies to mitigate the impact of fish pathogens, ensuring the health and sustainability of aquatic ecosystems (Dawood et al., 2021).

The antibacterial activity of EOs studied in this work can be attributed to their main chemical constituents. The results agree with earlier reports that the activity of essential oils is linked to the presence of bioactive phenolic and alcohol components. Compounds such as thymol, carvacrol, eugenol, and cinnamaldehyde are known to damage bacterial membranes and interfere with enzyme activity, leading to cell death (Ultee et al., 2000; Burt and Reinders, 2003). Other constituents, including Linalool and Terpinen-4-ol, have similar disruptive effects on bacterial membranes (Lee et al., 2009; Nazzaro et al., 2013).

While EOs generally require higher concentrations to achieve bacteriostatic or bactericidal effects compared to antibiotics, their multi-target modes of action, effectiveness against biofilms, and reduced risk of resistance development make them valuable complementary agents in aquaculture settings where antibiotic resistance is a growing concern. For instance, oregano, thyme, cinnamon, and absinthe oils have been reported to exert strong inhibitory effects against Aeromonas veronii and Vibrio harveyi when tested by agar dilution and well diffusion methods (Starliper et al., 2015; Tural et al., 2019; Domínguez-Borbor et al., 2020). These findings are consistent with our results and highlight the potential of selected essential oils as alternatives to conventional antibiotics, for example oregano, absinthe, cinnamon, and thyme, which exhibited the highest antimicrobial activity against the pathogens tested in this study. This ranking was substantiated by studies assessing EOs from the same plant species against A. veronni and V. harveyi, utilizing the agar dilution method and well tests (Lee et al., 2009; Starliper et al., 2015; Tural et al., 2019; Domínguez-Borbor et al., 2020; Dawood et al., 2021). Our findings are in line with the work of Bektaş and Özdal (2022) (Athanassopoulou et al., 2018) who investigated the effectiveness of essential oils from Eucalyptus camaldulensis against fish pathogens, including Aeromonas caviae. Furthermore, Kot et al. (2019). explored the antimicrobial activities of specific phytochemicals, including trans-cinnamaldehyde and thymol, against Aeromonas species, indicting the broader potential of plant-derived compounds in combating fish pathogens.

A further crucial consideration revolves around the impact of lecithin as an emulsifier. A number of authors propose that, despite its favorable emulsifying qualities, lecithin could diminish the in vitro antibacterial effectiveness of EOs. This could be due to lecithin’s potential to physically obstruct the interaction between EOs and bacterial cells, possibly by augmenting the availability of phospholipids (Ismaiel and Pierson, 1990; Keweloh et al., 1991; Ultee et al., 2000; Burt and Reinders, 2003). Thus, it’s conceivable that the antibacterial and bactericidal potential of EOs might be even more robust without the presence of lecithin.

As emphasized earlier, variations in EO composition within the same plant species, influenced by factors like harvesting season and geographic location, are acknowledged. Nonetheless, studies performed with specific EO batches should consistently demonstrate reliable reproducibility. The findings of the present work are encouraging and provide support for future research on the potential use of essential oils (EOs) to enhance the antibacterial and bactericidal effects in Mediterranean farmed fish species. This concept is consistent with the findings of Zhang et al (Dawood et al., 2021), who reviewed the use of essential oils in animal production for the purpose of managing food-borne pathogens.

The results of the present work are relevant to the Mediterranean aquaculture industry as they underscore the substantial in vitro antibacterial properties of Absinthe, Thyme, Oregano, and Cinnamon EOs. In fact, these EOs stand out as the most consistently effective across aquaculture studies, with antimicrobial properties which have been widely reported in a range of pathogens (Firmino et al., 2021; Poudel et al., 2021; Dawood et al., 2021). The stronger antimicrobial activity of oregano, thyme, cinnamon, and absinthe oils can be explained by their high content of compounds such as thymol, carvacrol, eugenol, and cinnamaldehyde. This is consistent with earlier work by Mandalakis et al. (2021), who reported strong activity of oregano- and carvacrol-rich oils against Aeromonas veronii bv. sobria.

Particularly intriguing is the prospect of amplifying the antibacterial and bactericidal effects of EOs through simultaneous administration, an area ripe for exploration in future research. Moreover, essential oils can reduce production costs and enhance market appeal for antibiotic-free aquaculture products (Karagouni et al., 2005).

The relatively high MIC values of essential oils compared to antibiotics (Chouhan et al., 2017) may restrict their direct use in aquaculture, since larger doses raise concerns of toxicity and cost-effectiveness. Nevertheless, the growing problem of antibiotic resistance underscores the need to explore such natural alternatives. Although EOs may not always reach the low MIC levels of antibiotics, their activity against resistant strains and eco-friendly profile make them promising candidates for sustainable disease management. Although stability of the tested essential oils was not a limiting factor in the present study due to the in vitro and short incubation periods, long-term application in aquaculture systems may be affected by the degradation of certain compounds. Ganosi et al. (2023) showed that while major constituents such as carvacrol and thymol remain relatively stable for several months, many minor components degrade within 10–70 days, particularly under elevated temperature or sunlight. This indicates that although the main antibacterial activity can be retained, the overall chemical profile of the oils changes over time. This highlights the need for further research aimed at enhancing their efficacy, for example through nanoencapsulation, which improves stability, bioavailability, and sustained release of active compounds, or through synergistic combinations of essential oils with each other as a combination of strategies to enhance stability, bioavailability, and antimicrobial potency of EOs (Chouhan et al., 2017; Valdivieso-Ugarte et al., 2019; Firmino et al., 2021). It should be stated that the effectiveness of EOs is expected to vary according to dosage, species, and exposure time, and acute toxicity tests are necessary to define safe margins before use in live fish. Sublethal effects, such as behavioral stress or immunosuppression, may also occur if concentrations are not carefully controlled (Lanzerstorfer et al., 2021). Therefore, following the results of the present in vitro study, further in vivo testing is required to establish safe and effective application protocols for aquaculture systems.

To our knowledge, the results of the present work constitute a maiden effort in investigating the antibacterial potential of the tested essential oils against Tenacibaculum maritimum, a significant pathogen affecting European sea bass (Dicentrarchus labrax). Infections occur mainly in small fingerlings, creating serious problems in nursery stations (Avendaño-Herrera et al., 2008). At present, no commercial vaccine is available, and trials with autogenous vaccines in Mediterranean farms have provided limited protection, resulting in continued reliance on antibiotic treatments (Kolygas et al., 2012; Mabrok et al., 2022). Moreover, the persistence of T. maritimum remains a concern even after sea bass are transferred to open sea cages.

As a potential solution, the use of oral essential oil-based nutraceutical supplements could help reduce the overall dependence on antibiotics. The prospects for scaling up the use of essential oils in commercial aquaculture are promising, given the available research reports on the benefits in enhancing growth, feed efficiency, immunity, and disease resistance in fish, but successful application will depend on optimizing delivery methods and ensuring cost-effectiveness, for example through microencapsulation or incorporation into aquafeeds (Valdivieso-Ugarte et al., 2019; Liu et al., 2023). An interesting observation of the present study is that Tenacibaculum maritimum is less susceptible to the inhibitory and killing effects of EOs compared to the other fish pathogens tested. It should also be noted that soy lecithin, which was included in the broth assays as an emulsifier to disperse the hydrophobic essential oils, may have interacted with some of their active compounds and reduced their apparent antimicrobial activity. This possible interference represents a limitation of the experimental design and may partly explain the lower susceptibility observed in T. maritimum. In addition, the reduced sensitivity could also reflect resistance mechanisms inherent to this bacterium, including intrinsic resistance, efflux pumps, biofilm formation, or altered cell membrane composition, which are consistent with its documented tolerance to several antibacterial substances (Avendaño-Herrera et al., 2008; Kolygas et al., 2012; Mabrok et al., 2022).