Therapeutic potentials of Excoecaria agallocha against gram-positive and gram-negative fish bacterial pathogens

Background: The present investigation of a mangrove plant, Excoecaria agallocha, which is a popular medicinal substitute for the treatment of microbial ailments, were evaluated for potential antimicrobial activity against pathogenic bacteria Escherichia coli and Streptococcus agalactiae in tilapia, Oreochromis niloticus. Methods: Antibacterial activity was performed using agar diffusion method, disc diffusion method, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and antibiotic susceptibility assays. Experimental fish fed diet containing 0 (control), 5, 25, and 50 mg kg-1 E. agallocha leaf methanol extract for 28 days then challenged individually with E. coli or S. agalactiae and mortalities were recorded over a ten-day postinfection period. Results: Results indicated that both bacterial species are sensitive to tetracycline, ampicillin, and amoxicillin. E. coli was found to be resistant to neomycin. E. agallocha extract concentration of 50 mg/ml produced a zone of inhibition of 18 mm against E. coli, in contrast to 13 mm against S. agalactiae. E. agallocha showed bactericidal activity against E. coli and bacteriostatic activity against S. agalactiae. The highest E. agallocha LC50 activity was 83 mg/ml. The highest cumulative mortality was 90.0 ± 10.0% in control as compared to 26.7 ± 11.5% in the group fed with 50 mg kg-1 E. agallocha extract, significant differences (P < 0.05). Conclusion: Hence, E. agallocha showed antibacterial activity against fish pathogens Escherichia coli and Streptococcus agalactiae in tilapia, Oreochromis niloticus; therefore, E. agallocha may be used as an alternative therapeutic agent against fish pathogenic bacteria as an additive to feed at a concentration depend, safe, noncytotoxic doses.


Background
The Bacterial infections in aquaculture are a rising concern across the globe. The effectiveness of conventional antibiotics is decreasing due to the global emergence of multi-drug resistant (MDR) bacterial pathogens. The use of broad-spectrum antibacterial agents has led to one of the largest recent global health problems as a result of the development of bacterial resistance. Several bacterial species have developed multidrug resistance, including Escherichia coli [1].
E. coli is a common disease of freshwater fish, especially under cultured conditions [2], and has an important role in economic losses among the fish industry. This pathogen has a variable clinical course ranging from mild to severe infection, possibly leading to death due to septicemia, depending on the bacterial strain and its virulence as well as host-related factors such as age and immunity [3]. Likewise, Streptococcus agalactiae is an important human and animal pathogen associated with a variety of diseases in fish [4]. Septicemia is a common condition in tilapia fish infected with Streptococcus agalactiae [5] becoming highly problematic in freshwater fishes, constantly threatening the sustainability of the global tilapia aquaculture industry [6]. Therefore, efforts have been now shifted to focus on identifying novel drugs of natural plant sources to be used as an alternative to antibiotics in disease management in aquaculture [7].
Excoecaria agallocha contains a copious number of polyphenols and terpenoids, which have been reported to have an epidemic and endemic disease control as an antimicrobial, anti-cancer, and anti-diabetic agent [8]. However, there is sparse information regarding the capability of E. agallocha for the treatment of infections caused by aquaculture bacterial pathogens [9][10][11]. Thus, new compounds that target bacterial virulence can be developed to control the threat posed by multidrug resistance found in economically valuable fish. The search for a new treatment is urgently needed predominantly in those who are non-therapeutic in nature as well as being environmentally friendly. Therefore, the objective of this study was conducted to evaluate the effect of food additive with E. agallocha leaf methanolic extract on artificially infected tilapia, Oreochromis niloticus, with zoonotic bacteria Escherichia coli and Streptococcus agalactiae.

Preparation of plant and extraction
Excoecaria agallocha leaves were collected from the east coast in Terengganu and was identified at the Plant Taxonomy Laboratory in University Malaysia Terengganu (UMT). The leaves were washed and air-dried in the oven overnight at 40°C. The leaves were then ground to a fine powder using a blender, weighing 1 kg. Then, 25 g of plant sample was Soxhlet extracted with 250 mL of MeOH at 60°C for 12 h [12]. Extracts in methanol solvents were evaporated and concentrated to dryness using a rotary evaporator at 45°C. The weight after extraction was determined, and the percent yield (%) for extraction was calculated [11].

Microbial assessment
Fish pathogens as One Gram-negative bacteria Escherichia coli and One Gram-positive bacteria Streptococcus agalactiae, deposited in GenBank (Accession No. KT869025), were used in this study. Both were obtained from Fish Disease Laboratory, School of Fisheries and Aquaculture Sciences, University Malaysia Terengganu (UMT), Malaysia.

Antibiotics susceptibility test
Antibiotics resistance patterns were determined using the disk diffusion method, according to CLSI [13].

Antibacterial assay
Disk diffusion assay was carried out on Mueller Hinton Agar (MHA) following the methods described by Barker [14], and the agar well diffusion method, according to Perez [15].

Determination of MIC and MBC
The minimum inhibitory concentration (MIC) was determined using the broth dilution assay technique in 96 wells microtiter plates. This method was carried out according to CLSI [13], while the minimum bactericidal concentration (MBC) extract was determined according to Ilavenil [16].

Toxicity test
Brine shrimp lethality assay was carried out to study the general toxicity of the extract of E. agallocha according to the methods of Pisutthanan [17].

Determination of the Median Lethal Dose (LD50)
All assays in experimental fish were divided into two main groups. The first group was challenged with E. coli, and the second group challenged with S. agalactiae, individually. Both were challenged under the same environmental laboratory conditions.

Fish collection and acclimatization
Healthy juvenile tilapia, O niloticus, (3.5 ± 0.2 g) were obtained from UMT hatchery. Fish were acclimatized to laboratory conditions for 14 days and then stocked into 16 composite tanks (20 L) in a flow-through freshwater supply system, fed twice a day a commercial feed (30% crude proteins). Water temperature (26 ± 2°C), dissolved oxygen (DO) (5.57 ± 0.01 mg/L) and pH was (7.2 ± 0.2) ranged on acceptable values throughout the experimental period. These were measured with (YSI -Yellow Springs Instruments -model 85/10).

Prepare Bacteria Inoculum
The stock bacteria of E. coli or S. agalactiae were first passed through healthy fish to potentiate its virulence. Then they were grown on blood agar (Oxoid, U.K.) at 28°C for 24 to 48 hours. Bacterial cells were washed twice with physiological saline and then re-suspended in the same solution to obtain a bacterial suspension. The bacteria suspension was adjusted to McFarland turbidity standard No.5 equivalent to 15x108cfu mL 1. Ten-fold serial dilutions were done to obtain a different concentration of bacteria. Intra-peritoneal injection method (i.p.) inoculated into tilapia, O niloticus.

Experimental design
The LD50 value was determined to obtain the lowest bacterial dose of E. coli or S. agalactiae (individually) that cause 50 % mortality in tilapia, O niloticus. Six groups of tilapia, O niloticus, with ten fishes per group, were housed in a 20 L aquarium supplied with proper aeration. The environmental conditions were maintained as optimum as possible. The temperature was kept at 26 ± 2°C, dissolved oxygen (DO) was 5.4 ± 0.5 mg L -1 , and pH was 7.2 ± 0.2. Fish were kept in a fasting state for 24 h before beginning the experiment.
The fishes were anesthetized using Tricaine Methane sulfonate (MS-222) (Sigma, Aldrich). The fish in groups 1, 2, 3, 4 and 5 were artificially injected intraperitoneally (i.p.) with 0.1 ml of culture suspension of pathogenic E. coli or S. agalactiae containing 15 x 108, 15 x 107, 15 x 106, 15 x 105 and 15 x 104 CFU mL -1 , respectively. The 6th group, control group, was injected intraperitoneally (i.p.) with 0.1 ml with physiological saline. Fish mortality was recorded every 24 h for ten days. Dead fish were removed from the aquarium daily.

Preparation of E. agallocha diets
During the acclimation period of 14 days, fish were fed with commercial diets (Analytical Laboratories of UMT hatchery) at 3% BW d -1 twice a daily between the hours of 8.00-9.00 and 15.00-16.00. During the experimental period, the fish were fed for 28 days with 5, 25, and 50 mg extract per -1 kg food, respectively, and the basal diet acted as controls. The extract at a concentration of 5, 25, and 50 mg was dissolved in distilled water and sprayed on the thin layer of basal food. The pellets were dried in an oven at 30°C for 18 h, packed in polythene bags and stored in a freezer at -20°C until used. All fish were deprived of food for 24 h before the challenge test.

Experimental design
A total of 300 healthy juvenile tilapia, Oreochromis niloticus, (3.5 ± 0.2 g) were obtained from UMT hatchery. Fish were acclimatized for 14 days before being used for experiments. The experimental fish were divided into two groups; the first group was the control group, included positive and negative fed basic diet (no additive of plant extract). The second group acted as a treatment group, which consisted of three subgroups which received three different levels of E. agallocha leaf methanol extract at concentration 5, 25, and 50 mg kg -1 , respectively. Each group for three replicates (10 fish per 20L tank). Fish were fed at 3% BW d -1 twice daily (9:00 and 18:00) for 28 days. On the 29th day, all fish in the treatment group were injected intraperitoneally (i.p.) with 100 μl bacteria S. agalactiae at dose 15×105 CFU mL -1 (This dose was calculated previously from the determination of Lethal dose LD50). Meanwhile, negative control groups were injected intraperitoneally (i.p.) with 100 μl bacteria S. agalactiae at dose 15×105 CFU mL -1 . The positive control group was injected intraperitoneally (i.p.) with 100 μl physiological saline. The same design applied but a different type of bacteria, using E. coli at dose 15×108 CFU mL -1 . The cumulative mortality was monitored for a group of fishes in all experiments for ten days post-infection.

Disease resistance
The effect of E. agallocha leaf extract incorporated feed for the disease resistance (survival percentage) on fish (n = 10/group) was determined. The fish were artificially challenged with a dose of 15×105 CFU mL -1 , 15×108 CFU mL -1 of live virulent pathogen S. agalactiae, and E. coli, respectively.
The mortality was observed for ten days, and the average of the triplicate set was used to express in terms of percentage of survival.

Statistical analysis
Effects of E. agallocha diets on survival were expressed as the arithmetic mean ± standard division (SD) by using Tukey statistical analysis using one-way analysis of variance (ANOVA) to reveal the significant difference between the treatments at the 5% (P < 0.05) level of significance by using SPSS version 20.0 for windows. Data were expressed as concentration ± mortality (%) of triplicate measurements. Probit analysis was used to determine 120 h (LD50) and (LC50) using SPSS 20.0.

Clinical signs
There were no clinical signs apparent on the experimental fish infected with E. coli and S. agalagtiae except for sudden death and petechial hemorrhages on the site of injection in some fish.

Antibiotic assay
Antibiotic susceptibility test was done on selected samples of gram-positive S. agalactiae and gram-negative bacteria E. coli. The inhibition zone (mm) was recorded for five types of antibiotics. Both isolates showed 100% sensitivity to amoxicillin, ampicillin, and tetracycline. However, gramnegative bacteria E. coli isolates showed resistance to neomycin (Table 1 & Fig. 1, 2).

Toxicity test
The toxicity of the brine shrimp test was analyzed by Excel to obtain the 50% lethality concentration (LC50), showing the value of LC50 to be 83 mg/ml (Fig. 5).

Disease Resistance
Total mortalities recorded during the experiment were attributed to the challenge infection with pathogenic bacteria E. coli and S. agalactiae. These mortalities were observed from the first day until the fifth day in fish injected with S. agalactiae and sixth day in fish injected with E. coli post-challenge. There were no mortalities up to 10 days post-challenge. Among the treatment groups that were challenged with S. agalactiae, the highest cumulative mortality was 90.0±10.0 % in the control group as compared to 26.7±11.5%, with the group fed with 50 mg kg-1 of E. agallocha, a significant difference (p<0.05) ( Table 5 & Fig  6). On the other hand, the cumulative mortality was 70.0±17.3%, and 40.0±0.0% in the group fed with 5 mg kg-1 and 25 mg kg-1 of E. agallocha leave crude extract, respectively, with significant difference (p<0.05) ( Table 6 & Fig. 7). In contrast, the control group challenged with E. coli showed the highest cumulative mortality was 100.0 ±0.0 % as compared to 37.0±5.7% in the group fed at 50 mg kg-1 of E. agallocha extract, a significant difference (p<0.05). Moreover, the cumulative mortality was 73.3±5.7% and 47.0±5.7% in the group fed with 5 mg kg-1 and 25 mg kg-1 of E. agallocha, respectively, with significant difference (p<0.05) ( Table 6 & 5). To best our knowledge, this study was the first, which discussed the gender differences in the domains of emotional exhaustion among a sample of medical doctors in Iraq and Arab region using the validated MBI questionnaire.

Discussion
Mangroves are biochemically unique, producing a wide range of novel natural products. In this study, the antimicrobial activity of Excoecaria agallocha was established on pathogenic bacteria Escherichia coli and Streptococcus agalactiae in fish. Antimicrobial agents can be defined as substances that can kill and/or inhibit the growth of microorganisms. The antimicrobial action of such compounds is related to the inactivation of cellular enzymes or by membrane permeability changes causing a loss of cellular integrity and eventual cell death [18]. Mangrove plants are a rich source of bioactive composites and are utilized in many parts of the world as a renewable resource and as a treatment option to medical illness in various cultural communities globally. Nowadays, mangrove plants play a significant role in the treatment of aquatic and fish disease because of the minimal side effects and therapeutic potential [11]. The present study focuses on the use of E. agallocha leaf extract to control bacterial infection in fish. E. agallocha extract exhibited significant in vitro antibacterial activity against bacteria. Similarly, Laith [11] observed in previous studies that E. agallocha extract exhibited antibacterial activity against fish pathogens bacteria, including Flavobacterium Indicum, Chryseobacterium indologenes, Chryseobacterium gleum, and Elizabethkingia meningoseptica. Moreover, our findings receive support from the works of Bakshi [19] who recorded the antimicrobial activity of leaf extracts of mangrove plants: Avicennia alba, Avicennia marina, Avicennia officinalis, Excoecaria agallocha, Sonneratia caseolaris, Sonneratia apetela, Aegiceres corniculatum, Acanthus ilicifolius, Nypa fruticans, and Ceriops decandra to have inhibitory effect against Escherichia coli with the most active extracts being from Excoecaria agallocha plant with a zone of inhibition of 10.3 mm.    In vitro assays indicated that the growth of the tested fish pathogens was suppressed by E. agallocha extract. Gramnegative bacteria E. coli showed a high inhibition zone of 18 mm as compared to 13 mm from Gram-positive bacteria S. agalactiae. These results were confirmed by in vivo assays which showed cumulative mortality to be 26.7 ± 11.5% in the group fed with 50mgkg-1 of E. agallocha leave crude extract injected with gram-positive S. agalactiae, while this percentage was 37.3 ± 5.7% of the same concentration dose of plant extract but injected with gram-negative E. coli. Jayanta [20] reported that both methanol and aqueous extract of E. agallocha were effective against E. coli. These findings are in agreement with Agoramoorthy [21], who determined that antimicrobial compounds extracted from plant sources are considered to be more active on Gram-negative than Gram-positive bacteria due to cell wall structure differences among the two. Similar results were also obtained by Laith [22] in that active constituents of plants interfere with the growth and metabolism of microorganisms in a negative manner. Moreover, Packialakshmi [23] evaluated the antibacterial activity of aqueous E. agallocha leaf extracts using the disc diffusion method against gram-negative and positive bacteria. The result revealed that the gram-negative bacteria: Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumonia had a higher inhibition zone of 17, 15, and 16 mm, respectively, as compared to gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with zones of 15, 14 mm, respectively. This antibacterial activity that is possessed by E. agallocha against infectious pathogens, as shown in this study, may be attributed to the presence of diterpenes and benzoxazole derivatives and other bioactive compounds found in this mangrove plant [24]. In contrast, our results were not in agreement with the findings of Rajia [25] who stated that methanol extract of E. agallocha did not show any antibacterial activity against the clinical isolates; gram-positive bacteria, Staphylococcus aureus and Bacillus subtilis, or gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and Pseudomonas Vulgaris at different concentrations of 1, 2 and 3 mg/ml due to bacterial resistance which could cause this ineffectiveness of the mangrove extract. Nonetheless, Laith [11] indicated that E. agallocha leaf extracts displayed antimicrobial activity due to secondary metabolites, and the potential mechanisms of these components on bacteria may be related to the alteration in bacteria cell wall structures and inhibition of bacterial proteins. MIC and MBC values are used to assess the antimicrobial efficacy of metabolites or synthetic compounds of E. agallocha [26]. Therefore, MIC and MBC values are useful as guidelines to choose the appropriate and effective concentrations for therapeutic substances [27]. In this study, it was determined that the lowest MIC and MBC values for the E. agallocha against E. coli were 3.12 and 6.25 mg/ml, and 6.25 and 25 mg/ml against S. agalactiae, respectively. Similar results observed by Jayanta [20] who determined the MIC values of E. agallocha against Streptococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Bacillus subtilis, and E. coli ranged from 5 to 7 mg/ml. Similar observations were recorded by Selvam [28] in that the MIC of mangrove plants tested against bacterial pathogens ranged between 20 mg/ml to 640 mg/ml. According to Manilal [29], a substance is considered bactericidal when the ratio of MBC/MIC ≤ 2, is bacteriostatic if the ratio of MBC/MIC ≥ 2 and is ineffective if the ratio of MBC/MIC ≥ 16. Therefore, it was concluded that E. agallocha leaf extract was bactericidal against E. coli and bacteriostatic against S. agalactiae. Additionally, the toxicity of plant extract plays an important role in medicine; therefore, brine shrimp larvae bioassay, which is a simple and useful tool for the isolation of potentially cytotoxic compounds from plant extracts [10], was used to determine the toxic level of extract substance. In this study, the LC50 value of E. agallocha was 83 mg/ml, similar to the results observed by Laith [10], who recorded the LC50 value of E. agallocha to be 94.19 mg/ml. The difference in values of LC50 may be attributed to the different locations of the mangrove plant. Antimicrobial resistance is a progressively global problem, and the emerging antimicrobial resistance has become a major public health issue [30]. Our result revealed E. coli was resistant to neomycin and susceptible to tetracycline, which is similar to observations recorded by Noryawati [31] in that isolated E. coli was the most sensitive to tetracycline. Also, our finding is supported by the works of Sabarinath [32], who stated that E. coli was susceptible to neomycin and even to Kibret [33], who said that E. coli was resistant to tetracycline. Bacteria resistance towards antibiotics might be due to their close genetic relatedness [34]. Moreover, sudden death was the most common sign of tilapia fish infected with E. coli and S. agalactiae. This can be attributed to septicemia caused by theses pathogenic bacteria [3,5]. Regarding the overall survival rate, the present study revealed that E. agallocha leaf extracts increased the survival rate of fish after a challenge trial with E. coli and S. agalactiae. The highest survival rate was in the group fed at 50 mg kg-1, and the lowest survival was observed in control. This finding is supported by the works of Pavaraj [35] in common Carp (Cyprinus carpio) administered with plant extract of Ocimum sanctum and challenged with Aeromonas hydrophila showed no mortality as compared to control group which showed 40% mortality. Decreased cumulative mortality of fish fed with E. agallocha diets and infected with E. coli and S. agalactiae are in agreement with the previous study by Laith [10,22] in that E. agallocha has the potential as a dietary additive to mitigate mortality of fish infected with pathogenic bacteria. Therefore, the efficacy of in vivo assay may establish the antimicrobial action properties and provide a tailor-made alternative source to combat infectious agents. Hence, our findings revealed a potent in vitro and in vivo activity of the E. agallocha leaves extracts on pathogenic fish bacteria E. coli and S. agalactiae.

Conclusion
The results of the present study conclude crude extract of mangrove plants is a suitable agent in providing a nonmedicinal antimicrobial activity to combat bacterial infections in fish. Further work on the purification of bioactive compounds is currently underway to discover additional effective solutions for treating fish diseases. Therefore, we recommend the use of E. agallocha as an alternative therapeutic agent against both gram-negative and gram-positive bacterial infections in the aquaculture industry.