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Introduction
The Mexican oregano is a generic group of three different species of aromatic plants: Lippia graveolens, also known as L. berlandieri, Poliomintha longiflora, and Monarda fistulosa var. menthifolia (Cid-Pérez et al., 2015). The genus Lippia is the most cultivated of these oregano plants. This genus belongs to the Family Verbenaceae, which contains 35 genera, and around 1000 species that are mainly distributed in temperate and warm regions of the northern and southern hemispheres of the American continent (López-Villafranco et al., 2017). The genus Lippia includes about 200 species that are predominant in the tropical and subtropical regions of the American continent (González-Elizondo et al., 2011). In México, Lippia has 31 species (Huerta, 1997). It has been proposed that oregano plants of the genus Lippia are a complex of various close species including L. graveolens, L. berlandieri, L. palmeri, L. alba and other infraspecies located in the north and northwest, to the south of Mexico and even Central America (Pascual et al., 2001; Gonzalez-Elizondo et al., 2011; Cid-Pérez et al., 2015; Orona-Castillo et al., 2017; Calvo-Irabien, 2018). The species L. graveolens and L. berlandieri are considered as one species, or variants of different populations (Huerta, 1997; Gonzalez-Elizondo et al., 2011; Cid-Pérez et al., 2015; Calvo-Irabien, 2018). It is possible that this species is polymorphic, comprised of several populations with various habitat preferences and distinct morphological, phenological and phytochemical features (González-Elizondo et al., 2011). It also seems that this genus has a consistent chemical profile and pharmacological activities (Pascual et al., 2001).
Plants of the genus Lippia have different habitat preferences, going from a warm climate, gradients of dry and semi-dry climates, and a part of temperate climates. In México, they are found through the Gulf Coast, the Pacific region, the Yucatan peninsula, the arid zones of Tamaulipas and Hidalgo, and the Chihuahua desert. The form of the plant is variable, from xerophytic scrub, subtropical scrub, tropical deciduous forest (low jungle), chaparral, and acahuales derived from the tropical deciduous forest (medium jungle). They can also be the dominant form in the subtropical scrub at southern Durango and northern Zacatecas (González-Elizondo et al., 2011). The variety L. berlandieri is distributed in the central-east part of Mexico; the variety L. graveolens, is found in the north, and the variety L. palmeri locates in the northwest of the country (Huerta, 1997; Cid-Pérez et al., 2015).
The oregano essential oils (OEO) have recently been found to have antimicrobial activity due to the presence of chemicals such as -cymene, which is the precursor of compounds such as thymol and carvacrol, that may be effective antimicrobials (Skandamis & Nychas, 2001; Gracia-Valenzuela et al., 2012; Gracia-Valenzuela et al., 2014; Quiroz-Velázquez et al., 2017). These compounds make up most of the antimicrobial molecules in this type of essential oils (Paredes-Aguilar et al., 2007), and they have shown potential to control pathogenic microorganisms affecting various farmed species of fish and crustaceans (Gracia-Valenzuela et al., 2012; 2014; Morales-Covarrubias et al., 2016; de Souza et al., 2017; Majolo et al., 2017; 2018; Marasca et al., 2020).
At present, most farmed fish, mollusks, and crustaceans can be affected by several types of pathogens causing disease and mortality (Sindermann, 1984; Lightner et al., 2012). Of these, bacteria are one of the most damaging, since they can be highly infectious and can cause massive mortalities. Hence, one main goal in aquaculture is to reduce the impact of infectious diseases and at the same time, diminishing the impact of chemicals on the aquatic environment and communities of animals and microorganisms living in it.
The use of chemicals and antibiotics to control microbial pathogens poses many risks to environmental and human health, including antibiotic resistance issues and environmental pollution (Pridgeon & Klesius, 2012). In aquaculture, the impact of drug-resistant bacteria on farmed tilapia in China has caused losses of 400 million US dollars, whereas, in Costa Rica, drug-resistant bacteria has caused 2.5 million US dollars losses in tilapia cultures (Oviedo-Bolaños et al., 2021). Moreover, various bacterial species have been reported to show resistance to antibiotics, including Enterobacter (Cohen et al., 2020), Salmonella, and Vibrio (Banerjee et al., 2012). To fight bacterial pathogens affecting shrimp cultures, it is necessary to develop and test substances from natural sources, that are friendly to the environment and represent an alternative to the use of chemicals and antibiotics that are hazardous to fauna and beneficial microorganisms in the aquatic environment. These premises make the case for the search for products of natural origin derived from plants such as essential oils which contain active compounds with several biological properties, including antimicrobial activity. These natural products may contribute to reduce the negative impact of infectious diseases in aquaculture, and also curb the damaging effects to the aquatic environment caused by the presently used chemicals (Escobedo-Bonilla, 2021). Therefore, it is important to find and evaluate natural compounds with effective antimicrobial activity that may be used in aquaculture to maintain both aquatic animal health and preserve the environment (Citarasu, 2012; Zhu, 2020).
The present study aimed to determine in vitro the antimicrobial effect of two OEOs with different proportions of carvacrol and thymol on naturally-occurring bacteria from farmed shrimp from Sonora, Mexico.
Materials and Methods
Morphological traits of Mexican oregano
Plants of the Mexican oregano Lippia complex, are aromatic shrubs measuring up to 2.5 m high and 1.20 m average foliage. They have branched stems with high numbers of leaves, which are the usable part of the plant. The smell and taste of the wild plants are robust and strong (Cid-Pérez et al., 2015). The leaves are 1 to 3 cm long, and 0.5 to 1.5 cm wide, they are opposed, alternate, with oval shape, dark green color, serrated edges rough texture, with villi. The flowers are white and small forming inflorescences in clusters. The fruits are small capsules containing brown seeds, not bigger than 0.25 mm (Huerta, 1997).
Production of oregano essential oils (OEOs)
Culture fields of Lippia berlandieri from Salaices, Chihuahua were the origin of the vegetal material. Leaves were dried and ground to prepare the essential oils, according to the methods described by Paredes-Aguilar et al. (2007). Briefly, dry oregano leaves were used for hydro-distillation using a Clevenger-type distiller to produce the essential oils. The oils were separated by density using a separation funnel. The fractions containing different concentrations of thymol and carvacrol were obtained by placing the resulting essential oil into a flask coupled to a distillation column, and heating until boiling. Distillation was done until about 30% of the initial volume was left in the flask. The distilled solution was again distilled under the same conditions until the two fractions with different concentrations of thymol and carvacrol were obtained.
Analyses of the resulting fractions showed that OEO1 had higher thymol content (48 - 64 % thymol, 23 - 26 % carvacrol), whereas OEO2 had higher carvacrol content (3 - 6 % thymol, 77 - 82 % carvacrol) (Paredes-Aguilar et al., 2007). These OEOs were stored at room temperature (28 ± 2 °C) until used in the experiments.
Bacteria isolates
Samples from shrimp, pond soil and water were collected in 13 shrimp farms in Sonora, México. On each farm, 130 shrimp with signs of bacterial infection were collected and transported to the laboratory to be processed for bacteriological analyses. At the laboratory, shrimp were externally sanitized with 70 % ethanol, and the hepatopancreas was aseptically removed. Organ tissues (≈ 50 mg) were homogenized and diluted with 45 ml of sterile 0.1% peptone water. Tenfold dilutions were prepared and plated in triplicate on Thiosulfate-Citrate-Bile salts-Sucrose Agar (TCBS). Plates were incubated at 30 ± 2 °C for 24 h according to the method proposed by Gracia-Valenzuela et al. (2014). Sediments and water samples (one sample per pond) were also used to isolate bacteria. This type of sample was collected in sterile plastic bags, placed in coolers at 4 (C and transported to the laboratory, where the samples were processed to obtain bacterial cultures as described above.
Determination of bacteria species
The genera and species of the cultured bacteria were determined by using the commercial kit API20E (BioMérieux, France) according to the manufacturer’s instructions. Briefly, the API 20E system involves 23 standardized biochemical tests to identify Gram-negative Enterobacteriaceae. This system can identify 108 genera and 104 species of bacteria. The system works as follows: a bacterial suspension is inoculated to each of the test tubes of the system, inducing different reactions to determine a specific biochemical property, revealed by a change of color (or not) in the reaction (after adding catalysts or not), giving a colorimetric interpretation of the bacterial species.
OEOs antibacterial activity
The evaluation of the antimicrobial activity of OEOs compared to antibiotics was done by the inhibition zone test as follows (Roque et al., 2001). Plates with Mueller-Hinton medium added with 2.5% NaCl were inoculated with 100 μL of each bacterial suspension (n = 19) and evenly spread with a glass rod in order to get a uniform bacterial layer. Then, sterile paper circles (5 mm diameter) were individually saturated with 20 μL of each of the OEOs and placed on top of the culture medium. The same procedure was done with the antimicrobials: avifloxTM (AF), florfenicolTM (FF), magnamycineTM (MC), enrofloxacineTM (EF) and oxytetracyclinTM (OTC). For these antibiotics, a concentration of 50 μg/mL was used to saturate each paper disk. A saline solution (2.5 % NaCl) was used as negative control. The plate was incubated at 30 °C overnight and it was observed at 24 and 48 h post-incubation to determine the inhibition zones. The measurement of the diameter (mm) of the inhibition zones was done in triplicate and recorded. It was not possible to include positive controls of pure thymol or carvacrol molecules.
Statistical analyses
Differences between inhibition zone (mm) of the different antimicrobials tested were analyzed by the non-parametric Kruskal-Wallis test. Multiple comparison analysis between treatments was done with the Mann-Whitney U test (Zar, 1998), using the online Kruskal-Wallis calculator (https://www.statskingdom.com/kruskal-wallis-calculator.html).
Results
Bacterial isolates
A total of 19 bacterial isolates belonging to seven genera (Klebsiella, Pasteurella, Pseudomonas, Salmonella, Serratia, Enterobacter and Proteus) and 10 species were obtained from shrimp hepatopancreas, water and/or sediments from the sampled farms (Figure 1). All the isolated bacterial species were gram-negative and were identified using the API20E system. The genera Pseudomonas and Salmonella had the highest frequency in the samples. These genera were mainly isolated from hepatopancreas. Conversely, Proteus was the genus with the lowest frequency. The frequency of these genera or species corresponds to the number of farms where they were found (Table 1 and Figure 1).
Table 1 Bacterial species found using the API20E system.
| Isolate code | Genus and species | Species number | Frequency (number of farms found) |
|---|---|---|---|
| C-17 | Enterobacter cloacae | 1 | 2 |
| C-15 | Klebsiella ozaenae | 2 | 2 |
| C-19 | Pasteurella spp | 3 | 3 |
| C-46 | Proteus vulgaris | 4 | 1 |
| C-40 | Pseudomonas aeruginosa | 5 | 4 |
| C-6 | Salmonella spp. | 6 | 1 |
| C-4 | Salmonella paratyphi A | 7 | 2 |
| C-5 | Salmonella typhi | 8 | 1 |
| C-18 | Serratia marcescens | 9 | 2 |
| C-53 | Not identified | 10 | 1 |
Evaluation of OEOs antimicrobial activity
The antimicrobial effect of OEOs evaluated as inhibition zone is shown in Table 2. Overall, the average inhibition halo size (mm) against all the bacteria species for each treatment were: OEO1 26.02, OEO2 23.30, florfenicol 22.89, enrofloxacin 21.85, oxytetracycline 8.35, magnamycin 16.96 and aviflox 22.19, respectively. The Kruskal-Wallis analysis showed significant differences between treatments, although no significant differences were found between the antibacterial proficiency of thymol-rich OEO and carvarol-rich OEO. Multiple comparisons using the Mann-Whitney U test showed significant differences (p < 0.007143) (using the Bonferroni correction) in size of inhibition halos between OEO1 and enrofloxacine, oxytetracycline, magnamycin and aviflox; whereas OEO2 had significant differences with oxytetracycline and magnamycin (Table 3).
Table 2 Inhibition zones (average ± standard deviation in mm) of bacteria isolates exposed to different antimicrobial products.
| Isolate/species | OEO 1* | OEO2* | FF* | EF* | OTC* | MC* | AF* |
|---|---|---|---|---|---|---|---|
| Enterobacter cloacae 1 | 24 ± 1 | 20 ± 1 | 28 ± 0.6 | 21 ± 1 | 10 ± 0.6 | 19 ± 1 | 23 ± 1 |
| Enterobacter cloacae 2 | 27 ± 0.6 | 28 ± .6 | 24 ± 1 | 27 ± 1 | 10 ± 0.6 | 15 ± 1 | 24 ± 0.6 |
| Klebsiella ozaenae 1 | 28 ± 0.6 | 23 ± 1 | 23 ± 1 | 21 ± 0.6 | 6 ± 0.6 | 15 ± 0.6 | 22 ± 1 |
| Klebsiella ozaenae 2 | 28 ± 1 | 23 ± 0.6 | 20 ± 0.6 | 22 ± 1 | 8 ± 0.6 | 10 ± 1 | 23 ± 0.6 |
| Pasteurella spp 1 | 22 ± 0.6 | 19 ± 1 | 24 ± 1 | 25 ± 0.6 | 11 ± 1 | 16 ± 0.6 | 26 ± 1 |
| Pasteurella spp 2 | 24 ± 1 | 24 ± 0.6 | 22 ± 0.6 | 21 ± 1 | 12 ± 0.6 | 18 ± 1 | 20 ± 1 |
| Pasteurella spp 3 | 26 ± 1 | 22 ± 1 | 21 ± 1 | 25 ± 0.6 | 9 ± 1 | 20 ± 1 | 19 ± 0.6 |
| Proteus vulgaris | 30 ± 1 | 24 ± 1 | 20 ± 0.6 | 17 ± 1 | 7 ± 1 | 17 ± 0.6 | 25 ± 1 |
| Pseudomonas aeruginosa 1 | 22 ± 0.6 | 19 ± 1 | 23 ± 1 | 26 ± 0.6 | 8 ± 0.6 | 16 ± 1 | 22 ± 1 |
| Pseudomonas aeruginosa 2 | 22 ± 0.6 | 23 ± 0.6 | 26 ± 1 | 17 ± 0.6 | 10 ± 0.6 | 24 ± 1 | 21 ± 1 |
| Pseudomonas aeruginosa 3 | 22 ± 1 | 23 ± 1 | 22 ± 0.6 | 17 ± 1 | 8 ± 0.6 | 16 ± 0.6 | 24 ± 0.6 |
| Pseudomonas aeruginosa 4 | 22 ± 0.6 | 19 ± 1 | 25 ± 0.6 | 20 ± 1 | 9 ± 0.6 | 17 ± 1 | 20 ± 1 |
| Salmonella spp. | 29 ± 0.6 | 25 ± 0.6 | 17 ± 0.6 | 28 ± 0.6 | 7 ± 0.6 | 17 ± 0.6 | 20 ± 0.6 |
| Salmonella paratyphi 1 | 28 ± 1 | 25 ± 1 | 23 ± 1 | 20 ± 1 | 10 ± 1 | 16 ± 0.6 | 20 ± 1 |
| Salmonella paratyphi 2 | 28 ± 0.6 | 23 ± 0.6 | 20 ± 1 | 20 ± 1 | 9 ± 0.6 | 20 ± 0.6 | 25 ± 1 |
| Salmonella typhi | 24 ± 1 | 26 ± 1 | 25 ± 0.6 | 15 ± 1 | 6 ± 0.6 | 11 ± 0.6 | 28 ± 0.6 |
| Serratia marcescens 1 | 31 ± 0.6 | 28 ± 0.6 | 22 ± 1 | 26 ± 0.6 | 6 ± 1 | 19 ± 1 | 20 ± 1 |
| Serratia marcescens 2 | 31 ± 1 | 26 ± 0.6 | 28 ± 0.6 | 20 ± 1 | 7 ± 0.6 | 22 ± 1 | 18 ± 0.6 |
| Not identified | 23 ± 0.6 | 19 ± 0.6 | 25 ± 1 | 26 ± 1 | 7 ± 0.6 | 15 ± 1 | 20 ± 1 |
* OEO 1: OEO rich in thymol; OEO 2: OEO rich in carvacrol; FF: florfenicol; EF: enrofloxacine; OTC: oxytetracycline; MC: magnamycin; AF: aviflox.
The thymol-rich OEO1 had highest antimicrobial activity against Serratia marcescens (31 ± 1 mm), Proteus vulgaris (30 ± 1 mm), Salmonella spp. (29 ± 0.6 mm), Salmonella paratyphi (28 ± 1 mm), Klebsiella ozaenae (28 ± 1 mm), Enterobacter cloacae (27 ± 0.6 mm) and Pasteurella spp. (26 ± 1 mm). Likewise, the carvacrol-rich OEO2 had high antimicrobial activity against Enterobacter cloacae (28 ± 0.6 mm), Serratia marcescens (28 ± 0.6 mm), Salmonella typhi (26 ± 1 mm), Salmonella spp. (25 ± 0.6 mm), Salmonella paratyphi (25 ± 1), Proteus vulgaris (24 ± 1 mm), Pasteurella spp. (24 ± 0.6 mm) and Klebsiella ozaenae (23 ± 1 mm). The antibiotic aviflox had the highest activity against Salmonella typhi (28 ± 0.6 mm) and Pasteurella spp (26 ± 1). Enrofloxacin showed highest antibacterial activity against Salmonella spp. (28 ± 0.6 mm) and Enterobacter cloacae (27 ± 1 mm). Last, the antibiotic florfenicol showed highest activity against Enterobacter cloacae and Serratia marcescens (28 ± 0.6 mm, respectively) and Pseudomonas aeruginosa (26 ± 1 mm) (Table 2).
Discussion
The present study examined the antibacterial effect of two OEOs, one with higher thymol content and the other with higher carvacrol content on 10 bacteria species belonging to the gram-negative Enterobacteriaceae living in the aquatic environment of shrimp ponds. These extracts showed higher antibacterial activity than some commonly used antibiotics (Table 3).
Table 3 Mann-Whitney U multiple comparisons between statistically significant test pairs.
| Treatment pair | Difference | H value | Critical value | P value | Conclusion |
|---|---|---|---|---|---|
| OEO1 >> EF | 5 | 10.0394 | 7.2367 | 0.001532 | significant differences |
| OEO1 >> OTC | 18 | 27.9712 | 7.2367 | 1.23E-07 | significant differences |
| OEO1 >> MC | 9 | 24.9826 | 7.2367 | 5.79E-07 | significant differences |
| OEO1 >> AF | 4 | 10.2807 | 7.2367 | 0.001344 | significant differences |
| OEO2 >> OTC | 15 | 27.9712 | 7.2367 | 1.23E-07 | significant differences |
| OEO2 >> MC | 6 | 19.3575 | 7.2367 | 0.00001084 | significant differences |
| FF >> OTC | 15 | 27.922 | 7.2367 | 1.26E-07 | significant differences |
| FF >> MC | 6 | 19.9707 | 7.2367 | 0.000007864 | significant differences |
| EF >> OTC | 13 | 27.9374 | 7.2367 | 1.25E-07 | significant differences |
| EF >> MC | 4 | 12.4108 | 7.2367 | 0.0004269 | significant differences |
| OTC >> MC | 9 | 26.2839 | 7.2367 | 2.95E-07 | significant differences |
| OTC >> AF | 14 | 27.9867 | 7.2367 | 1.22E-07 | significant differences |
| MC >> AF | 5 | 17.1745 | 7.2367 | 0.0000341 | significant differences |
The bacterial genera found using the API20E system included Klebsiella, Pasteurella, Pseudomonas, Salmonella, Serratia, Enterobacter and Proteus. Most of these genera have previously been reported to occur in different aquatic environments such as coastal and brackish water (Cohen et al., 2020), coastal and marine waters (Joyner et al., 2014), fresh- and marine water (Singh & Kulshreshtha, 1992), water and soil (Wareth & Neubauer, 2021), shrimp digestive tract (Buglione et al., 2010), and digestive tract of other animals (Drzewiecka, 2016).
Many of these bacteria genera and/or species have been reported to display some degree of antibiotic resistance. Such species have developed and/or acquired various extended-spectrum β-lactamase enzymes, which inhibit β-lactam antibiotics such as penicillins, cephalosporins, and carbapenems. Genes responsible for enzyme inactivation are often located on mobile genetic elements and provide a risk of transfer to other bacteria (Wareth & Neubauer, 2021).
Among the species reported to contain or produce the carbapenemase- and extended-spectrum β-lactamase include Enterobacter cloacae and Klebsiella oxytoca (Cohen et al., 2020). Other species are Klebsiella pneumoniae, Enterococcus faecium, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (Wareth & Neubauer, 2021). In addition, other genera reported in the present study have also been shown to display antibiotic resistance. Examples are the Salmonella enterica serovar Corvallis isolated from water in two shrimp farms and showed resistance to erythromycin. Two other S. enterica serovar Corvallis isolates from another shrimp farm were resistant to oxolinic acid, sulphonamides, tetracycline, sulfamethoxazole/trimethoprim, ampicillin, doxycycline hydrochloride, erythromycin and chloramphenicol. Nonetheless, they were sensitive to nitrofurantoin and norfloxacin (Banerjee et al., 2012).
Several Vibrio species have also been reported to display antibiotic resistance. Water-isolated V. mimicus was resistant to sulfamethoxazole/trimethoprim, sulphonamides, and ampicillin. Isolates of V. cholerae were resistant to ampicillin; whereas isolates from shrimp were resistant to doxycycline hydrochloride, ampicillin and tetracycline. An isolate of V. cholerae was resistant to doxycycline hydrochloride and tetracycline. Isolates of V. mimicus from shrimp and water, and V. vulnificus from shrimp, were resistant to ampicillin. Likewise, most Vibrio isolates showed resistance to ampicillin, followed by resistance to tetracycline and doxycycline. Two farms had a higher presence of antibiotic-resistant bacteria (one to four antibiotics) than another farm (one to two antibiotics). The species V. cholerae showed the widest antibiotic-resistant pattern (Banerjee et al., 2012). Another foodborne bacterial disease of concern is caused by Salmonella enterica, not only for its impact on human health but also for the emergence and spread of antibiotic resistant variants worldwide (Santos et al., 2019).
Since the number of multidrug-resistant bacteria species continues to rise and are present in various aquatic environments, strategies to reduce their impact on aquaculture and to restrict their threat to aquatic animal health are urgently needed. Some of such strategies include the use of natural products from plants or seaweeds, which have antimicrobial and growth-promoting properties. Moreover, they represent environmental-friendly alternatives to antibiotic use (Menanteau-Ledouble et al., 2015; Escobedo-Bonilla, 2021). For example, various plant-derived extracts have been tested against several bacterial pathogens. Such plant extracts include concoctions from barks, stems and roots of Zingibe officinale, Mentha piperita, Abarema cochliacarpos, Syzygium cordatum, among others. The precise antimicrobial mechanisms of most plant bioactive components are not yet clarified. Nonetheless, some mechanisms have been suggested such as the disruption of pathogen membranes, interruption of DNA/RNA synthesis and function, interference with intermediary metabolism, coagulation of cytoplasmic constituents, and the interruption of normal cell communication (Santos et al., 2019).
Previously, other studies have used OEOs to evaluate their antibacterial activity in vitro against bacteria of the genera Aeromonas, Pseudomonas and Vibrio (Gracia-Valenzuela et al., 2012). Their efficacy was assessed in vivo inoculating shrimp with different species of Vibrio and then treating them with each of the OEO obtaining promising results (Gracia-Valenzuela et al., 2014). In the present study, these OEOs showed high efficacy against Enterobacteriaceae in vitro. The size of the inhibition halos produced by the two OEOs was similar according to the statistical analysis. The OEO with higher thymol content showed higher average inhibition halos (26.02 mm) than the OEO containing a higher amount of carvacrol (23.30 mm). These two products had higher mean inhibition halo size than the commercial antibiotics tested. The least effective antibiotics were oxytetracycline and magnamycin (Table 3). These results suggest that the two OEOs assayed in this work had antibacterial properties against the tested species of the Enterobacteriaceae, many of which are becoming global threats to human and animal health, due to their multidrug resistance variants.
In conclusion, the antibacterial effect of two OEOs, one with higher thymol content and other with higher carvacrol content on 10 gram-negative species of the Enterobacteriaceae, showed higher antibacterial activity in vitro than some commonly used antibiotics in aquaculture. The bacterial genera and species found in this study are normally found in marine and brackish aquatic environments, including the shrimp digestive tract. Many of these bacteria have been reported to display some degree of multidrug resistance to antibiotics such as penicillins, cephalosporins, and carbapenems. Therefore, strategies based on natural products with antimicrobial properties to reduce their impact on aquatic animal health are urgently needed. The antimicrobial activity of two OEOs in vitro was very effective against 10 bacterial species of Enterobacteriaceae. These natural compounds were significantly efficient to inhibit bacterial growth compared to antibiotics commonly used in aquaculture operations and human health.
