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Introduction
Tilapia (Oreochromis spp.) is considered worldwide as the second-largest aquaculture finfish species group by volume 7,20. In Guatemala, the tilapia aquaculture industry, according to the Food and Agriculture Organization of the United Nations (FAO), produced approximately 10,914 MT of tilapia in 2018. This industry has shown an annual growth of 10.53% since 2011, supplying an alternative source of animal protein and providing an income to Guatemalans with different economic development levels. In recent years, the tilapia culture industry has been significantly affected worldwide by the breakout of infectious diseases, especially from viral and bacterial origin, being the most frequently cited: Streptococcus agalactiae, S. iniae, S. dysgalactiae, Lactococcus garvieae, Flavobacterium columnare, Francisella noatunensis subsp. orientalis, Edwardsiella ictaluri, E. tarda, E. anguillarum, E. piscicida, Aeromonas hydrophila, A. veronii, A. jandaei, A. shuberti, A. dhakensis16,17,18. Moreover, the conventional approaches to control fish diseases have limited success in fish disease prevention, control and treatment. In aquaculture traditional health management, to avoid mortalities, to control and prevent the infection from several bacterial pathogens, a broad-spectrum of veterinary drugs are used 19,33,41. Fish diseases related to parasites and fungal pathogens have mostly been treated and controlled by using formalin, quaternary ammonia and hydrogen peroxide 38. While, for treating bacterial diseases, the antibiotics most widely used are florfenicol, oxytetracycline, enrofloxacin, chloramphenicol, erythromycin and trimethoprim + sulfametoxazol 33. It is well known that chemical and antibiotic drugs can have negative effects not only in the environment, through all trophic levels, but also on human health by consumption of contaminated food and water 29 and most importantly, their use can result in drug-resistant pathogenic microbes. Thus, their routine use is now restricted or banned in some countries.
Therefore, medicinal plants and their extracts have been widely used as antibacterial, antiparasitic, antiviral and immunostimulants 10,51,54. Due to their phytochemical components, such as flavonoids, terpenes, tannins, alkaloids and coumarins, among others, they have been assessed for the treatment and prophylaxis of different pathogens in the aquaculture industry 3,46,47. Moreover, most of these natural products are environmentally friendly, because they are biodegradable compared to synthetic products such as antibiotics, and they are less likely to generate drug resistance on bacteria due to their molecule’s diversity 36.
Several plant extracts have been shown to display antibacterial effects in aquaculture, including those from Lippia spp. Lippia belongs to the Verbenaceae family, comprising 200 species widely distributed in America 56. In Guatemala, approximately 13 species have been identified, being the most common L. graveolens (Khunth) and L. alba (Mill). They are also frequently used for medical purposes in public health 11,50. Among the phytochemical components in Lippia spp., the most frequently found are p-cymene, cis-sabinene hydrate, terpinen-4-ol, carvacrol, thymol, caryophyllene oxide, citral, geranial, neral and linalool 21,37. Most of these major components from different Lippia species have an antibacterial capacity in different fish bacterial pathogens, such as Aeromonas spp., Vibrio spp., Streptococcus spp., Edwardsiella spp. and Flavobacterium columnare, among others, but there is no evidence for L. graveolens against fish bacteria pathogens 3,48.
The efficiency of these metabolites against different bacterial pathogens depends on the method of extraction and solvent. Although several studies have evaluated the efficacy of herbal medicine 24,40,57, limited information exists on the efficacy of extracts obtained using different extraction methods and solvents. Therefore, this study aimed to determine the (in vitro) antimicrobial capacity of different types of extracts and essential oils of L. alba and L. graveolens against nine pathogenic strain bacteria isolated from tilapia cultures in Guatemala.
Material and methods
Plant collection and extraction methodology
Two Lippia species (Verbenaceae) were sampled in Guatemala during April 2019. Fresh and healthy leaves of mature L. graveolens (LG) and L. alba (LA) plants were collected from wild suburbs in Zacapa department (14°59' 50.2'' / 89° 40' 26.2'') and from the medical plant collection from Centro Experimental Docente de Agronomía, Facultad de Agronomía, Universidad de San Carlos de Guatemala (CEDA-FAUSAC) in Guatemala City (14° 35' 6.45'' / 90° 33' 24.34''); respectively. The plant species were identified at Herbario de la Escuela de Biología, Facultad de Ciencias Químicas y Farmacia, Universidad de San Carlos de Guatemala. The leaves were oven-dried at 45 ⁰C for 48 h, crushed into powder using an electric grinder, sieved through a 300-500 µm mesh size and finally stored at room temperature until use.
Once the plant-powder was prepared, three methods of extraction with different solvents were used to obtain the crude extracts and essential oils from L. graveolens and L. alba. The extraction methods were the following:
Percolation technique. Crude extracts were obtained using the percolation technique (PE) through consecutive extractions with three different solvents: cyclohexane (CLH), ethyl acetate (ACE), and ethanol 95% (ETH) 44. Sequential extractions were carried in amber glass bottle (5 L), filled with 300 g of plant-powder and 3,000 mL of the selected solvent (ratio 1:10 w/v). Each solution was maintained for 48 h at room temperature (28 ± 2 ⁰C). Afterwards, the solution was separately filtered through a Whatman No. 1 filter paper (Whatman, USA). Finally, the filtered solution was evaporated using a rotary vacuum evaporator at 45 ⁰C and 115 mB until the solvent faded completely. The resulting crude extracts were separately stored at 4 ⁰C in 150 mL amber glass bottles.
Supercritical fluid technique. The supercritical fluid extracts (FSC) were obtained using a homemade apparatus based on the model according to Loaiza-Salguero, Ortíz & Robles (1994). For each plant, the extraction vessel (2,000 mL) was filled with 1,000 g of plant-powder and 1,000 mL of ethanol 95% (ratio 1:1). The extractions were done at 45 ⁰C and 1,300 psi of CO2 for 45 min. The extracts obtained were collected in a glass flask. To improve the efficiency of this extraction method, the procedure was repeated three times with the same plant material and the same solvent ratio. Finally, the extracts were evaporated using a rotary vacuum evaporator at 45 ⁰C and 115 mB.
The crude extracts were separately stored at 4 ⁰C in 150 mL amber glass bottles Essential oil. Essential oils (EOS) were extracted by hydro-distillation using a Clevenger type apparatus 30. Each extraction used 150 g of plant-powder, placed in a 2,000 mL volumetric flask and covered with tap water until submerged. The extraction lasted 2 h after water boiling. The essential oils were collected and stored at 4 ⁰C in 5 mL amber glass bottles.
For each of the extraction methods, the extract yield was estimated as Y (%) = M/MV x 100, where M is the mass of extract obtained with each method and MV is the amount of ground dried plant material used.
Microorganisms and culture conditions
Nine bacterial pathogens (six strains of Aeromonas sobria and three of A. hydrophila) were obtained from the bacterial collection of Centro de Estudios del Mar y Acuicultura, Universidad de San Carlos de Guatemala, and used in the study. These bacteria strains were isolated from diseased tilapia (Oreochromis spp.) and identified using the API 20NE (BIOMERUX, France). The bacteria were cultured in trypticase soy agar at 28 ⁰C for 24 h before used in the antimicrobial susceptibility tests.
Antimicrobial screening
The effect of the different extracts obtained from L. graveolens and L. alba were assayed on the nine bacteria strains using the disc diffusion method as described by Tkachenko et al. (2016). Each bacterial suspension was adjusted to a concentration of 1-2 x 108 CFU/mL in sterile phosphate buffer solution (PBS), then inoculated on the surface of Muller Hinton agar and spread all around. Subsequently, three blank discs were permeated with 10 mg of each extract, and in the case of EOS 10 µL were used. For positive control, standard discs of oxytetracycline (OXI: 40 µg) and florfenicol (FLC: 40 µg) were obtained from AVIMEX®. For negative controls, blank discs individually impregnated with 20 µL of cyclohexane, ethyl acetate, and ethanol 95% were used. All discs were placed in an incubator for 10 min at 30 ⁰C to volatilize the solvents. Then, the discs for each treatment and controls were separately set upon the surface of the plates containing the bacteria strains. Each of the extracts were tested twice in triplicate for the nine bacteria strains. The plates were incubated at 28 ⁰C for 24 hours. The inhibition zone diameters (IZD) were measured to an accuracy of 0.01 mm after 24 h of incubation. The interpretation of the IZD was done according to García-Pérez & Marroquin-Mora, (2020) for the following criteria: extracts or essential oils were considered effective when their IZD was equal or greater than half of the IZD measured for each bacteria strain against the antibiotics. In this case, the most widely used in Guatemala aquaculture, which is oxytetracycline.
Analysis of the plant’s mayor components by Gas chromatography-mass spectrometry (GC/MS)
Due to their high effectiveness in the antimicrobial screening, the EOS chemical components of L. graveolens and L. alba were determined. The GC/MS analyses were performed using a Shimadzu 2010 Plus system coupled with a Shimadzu QP-2010 Plus selective detector (MSD), equipped with a DB5-MS capillary fused silica column (60 m, 0.25 mm I.D., 0.25 µm film thickness). The oven temperature was programmed from 60 ºC to 246 ºC at 3 ºC/min and then held isothermally at 246 ºC for 20 min. He (99.999%) was used as carrier gas with a flow rate of 1.03 mL/min; split ratio of 1:50. Mass spectra were taken at 70 eV. The m/z values were recorded in the range of m/z 40-700 Da. The components were identified by their mass spectra and retention indices compared to values reported in the literature 1. Relative amounts of components were calculated based on GC peak areas without using correction factors.
Data analysis
All experiments were conducted twice in triplicate. Mean values and standard deviations of the inhibition zone diameters in disk diffusion assay were calculated from the experimental data obtained. Mean significance of inhibition zone diameter for different bacterial strains, plant material and type of extraction were analyzed using Generalize Liner Mixed Model (GLM: Negative binomial), to predict the effect of different plant and extraction methods. The differences between the mean values were considered significant when p < 0.05. The statistical analysis was carried out using R statistic software 39.
Results
Among the different extraction methods used, the highest yield in both plants was obtained with the FSC method (5.29% for L. alba and 5.06% for L. graveolens), followed by PE extraction (solvent: CLH + ACE + ETH). Of the PE methods, 95 % ethanol produced the highest yield (4.16 % for L. graveolens and 3.28% for L. alba); while cyclohexane (2.48% for L. graveolens and 0.73% for L. alba) was the least efficient. The lowest yield was obtained with the essential oil extraction using water as solvent for L. alba with 0.53% (Table 1).
Table 1 Extraction yield (%) for L. graveolens and L. alba obtained with different extraction methods.
Note: derived from research.
Concerning the aroma displayed by the plants and extracts, the difference was evident. L. graveolens had a spicy and intense pungent odor, while L. alba had a distinctly penetrating herb-like and an intense lemon citrus odor. For both plants, the supercritical fluid and essential oil extraction had similar aroma, as well as the extract obtained by PE with 95% ethanol. In contrast, the extracts obtained with cyclohexane and ethyl acetate showed a milder aroma.
The results of antimicrobial activities are presented in table 2 and figure 1. No inhibition zones were recorded in the negative controls (cyclohexane, ethyl acetate, and ethanol 95%), while the positive controls (oxytetracycline: OXI and florfenicol: FLC) showed strong antibacterial activity against Aeromonas spp. Their inhibition zones diameter ranged from 9.58 to 31.79 mm (Figure 1c). The inhibition zone diameter for OXI ranged from 9.74 to 27.17 mm. To be considered effective, the inhibition zone of plant extracts must be equal or greater than 5.10 - 13.59 mm, depending on the bacteria strain. A lower value was considered ineffective in this study.
Table 2 Antimicrobial potential of L. graveolens and L. alba against Aeromonas spp.
Note: Values (mean ± standard error) of inhibition zones diameter of oxytetracycline (OXI), florfenicol (FLC), ethyl acetate (ACE), cyclohexane (CLH), essential oil extraction (EOS), ethanol 95% (ETH), Supercritical fluid extraction (FSC).
Among all tested extracts and EOS, L. graveolens (LG) exhibited a higher inhibitory effect. The EOS-LG was the most effective, which inhibited 100% of bacteria strains and presented inhibition zone diameters that ranged from 25.2 to 36.94 mm (Table 2, Figure 1a). The other extracts showed lower inhibitory effects, FSC-LG, CLH-LG, ACE-LG (77.78%) respectively and ETH-LG (22.22%), respectively. In the case of ETH-LG, its inhibition zone diameters ranged from 8.34 to 9.57 mm, for this reason, it was classified as an extract with limited antibacterial effect according to the criteria established in this study. The crude extracts from L. alba did not show any antibacterial activity, while its EOS inhibited 66.67% of the Aeromonas spp. strains, presenting inhibition zone diameters ranging from 10.68 to 16.29 mm (Figure 1b).
Figure 1 Antimicrobial activity of different organic extracts and essential oils from L. graveolens and L. alba. .
According with the GLM analysis, the glmer.nb (Y~ (PLANT/EXTRACT), with two random effects (1| SAMPLES) + (1| PLANT), was the best model to predict the extracts strong dependent activity against all bacteria species evaluated. L. graveolens, as essential oils, is the best predictor to inhibit Aeromonas spp. (p < 0.001), compared with the antibiotic and L. alba. Although the inhibition zone diameter of L. alba essential oils was smaller than L. graveolens, these inhibited 67% of bacteria strains, also proving to be effective.
Among the samples, oxytetracycline showed resistance in 44.44% (A. hydrophila = 1 strain / A. sobria = 3 strain) of the bacteria strains. This study showed that L. graveolens and L. alba antimicrobial activity does not depend on the antibiotic susceptibility pattern (Figure 2).
Figure 2 Antimicrobial activity of L. graveolens and L. alba against oxytetracycline resistant Aeromonas spp.
The volatile components identified in the essential oil of the L. graveolens and L. alba essential oils are reported in table 3. For L. graveolens 10 components were identified, representing nearly 98.22% of the total essential oil’s composition. The major compound was thymol (81.31%), follow by ο-cymene (8.44%), while the remainder components were below 2%. Moreover, in L. alba 20 components were identified, representing nearly 97.74% of the total essential oil’s composition. The major compounds were geranial (36.8%), followed by neral (25.7%) and 1,8-cineole (13.45%), while the rest of the compounds were below 4%.
Discussion
The essential oils from L. graveolens and L. alba offer a promising alternative for the control of Aeromonas spp. growth. The oil extracted from L. graveolens exhibited better antibacterial properties than L. alba, which included a significantly larger inhibition zone diameter against Aeromonas spp. and inhibition against all bacteria strains. It is well documented that L. graveolens have an antibacterial effect against a broad group of pathogens, especially against Gram-negative bacteria 2,6,42, however, until now there was no evidence for the effectiveness of L. graveolens against fish bacteria pathogens. There are many studies with plants with similar metabolites profile as L. graveolens such as Origanum vulgare. This plant known as European oregano has well documented 4,26,49 uses against fish bacterial pathogens especially Gram-negative bacteria. These properties may be attributed to several active metabolites, such as carvacrol, thymol, c-terpinene and ο-cymene 25 that have antibacterial activity and have been detected in various species of Origanum.
The chemical composition of L. graveolens in this study was slightly different compared to other studies from Guatemala and Mexico 9,32,43. For example, Pérez-Sabino et al., (2012) also reported thymol (67.4 - 73.5%) and ο-cymene (1.9 - 5.9%) as the major constituents in L. graveolens from Zacapa department, but did not report the presence of Myrcene, Humulene epoxide II and α-Humulene. Also, the same authors reported that L. graveolens presented three different chemotypes (Thymol, Carvacrol, and Mixed), indicating that these volatile compounds were affected by the prevailing geographical region in Guatemala, genetic factors, nutrients in the soil, harvest time and method of extraction among other factors.
In addition, there are other species of Lippia with similar chemical profile as L. graveolens which have a strong antibacterial effect, such as L. sidoides and L. origanoides23,27,30, which have thymol (9-76.6%) and carvacrol (49.7%) as major components. The essential oils of L. origanoides and L. sidoides had inhibitory effects against A. hydrophila with a minimal inhibitory and bactericidal concentration ranging from 1250 to 5000 µg mL -1. This indicates that probably those components, thymol and carvacrol, are responsible for the antimicrobial activity, showing an inhibitory effect against Gram-negative such as A. hydrophila, but also Gram-positive bacteria. It should be noted that both compounds (thymol and carvacrol) have a similar chemical structure. Although, the chemical composition does not appear to have any major effect on the antibacterial activity, both metabolites provoke the disintegration of the external membrane of bacteria 27.
In the present study, thymol, which is responsible for the inhibition of Aeromonas spp, was found to be the major compound in the L. graveolens essential oils. This phenolic terpene exhibits strong antimicrobial activity capable of rupturing the external membrane of bacteria and increasing its permeability to ATP 27. On the other hand, the efficiency of L. graveolens extracts could be related to the extraction methodology and solvent used, which can affect the amount of thymol extracted. In the present study, L. graveolens showed antibacterial activity with FSC-LG extract, which indicated that the polarity of CO2 combined with ethanol 95% as co-solvent might increase the extraction of polar compounds such as thymol 12. However, this method probably didn’t extract the total amount of the polar compounds, since its efficiency was lower than the EOS, which could contain higher concentration of thymol. Moreover, the efficiency of the PE method was related to the type of solvent used, varying the amount of the major component with the higher antibacterial activity. In this case, CLH-LG and ACE-LG were the solvents with the higher affinity to extract the thymol, however, they apparently did not extract enough of that compound. Consequently, those extracts showed lower antibacterial activity compared to FSC and EOS. This indicates that it is necessary to properly select the type of solvent and technique for extracting a target compound efficiently. Another important factor to be considered is the amount of extract used to permeate the discs in the assays. In this case, 10 mg for the crude extract and 10 µL for the EOS appears to be enough to incorporate a specific proportion of the compounds to show antibacterial activity.
Lippia alba EOS inhibited the growth of A. hydrophila and A. sobria agreeing with results found by other authors, who reported the effectiveness of L. alba EOS in tests against several Aeromonas spp. strains and other Gram-negative and positive bacteria 15,30,35, indicating that the volatile compounds with antibacterial activity were geranial, neral and 1,8-cineole. Other authors reported that the antibacterial activity of L. alba is mainly against Gram-positive bacteria 5,31,45. This is due to the morphological structure of the bacterial cell wall of Gram-positive bacteria, which is less complex than Gram-negative bacteria 55. The present study suggests that the lipophilic character of essential oils of L. alba may interact with lipopolysaccharides, therefore, reducing its efficiency to cause changes in Gram-negative bacteria structure, which could result, in some cases, in no bacterial lysis. Moreover, the chemical composition of L. alba EOS could vary depending on location, soil type, weather conditions, among other factors, showing different chemotypes with varying proportions of major compounds (e.g., geranial, neral, carvone, linalool, myrcene) 13,21,52. The antimicrobial activity of these compounds may be correlated with a specific chemotype.
Finally, bacteria can develop resistance to antimicrobials due to the continuous and prolonged exposure to them 14,34,40. Contrarily, due to the complex composition of L. graveolens and L. alba essential oils, it is expected that bacteria rarely develop a resistance because plants have different mechanisms of action that implies several cell targets 8. This work confirms that L. graveolens (extracts and EOS) and L. alba (EOS) have a high antimicrobial activity, indicating that this is independent on the antibiotic susceptibility pattern, since at least one byproduct of Lippia spp. influences the most resistant bacteria. The efficiency of the plant is related to the plant´s chemical composition, but also to the extraction procedure. The effectiveness of L. alba was limited compared to L. graveolens, due to 1) a lower content of the major metabolites, 2) different types of metabolites. This research also addresses the importance of choosing the proper extraction method and solvent to reach a high effectiveness in the extracts.
Conclusion
This study shows that essential oils from Lippia spp. have high antibacterial activity against Aeromonas spp., isolated from tilapia. The greatest inhibitory effect was obtained with L. graveolens EOS. The results suggest that the antibacterial activity of EOS is attributed to the abundance of monoterpenes and terpenoids compounds. However, further investigations are required to evaluate the mechanisms of the antibacterial activity of the essential oils from Lippia spp. Additionally, future studies should include the isolation of major bioactive components of each EOS and assess their individual effects. In this sense, special attention should focus on thymol for L. graveolens and geranial for L. alba, to evaluate their efficiency in controlling pathogens in aquaculture.
