Utilization of Plant-Derived Essential Oils as Natural Alternatives for Controlling Fish Pathogens: A Critical Review of Their Use Against Aeromonas hydrophila

Abstract

Aeromonas hydrophila infection is typically linked to outbreaks of diseases and its pathogenicity seems to be connected to environmental stress in hosts whose immune systems are compromised. Fish diseases have long been treated using synthetic antibiotics, but there are environmental and health concerns regarding their use due to the development of bacterial resistance. It has been suggested that essential oils (EOs), which are alternative antibacterial agents, could be applied in the aquaculture industry. Their biodegradability, affordability, efficiency, and quick metabolism minimize the likelihood of them building up in bodily tissues and provoking bacterial resistance. EOs can directly influence bacteria, altering the lipid composition and structure of bacterial cell membranes, enhancing their permeability, and disrupting their structure. In addition, they can boost fish immunity and increase resistance to contagious bacteria. EOs can be incorporated into fish diets as nutritional additives and/or applied in baths. This review discusses the in vitro and in vivo methodologies used to study the effects of EOs on A. hydrophila and the results obtained so far, as well as perspectives for new studies.

1. Introduction

The natural kingdom has thousands of medicinal plants which are used in various sectors, including as flavorings, fragrances, cosmetics, perfumery and pharmaceuticals. Among the most valued products derived from these plants are essential oils (EOs). These EOs, are often registered in a pure form and encapsulate the very essence of a plant, capturing its flavors and aromas. Therefore, they are known as “essential.” As highlighted by Raut and Karuppayil (2014) [1], more than 3000 EOs have been distilled from at least 2000 botanical species, many of which hold considerable industrial significance. Demonstrating a plethora of biological activities, EOs exhibit antibacterial, antioxidant, antiviral, and insecticidal properties [2,3,4,5]. Ancient techniques to eradicate a number of infectious diseases by using EOs date back thousands of years [6] and contemporary research continues to underscore their antimicrobial efficacy and the active compounds they possess.

Plant-derived medications are increasingly recognized as safer alternatives to synthetic treatments in terms of acute toxicity, carcinogenic potential, and environmental impact [7,8]. The use of synthetic antimicrobials in aquaculture, including antibiotics and anthelmintic drugs, may not only pose health risks to consumers of treated fish but also incur high costs [9,10]. Given that infections affect entire populations, addressing fish diseases requires community-wide responses rather than individual-focused interventions. Consequently, interest in exploring medicinal plants and their EOs as substitutes for conventional medicines in fish disease treatment has been growing [11,12].

One of the most resistant fish pathogens, Aeromonas hydrophila, show high susceptibility to aminoglycosides [13,14,15] (amikacin, gentamycin, and neomycin), fluoroquinolones (ciprofloxacin and ofloxacin), and several others like chloramphenicol and trimethoprim/sulfamethoxazole [16]. However, it exhibits high resistance to many common antibiotics including third- and fourth-generation cephalosporins, β-lactams (penicillin and amoxicillin), and certain quinolones [17,18,19]. Resistance patterns vary based on the source of the isolates, and continuous increases in resistant strains [20,21] necessitate careful monitoring and tailored antibiotic use [22,23].

Motile aeromonads are a group of bacteria abundant in warm water environments and include species like A. hydrophila [24], A. veronii, [25,26], A. caviae, [27,28], A. jandaei, [29,30], and A. schubertii [31,32]. On the other hand, A. salmonicida belongs to the non-motile aeromonad group and is typically found in cold water environments [33,34,35]. A. hydrophila has been identified as the most prevalent motile aeromonad pathogen in at least 15 freshwater species [36,37].

Global aquaculture businesses have suffered greatly due to outbreaks of a pathotype called hypervirulent A. hydrophila (vAh) [37,38]. Warm-water habitats are ideal for A. hydrophila’s growth and wide host range, which includes mammals, birds, fish, reptiles, and amphibians. Among the illnesses brought on by A. hydrophila, Motile Aeromonas Septicemia (MAS) causes significant distress to fish. Infection caused by A. hydrophila is linked to a wide range of clinical symptoms, including meningitis, septicemia, gastroenteritis, and soft tissue infections, especially skin and gill infections in aquatic animals [39,40,41]. Serious internal and external bleeding together with exophthalmia are the signs of MAS, which frequently leads to mortality within a few hours of disease exposure [42,43,44,45,46,47]. In addition, several studies have stated that A. hydrophila is a potent foodborne pathogen that causes zoonotic diseases that transmit via infected animals to humans [48,49,50]. Thus, it causes numerous health concerns in people who consume diseased fishes, which include intestinal and extra-intestinal allergies such as diarrhea, septic arthritis, gastroenteritis, meningitis, and skin and wound ulcers. Nevertheless, toxins (aerolysin, lipase, DNase protease and hemolysin) tethered with the pathogenicity of A. hydrophila may enhance its virulence against humans as well as its impact on animal welfare [51,52].

International outbreaks of Aeromonas infections have been predicted since the identification of A. hydrophila J-1 in 1989 throughout the epidemics of MAS in Jiangsu Province, China, where the first vAh pathotype of A. hydrophila was discovered [53]. Sequence type 251 (ST251), to which this strain belongs, was identified for its ability to induce high mortality rates in grass carp (Ctenopharyngodon idella) [54]. Annual fish losses in China’s farmed carp sector have been around 2200 tons due to recurrent MAS eruptions every year [54,55,56,57]. The vAh isolate called A. hydrophila NJ-35 was the causative agent in the 2010 resurgence of ST251-associated MAS outbreaks in the same country [58,59].

Farmers from Mississippi and Washington state isolated A. hydrophila S04-690 from infected Ictalurus punctatus (channel catfish) in 2004. This was the most renowned instance of ST251-combined MAS in the United States [60,61]. Subsequently, in early 2009, strains of vAh were consistently linked to MAS outbreaks in the fish farms of western Alabama. The beginning of the epidemic year resulted in the loss of around 2000 tons of fish [62]. Over time, this amount has risen to surpass an estimated 10,500 tons, with most disease outbreaks (35%) occurring at the Alabama Fish cultivation Farm being attributed to vAh isolates [63]. While it may be difficult to gather accurate statistics specifically on production losses caused by vAh, it is clear that vAh poses a substantial risk to warm-water aquaculture sectors [60].

The growing interest in EOs as antibacterial agents has led to a huge number of studies investigating their antibacterial nature against a wide variety of pathogens. However, due to the vast variety of plants that can produce EOs, there are differences in the compositions of EOs, even from same species, and the diversity of testing methods make it challenging to generalize. This review, uniquely, explores the antibacterial properties of plant-derived EOs and their active compounds against A. hydrophila, offering a comprehensive analysis that bridges the gap between in vitro and in vivo efficacy. Treatment/prevention with EOs is anticipated to be one of the most effective methods for medicating aquatic animals suffering from MAS [64]. This comprehensive review has shown that a wide variety of highly efficient EOs oils are available for the eradication/management of MAS [65,66]. The findings highlight the potential of EOs as a promising solution for mitigating the impact of MAS on various aquatic species, offering a natural and potentially sustainable approach to disease management in aquaculture [67,68].

This review goes beyond previous studies by providing detailed mechanistic insights into how EOs disrupt bacterial processes, and highlights their potential as alternatives to antibiotics, especially against resistant strains of A. hydrophila [69,70,71,72,73,74]. Additionally, the review considers the environmental impact of EO use, discussing sustainability, biodegradability, and effects on non-target organisms. By identifying research gaps and suggesting future directions, thus, this review highlights the effectiveness of plant-derived EOs identified through existing research showcasing low MIC and MBC values along with successful in vivo applications in aquatic habitats.

2. Search Strategy

We performed a comprehensive assessment of the literature published from 1990 to 2024 by utilizing the Google Scholar, Web of Science, Research Gate, PubMed, and Scopus databases, in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) standards. The search included relevant English keywords such as ‘plants’ ‘essential oil’, ‘compounds of essential oil’, ‘Antibacterial effects’, ‘Aeromonas hydrophila’, ‘international status’, ‘in vitro mechanism’, ‘resistant patterns’, and ‘treatment with animals’. There were no limitations on performing searches in databases.

2.1. Study Selection and Data Extraction

The exclusion criteria for MAS therapy differ from those applied to resistance mechanisms and therapeutic techniques. Additionally, review papers and other publications that did not describe new or modified processes were ignored. The authors independently assessed the abstracts and titles using the inclusion and exclusion criteria. Additional papers were selected by reviewing the references in the retrieved articles. Disputes among reviewers were resolved through discussion until a consensus was reached. This review now includes data collected for the primary aims of the research, such as revised treatment objectives and resistance to antibiotics. Emphasis was also placed on diagnosis and treatment in the original publications related to the MAS illness model.

2.2. Outcome

A total of 665 papers were initially identified in the search, and an additional 50 publications were subsequently included, bringing the total number of citations for the first study to 715. After reviewing the titles and abstracts, a total of 425 publications were chosen for additional examination. Afterwards, 216 references were carefully examined to assess the efficacy of the EOs against A. hydrophila and the methodologies used.

3. Chemical Properties of Plant-Derived EOs

Plants can generate an extensive series of chemical substances with antibacterial properties. While certain compounds are consistently found in plants, others are triggered by stressors like infections, injuries, predators, and changes in weather patterns. The factors that influence the composition of EOs can differ depending on the plant species, period of the plant being collected, place of cultivation, and the extraction technique employed [75,76]. Owing to the wide variations in chemical composition, EOs require thorough characterization and standardization for effective and reliable use in aquaculture [77,78,79,80,81,82]. Steam and hydro distillation methods are often utilized for mass production of EOs [83]. High-performance liquid chromatography (HPLC) [84,85,86], liquid chromatography coupled to mass spectrometry (LC–MS) [87], gas chromatography (GC), and gas chromatography–mass spectrometry (GC–MS) [88,89,90] stand out as conventional methods to analyze and investigate the chemical compositions of volatile EOs.

Terpenes and phenolic compounds are the principal chemical constituents of EOs. The molecular structures of terpenes are composed of multiple isoprene units (C₅H₈) that join to form hydrocarbon molecules. They originate from both mevalonate and mevalonate-independent pathways within EOs [91]. Terpenes are predominantly found as monoterpenes (C₁₀H₁₆) or sesquiterpenes (C₁₅H₂₄), although trace quantities of long-chain molecules like diterpenes (C₂₀H₃₂), triterpenes (C₃₀H₄₈), and tetraterpenes (C₄₀H₆₄) can also be present depending on the extraction method used to obtain the EOs. The most common terpene hydrocarbons found in EOs include α-caryophyllene, p-cymene, α- and β-pinene, limonene, sabinene, terpinene, myrcene, cinnamyl alcohol, and 3-carene [92]. Terpenoids, which are oxygenated derivatives of terpene compounds, can be further classified into alcohols, ethers, ketones, aldehydes, esters, and epoxides [93,94]. Examples of terpenoids are linalyl acetate, geraniol, citronellol, citronellal, thymol, terpineol, menthol, carvacrol, and linalool [95,96,97] (Figure 1).

4. Antibacterial Mechanism of EOs Against A. hydrophila Cells

4.1. Complement-Mediated Defense and Antibiotic Tolerance in A. hydrophila

Non-immune serum’s bactericidal effect is vital to the immediate defense of the host against bacterial infections. This term is called complement-mediated and it has been the focus of intensive research [98,99,100,101]. Previous studies have indicated that the ability of antibodies to kill bacteria can be hindered by several components found in cell walls, including surface proteins and lipopolysaccharides (LPS) [102,103,104,105,106] along with capsular polysaccharides [107,108,109,110]. A. hydrophila is a Gram-negative bacterium that can activate complement fixation pathways by either the classical complement pathway (CCP) or the alternative complement pathway (ACP). Possibly, the bacterial surface antigen and antibody interactions trigger the CCP, or the lipid A moieties directly from LPS can activate the CCP in some circumstances [111,112]. On the other hand, the ACP typically responds to bacterial polysaccharides in the absence or lack of antibodies [113,114,115,116]. The classification of the virulence categories of A. hydrophila is possible based on surface attributes, including the nature of LPS and the existence of an S-layer [117,118].

Generally, the anti-bacterial mechanism of EOs is largely attributed to their high hydrophobicity, which disrupts the cell permeability of A. hydrophila by breaking down its LPS and results in the loss of vital cellular components (Figure 2). This disruption can cause irreversible damage to cell membranes, particularly when the EOs possess potent active compounds in sufficient quantities and strength. Eventually it leads to bacterial cell lysis (cytolysis) and results in cell death. Another profound mechanism is inhibition of toxin production through the suppression of protease and amylase secretion, which leads to coagulation of bacterial components [119]. Among the EOs, those from the Lamiaceae family have demonstrated strong antibacterial activity, primarily due to the presence of a few key phenolic and terpenoid compounds such as carvacrol and thymol. Other components, like 1,8-cineole, citral, eugenol, geraniol, α-pinene, perillaldehyde, and terpinen-4-ol, also play significant roles in enhancing antibacterial activity [93,120]. The antibacterial mechanisms can be evaluated through several techniques as follows:

4.2. In Vitro Bioassays

Various bioassays, such as disk diffusion, well diffusion, and agar or broth dilution, may be used. After incubation the diameter of the zone of inhibition or the values of minimum inhibitory concentration (MIC) are calculated to assess antimicrobial activities against A. hydrophila [121,122,123,124,125]. Determining the minimum bactericidal concentration (MBC) is one of the initial steps for evaluating antimicrobial compounds [67,122,126,127], but a previous review demonstrated that there is no relationship between the results of MIC or MBC tests for EOs and their in vivo results. This outcome may be related to the immunomodulatory activity of the EOs, which increases the host’s immune defense [128]. The ratio of MBC to MIC serves as a parameter for evaluating the antimicrobial effect of EOs: compounds with an MBC/MIC ratio ≤ 4 are considered bactericidal, while those with an MBC/MIC ratio > 4 are deemed bacteriostatic [64,129,130,131,132,133,134].

4.3. Determination of the Permeability of the Cell Membrane

Cell Membrane Integrity Assessment

The extracellular membrane is composed mainly of proteins and phospholipids, and it is essential for maintaining the integrity of the bacterium, facilitating material exchange between cells and the environment [135,136]. EOs may disrupt membrane structure, alter membrane potential, increase permeability, and cause intracellular substance leakage, ultimately affecting microbial survival. The crucial role of cell membrane integrity in typical bacterium activity and survival is well recognized [137,138].

Cell membrane integrity is assessed by quantifying the release of cell organelles, including DNA and RNA, through absorbance measurements at 260 nm in the bacterial supernatant [139]. For example, the exposure of A. hydrophila for 4–12 h at ½, 1, and 2 MIC to Citrus unshiu EO resulted in increases in DNA and RNA levels in the supernatant as exposure time increased [140]. Therefore, the higher concentrations and elevated time of incubation of EOs enhanced RNA and DNA interruption, resulting in irreversible damage to cytoplasmic membranes and subsequent cell death [141]. One of the most promising bioactive compounds is thymol, from T. vulgaris EO. Exposure of A. hydrophila in vitro to this compound promoted significant structural alterations, including collapsed cell walls, loss of membrane integrity and the presence of wrinkles on the surface, which were observed by scanning electron microscopic (SEM) and transmission electron microscopic (TEM). A. hydrophila exhibited separated cell membranes and cytoplasm leakage after thymol treatment, resulting in the destruction/denaturation of macromolecular constituents such as proteins and DNA, impacting cell development [142]. Similarly, treatment with linalool, a monoterpenoid mainly derived from Lamiaceae plants, leads to a sequence of antibacterial mechanisms, observed through SEM and TEM. Initially the compromised cell wall and membrane became permeable and allow intracellular components to be discharged outward. This deformation causes the cell to collapse. Subsequently, external water enters the damaged cell, leading to swelling. This sequence of events highlights the progressive nature of cell damage following membrane disruption, starting with leakage of cell components and ending with deformation caused by osmotic pressure [143]. These observations suggest that many plant EOs have the potential to disrupt the morphological structure of bacterial cells, contributing to cell death [144,145]. Furthermore, membrane integrity was evaluated through flow cytometry analysis with a different staining method using thiazole orange and propidium iodide, bis-1,3-dibutylbarbutiric acid (BOX), propidium iodide and ethidium bromide staining after treatment with the MIC of EOs. The percentage of membrane integrity was significantly lower compared to the control group: 75.6% and 11.9% were positioned in Q2 and Q4 plots respectively, which indicated that the DNA and RNA of the bacteria were denatured [140].

4.4. Interruption of Cellular Proteins

A. hydrophila exposed for 4–24 h to several EO concentrations (¼, ½, 1, and 2 MIC) showed reduced intracellular protein content compared to the untreated bacteria, indicating changes in protein metabolism [146]. For example, at subinhibitory concentrations, thymol affected the intracellular protein content and changed the protein profile in A. hydrophila, suggesting possible disruptions in RNA and protein metabolism and inhibiting bacterial growth [147]. Inhibitory concentrations of cinnamaldehyde also reduced intracellular soluble protein, demonstrated by gradual fading of protein bands in gel electrophoresis analysis in a time dependent manner, indicating inhibition of protein biosynthesis [148]. The researchers reported that cinnamon oil and tea tree [80,81] (Melaleuca alternifolia and Cinnamomum cassia) EO showed strong and synergic antimicrobial and bactericidal activity against the A. hydrophila K894 strain when compared to the pure compound trans-cinnamaldehyde. The mixture of both agents shows reduced MIC of from 80% to 87% [149,150]. The MIC values varied based on the ratio of components present in the EOs. Most probably, thymol or carvacrol may enhance the permeability of cytoplasmic membrane, which allows the EOs to pass through and bind with protein molecules to be disintegrated. This membrane alteration leads to the loss of functional proteins transporting molecules and eventually there is denaturation of several enzymes and proteins and loss of ions and metabolites, as depicted in Figure 2 [146,151,152].

4.5. Antibiofilm Effect

A. hydrophila is one of the strongest pathogens that produces biofilms on stainless steel surfaces [153]. Small signaling molecules acyl homoserine lactones (AHL) induce large bacterial colonization through cell–cell communication systems mainly regulated by quorum sensing (QS) [154]. Several studies showed that the pathogenicity of A. hydrophila could be suppressed by affecting the QS; therefore, it can be a combat method to control A. hydrophila infections [155]. In addition, the complex biofilm matrix that protects the structural integrity of bacteria from several stress factors [156,157,158,159] can be damaged by antimicrobial agents. Most of the researchers in the reviewed studies adopted O’Toole et al.’s (1999) [160] modified protocol and Pa (2002) [161] to analyze the antibiofilm activity of EOs from various plants against A. hydrophila. For example, [161] found that oregano and thyme EOs exhibited the strongest antibiofilm activity against A. hydrophila, followed by tea tree and peppermint oils. Additionally, they observed that biofilm formation increased as EO concentrations decreased. The lowest concentrations capable of significantly suppressing biofilm formation were 0.0078 µL/mL for oregano, thyme, and peppermint EOs, and 0.015 µL/mL for tea tree EOs [162]. Husain et al. (2013) [163] reported a decrease in A. hydrophila biofilm formation dependent on the concentration of clove oil. Oil concentrations ranging from 0.05 to 0.4% v/v led to a 35–66% decrease in biofilm development. This reduction was associated with a decrease in QS-regulated virulence factors such as LasB, total protease, chitinase, and pyocyanin production, as well as swimming motility and exopolysaccharide production [64,164]. Cinnamaldehyde could downregulate AHLs in a concentration-dependent manner because the main signal molecule of the QS system in A. hydrophila can inhibit biofilm formation through QS regulation. The MIC of 1 μg/mL could inhibit the biofilm significantly [141].

4.6. Anti-Hemolytic Effect

According to Sutili et al. (2014) [165] the hemolysis assay is a method used to assess the anti-hemolytic activity of EOs on red blood cells. In this assay, a β-hemolytic bacterial strain is cultured in Mueller–Hinton broth (MHB) with subinhibitory concentrations of EOs diluted in ethanol. The hemolytic activity of eugenol (50 µg/mL) tested by Sutili et al. (2014) [165] showed that a significant reduction had been achieved (35%) using a subinhibitory concentration with fish blood cells. It is likely that the EOs of Hesperozygis ringens, Ocimum gratissimum, and Ocimum americanum showed efficient anti-hemolysis effects against A. hydrophila. Nevertheless, a higher concentration (200 µg/mL) increased hemolysis because the existence of EOs in the supernatant of the bacteria may lead to cytotoxicity in the blood cell membrane [132]. Li et al. (2023) [166] determined that aerolysin production decreased with increasing cinnamaldehyde concentrations. Therefore, cinnamaldehyde may reduce the hemolytic activity of A. hydrophila supernatants by inhibiting aerolysin production. The extent of hemolysis is determined by measuring optical density at 540 nm, and the percentage of hemolysis is calculated by comparing the results to total and no-hemolysis controls [165].

4.7. Morphological Effects of Essential Oils on Aeromonas hydrophila

SEM and TEM analyses of A. hydrophila treated with EOs revealed significant morphological alterations [167], including cell wall and membrane damage, cytoplasmic disintegration, and leakage of cell contents. These findings suggest that EOs may primarily target the cell membrane, contributing to their antibacterial effects [168]. SEM and TEM analysis revealed significant modifications in the ultrastructure of bacterial cells treated with thymol, indicating its efficacy against A. hydrophila [146,169]. While control group cells exhibited intact structures and smooth edges, treatment group cells showed collapse, wrinkle formation, and membrane integrity loss [170].

4′,6-diamidino-2-phenylindole (DAPI), a fluorescent dye binding to DNA and RNA, exhibits stronger fluorescence with higher nucleic acid levels [151,171]. Upon penetrating the cell membrane, DAPI binds to nucleic acid within the cell. Observation under a fluorescence microscope revealed that A. hydrophila treated with inhibitory concentrations of cinnamaldehyde exhibited significantly weaker fluorescence compared to control groups [148]. Cinnamaldehyde also showed concentration-dependent inhibition of DNA synthesis, with fluorescence intensity gradually decreasing with increasing cinnamaldehyde concentration, possibly due to altered cell membrane permeability [172].

5. Treatment of A. hydrophila Infection in Fish with Essential Oils

Numerous fish species, such as Rhamdia quelen [123,132,173], Colossoma macropomum [174,175], Oreochromis niloticus [65,66], Ictalurus punctatus [176,177], Oncorhynchus mykiss [178,179], Rutilus caspicus [180], Cyprinus carpio, etc. [80,81], have been significantly affected by MAS. EOs have the potential to be added to fish diets as nutritional supplements, and they have shown promising results in terms of improving health and welfare [81,166,181,182,183,184]. These benefits include enhancing resistance against ulcerative dermatitis caused by A. hydrophila [163], enhanced functionality of digestive enzymes in the intestine [185], positive effects on intestinal microbiota [186], and enhanced immune system function [173]. In addition, EO-treated fish show less morpho-pathological alterations than non-treated ones [123,132,173,187].

The compounds carvacrol and thymol have exceptionally low MIC and MBC values (0.06 mg/mL and 0.12 mg/mL respectively) (Table 1) and may play crucial roles in combating A. hydrophila. Though EOs are often anticipated to be harmless substitutes for synthetic drugs, their biological safety in aquatic fauna is extremely dose dependent [188]. According to de Sousa et al. (2023) and Makeri et al. (2023), the compounds of jasmine oil are mainly terpenoids, and these are completely safe chemicals for humans at smaller quantities, whereas they can be toxic to aquatic organisms at prominent quantities [189,190]. Terpenoids mainly affect kidney infiltrations, and reabsorption may lead to dysfunction [10]. In addition, the compounds thymol and carvacrol have been shown to induce adverse effects (epithelial cell interruption, hepatic stress, oxidative imbalance, and behavioral changes) when the animals are administered high dosages [191]. Summarily, the in vivo studies showed positive findings among the EOs, such as for survival rate, immunity enhancement, and pathogen load reduction, while comprehensive toxicity endpoints are less consistently reported [192,193]. The juveniles of C. macropomum were anesthetized using thyme oil nanoemulsion (≤100 nm), which caused changes in physiological biomarkers without inducing mortality [193]. Bandeira et al. (2017) utilized the EOs from O. gratissimum and H. ringens to predict acute toxicity in Daphnia pulex, with lethal concentrations (LC50–24 h) of 37.8 mg/L and 61.5 mg/L respectively. In prior studies, the researchers studied antioxidant and antiparasitic activities with same concentrations, which were proved non-toxic to the host and the survival rate was likewise greater, ranging from 80% to 90% [65].

Owing to volatility, the bioactivity and immiscibility of EOs can be compromised when exposed to air, temperature, irradiation, and UV illuminations during feed preparation and processing [194]. To overcome this and to improve absorption, controlled release of EOs and feed encapsulation are incorporated with various processes such as nanoemulsion [195], nanoliposomes [196], solid lipid carriers, and nanostructured lipid carriers [197] using hassle-free solvents and chemicals, viz., PEG, DMSO, ENRO, FFC, OTC and TAP [10,198,199]. These coatings help to prevent thermolabile EOs from volatilization and oxidative degradation, whereas carrier-based formulations enhance dispersion and bioavailability to replace organic solvents [131,200]. Even though solvents like DMSO are sometimes used in laboratory studies for uniform mixing, they are not applicable to large-scale aquaculture. Industrial approaches could instead rely on water-dispersible formulations that would minimize solvent usage, thereby decreasing environmental load and operational costs [31]. The use of polyethylene glycol (PEG 400) and dimethyl sulfoxide (DMSO) to dilute T. vulgaris EOs yields an MIC on A. hydrophila (0.553 mg/mL and 0.30 mg/mL) with PEG400 and an MBC (1.176 mg/mL and 0.588 mg/mL) with DMSO (Table 2) [201]. The mixture of carvacrol and thymol (major active compounds from T. vulgaris EOs) stands out as a potent combination. Similarly, the tethered chemicals PEG 400 and DMSO with A. sativum EOs showed efficient MIC and MBC values of 0.338 mg/mL and 1.353 mg/mL, respectively (Table 2) [201]. Research revealed that a combination of some hassle-free chemicals can suppress bacterial infections, such as PEG, DMSO, enrofloxacin (ENRO), florfenicol (FFC), oxytetracycline (OTC), and thiamphenicol (TAP) [10,199]. Secondly, the compounds cinnamaldehyde, o-methoxycinnamaldehyde and cinnamyl acetate derived from Cinnamomum cassia showed remarkable effectiveness against MAS in C. carpio, with 98.66% survival rate and very low MIC and MBC values (0.039 and 0.50 mg/mL respectively) [80,81]. Furthermore, EOs from Lippia sidoides and O. gratissimum achieved 100% survival rate in in vivo experiments with C. macropomum [163]. Eugenol, 1,8-cineole and β-selinene contribute to effectiveness against A. hydrophila. However, the MIC and MBC values are significantly higher and more variable compared to other EOs [174,202]. The EOs from Aloysia triphylla, Satureja hortensis, Coriandrum sativum L., Zingiber officinale exhibited over 90% of survival rates in in vivo experiments with R. quelen, R. caspicus, O. niloticus, and C. macropomum, in spite of showing high in vitro MIC values [180,187,203,204]. In a nutshell, the compounds carvacrol, thymol, cinnamaldehyde, eugenol, terpinene, and p-cymene present in EOs profoundly enhanced antibacterial activity against A. hydrophila and exhibited more than 90% survival rate in the in vivo studies (Table 3). Also, the formulation stratagems support the scalability of EO-based intrusion by improving stability during feed manufacturing and enabling efficient, low burden application in immersion treatments [31].

Table 1. In vitro antibacterial efficacy of essential oils from various plants and their main compounds against A. hydrophila.

Plant Species	Major Compounds (%)	MIC (mg/mL)	MBC (mg/mL)	Activity Level	Reference
Artemisia annua	Camphor (29.2), 1,8-cineole (13.27), and tetradecanol (6.16),	3.2	>6.4	W	[205]
Cinnamomum cassia	Cinnamaldehyde (84.8), o-methoxycinnamaldehyde (10.07), cinnamyl acetate (2.17)	0.039	0.5	S	[80,81]
Coriandrum sativum L.	Linalool and geranyl acetate.	8.68 0	-	W	[204]
Cymbopogon citratus	α-citral (31.80), β-citral (27.75), isoneral (5.36), α-myrcene (4.44), linalool (3.48) and epoxy-linalooloxide (3.11)	50	-	W	[206]
Cymbopogon flexuosus	Geranial (48.89), neral (40.32), cis-verbenol (2.38), and camphene (0.89)	0.195	0.195	M	[65,66]
	Geranial 41, neral 32, geraniol 6.7, geranyl acetate 3.2	>15.60	>15.60	W	[174]
Cymbopogon nardus	Citronellal, citronellol, and geraniol	-	27.8	W	[207]
Eugenia caryophyllus	Not provided	0.19–1.56	0.38–3.12	M	[149]
Hesperozygis ringens	Monoterpenoid pulegone (47) and menthone (5.82)	0.8	3.2	W	[132,208]
Lippia alba	Geranial (25.4) and neral (16.6)	2.862	5.998	W	[122,123]
Lippia graveolens chemotype II,	Carvacrol (51.82) and thymol (79.62)	0.092	0.184	S	[68]
Lippia origanoides	Carvacrol (40.4) and p-cymene (11.4)	0.625	1.25	W	[122,201]
Lippia sidoides	Eugenol (43.3), 1,8-cineole (28.2) and β-selinene (5.5)	0.625	1.25	W	[134,202]
	Carvacrol (44.50), p-cymene (14.06), γ-terpinene (12.43) and thymol (7.99)	0.625	3.125	W	[64]
Melaleuca alternifolia	Not provided Terpinen-4-ol (38.44)	0.78–12.5 0.039	1.56–50 -	W S	[149,150]
Ocimum americanum	β-linalool (46.61), camphor (9.5), 1.8-cineole (8.43) and germacrene D (4.76)	6.4	6.4	W	[173]
Ocimum gratissimum	Geranial (23.2), neral (16.7) and 1,8-cineole (15.8)	1.25	5	W	[132]
Origanum majorana	Terpinen-4-ol (20.55), α-terpineol (4.396), terpinene (13.136), terpineol, trans (12.668), α-terpineol (4.396)			-	[128]
Pelargonium graveolens	Citronellol (32.54), 3-methylpentane (11.96) and isomenthone (10.64)	50	-	W	[206]
Plectranthus amboinicus	Cymenederivatives (22.93), y-terpinene (8.04) and carvacrol (65.36)	0.062	0.125	S	[67]
Salvia pisidica	Camphor (23.8), sabinol (19.2), α-thujene (14.2)	>20	>20	W	[209]
Schinus terebinthifolius	δ-3-careno (56.00), α-pinene (16.89)	20	20	W	[210]
Thymus vulgaris	Thymol (36.3), p-cymene (18.5), γ-terpinene (10.9), linalool (7.1), carvacrol (5.2), β-caryophyllene (4.5) Not provided	0.06 0.09–1.56	0.12 0.09–6.24	S S	[178]
Zingiber officinale	Geranial (23.2), neral (16.7) and 1,8-cineole (15.8)	1.25	5	W	[164]

Strong (S) < 100; Moderate (M) 100–500; Weak (W) > 500 (µg/mL).

Table 1. In vitro antibacterial efficacy of essential oils from various plants and their main compounds against A. hydrophila.

Plant Species	Major Compounds (%)	MIC (mg/mL)	MBC (mg/mL)	Activity Level	Reference
Artemisia annua	Camphor (29.2), 1,8-cineole (13.27), and tetradecanol (6.16),	3.2	>6.4	W	[205]
Cinnamomum cassia	Cinnamaldehyde (84.8), o-methoxycinnamaldehyde (10.07), cinnamyl acetate (2.17)	0.039	0.5	S	[80,81]
Coriandrum sativum L.	Linalool and geranyl acetate.	8.68 0	-	W	[204]
Cymbopogon citratus	α-citral (31.80), β-citral (27.75), isoneral (5.36), α-myrcene (4.44), linalool (3.48) and epoxy-linalooloxide (3.11)	50	-	W	[206]
Cymbopogon flexuosus	Geranial (48.89), neral (40.32), cis-verbenol (2.38), and camphene (0.89)	0.195	0.195	M	[65,66]
	Geranial 41, neral 32, geraniol 6.7, geranyl acetate 3.2	>15.60	>15.60	W	[174]
Cymbopogon nardus	Citronellal, citronellol, and geraniol	-	27.8	W	[207]
Eugenia caryophyllus	Not provided	0.19–1.56	0.38–3.12	M	[149]
Hesperozygis ringens	Monoterpenoid pulegone (47) and menthone (5.82)	0.8	3.2	W	[132,208]
Lippia alba	Geranial (25.4) and neral (16.6)	2.862	5.998	W	[122,123]
Lippia graveolens chemotype II,	Carvacrol (51.82) and thymol (79.62)	0.092	0.184	S	[68]
Lippia origanoides	Carvacrol (40.4) and p-cymene (11.4)	0.625	1.25	W	[122,201]
Lippia sidoides	Eugenol (43.3), 1,8-cineole (28.2) and β-selinene (5.5)	0.625	1.25	W	[134,202]
	Carvacrol (44.50), p-cymene (14.06), γ-terpinene (12.43) and thymol (7.99)	0.625	3.125	W	[64]
Melaleuca alternifolia	Not provided Terpinen-4-ol (38.44)	0.78–12.5 0.039	1.56–50 -	W S	[149,150]
Ocimum americanum	β-linalool (46.61), camphor (9.5), 1.8-cineole (8.43) and germacrene D (4.76)	6.4	6.4	W	[173]
Ocimum gratissimum	Geranial (23.2), neral (16.7) and 1,8-cineole (15.8)	1.25	5	W	[132]
Origanum majorana	Terpinen-4-ol (20.55), α-terpineol (4.396), terpinene (13.136), terpineol, trans (12.668), α-terpineol (4.396)			-	[128]
Pelargonium graveolens	Citronellol (32.54), 3-methylpentane (11.96) and isomenthone (10.64)	50	-	W	[206]
Plectranthus amboinicus	Cymenederivatives (22.93), y-terpinene (8.04) and carvacrol (65.36)	0.062	0.125	S	[67]
Salvia pisidica	Camphor (23.8), sabinol (19.2), α-thujene (14.2)	>20	>20	W	[209]
Schinus terebinthifolius	δ-3-careno (56.00), α-pinene (16.89)	20	20	W	[210]
Thymus vulgaris	Thymol (36.3), p-cymene (18.5), γ-terpinene (10.9), linalool (7.1), carvacrol (5.2), β-caryophyllene (4.5) Not provided	0.06 0.09–1.56	0.12 0.09–6.24	S S	[178]
Zingiber officinale	Geranial (23.2), neral (16.7) and 1,8-cineole (15.8)	1.25	5	W	[164]

Strong (S) < 100; Moderate (M) 100–500; Weak (W) > 500 (µg/mL).

Table 2. In vitro antibacterial efficacy of essential oils from various plants with tethering chemicals (PEG 400 and DMSO) against A. hydrophila [14].

Plant Species	Major Active Compounds (%)	PEG 400		DMSO
		MIC (mg/mL)	MBC (mg/mL)	MIC (mg/mL)	MBC (mg/mL)
Allium sativum	Not provided	0.338	1.353	0.338	1.353
Artemisia vulgaris	Limonene (14.1), α-terpineol (8.1), camphor (17.3), 1,8-cineole (14.2), β-caryophyllene (8.4), β-himachalene (7.3)	9.3	9.3	18.61	18.61
Boswellia carterii	α-pinen (65.1), p-cymene (6.8), limonene (6.4), estragole (5.2)	8.47	8.47	16.95	16.95
Cananga odorata	Germacrene D (24.9), aromadendrene (5.2), β-caryophyllene (19.8), β-farnesene (10.8), benzyl benzoate (7.3)	36.6	36.6	>36.6	_
Cinnamon cinnamomum verum	Cinnamaldehyde (84.8), o-methoxycinnamaldehyde (10.07), cinnamyl acetate (2.17)	0.662	1.325	0.625	0.125
Citrus aurantium var. amara	Limonene (13.5), α-Pinen (8.6), linalool (16.4), linalyl acetate (35.3), β-caryophyllene (10.5)	35.56	35.56	>35.56	_
Citrus bergamia	p-cymene (6.3), linalool (10.7), geranyl acetate (6.1), linalyl acetate (33.1), eugenol (5.3)	8.74	8.74	17.26	17.48
Corymbia citriodora	Cis-sabinene hydrate (7.7), citronellal (77.2), citronellyl acetate (7.0),	4.31	4.31	17.26	34.52
Cupressus sempervirens	δ-3-carene (20.2), α-pinen (52.5), limonene (5.4)	17.26	17.26	34.52	34.52
Cymbopogon citratus	Myrcene (13.8), geranial(40.0), neral (30.4)	2.18	4.37	2.18	4.37
Eucalyptus globulus	Limonene (12.1), 1,8-cineole (73.0), p-cymene (4.4)	18.24	18.24	18.24	18.24
Mentha x piperita	Menthol (70.0), linalool (6.9), menthone (6.5),	4.49	8.98	8.98	17.96
Ocimum basilicum	Linalool (16.9), estragole (77.4)	1.111	2.222	1.111	22.5
Pelargonium graveolens	Citronellol (47.0), geraniol (10.1), citronellyl formate (10.6)	4.44	4.44	8.89	8.89
Thymus vulgaris	p-cymene (34.0), carvacrol (6.6), linalool (6.7), thymol (40.4)	0.588	1.176	0.3	0.588

Table 2. In vitro antibacterial efficacy of essential oils from various plants with tethering chemicals (PEG 400 and DMSO) against A. hydrophila [14].

Plant Species	Major Active Compounds (%)	PEG 400		DMSO
		MIC (mg/mL)	MBC (mg/mL)	MIC (mg/mL)	MBC (mg/mL)
Allium sativum	Not provided	0.338	1.353	0.338	1.353
Artemisia vulgaris	Limonene (14.1), α-terpineol (8.1), camphor (17.3), 1,8-cineole (14.2), β-caryophyllene (8.4), β-himachalene (7.3)	9.3	9.3	18.61	18.61
Boswellia carterii	α-pinen (65.1), p-cymene (6.8), limonene (6.4), estragole (5.2)	8.47	8.47	16.95	16.95
Cananga odorata	Germacrene D (24.9), aromadendrene (5.2), β-caryophyllene (19.8), β-farnesene (10.8), benzyl benzoate (7.3)	36.6	36.6	>36.6	_
Cinnamon cinnamomum verum	Cinnamaldehyde (84.8), o-methoxycinnamaldehyde (10.07), cinnamyl acetate (2.17)	0.662	1.325	0.625	0.125
Citrus aurantium var. amara	Limonene (13.5), α-Pinen (8.6), linalool (16.4), linalyl acetate (35.3), β-caryophyllene (10.5)	35.56	35.56	>35.56	_
Citrus bergamia	p-cymene (6.3), linalool (10.7), geranyl acetate (6.1), linalyl acetate (33.1), eugenol (5.3)	8.74	8.74	17.26	17.48
Corymbia citriodora	Cis-sabinene hydrate (7.7), citronellal (77.2), citronellyl acetate (7.0),	4.31	4.31	17.26	34.52
Cupressus sempervirens	δ-3-carene (20.2), α-pinen (52.5), limonene (5.4)	17.26	17.26	34.52	34.52
Cymbopogon citratus	Myrcene (13.8), geranial(40.0), neral (30.4)	2.18	4.37	2.18	4.37
Eucalyptus globulus	Limonene (12.1), 1,8-cineole (73.0), p-cymene (4.4)	18.24	18.24	18.24	18.24
Mentha x piperita	Menthol (70.0), linalool (6.9), menthone (6.5),	4.49	8.98	8.98	17.96
Ocimum basilicum	Linalool (16.9), estragole (77.4)	1.111	2.222	1.111	22.5
Pelargonium graveolens	Citronellol (47.0), geraniol (10.1), citronellyl formate (10.6)	4.44	4.44	8.89	8.89
Thymus vulgaris	p-cymene (34.0), carvacrol (6.6), linalool (6.7), thymol (40.4)	0.588	1.176	0.3	0.588

Table 3. In vivo efficacy of essential oils from various plants against A. hydrophila.

Plant Species	Major Compounds (%)	Survival (%)	Significant Impacts of the Study	Reference
Bath treatment (μL/L or mg/L)
Gaultheria procumbens	Methyl salicylate (99.5)	5–10—Rhamdia quelen	It improved survival rates in infected fish.	[211]
Hesperozygis ringens	Monoterpenoid pulegone (47) and menthone (5.82)	20–70—Rhamdia quelen	It suppressed apoptosis triggered by infection and increased cell viability. It diminishes bacteria-induced oxidative stress and stimulates the antioxidative responses in muscle of A. hydrophila-infected silver catfish.	[132,208]
Lippia alba	Geranial (25.4) and neral (16.6)	20—Rhamdia quelen—90	Lower anesthetic concentrations could promote survival of infected fish.	[123]
Lippia origanoides	Carvacrol (40.4) and p-cymene (11.4)	5—Rhamdia quelen—58	Therapeutic efficacy of EO against A. hydrophila was enhanced, with low mortality rates.	[201]
	Carvacrol (49.7), p-cymene (13.3) and thymol (9.9)	10—Colossoma macropomum—80	Sublethal concentrations altered a few biochemical and hematological changes and showed significant effects of EO treatment on tambaqui.	[212]
Lippia sidoides	Eugenol (43.3), 1,8-cineole (28.2) and β-selinene (5.5)	0.625—Colossoma macropomum—40	The elevated respiratory activity of leukocytes induced by the innate immune system in post-challenge test.	[174]
Ocimum americanum	β-linalool (46.61), camphor (9.5), 1.8-cineole (8.43) and germacrene D (4.76)	20—Rhamdia quelen -75	The potential usage of EO against the pathogens A. hydrophila and Gyrodactylus sp. in silver catfish (Rhamdia quelen).	[173]
Ocimum gratissimum	Geranial (23.2), neral (16.7) and 1,8-cineole (15.8)	20—Rhamdia quelen -75	It decreases the virulence of A. hydrophila and supports therapeutic applications and anesthetics.	[132]
	Eugenol (43.3), 1,8-cineole (28.2) and β-selinene (5.5)	5—Colossoma macropomum—89.5	Reduced hematocrit percentage, RBC and Rh factors and also mild liver damage.	[134]
Origanum majorana	Terpinen-4-ol (20.55), ɑ-terpineol (4.396), terpinene (13.136), terpineol, trans (12.668), ɑ-terpineol (4.396)	20—Rhamdia quelen—100	Nanoencapsulated formulae endorsed an improved persistence of silver catfish infected with A. hydrophila.	[128]
Zingiber officinale	Citrol (39.9), geranial (23.2), neral (16.7) and 1,8-cineole (15.8)	10—Colossoma macropomum—52.1	Showed week and moderate antimicrobial activity and survival of the animal.	[134]
Preventive treatment (g/kg feed or mL/kg feed)
Aloysia triphylla	-	2.0—Rhamdia quelen > 90	EO diet improved oxidative status and lowered stress response in silver catfish.	[213]
Citrus sinensis		0.4–0.8—Colossoma macropomum	Lower mortality and enhancement on growth performance and hematological parameters.	[199]
Coriandrum sativum L.	Linalool and geranyl acetate	10–20—Oreochromis niloticus—100	Enhancement of antioxidant and anti-inflammatory activities and innate immunity against A. hydrophila.	[204]
Croton sonderianus	-	Up to 1.5—Colossoma macropomum—no effect on survival	Enhancement in growth performance, hematology and biochemical parameters against bacterial resistance in tambaqui juveniles.	[184]
Cymbopogon citratus	α -citral (31.80), β-citral (27.75), isoneral (5.36), α-myrcene (4.44), linalool (3.48) and epoxy-linalooloxide (3.11)	0.20—Oreochromis niloticus—96	Dietary supplementation of EO in fish meal enhancing growth performance, feed utilization, oxidative status, immune responses and disease resistance.	[206]
	α-citral (73.56), myrcene (12.65), linalool (1.00), isogeranial (2.91), geraniol (3.51)	0.25—Colossoma macropomum—50	Treatment with EO against infected fish showed improvement in hematological variables, muscle glycogen levels and intestinal alkaline protease activity in tambaqui.	[175]
Cymbopogon flexuosus	Geranial—(48.89), neral—(40.32), cis-verbenol—(2.38), and camphene—(0.89)	2.0—Oreochromis niloticus	Improved growth rate, biochemical and physiological parameters and reduced mortality of Nile tilapia after treatment.	[66]
Lippia alba	Geranial (25.4) and neral (16.6)	0.25—Oreochromis niloticus—100	It ameliorated feed conversion, hematocrit and adaptive immunity, and improved survival rate after infection.	[172]
Lippia sidoides	Thymol (76.6), carvacrol (26.4) and 1.8 cineole (22.63) ortho-cimene (6.3) and beta-cariofilene (5.0)	0.125—Cyprinus carpio—0.65	Each concentration showed specific benefits to the animal, such as increased zootechnical performance, highest concentration of lactic acid bacteria in the intestine and the greatest post-challenge survival.	[202]
Ocimum basilicum	Linalool	2.0—Oreochromis niloticus—30	Improvement in growth performance and increased hematological variables and activity of intestinal enzymes; however, the survivability of fish after infection was not influenced by the addition of EO in the diet.	[214]
Origanum heracleoticum L.	Carvacrol (54.9) p-cymene (22.0) Thymol methyl ether (1.6) γ-terpinene (3.5)	0.50—Ictalurus punctatus—95	The mixed compounds increase growth rate of cat fish.	[176]
Pelargonium graveolens	Cymenederivatives (22.93), y-terpinene (8.04) and carvacrol (65.36)	0.40—Oreochromis niloticus—95	Stimulate lysozyme activity in the innate immune system and increased beneficial microbial flora in gut region.	[206]
Satureja hortensis		0.20—Rutilus caspicus—96	EO-treated fish showed significant increase in serum total immunoglobulin, lysozyme and alternative complement pathway hemolytic activity.	[180]
Thymus vulgaris	Thymol (36.3), p-cymene (18.5), γ-terpinene (10.9), linalool (7.1), Carvacrol (5.2), and β-caryophyllene (4.5)	0.5—Oncorhynchus mykiss—31.5	Increased growth rate of rainbow trout, improved immunity and developed resistant to MAS.	[179]

Table 3. In vivo efficacy of essential oils from various plants against A. hydrophila.

Plant Species	Major Compounds (%)	Survival (%)	Significant Impacts of the Study	Reference
Bath treatment (μL/L or mg/L)
Gaultheria procumbens	Methyl salicylate (99.5)	5–10—Rhamdia quelen	It improved survival rates in infected fish.	[211]
Hesperozygis ringens	Monoterpenoid pulegone (47) and menthone (5.82)	20–70—Rhamdia quelen	It suppressed apoptosis triggered by infection and increased cell viability. It diminishes bacteria-induced oxidative stress and stimulates the antioxidative responses in muscle of A. hydrophila-infected silver catfish.	[132,208]
Lippia alba	Geranial (25.4) and neral (16.6)	20—Rhamdia quelen—90	Lower anesthetic concentrations could promote survival of infected fish.	[123]
Lippia origanoides	Carvacrol (40.4) and p-cymene (11.4)	5—Rhamdia quelen—58	Therapeutic efficacy of EO against A. hydrophila was enhanced, with low mortality rates.	[201]
	Carvacrol (49.7), p-cymene (13.3) and thymol (9.9)	10—Colossoma macropomum—80	Sublethal concentrations altered a few biochemical and hematological changes and showed significant effects of EO treatment on tambaqui.	[212]
Lippia sidoides	Eugenol (43.3), 1,8-cineole (28.2) and β-selinene (5.5)	0.625—Colossoma macropomum—40	The elevated respiratory activity of leukocytes induced by the innate immune system in post-challenge test.	[174]
Ocimum americanum	β-linalool (46.61), camphor (9.5), 1.8-cineole (8.43) and germacrene D (4.76)	20—Rhamdia quelen -75	The potential usage of EO against the pathogens A. hydrophila and Gyrodactylus sp. in silver catfish (Rhamdia quelen).	[173]
Ocimum gratissimum	Geranial (23.2), neral (16.7) and 1,8-cineole (15.8)	20—Rhamdia quelen -75	It decreases the virulence of A. hydrophila and supports therapeutic applications and anesthetics.	[132]
	Eugenol (43.3), 1,8-cineole (28.2) and β-selinene (5.5)	5—Colossoma macropomum—89.5	Reduced hematocrit percentage, RBC and Rh factors and also mild liver damage.	[134]
Origanum majorana	Terpinen-4-ol (20.55), ɑ-terpineol (4.396), terpinene (13.136), terpineol, trans (12.668), ɑ-terpineol (4.396)	20—Rhamdia quelen—100	Nanoencapsulated formulae endorsed an improved persistence of silver catfish infected with A. hydrophila.	[128]
Zingiber officinale	Citrol (39.9), geranial (23.2), neral (16.7) and 1,8-cineole (15.8)	10—Colossoma macropomum—52.1	Showed week and moderate antimicrobial activity and survival of the animal.	[134]
Preventive treatment (g/kg feed or mL/kg feed)
Aloysia triphylla	-	2.0—Rhamdia quelen > 90	EO diet improved oxidative status and lowered stress response in silver catfish.	[213]
Citrus sinensis		0.4–0.8—Colossoma macropomum	Lower mortality and enhancement on growth performance and hematological parameters.	[199]
Coriandrum sativum L.	Linalool and geranyl acetate	10–20—Oreochromis niloticus—100	Enhancement of antioxidant and anti-inflammatory activities and innate immunity against A. hydrophila.	[204]
Croton sonderianus	-	Up to 1.5—Colossoma macropomum—no effect on survival	Enhancement in growth performance, hematology and biochemical parameters against bacterial resistance in tambaqui juveniles.	[184]
Cymbopogon citratus	α -citral (31.80), β-citral (27.75), isoneral (5.36), α-myrcene (4.44), linalool (3.48) and epoxy-linalooloxide (3.11)	0.20—Oreochromis niloticus—96	Dietary supplementation of EO in fish meal enhancing growth performance, feed utilization, oxidative status, immune responses and disease resistance.	[206]
	α-citral (73.56), myrcene (12.65), linalool (1.00), isogeranial (2.91), geraniol (3.51)	0.25—Colossoma macropomum—50	Treatment with EO against infected fish showed improvement in hematological variables, muscle glycogen levels and intestinal alkaline protease activity in tambaqui.	[175]
Cymbopogon flexuosus	Geranial—(48.89), neral—(40.32), cis-verbenol—(2.38), and camphene—(0.89)	2.0—Oreochromis niloticus	Improved growth rate, biochemical and physiological parameters and reduced mortality of Nile tilapia after treatment.	[66]
Lippia alba	Geranial (25.4) and neral (16.6)	0.25—Oreochromis niloticus—100	It ameliorated feed conversion, hematocrit and adaptive immunity, and improved survival rate after infection.	[172]
Lippia sidoides	Thymol (76.6), carvacrol (26.4) and 1.8 cineole (22.63) ortho-cimene (6.3) and beta-cariofilene (5.0)	0.125—Cyprinus carpio—0.65	Each concentration showed specific benefits to the animal, such as increased zootechnical performance, highest concentration of lactic acid bacteria in the intestine and the greatest post-challenge survival.	[202]
Ocimum basilicum	Linalool	2.0—Oreochromis niloticus—30	Improvement in growth performance and increased hematological variables and activity of intestinal enzymes; however, the survivability of fish after infection was not influenced by the addition of EO in the diet.	[214]
Origanum heracleoticum L.	Carvacrol (54.9) p-cymene (22.0) Thymol methyl ether (1.6) γ-terpinene (3.5)	0.50—Ictalurus punctatus—95	The mixed compounds increase growth rate of cat fish.	[176]
Pelargonium graveolens	Cymenederivatives (22.93), y-terpinene (8.04) and carvacrol (65.36)	0.40—Oreochromis niloticus—95	Stimulate lysozyme activity in the innate immune system and increased beneficial microbial flora in gut region.	[206]
Satureja hortensis		0.20—Rutilus caspicus—96	EO-treated fish showed significant increase in serum total immunoglobulin, lysozyme and alternative complement pathway hemolytic activity.	[180]
Thymus vulgaris	Thymol (36.3), p-cymene (18.5), γ-terpinene (10.9), linalool (7.1), Carvacrol (5.2), and β-caryophyllene (4.5)	0.5—Oncorhynchus mykiss—31.5	Increased growth rate of rainbow trout, improved immunity and developed resistant to MAS.	[179]

6. Conclusions and Prospects

Several EOs exhibit promising antibacterial activity against A. hydrophila according to in vitro and in vivo investigations. However, their industrial application for the treatment of A. hydrophila infections is limited due to the necessity of high concentrations for effective antimicrobial action in baths, and the optimization of quantity, source, and active composition profiles on fish diseases remains unresolved. On the other hand, dietary supplementation with EOs is a viable alternative as a preventive treatment against A. hydrophila infections. Based on the findings outlined in the review, it was observed that T. vulgaris, C. cassia and L. sidoides EOs displayed noteworthy MIC and MBC values and showed >90% success rates in in vivo experiments against A. hydrophila when compared to previous investigations. Furthermore, the effectiveness of EOs in inhibiting the growth of A. hydrophila highlights their potential as natural antimicrobial agents for combating bacterial infections in aquaculture. Additional studies into the precise bioactive materials and mechanisms responsible for their antibacterial properties may provide valuable insights for the advancement of innovative treatments for contagious diseases. Nonetheless, challenges including lipophilic characteristics, vulnerability to degradation, high volatility and oxidation are considerable limitations that affect the therapeutic efficacy associated with pharmaceutical products. Future initiatives may investigate innovative technology strategies to optimize essential oil delivery systems, with the objective of enhancing bioavailability and therapeutic efficacy in aquaculture.