Oregano essential oil has the potential to reduce the virulence of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) in Penaeus vannaei

doi:10.21203/rs.3.rs-5276769/v1

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Oregano essential oil has the potential to reduce the virulence of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) in Penaeus vannaei

https://doi.org/10.21203/rs.3.rs-5276769/v1

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Acute Hepatopancreatic Necrosis Disease (AHPND), caused by various species of Vibrio, results in significant mortality rates in global penaeid shrimp aquaculture. While antibiotics are commonly employed to combat this disease, concerns about the rapid emergence of resistant strains and their potential adverse effects on human health and the environment are rising. In this context, plant-derived essential oils have shown promising potential for controlling vibriosis in shrimp farming. This study investigated the antivirulence effects of oregano essential oil (OEO) at sub-minimum inhibitory concentrations (sub-MIC) on three virulence phenotypes (exopolysaccharide production, biofilm formation, and swarming motility) in V. parahaemolyticus the causative agent of AHPND. The antivirulence effect of OEO was validated through a challenge test by infecting Penaeus vannamei with V. parahaemolyticus cultured with and without OEO. Additionally, a transcriptomic analysis was performed to compare the transcriptional regulation of V. parahaemolyticus with and without OEO addition to identify therapeutic targets at the molecular level. The three virulence phenotypes were significantly inhibited (P < 0.05), with their effect being concentration-dependent. In the in vivo challenge, shrimp mortality infected with V. parahaemolyticus cultured with OEO (56 ± 5%) was significantly (P < 0.05) lower than in the control group (93 ± 5%). Transcriptomic analysis revealed 89 differentially expressed genes (DEGs) downregulated, associated with membrane integrity, ribosomes, motility, transport, type VI secretion system (T6SS), and enzymes involved in energy metabolism, and 94 DEGs upregulated, linked to biosynthetic processes, regulatory proteins, osmoadaptation, multidrug efflux transporters, ABC transporters, and zinc and nitrogen transport. These findings suggest that OEO targets multiple therapeutic pathways, inhibiting V. parahaemolyticus virulence. Our study demonstrates the significant antivirulence effect of OEO on V. parahaemolyticus, underscoring OEO's potential as a viable and eco-friendly alternative to traditional antibiotics for controlling AHPND in shrimp aquaculture.

Aquaculture and Mariculture

Animal Science

Veterinary Epidemiology

Antimicrobial

Antivirulence strategy

Aquaculture

Vibriosis

biofilms

Penaeus vannaei

Globally, several Vibrio species have been identified as causative agents of acute hepatopancreatic necrosis disease (AHPND) in shrimp farming [1–4]. These vibrios carry a large 70 kbp extrachromosomal plasmid (pVA1) that encodes highly virulent PirA^Vp and PirB^Vp toxins for penaeid shrimp [5–7]. AHPND can cause massive mortalities, reaching 100% in shrimp farming systems [8], resulting in substantial economic losses [8, 9]. Antibiotics are a common treatment for vibriosis in aquaculture [10, 11]. However, antibiotic use imposes selective pressure that favors bacterial resistance [12–14]. It also disrupts beneficial microbiota [8, 15], induces biofilm formation essential for bacterial persistence [16], and causes irreversible environmental changes [17]. Antibiotic residues in animals contribute to the development of multidrug-resistant strains [18, 19], posing a significant public health risk [20, 21]. Several studies highlight the potential of antivirulence strategies for controlling pathogenic vibrios in aquaculture [22–26].

Antivirulence strategies focus on interfering with the mechanisms by which pathogenic bacteria initiate infection and cause disease [27]. These include cell adhesion and invasion, intracellular replication, biofilm formation, disruption of bacterial communication (QS) [28, 29], and evasion of host immune defenses. Considering the increase in microbial resistance to antibiotics, these strategies have gained global relevance in the last decade. Unlike antibiotics, which eliminate bacteria and generate high selective pressure favoring resistance, antivirulence therapy seeks to neutralize virulence factors without affecting their viability [28], thereby reducing the risk of resistance [30, 31]. Sublethal concentrations of essential oils (EOs) have been reported to inhibit the expression of virulence genes [32–36], making them promising candidates for antivirulence strategy. Using this approach, essential oils are expected to exert less selective pressure, thus reducing the risk of bacterial resistance development [31].

EOs are complex mixtures of low molecular weight molecules, primarily monoterpenes and sesquiterpenes with their oxygenated derivatives. The composition of EOs varies based on cultivation location, plant harvests, and extraction methods, which in turn influence their biological activity [37]. Monoterpenes are considered to exhibit the highest antivirulence activity, potentially targeting multiple therapeutic sites, including disrupting outer and inner membranes, inhibiting protein synthesis, or binding to structural proteins, thereby affecting their function [38]. Moreover, monoterpenes interfere with the synthesis of structural proteins of appendages, which are essential for motility, adherence, colonization, and biofilm formation [39–41]. They can also interfere with efflux pumps, a primary bacterial resistance mechanism [34, 42, 43]. Additionally, monoterpenes can disrupt signaling cascades such as the quorum sensing (QS) system by binding to acyl-homoserine lactone (AHL) receptors due to structural similarity, thereby affecting bacterial virulence [43, 44]. EOs have demonstrated low toxicity in vertebrates and are classified as Generally Recognized as Safe (GRAS) by the Food and Drug Administration (FDA, 2016) [45], and are deemed safe for consumers by the European Commission [46]. Furthermore, the active compounds in EOs naturally degrade within a few days, diminishing their biocidal effect and resulting in a low ecological impact [47]. These attributes make EOs ideal candidates for use as therapeutic agents in controlling pathogenic vibrios in aquaculture. In this last decade, various molecular tools and bioinformatics techniques have been widely used to determine the molecular mechanisms of action of EOs, among other phytochemicals [48].

Omics techniques, including transcriptomics, have been key to identify and analyzing how antibacterial agents with high precision and efficiency [49–51]. In this sense, omics play an important role in understanding the mechanisms that microbes exhibit when treated with antibacterial agents [49]. Furthermore, they allow the detection of compounds and molecules as potential inhibitors of pathological targets and can also help us discover and optimize more effective next-generation antimicrobials. Studies involving the analysis of the transcriptome of V. parahaemolyticus causing AHPND in shrimp exposed to sub-MIC doses of oregano essential oil (OEO) are lacking. However, transcriptional analysis performed in other bacteria exposed to sublethal concentrations of EOs has provided clues regarding the antibacterial mechanism [52–55], suggesting that OEO mainly affect bacterial motility, flagella formation, and quorum sensing [56–62]. OEO has also been reported to induce adaptive responses to cope with stress in Y. enterocolitica [63], as well as energy metabolism, fatty acid degradation, TCA cycle and oxidative phosphorylation in Salmonella [58].

OEO has been widely studied for its various biological properties, particularly in aquaculture, demonstrating antibacterial activity [64–67], anti-QS [26], antifungal [68], antiparasitic [69], anesthetic [70], antioxidant, and immunostimulant properties [71–73]. In a previous study, we reported the antivirulence activity of oregano essential oil (OEO) against aquaculture pathogenic vibrios [26]. However, the antivirulence effect of OEO on Vibrio parahaemolitycus, the causative agent of AHPND in farmed shrimp, has not yet been reported nor characterized. The present study initially evaluated the in vitro effects of OEO at different sublethal concentrations on total exopolysaccharide production, biofilm formation, and swarming motility in V. parahaemolyticus (strain BA94C2) AHPND- causing. Subsequently, OEO concentrations were evaluated in vivo through a challenge test by infecting Pennaeus vannamei shrimp with bacterial inoculum cultured with or without OEO to confirm the impact on V. parahaemolyticus virulence. Additionally, we assessed the transcriptional effects on V. parahaemolyticus exposed to an active sublethal dose of OEO to elucidate potential therapeutic targets and the molecular-level antivirulence effect of OEO. Our results indicate a profound negative effect of OEO on the expression of V. parahaemolyticus virulence genes and its ability to cause mortality in P. vannamei shrimp.

2.1 Growth Conditions of V. parahaemolyticus causing AHPND

In this study, we used an isolate of V. parahaemolyticus (strain BA94C2) harboring genes encoding the PirA/B^Vp toxins responsible for AHPND in shrimp farming [74, 75]. BA94C2 was grown aerobically on Tryptic Soy Agar supplemented with 2% NaCl (TSA + 2% NaCl) by streaking and incubating for 24 hours at 28°C. Eight colonies were selected and transferred to a screw-cap glass bottle containing 100 mL of Tryptic Soy Broth supplemented with 2% NaCl (TSB + 2% NaCl) and incubated at 28°C with constant agitation (110 rpm) overnight. One mL of the grown culture was transferred to another flask containing 99 mL of TSB + 2% NaCl broth and allowed to grow for 8 hours at 28°C and 150 rpm. The exponentially growing BA94C2 culture was resuspended in TSB + 2% NaCl broth to obtain bacterial suspensions of 2 × 10⁸ colony forming units (CFU) mL^-1 at an optical density (OD) of 0.32 nm. Serial dilutions were made in sterile saline solution (SS-2% NaCl) for subsequent assays. Additionally, the presence of PirA/B genes in BA94C2 bacterial cultures was confirmed by nested PCR using the AP4 method [76] on ten randomly selected colonies.

2.2 Composition and Emulsion of Oregano Essential Oil

Components in the highest percentages of Oregano essential oil (OEO) included carvacrol (69.1%), o-cymene (5.9%), thymol (4.4%), terpinene (3.7%), caryophyllene (2.9%) and β-pinene (1.6%). OEO was emulsified in SS-NaCl and Tween-80 following the methodology described by Deng et al. [77] with slight modifications. Briefly, the emulsifying solution was prepared with Tween-80 (1% v/v) in SS-2% NaCl, stirred at 150 rpm for 15 minutes using a vortex mixer, and then OEO was added (10% v/v) and stirred again for 15 minutes to achieve a homogeneous suspension. From this suspension, different dilutions were made to obtain the desired concentration. Additionally, the emulsifying solution was included in each assay to rule out its impact on the evaluated virulence phenotypes.

2.3 Bactericidal Activity of OEO against V. parahaemolyticus causing AHPND

To determine sublethal concentrations for the virulence assays of V. parahaemolyticus, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the microdilution assay technique described by Sokovicx́ et al. [78], with slight adaptations. Briefly, in 96-well microplates, 160 µL of sterile TSB + 2% NaCl broth, 20 µL of OEO at different final concentrations (0-2000 µg mL⁻¹) in TSB + 2% NaCl, and 20 µL of V. parahaemolyticus inoculum (10⁶ CFU mL⁻¹). were added to each well. The microplates were incubated at 28°C for 24 hours, and MIC values were determined as the lowest dose at which wells showed no turbidity due to bacterial growth, read at OD₆₀₀ nm (VarioskanLux_0315). To determine the MBC, a total of 100 µL per well from the selected MIC wells and the subsequent wells containing the three highest concentrations of OEO were plated on TSA + 2% NaCl Petri dishes and incubated at 28°C for 24 hours. Petri dishes without bacterial growth corresponding to the OEO concentrations were considered as MBC and expressed in µg mL⁻¹ OEO.

2.4 Validation of Sublethal OEO Concentrations on V. parahaemolyticus Growth and Viability

Fresh cultures of V. parahaemolyticus grown for 8 hours were adjusted by OD₆₀₀ nm to 10⁸ CFU mL^− 1. One mL of the adjusted inoculum was transferred to eight different flasks containing 999 mL of fresh TSB 2% NaCl broth and OEO at different concentrations (0; 0.1; 0.25; 0.5; 1.0; 2.5; 5.0 and 10.0 µg mL^− 1). Cultures were incubated for 48 hours at 28°C, and every 2 hours, 200 µL were dispensed in triplicate into 96-well microplates and read at OD₆₀₀ nm. The maximum growth of V. parahaemolyticus was established in the culture without OEO addition. Simultaneously, the same cultures were used to determine viability following the methyl thiazole tetrazolium (MTT) reduction method described by Constante et al. [79]. Briefly, after 12 hours of culture from each flask, 200 µL per well were transferred to microplates, six replicates for each evaluated concentration, and immediately 10 µL of MTT solution at a concentration of 5.0 mg mL^− 1 diluted in PBS was added per well and incubated for 2 hours at 30°C with shaking. The microplates were centrifuged for 20 minutes at 2500 g at 24°C in a centrifuge (ThermoScientific™ Megafuge 40R). The supernatant from each well was carefully removed, 200 µL of absolute ethanol were added per well to dissolve the formazan crystals, and OD_620nm absorbance was read using a microplate reader. The results were transformed into a percentage of cell viability considering 100% viability in the culture not exposed to OEO.

2.5 Effect of OEO on Exopolysaccharide Production in V. parahaemolyticus causing AHPND

To determine the effect of OEO on exopolysaccharide (EPS) production, the methodology described by Zhang et al. [80] was followed. From a fresh culture of V. parahaemolyticus, adjusted to 10⁸ CFU mL^− 1 by OD₆₀₀ nm, 100 µL were transferred to test tubes containing 10 mL of TSB + 2% NaCl with OEO at different final concentrations (0; 0.1; 0.25; 0.5; 1.0; 2.5; 5.0 and 10.0 µg mL^− 1). A treatment with the antibiotic oxytetracycline at a single concentration equal to the MIC = 10 µg mL^− 1, and another treatment with the emulsifying solution used for OEO emulsion were also included, six replicates per treatment. All tubes were incubated under static conditions for 48 hours at 28°C. They were then centrifuged at 5000 × g for 30 minutes at 4°C, and the supernatant was filtered (0.22 µm). The total EPS was precipitated by adding three volumes of 95% ice-cooled ethanol (-20°C) and incubated at 4°C overnight. The precipitated EPS was collected by centrifugation at 5000 × g for 30 minutes at 2°C, and the sediment was dissolved in 1 mL of deionized water and stored at -40°C until use. The total carbohydrate content in the EPS was quantified using the phenol-sulfuric acid method with glucose as a standard as described by Dubois et al. [81]. Absorbance was measured at 490 nm. The production of 100% EPS was considered for cultures without OEO (control), and the level of EPS inhibition was calculated using the formula: Percentage of inhibition = [(OD of control - OD of test) / OD of control] × 100.

2.6 Effect of OEO on Biofilm Formation in V. parahaemolyticus causing AHPND

Biofilm formation was quantified using crystal violet (CV) staining as described by Stepanović et al. [82]. Overnight cultures of V. parahaemolyticus were adjusted to 10⁶ CFU mL^− 1 in TSB + 2% NaCl. In 96-well microplates, 160 µL of sterile TSB + 2% NaCl, 20 µL of OEO at the specified concentrations, and 20 µL of adjusted inoculum were added to each well. Treatments with antibiotics and the emulsifying solution were also included, with six replicates for each treatment. Bacterial cultures were allowed to adhere to the well surface and grow without shaking for 72 hours at 28°C. Planktonic cells were removed, and biofilms formed in the wells were carefully washed with 200 µL of PBS buffer (pH 7.2) to remove non-adherent bacteria. Biofilms were fixed by heating the microplates at 50°C for 30 minutes and then stained with 220 µL per well of 0.1% (w/v) CV (Merck_C0775) for 15 minutes. Plates were rinsed with tap water until the stain was removed and dried at room temperature (27°C). The CV was solubilized with 220 µL of 95% ethanol per well, and 150 µL per well were transferred to a new microplate and read at OD₅₉₀ nm. Biofilm production was considered 100% in cultures without OEO, and the level of biofilm inhibition was calculated using the formula: Percentage of inhibition = [(OD Control − OD Test) / OD Control] × 100.

The effect of OEO on the biofilms of V. parahaemolyticus was also visualized using an epifluorescence microscope. Biofilms treated with or without OEO were stained with acridine orange (AO, Sigma). Briefly, in SlideFlask (Nunc cat. No. 170920) for cell culture, 4 ml of the inoculum previously adjusted to 10⁶ UFC mL^− 1 were transferred, along with 1 mL of OEO at different final concentrations (0; 0.5; 1.0; 2.5; 5.0; 10 µg mL-¹). The SlideFlasks were incubated at 28°C for 72 hours. Floating cells were discarded, and washes were performed with PBS buffer (pH 7.2). Biofilms treated with and without OEO were stained with 1 mL of acridine orange solution (0.25%) for 10 minutes at room temperature (27°C) in the dark. Afterward, two more washes with PBS were performed, and the slides from the SlideFlask were removed for observation under an epifluorescence microscope (Nikon Eclipse E200) with the appropriate filter sets. Photographs of the biofilms were taken with a camera (MshOt MS60) and using the professional image analysis software iWorks v.2.0 (Nahwoo Trading Co.®), 3D images were created, and the average thickness of the biofilms for each OEO concentration was determined.

2.7 Effect of OEO on the Swarming Motility of V. parahaemolyticus

The swarming motility assay was conducted following the methodology described by Fünfhaus et al. [83], with slight adaptations. Petri dishes with soft agar were prepared using LB medium supplemented with 0.7% agar and 2% NaCl. Briefly, seven flasks containing the medium were sterilized by autoclaving. After cooling the medium to 45 ± 3°C, OEO was added to each flask at different final concentrations (0, 0.1; 0.5; 1.0; 2.5; 5.0; 10.0 µg mL^− 1). The treatment with antibiotics and the emulsifying solution was also included, with six replicates for each treatment. The medium was dispensed into Petri dishes, and the plates were dried for 15 minutes inside a biosafety cabinet (Thermo Scientific 1300, Series Class II). Then, 5 µL of V. parahaemolyticus adjusted to 10⁶ CFU mL^− 1 was inoculated in the center of the plates, and they were incubated at 26°C for 72 hours, with six plates for each evaluated concentration and controls. The migration of swarming motility was measured in millimeters (mm), and the swarming motility of Vibrio treated with OEO was compared to those not treated.

2.8 In Vivo Effect of OEO on the Virulence of V. parahaemolyticus

The effect of OEO on the virulence of V. parahaemolyticus was verified through a challenge test, healthy P. vannamei shrimp with an average weight of 2.52 ± 0.36 g were used. following the methodology described by Zhang et al. [84], with slight adaptations. The shrimp were acclimated for 5 days, and after confirming the absence of AHPND by PCR and histopathology, they were randomly distributed into glass aquariums (15 shrimp per aquarium) to be exposed to V. parahaemolyticus cultured in the presence or absence of OEO at different sublethal concentrations (0; 0.1; 0.25; 0.5; 1.0; 2.5; 5.0; 10.0 µg mL^− 1), with six repetitions for each treatment. Filtered and UV-sterilized seawater was used, continuous aeration was provided, and the temperature was set at 31.2 ± 0.5°C. The juvenile shrimp were fed a commercial diet every 8 hours throughout the bioassay. The bacterial inoculum was prepared as follows: a fresh culture of V. parahaemolyticus grown for 8 hours was adjusted by OD₆₀₀ nm to 10⁸ UCF mL^− 1. One mL of the adjusted inoculum was transferred to eight different flasks containing 1000 mL of fresh TSB 2% NaCl broth. OEO was added to each flask at the previously stated concentration. The flasks with the cultures were incubated for 9 hours at 30°C with constant agitation. Subsequently, the culture was centrifuged (4000 g for 10 minutes at 25°C), the supernatants were discarded, and the cell sediments were resuspended in sterile saline solution (2% NaCl), adjusted to a concentration of 10⁸ CFU mL^− 1, and immediately inoculated into each assigned treatment at a final concentration of 10⁶ CFU mL^− 1. The water in the experimental units was replaced (50%) at 24 hours post-exposure (hpe). Cumulative survival was determined at 120 hpe by counting and removing dead shrimp every four hours. This bioassay considered 100% virulence in the BA94C2 inoculum cultured without OEO, used to challenge the shrimp in the control group. Additionally, during and at the end of the bioassay, shrimp were fixed in Davison solution for classical histological analysis to corroborate the histopathological signs characteristic of AHPND.

2.9 Transcriptomic Analysis of V. parahaemolyticus Exposed to OEO

A global gene expression analysis was conducted with isolates of V. parahaemolyticus exposed and not exposed to a single dose of OEO (1.0 µg mL^− 1) as described previously. Samples were collected at 72 hours, including four replicates for the treatment and four for the control. Bacterial cells growing on the edges of the plates from each replicate were collected with a sterilized platinum loop and deposited in a microcentrifuge tube containing ultrapure distilled water (Invitrogen ref. 10977-015). Total RNA was extracted from these bacterial biomasses using the MasterPure™ RNA Purification Kit (Cat. No. MCR85102, Epicenter, an Illumina company) following the manufacturer's instructions. Briefly, bacterial cells were centrifuged at 10,000 g for 10 minutes at 4°C, the supernatant was discarded, and tissue/cell lysis solution containing proteinase K was added to the bacterial sediment. MPC Protein Precipitation Reagent was added, and centrifugation was repeated under the same conditions. The supernatant was transferred to clean microcentrifuge tubes, isopropanol was added, and centrifugation was repeated to precipitate nucleic acids. DNase solution (DNase-free of RNase) was added to all samples. Subsequently, 2X T and C lysis solution and MPC protein precipitation reagent were added. The samples were centrifuged, and the supernatants containing RNA were transferred to clean microcentrifuge tubes. Isopropanol was added, and the samples were centrifuged, the sediments were rinsed twice with 70% ethanol. Finally, the samples were centrifuged, the ethanol was removed, and the residue was evaporated. Total RNA was suspended in ultrapure water and stored at -80°C until analysis.

Complementary DNA (cDNA) libraries from all samples were constructed and sequenced by Macrogen (Korea) using the Illumina sequencing platform (HiSeq™ 400), producing paired-end reads of 100 bp. The filtered and trimmed reads were aligned against the reference genome of Vibrio parahaemolyticus (GCF_003119375.1) using the STAR software version 2.7. Differential expression analysis of the samples exposed and not exposed to OEO was performed using the EdgeR software, described by Robinson et al. [85]. A significant difference was considered if the adjusted p-value (FDR) was less than 0.05 and if the FoldChange took an absolute value greater than 2. Gene set enrichment analysis (GSEA) was performed for the group of differentially expressed genes (DEGs) against the Gene Ontology (ont = ALL) and the Kyoto Encyclopedia of Genes and Genomes (organism = vpa) databases using the R package cluster Profiler, described by Robinson Yu et al. [86]. An overrepresentation test was performed using a Fisher's Exact Test for Count Data, considering a significance threshold of adjusted p-value less than 0.1. The reference gene list of Vibrio parahaemolyticus was constructed based on the NCBI annotation file and on the KEGG orthology database by R (Reference: Vibrio parahaemolyticus RIMD 2210633 V7).

2.10 Statistical analysis

All graphs were created using the ggplot2 package in R software (R Core Team, 2023). An ANOVA followed by Tukey's test was performed to compare the effect of different concentrations of OEO on various virulence phenotypes, including the production of total exopolysaccharides and the production and thickness of biofilms in V. parahaemolyticus (BA94C2). For swarming motility, the variable was transformed to a logarithm to meet the assumptions of ANOVA. For the mortality of shrimp exposed to V. parahaemolyticus, the variable was transformed using arcsine to meet the assumptions of ANOVA. A significance level of p < 0.05 was considered for all statistical tests.

3.1 Bactericidal Activity and Sublethal Concentrations of OEO against V. parahaemolyticus

To avoid affecting the viability of V. parahaemolyticus by exposure to OEO, the MIC and MBC values were determined. The MIC was established at 500 µg mL-¹, while the MBC was 600 µg mL^− 1 of OEO for V. parahaemolyticus (strain BA94C2). These values were used as references for the virulence phenotype inhibition assays of V. parahaemolyticus, and concentrations well below the MIC (0.1–10.0 µg mL^− 1) were selected. The results show that the selected concentrations of OEO, evaluated in this study, did not affect the growth and viability of V. parahaemolyticus cultures (Fig. 1). Figure 1A shows the growth of V. parahaemolyticus over 24 hours exposed to sub-MIC concentrations of OEO compared to the culture without OEO. The viability of V. parahaemolyticus was over 98% at all evaluated concentrations, with no significant differences (P > 0.05) compared to the culture not exposed to OEO (Table S1). Figure 1B illustrates the reduction of compound MTT to formazan resulting from the cell viability of V. parahaemolyticus exposed to different sub-MIC concentrations of OEO.

3.2 Effect of OEO on the Production of Total Exopolysaccharides in V. parahaemolyticus

OEO inhibited the production of total EPS of V. parahaemolyticus causing AHPND, as shown in Fig. 2. The production of EPS was dependent on the concentration of OEO added to the culture, significantly reducing (P < 0.05) the EPS from the third evaluated concentration (0.5 µg mL^− 1). Approximately 58%, 64%, 83%, and 89% of total EPS were inhibited when OEO was added at 1, 2.5, 5.0, and 10 µg mL^− 1), respectively. In the treatment exposed to oxytetracycline at the MIC concentration of 10 µg mL^− 1, 55% of total EPS production was observed, registering a higher value than when 1.0 µg mL^− 1 of OEO was added, which registered a production of 44%. Consequently, at this concentration of OEO, approximately 56% of total EPS of V. parahaemolyticus was reduced (Fig. 2).

3.3 Effect of OEO on Biofilm Formation in V. parahaemolyticus

The biofilm formation of the pathogen V. parahaemolyticus causing AHPND was progressively reduced with the increase in OEO concentration (Fig. 3). Figure 3 shows that at concentration of 1.0 µg mL^− 1 and 10 µg mL^− 1 of OEO reduced biofilm formation by 50% and 75%, respectively, compared to the culture without OEO. A similar pattern of the effect of OEO was observed in the thickness of biofilms (Fig. 4). For example, when 1.0 µg mL^− 1 of OEO was added, the biofilm thickness was 35 ± 5 µm, while when 10.0 µg mL^− 1 was added, the values were 8 ± 3 µm, thus reducing the average biofilm thickness by about four times.

3.4 Effect of OEO on Swarming Motility of V. parahaemolyticus

Another virulence phenotype inhibited by OEO in V. parahaemolyticus was swarming motility, as shown in Fig. 5. All concentrations of OEO evaluated in this study significantly (P < 0.05) inhibited swarming motility. Swarming motility migration zones of 10.8 ± 1.4 mm were recorded when 1.0 µg mL^− 1 of OEO was applied, and maximum inhibition was observed when 10.0 µg mL^− 1 of OEO was added, recording swarming motility zones of 4.8 ± 0.8 mm, resulting in ten times less swarming motility of V. parahaemolyticus in the control treatment without OEO. Contrary to what was observed previously, swarming motility significantly (P < 0.05) increased when the antibiotic was added, recording values of 75.8 ± 10.3 mm, which can be interpreted as a stress response mechanism or escape from a harmful environment (Fig. 5).

3.5 Virulence of V. parahaemolyticus Exposed to OEO

The virulence of V. parahaemolyticus was reduced because of exposure to OEO, significantly reducing (P < 0.05) the mortality of juvenile P. vannamei shrimp (Fig. 6). For example, V. parahaemolyticus in the control group produced a cumulative mortality of 93 ± 5% over the 4 days of the challenge. However, when 1.0 µg mL^− 1 of OEO was added, the cumulative mortality was only 56 ± 6%, achieving up to a 30% reduction compared to the control. The antivirulence effect of OEO was enhanced at higher evaluated concentrations of 2.5, 5.0, and 10 µg mL^− 1, recording cumulative mortality rates below 45% (Fig. 6), which translates to higher survival rates of the shrimp challenged with OEO-treated inoculum. Additionally, the histopathological analysis of the moribund shrimp confirmed the virulence of V. parahaemolyticus causing AHPND, as all shrimp analyzed by histopathology exhibited characteristic clinical signs of AHPND (Table 1), including cell detachment, generalized hemocyte infiltrations, and necrotic tubules in the terminal phase (Fig. 7A-C). Regarding the surviving shrimp analyzed by histopathology, 75% showed mild lesions, and 25% showed no characteristic histopathological damage of AHPND and appeared very healthy, including the cells of the hepatopancreatic tubules (Fig. 7D).

Table 1

Samples were analyzed by histopathology to identify characteristic signs of AHPND. Shrimps analyzed were taken randomly.
			AHPND		Degree of lesion
	Treatments OEO µg/mL	Shrimp analyzed	Positive	Negative	Absent	Very mild	Mild	Medium	Severe
Before the challenge test	0	12	0	12	12	0	0	0	0
During the challenge test	0	9	9	0	0	0	0	3	6
	0.1	9	9	0	0	0	0	4	5
	0.25	9	9	0	0	0	0	2	7
	0.5	9	9	0	0	0	0	3	6
	1.0	9	9	0	0	0	0	4	5
	2.5	9	9	0	0	0	0	2	6
	10.0	9	9	0	0	0	0	2	6
Surviving shrimp (120 hpe)	0	6	4	2	2	2	2	0	0
	0.1	6	5	1	1	4	1	0	0
	0.25	6	4	2	2	3	1	0	0
	0.5	6	4	2	2	2	2	0	0
	1.0	6	5	1	1	4	1	0	0
	2.5	6	4	2	2	3	1	0	0
	10.0	6	4	2	2	4	0	0	0

3.6 Transcriptomic Analysis

To elucidate at the molecular level how sublethal doses of OEO affect the virulence of V. parahaemolyticus, we performed a global comparative RNA-seq transcriptomic analysis of V. parahaemolyticus (strain BA94C2) cultured in the absence and presence of OEO (1.0 µg ml^− 1) using swarming motility evaluation as a study model. We obtained a total of 13.82 Gb of sequencing data, including 27,892,638,134 raw reads and 65,318,934 clean reads, with all data having an average error rate of less than 0.02%. The Q20 and Q30 percentages were above 98% and 94%, respectively. For the OEO treatment and the control without OEO, the average GC percentages were 49.28% and 49.60%, respectively (Table 2). The quality control results of the sequencing of all samples were of good quality, suggesting they can be used for bioinformatic analysis.

Table 2

Summary of RNA-seq data from *V. parahaemolyticus* RNA libraries exposed to sub-MIC concentration of oregano essential oil (1 µg mL^− 1).
Sample ID	Raw Reads	Clean Reads	Mapped reads	GC(%)	AT(%)	Clean Q20(%)	Clean Q30(%)
OEO_R1	12007442	11934790	8235630	49,80	50,20	98,15	94,56
OEO_R2	12518842	12447750	9565302	49,01	50,99	98,16	94,55
OEO_R3	13528057	13442256	10350955	48,56	51,44	98,05	94,33
CT_R1	13116217	13042813	4243336	51,55	48,45	98,19	94,71
CT_R2	11079574	11012483	6094976	49,51	50,49	98,11	94,50
CT_R3	14932108	14847559	10884389	49,11	50,89	98,15	94,55
CT_R4	10572090	10513152	8810697	48,21	51,79	98,14	94,50
Sample ID: Sample name. GC (%): GC content; AT (%): AT content. Q20 and Q30, respectively, represented the percentage of bases with Phred values greater than 20 and 30 in the total bases after data quality control. EO: treated group with 1.0 µg/mL oregano essential oil; CT: control group.

3.6.1 Differentially Expressed Genes in V. parahaemolyticus Treated with OEO

A total of 183 significant DEGs (|log₂FC| ≥ 2.0 and p-adjust < 0.05), were recorded, including 89 downregulated genes and 94 upregulated genes, when comparing the transcriptomic profile of V. parahaemolyticus causing AHPND exposed to OEO with the control group (Fig. 8 and Table S2, S3). A summary of key genes involved in the regulation of V. parahaemolyticus virulence is described in Table 2 according to their functionality. Based on gene identification and gene name from the NCBI database, OEO caused the downregulation of 36 genes related to the ribosome, including 19 genes associated with the 50S ribosomal subunit and 16 with the 30S ribosomal subunit. Another 32 genes were related to enzymes, including L-threonine dehydrogenase, succinate coenzyme A ligase, succinil-CoA, ribose-5-phosphate isomerase, glutamine synthetase, phosphoenolpyruvate synthetase, 3-oxoacyl-[acyl carrier protein] synthase 2, tRNA m(1)G37 methyltransferase, dihydrolipoyl dehydrogenase, RNA polymerase, fused DNA-binding transcriptional repressor/proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase PutA, NADH:ubiquinone reductase (Na(+)-transporting) subunit and 8 genes ATP synthase. OEO also induced the downregulation of genes associated with the expression of several functional proteins, including 2 genes involved in motility (lafA and motA); 7 genes related to transport, such as biotin carboxyl carrier protein, iron chelate ABC transporter ATP-binding protein VctC, oligopeptide ABC transporter permease OppC, NADH:ubiquinone reductase (Na(+)-transporting) subunit, proline Na(+) symporter; 3 genes associated with the type VI secretion system (T6SS), including T6SS contractile sheath small subunit, T6SS lipoprotein, and T6SS contractile sheath large subunit. Additionally, OEO negatively affected the expression of the ompW gene, associated with the synthesis of the outer membrane protein OmpW. Regarding the genes upregulated by the effect of OEO, 21 genes were associated with proteins involved in the response to adaptation and stress tolerance, including envelope stress response protein PspG, transcriptional regulator BetI, DNA polymerase IV, phage shock protein B and C, reduced glutaredoxin 2, RNA polymerase sigma factor RpoS, cadaverine/lysine antiporter, alkyl hydroperoxide reductase, AhpF component; 19 genes were related to enzymes, including heme O synthase, betaine aldehyde dehydrogenase, periplasmic nitrate reductase subunit NapA, choline dehydrogenase, acyl-CoA dehydrogenase FadE, type I pullulanase, glutamate synthase subunit GltB, type I pullulanase, NO-inducible flavohemoprotein; 9 genes were associated with electron transport, and 8 with ABC transporters, such as Zn(2+) ABC transporter membrane subunit, glycine betaine ABC transporter membrane subunit ProW, glycine betaine/L-proline ABC transporter ATP-binding protein ProV, thiamine/thiamine pyrophosphate ABC transporter permease ThiP, and ribose ABC transporter permease. Interestingly, OEO also upregulated 11 genes associated with the type III secretion system and five genes associated with efflux pumps, including multidrug efflux RND transporter periplasmic adaptor subunit VmeU, VmeT and efflux RND transporter permease subunit VmeQ.

3.6.2 GO Function Analysis of Significant DEGs

The identified DEGs were annotated according to GO classification criteria and enriched in the GO database, classified into biological processes (BP), cellular components (CC), and molecular functions (MF) (Fig. 9). The results show that the DEGs were annotated in one or more GO terms, resulting in a total of 35 secondary GO terms, 10 for MF, 8 for CC, and 17 for BP. In the MF category, the DEGs were mainly involved in nucleic acid binding, RNA binding, and transmembrane transporter activity. In the CC category, the DEGs were mainly involved with non-membrane-bounded organelle, organelle, and intracellular organelle. In the BP category, the DEGs were primarily associated with organic substance biosynthesis process, cellular biosynthetic process, and organonitrogen compound metabolic process.

3.6.3 KEGG Functional Analysis of Significant DEGs

To identify the biological processes involved in the annotated genes, the pathway analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) was carried out. The enriched KEGG pathways of the DEGs that changed most significantly in the OEO treatment in V. parahaemolyticus causing AHPND were ribosome, pyruvate metabolism, propanoate metabolism, carbon metabolism, and microbial metabolism (Fig. 10). The ribosome was the most statistically significant pathway identified in “OEO-vs-Cont” with 35 genes, almost all of which showed decreased transcription levels after exposure to OEO. The second most significant KEGG pathway in V. parahaemolyticus exposed to OEO was pyruvate metabolism with 6 downregulated genes. The third pathway was propanoate metabolism with 5 downregulated genes, followed by carbon metabolism with 14 downregulated genes. Finally, the fifth most significant KEGG pathway was microbial metabolism in diverse environments, with 19 genes, most of which showed upregulated transcription levels.

The emergence of new highly virulent and antibiotic-resistant strains of vibrios in shrimp farming [10, 87–92] has led to a continuous search for new sources of active molecules and alternative strategies for their control. Certain EOs or their components, at sublethal doses, have effectively inhibited the virulence of various pathogenic bacteria of clinical, veterinary, and agricultural interest, showcasing their potential in antivirulence therapy [93–96]. Here, we report the antibacterial activity of OEO at sub-MIC doses against V. parahaemolyticus (strain BA94C2) AHPND- causing in P. vannamei shrimp. The in vitro results indicate that OEO significantly reduced the virulence phenotypes of V. parahaemolyticus, including swarming motility, EPS production, and biofilm formation. In vivo, OEO negatively affected the virulence of V. parahaemolyticus, as evidenced by a lower cumulative mortality rate in shrimp challenged with OEO-cultured inoculum compared to the control group. Although no significant changes were recorded in the genes associated with master quorum regulators, The transcriptional analysis of V. parahaemolyticus exposed to OEO revealed 183 DEGs, with 89 genes downregulated, mainly involved in membrane integrity, ribosome function, motility, transport, T6SS secretion system, and enzymes involved in energy metabolism. Additionally, 94 genes were upregulated, related to the response to environmental stress and nutrient deprivation. Together, these results suggest that OEO is a promising candidate for treating pathogenic vibrios causing AHPND in shrimp farming using antivirulence strategy.

Motility allows vibrios to obtain nutrients, escape toxic substances, colonize, and disperse [97, 98]. It is considered a key virulence factor for natural infections, enabling access to the host organism [99, 100]. Motility in vibrios, including V. parahaemolyticus, is regulated by two systems: the polar flagellar system, which includes the polar flagellum for swimming in an aqueous environment, and the lateral flagellar system (laf), which includes lateral flagella for movement on solid surfaces or during host colonization [101, 102]. The presence of lateral flagella in V. parahaemolyticus is crucial for colonization and subsequent invasion of host cells [103], making interference with the swarming motility of vibrios essential for affecting their virulence. In our study, OEO significantly inhibited the swarming motility of V. parahaemolyticus causing AHPND in shrimp farming, and its effect was dependent on the concentration of OEO (Fig. 5).

The effect of OEO on swarming motility in other bacterial species, including aquaculture pathogenic Vibrio spp., has been reported [26], but this is the first report documenting the effect of OEO on the swarming motility of V. parahaemolyticus causing AHPND. Interestingly, at the end of the experiment, the antibiotic treatment recorded higher swarming motility migration zones compared to the control. While several reports indicate the negative effect of antibiotics on swarming motility [104–106]. Sun et al. [107] and, more recently Dominguez-Borbor et al. [26] reported similar results to those recorded in this study. It is important to note that these differences were recorded at 96 hours since at 48 hours, the swarming motility values of the antibiotic group and the control group were very similar (data not shown). Possible explanations could be that the antibiotic degrades over time, losing efficacy, or alternatively, that the bacteria attempt to escape the hostile environment, as it has been reported that swarming motility induces acquired antibiotic resistance [108–111].

The laf system mediates swarming motility in V. parahaemolyticus, adapted to cells that move over wet or viscous surface [101, 103]. When the cell encounters a sufficiently viscous surface or environment, flagellar rotation is impaired and laf induced, promoting the production of lateral flagella [112]. Expression of the lafA gene, encoding the lateral flagellin (LafA) protein [103] is directly related to swarming motility and successful host infection [113, 114]. Evidence in non-AHPND V. parahaemolyticus shows that repression of the lafA gene repressed swarming motility [115], and deletion of the lafA gene in another mutant strain resulted in cells unable to produce lateral flagella, affecting swarming motility and bacterial virulence [101]. In our study, transcriptional analysis in V. parahaemolyticus exposed to OEO revealed a significant effect on the expression of the lafA gene, being one of the most downregulated genes (Table S2), which could be associated with the observed inhibition of swarming motility in V. parahaemolyticus causing AHPND in shrimp. Similar to our study, citral, a natural compound present in several EOs, has been shown to repress the lafA gene in V. parahaemolyticus gene in V. parahaemolyticus non-AHPND, negatively affecting swarming motility in a concentration-dependent manner [113].

Another downregulated gene was motA, necessary for rotation associated with flagellar assembly [116]. Although studies on the deletion of the motA gene in V. parahaemolyticus, have not been reported, several studies in other bacterial species, such as Aliivibrio salmonicida and Pseudomona euroginosa, have shown that deletion of the motA gene resulted in malformed or completely absent lateral flagella, affecting swarming motility, adhesion, biofilm formation, and virulence[117, 118]. Natural products such as trans-cinnamaldehyde, gum arabic, carvacrol, and indole derivatives have been shown to regulate the expression of the motA gene in other bacterial species, affecting motility, adhesion, and biofilm formation [119, 120]. These observations would largely explain the reduced swarming motility observed in V. parahaemolyticus causing AHPND exposed to OEO. Swarming motility demands energy and responds to the cellular physiological state, nutrient concentration, and cation concentration. Twenty-two genes associated with enzymes related to ATP biosynthesis and transport were downregulated, probably altering metabolism and reducing intracellular ATP levels in V. parahaemolyticus AHPND-causing. This leads us to hypothesize that OEO limits nutrients and intracellular energy sources, a hypothesis reinforced by the upregulation of genes related to proline uptake as an energy source and siderophore production for iron uptake.

Once bacteria adhere, they begin to express EPS, which not only promotes adherence but also protects bacteria from desiccation and, due to their anionic nature, helps retain minerals and nutrients near the cell. EPS are considered the main component of biofilms, potentially representing up to 50% of the total biomass, making it important to suppress or reduce EPS production to control biofilm formation [121]. OEO affected the production of exopolysaccharides in V. parahaemolyticus causing AHPND in a dose-dependent manner. Biofilms enable vibrios to survive in hostile environments, tolerate dryness and toxic substances, including antibiotics [122]. In fact, bacteria within biofilms tolerate higher concentrations of antibiotics [123], potentially increasing the MIC value up to 16 times compared to planktonic bacteria [124], thereby enhancing bacterial resistance.

Disrupting biofilm formation reduces antibiotic resistance and pathogenic potential. OEO significantly inhibited biofilm formation of V. parahaemolyticus causing AHPND in a concentration-dependent manner, even at a concentration of 1 µg/mL, showing more effectiveness than the antibiotic used at the MIC concentration (Fig. 3). This study did not aim to establish the MIC of OEO and/or antibiotics to inhibit biofilm of V. parahaemolyticus, so it would be interesting to determine the MIC-biofilm values to apply efficient treatments with the aim of eradicating biofilms in shrimp culture systems, as biofilms constitute the main reservoirs of bacteria and ensure persistence [125–127]. Several EOs at sublethal doses have shown efficacy in inhibiting biofilm formation of Vibrio, whether human pathogens [128, 129] or aquaculture pathogens [26].

Transcriptional analysis revealed that OEO downregulated the expression of the ompW gene, associated with an outer membrane porin protein (OmpW), which is widely conserved in Gram-negative bacteria [130, 131] and has been considered important for adherence and biofilm formation in various Vibrio species [132]. Additionally, two genes associated with flagella formation and assembly, and three genes (tssB, tssC, tssJ) from the TSS6, as well as several genes related to substance transport associated with biofilm formation (Table S2), were also downregulated. From these results, we conjecture that OEO inhibits early adherence of V. parahaemolyticus by causing significant damage to flagella and altering the outer membrane of cells, limiting adherence, and resulting in a significant reduction in biofilm formation. Studies denoting the effect of OEO on the expression of the ompW gene in V. parahaemolyticus causing AHPND have not been reported until now. However, other natural products, including citral, lactobionic acid, and butyrate, have shown to inhibit biofilm formation in non-AHPND V. parahaemolyticus by repressing the ompW gene [128, 129].

In addition, OmpW plays an important role in antibiotic resistance, as antibiotic entry mechanisms are through the outer membrane, including general diffusion porins (Omps) responsible for hydrophilic antibiotics, and the lipid-mediated pathway for hydrophobic antibiotics. In effect, antibiotic susceptibility is related to the size of the OMP channel [133], so affecting the expression of genes associated with OmpW synthesis has been proposed as a possible target for developing new drugs against resistant strains (Vranakis et al., 2014). Additionally, OmpW is also considered a key virulence factor; Ye et al. (2017) compared the transcriptome of highly virulent vs. attenuated C. sakazakii strains, identifying that the ompW gene was overexpressed in the highly virulent strain. In our study, OEO at sublethal doses significantly repressed the expression of the ompW gene.

The activation of efflux pumps, which play a fundamental role in expelling antibiotics from the cell, allowing bacteria to survive [134, 135]. Upregulation of associated genes is an undesirable property for any administered treatment, while downregulation is considered a promising trait. Several studies have evidenced that certain EOs or their components at microbicidal doses inhibit the efflux pumps of certain bacteria [136, 137]. Studies evaluating OEO at sublethal doses in V. parahaemolyticus AHPND-causing are nonexistent. The transcriptomic analysis of V. parahaemolyticus exposed to OEO. The transcriptomic analysis of V. parahaemolyticus exposed to OEO, revealed upregulated the expression of five genes associated with efflux pumps, including multidrug efflux RND transporter periplasmic adaptor subunit VmeT, multidrug efflux RND transporter periplasmic adaptor subunit VmeU and efflux RND transporter permease subunit VmeQ (Table S3). We hypothesize that at sublethal doses, a hostile environment is created, activating efflux pumps to survive. Therefore, this adverse effect warrants further investigation. However, it should be considered that the bacterium neglects other key virulence phenotypes such as adherence, swarming, and biofilm formation.

OEO significantly downregulated a total of twenty-two genes involved in the energy metabolism of V. parahaemolyticus causing AHPND, encoding enzymes associated with ATP biosynthesis and transport. As illustrated in Fig. 10, regarding the KEGG pathway analysis, three out of five enriched pathways that significantly changed by downregulating DEGs were pyruvate metabolism, propanoate metabolism, and carbon metabolism, whereas the enriched pathway with upregulated DEGs was microbial metabolism in diverse environments. OEO also repressed the expression of other genes, such as putP/putA involved in proline transport [138–140], and gcvP/gcvH associated with cardiolipin biosynthesis, responsible for one of the biosynthesis pathways of this membrane lipid [141], and genes associated with nucleic acid binding, RNA binding, and transmembrane transporter activity (Fig. 9). Likewise, OEO downregulated 36 genes related to the ribosome, including genes associated with the 50S, 30S, and 16S ribosomal subunits. In fact, it was the most enriched KEGG pathway with 35 downregulated DEGs. Altogether, this demonstrates that the metabolism of V. parahaemolyticus was altered by exposure to OEO, causing general destabilization, as the cellular functions allowing RNA and protein production were affected, consequently affecting virulence phenotypes.

To explore the effect of EOO on the virulence of V. parahaemolyticus AHPND-causing in juvenile P. vannamei shrimp, an inoculum of V. parahaemolyticus pretreated with different sub-MIC concentrations of EOO were used to challenge P. vannamei. This type of challenge test has been previously used to demonstrate the antivirulence effect of EOs against other pathogenic bacteria of clinical interest. In a study on the nematode Caenorhabditis elegans, increased survival was observed when challenged with E. coli treated with clove EO [142]. Similarly, in a model with wax moths (Galleria mellonella) challenged with Staphylococcus aureus, EO from Eugenia brejoensis reduced the virulence of S. aureus, resulting in less than 30% mortality compared to 100% untreated inoculum [33]. In aquaculture, indole (a compound present in EOs) has shown antivirulence properties against two pathogenic Vibrio strains in oysters, increasing the survival of oysters challenged with indole-pretreated inoculum [84]. Similar results were obtained for V. parahaemolyticus causing AHPND when treated with OEO, and a lower cumulative mortality rate was recorded in shrimp challenged with V. parahaemolyticus pretreated with OEO, which was significantly reduced (P < 0.05) when treated with > 1 µg/ml of EOO (Fig. 6). This suggests that EOO has a significant impact on the virulence of V. parahaemolyticus AHPND-causing and that the use of EOO could be a promising target for antivirulence therapy.

OEO affected three genes associated with T6SS, which could explain the reduced virulence of AHPND-causing V. parahaemolyticus in P. vannamei. Even if the expression of PirA/B toxin genes were not affected, the downregulation of TSS6-associated genes tssJ, tssC, and tssB could confer fitness to V. parahaemolyticus over other competing bacteria and facilitate shrimp infection [143]. Studies including deletion of the T6SS secretion system in V. parahaemolyticus causing AHPND are nonexistent; however, in other pathogens such as C. jejuni and X. oryzae, it has been shown that T6SS deletion affects adherence, invasion, and colonization of the pathogenic bacteria in the host cell [144, 145]. Another study where the tssB gene was suppressed in R. solanacearum caused several effects, including defective biofilm formation, reduced flagella operon expression, reduced motility, and significantly attenuated virulence [146].

At sublethal concentrations, OEO or its main components, such as thymol, carvacrol, and trans cinnamaldehyde, have been documented to affect genes related to flagellar biosynthesis and function, chemotaxis, biofilm development, type III secretion systems (T3SS) and efflux pumps in Escherichia coli [147]. Similar results were evidenced in the present study. EOs are mixtures of up to 300 molecules and may contain a wide range of active molecules, such as terpenes, terpenoids and phenylpropanoids, which have shown antibacterial activity for different species of pathogenic bacteria [148–151]. The major compounds of the OEO were carvacrol, o-cymene and thymol. In future work, it would be interesting to evaluate the individual molecules to determine the mechanism of action of the molecules present in the OEO and to determine whether the antivirulent activity recorded is given by a molecule or the synergy between its components present in the OEO.

Although the microbicidal activity of EO is well established, in vivo application has been scarcely addressed. One explanation could be that the MIC is much higher for EO than for antibiotics, which makes it unattractive for producers, since considerable quantities are required to achieve the microbicidal effect. For example, in comparative terms for fish pathogenic Aeromonas hydrophila, MIC values of 0.25 and 0.125 µg/mL were recorded for florfenicol and oxytetracycline, respectively, while for cinnamon (Ocimum basilicum) and Eugenia caryophyllata EOs, the MIC was 235 µg/mL, 3 orders of magnitude higher than antibiotics [152]. High application concentrations of EOs can negatively affect feed palatability, when administered through feed [153]. It should also be considered that EOs prices are higher than antibiotics and could impact their implementation [154–156]. However, OEO reduced the virulence phenotypes and, in vivo virulence of AHPND-causing V. parahaemolyticus at concentrations below 1 µg mL^− 1, suggesting its potential as an additive in shrimp feeds. This would generate a protective effect against V. parahaemolyticus according to the antivirulence strategy without compromising palatability or significantly increasing costs.

The sublethal use of EOs can reduce this risk as their molecules action multiple targets. However, the adaptive behavior of Vibrio under the continuous use of EOs needs to be evaluated, as some bacteria have shown reduced susceptibility to their effects after prolonged exposure to low concentrations. By preventing the expression of virulence factors, bacteria have a lower capacity to colonize the host, and the host immune system is expected to complement this strategy [28]. Future studies should evaluate the immune response of shrimp to V. parahaemolyticus treated with OEO and the effects of OEO on the shrimp immune system. In addition, essential oils are unstable because of their thermolabile nature [157], which can limit their use; therefore, encapsulation processes should be evaluated to improve their stability and efficacy. The discovery of new compounds that can control bacterial pathogens in aquaculture remains limited. Although omics technologies have helped us understand drug targets, microbial infections remain a global challenge because of the emergence of resistance genes and the indiscriminate use of antimicrobials.

Conclusion:

OEO negatively affected the three evaluated virulence phenotypes of V. parahaemolyticus: EPS production, biofilm formation, and swarming motility, and reduced P. vannamei mortality in an in vivo challenge. The transcriptomic analysis of V. parahaemolyticus exposed to OEO revealed different therapeutic targets associated with the inhibition of V. parahaemolyticus virulence. These results illustrate the advantages of using OEO in shrimp farming systems as an eco-friendly alternative to antibiotics in aquaculture, and its application in shrimp farming should be considered when treating AHPND.

OEO

Oregano essential oil

AHPND

Acute Hepatopancreatic Necrosis Disease

Optical density

SS-2% NaCl

Saline solution, 2% sodium chloride

CFU

Colony forming units

MTT

Tetrazolium salts (3-(4,5-dimethylthiazol-2-yl) − 2,5-diphenyltetrazolium bromide)

Crystal violet

hpe

hours post-exposure

DEGs

Differentially expressed genes

Ethics approval and consent to participate

No ethical approval was required for this study.

Availability of data and materials

All phenotypic data and the codes for creating the figures and statistical analyses are available in the repository https://github.com/GenomicsLaboratory/TranscriptomaVibrio.git. The raw RNA-Seq data were deposited in the NCBI database with project number PRJNA778211 and biosample registration numbers SAMN23011464-SAMN23011483, with short read archive (SRA) numbers SRR17054511-SRR17054520

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This work was funded by the Secretaría de Educación, Ciencia y Tecnología e Innovación of Ecuador (SENESCYT) in the framework of the PIC-14-CENAIM-001. Project “Caracterización de la Biodiversidad Microbiológica y de Invertebrados de la Reserva Marina El Pelado a escala taxonómica, metabolómica y metagenómica para uso en Salud Humana y Animal”, and by the National Center for Aquaculture and Marine Research (CENAIM) with funds derived from the Escuela Superior Politécnica del Litoral (ESPOL). We would also like to thank the National Doctoral Scholarship Program (ANID).

Authors' contributions

Dominguez C., Sánchez A., and Rodríguez J.: Conceptualization, Methodology, validation and Formal analysis. Dominguez C., Gallardo J., Flores J.: data curation. Domínguez C., Gallardo J., Flores J., Rodríguez J.: writing—original draft preparation. Domínguez C., Gallardo J., Flores J., Sánchez A., Rodríguez J., Sonnenholzner S.: writing—review and editing. Domínguez C., Gallardo J., Flores J., Sánchez A., Rodríguez J., Sonnenholzner S.: visualization. Rodríguez J., Gallardo J., Sonnenholzner S.: Supervision. Rodríguez J: project administration. Rodríguez J., Gallardo J., Sonnenholzner S. funding acquisition. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

We thank Ramiro Solorzano and Ivan Gonzabay for the collaboration provided in the laboratory, Cecilia Tomala for her collaboration in the collection and sending of samples for transcriptomic analysis, Juan Munoz for their assistance in the challenge test, Fanny Panchana for the process and histopathological analysis of the samples, and specially to Dr. Washington Cárdenas and Mariuxi Miraba from the ESPOL biomedical laboratory, for kindly providing us with the RNA extraction kit.

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The authors declare no competing interests.

SupplementaryMaterialCDOEO2024.docx
Supplementary Material Table S1. Viability by MTT reduction in V. parahaemolyticus-causing AHPND in shrimp exposed to different sub-MIC concentrations of OEO. Table S2. List of unigenes of V. parahaemolyticus-causing AHPND in shrimp downregulated by exposure to OEO. Table S3. List of unigenes of V. parahaemolyticus-causing AHPND in shrimp upregulated by exposure to OEO.

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Oregano essential oil has the potential to reduce the virulence of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) in Penaeus vannaei

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Abstract

Figures

1. Introduction

2. Methods

2.1 Growth Conditions of V. parahaemolyticus causing AHPND

2.2 Composition and Emulsion of Oregano Essential Oil

2.3 Bactericidal Activity of OEO against V. parahaemolyticus causing AHPND

2.4 Validation of Sublethal OEO Concentrations on V. parahaemolyticus Growth and Viability

2.5 Effect of OEO on Exopolysaccharide Production in V. parahaemolyticus causing AHPND

2.6 Effect of OEO on Biofilm Formation in V. parahaemolyticus causing AHPND

2.7 Effect of OEO on the Swarming Motility of V. parahaemolyticus

2.8 In Vivo Effect of OEO on the Virulence of V. parahaemolyticus

2.9 Transcriptomic Analysis of V. parahaemolyticus Exposed to OEO

2.10 Statistical analysis

3. Results

3.1 Bactericidal Activity and Sublethal Concentrations of OEO against V. parahaemolyticus

3.2 Effect of OEO on the Production of Total Exopolysaccharides in V. parahaemolyticus

3.3 Effect of OEO on Biofilm Formation in V. parahaemolyticus

3.4 Effect of OEO on Swarming Motility of V. parahaemolyticus

3.5 Virulence of V. parahaemolyticus Exposed to OEO

3.6 Transcriptomic Analysis

3.6.1 Differentially Expressed Genes in V. parahaemolyticus Treated with OEO

3.6.2 GO Function Analysis of Significant DEGs

3.6.3 KEGG Functional Analysis of Significant DEGs

4. Discussion

Abbreviations

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References

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