Antibacterial activity in secondary metabolite extracts of heterotrophic bacteria against Vibrio alginolyticus, Aeromonas hydrophila, and Pseudomonas aeruginosa

Background: Disease causing bacteria such as Vibrio alginolyticus, Aeromonas hydrophila, and Pseudomonas aeruginosa present a problem for fish farming. Treatment to remove them are generally carried out using antibiotics which have side effects on fish, the environment and humans. However, the use of antibacterial compounds derived from heterotrophic bacteria serve as a good alternative for antibiotics. Therefore, this study aimed to explore antibacterial activity in the secondary metabolite extracts of heterotrophic bacteria against Vibrio alginolyticus, Aeromonas hydrophila, and Pseudomonas aeruginosa. Methods: Heterotrophic bacteria namely Bacillus sp. JS04 MT102913.1, Bacillus toyonensis JS08 MT102920.1, Bacillus cereus JS10 MT102922.1, Bacillus sp. JS11 MT102923.1, Pseudoalteromonas sp. JS19 MT102924.1, Bacillus cereus JS22 MT102926.1, and Bacillus sp. strain JS25 MT102927.1 were used in this study. The sequences of these bacteria have been deposited and are available from NCBI GenBank. Each heterotrophic bacterium was cultured on 6L nutrient broth for 8 days, and extracts produced using ethyl acetate to obtain their secondary metabolites. These extracts were tested for their phytochemical contents using FT-IR and also tested for their inhibitory property in pathogenic bacteria by agar diffusion method. Results: Phytochemical test results showed that the seven heterotrophic bacterial isolates produced terpenoid compounds. Based on the inhibitory test, the secondary metabolite extracts from Bacillus sp strain JS04 had the highest inhibitory effect on the growth of pathogenic bacteria namely, V. alginolyticus (17.5 mm), A. hydrophila (16.8 mm), and P. aeruginosa (17.3 mm). Conclusion: It was concluded that the secondary metabolite extracts of heterotrophic bacteria inhibit the growth of V. alginolyticus, A. hydrophila, and P. aeruginosa.

Bacteria disease treatment is generally carried out using antibiotics, however, these can have adverse effects on the fish and their environment 32-37 . The accumulation of antibiotics in the fish increase the risk of bacterial resistance 38,39 . Escherichia coli bacteria isolated from the digestive organs of catfish showed high resistance levels towards tetracycline, ampicillin, and chloramphenicol 40 . Therefore, it is necessary to explore natural compounds with antibacterial activity 41 . Sea water is a potential source of heterotrophic bacteria that produce antimicrobial compound 42 , and have probiotic activity 43 Sea bacteria such as, Bacillus sp. B. cereus, B. toyonensis, and Pseudoalteromonas sp., are known to inhibit the growth of pathogenic bacteria namely, V. alginolyticus, A. hydrophila, and Pseudomonas sp 43 . They also produce antimicrobial compounds such as, Pseudoalteromonas 44 . Pseudoalteromonas piscicida produces antimicrobial substances that inhibit the growth of different pathogenic bacteria namely, Vibrio vulniosis 45 , Bacillus sp 46 , B. pumilus 47 , and B. subtilis 48,49 . Bacillus amyloliquefaciens shows antibacterial activity towards pathogenic bacteria such as, Aeromonas hydrophila, Vibrio harveyi, V. vulnificus, and V. parahaemolyticus 50 . Meanwhile, Bacillus subtilis shows antibacterial activity towards the pathogens Vibrio parahaemolyticus, V. vulnificus, and Aeromonas hydrophila 51 .
Heterotrophic bacteria extracted from Riau sea waters were examined and found to able to inhibit the activity of pathogenic bacteria Aeromon as salmonicida, Edwarsiela tarda and Edwarsiela ictalurias previously reported by Setiaji et al. 52 . However, the antibacterial activity of these heterotrophic bacteria extracted from Riau sea waters on the pathogenic bacterial namely, Vibrio alginolyticus, Aeromonas hydrophila, and Pseudomonas aeruginos have never been examined for its potential against pathogenic bacteria. Therefore, this study aims to explore antibacterial activity in secondary metabolite extracts of heterotrophic bacteria isolated from Riau sea water, against pathogenic bacteria namely, V. alginolyticus, A. hydrophila, and P. aeruginosa.

Bacterial culture
The heterotrophic bacteria isolates were collected from sea waters in Sungai Pakning Bengkalis Regency Riau Province Indonesia (North latitude 01 o 21'36,8" and East longitude 102 o 09'34,1"). 1 liter of the sea water was collected at 50 cm depth by using a water sampler (Tiolan Lab, type: WSV-BIT22), then was transferred into a sample bottle and was put into a coolbox filled with ice at 15°C, before being transported by car for 1 hour to the laboratory. The heterotrophic bacteria was cultured using nutrient Agar (NA; Merck-1.05450.0500). The heterotrophic bacteria cultured were used for an antagonist test against pathogen bacteria. The antagonist test procedure is as follows, 1 ml of pathogenic inoculants (OD 600nm = 0.08-0.1) (OD measured with Thermo scientific, Genesys 10S UV-Vis) was added to 15 ml liquid nutrient Agar media at 50°C, then homogenized, and poured into a petri dish to solidify. Furthermore, Oxytetracycline antibiotic disc paper (Oxoid, CT0041B, OT30 mcg) was used as the positive control, while 30 µl aquades (Kimiapedia id-1720602804) was dripped to a disc paper (Macherey-nagel, MN827 ATD) as the negative control. 30 µl heterotrophic bacterial isolate taken from bacteria culture in nutrient Broth (NB; Merck-1.05443.0500) was dripped to a disc paper and incubated at 30°C for 24 hours. The inhibitory power of heterotrophic bacterial isolate was measured from the diameter of clear zone formed around the disc. From the antagonist test, eight isolates with the best inhibition were collected, and the heterotrophic bacteria was identified using 16S rDNA technique 43 . The sequenced products were run through BLAST (NCBI Basic Local Alignment Search Tool) and registered to GenBank.
The pathogenic bacteria were obtained from the collection at the Marine Microbiology Laboratory of the Faculty of Fisheries and Marine Science, University of Riau, Indonesia. The heterotrophic and pathogenic bacteria were cultured on the nutrient Agar (NA; Merck-1.05450.0500). The cultured medium was sterilized in an autoclave at a pressure of 15 psi and 121°C for 15 minutes. After 1 hour at room temperature, the medium was inoculated by the heterotrophic bacteria and the pathogenic bacteria. Then the bacteria were incubated in an incubator (Memmert, Model 30-1060) at 30° C for 24 hours.

Isolates test
Previous studies showed that eight heterotrophic bacterial isolates possessed the potential to produce pathogens. Seven of these species were used in this study namely, Bacillus sp. Then, each bacterium was cultured in a 6 L nutrient Broth (NB; Merck-1.05443.0500) diluted with sea water of salinity 29 ppt and aerated for 8 days. After this, the bacteria were mixed with ethyl acetate (P.a) at ratio 1:1 and shaken vigorously to homogenize. Subsequent filtering was performed until a clear filtrate was obtained using funnel and filter paper (Whatman 41, no. 1441-125), and evaporated with a rotary evaporator (Cole Parmer, N-1300) at 50°C and a speed of 50 rpm. This allowed thick secondary metabolite extracts to be obtained 53 .
Phytochemical test and Fourier-transform infrared spectroscopy (FT-IR) Phytochemical test was conducted on the secondary metabolite extracts of heterotrophic bacteria, which included tests for alkaloid, terpenoid, flavonoid, phenolic, and saponin compounds 54 .
Alkaloids were tested for using using 10 mg heterotrophic bacteria extract and 250 µL Mayer reagent.
The terpenoid was tested using 10 mg heterotrophic bacteria extract, 10 drops of CH 3 COOH, and 3 drops of H 2 SO 4 .
Flavonoid tests were performed using 10 mg heterotrophic bacteria extract added to 5 mL distilled water. This was then boiled before adding 0.05 g Mg (Merck 1.05815.1000) and 10 drops of 37% HCl (Merck 1.00317. 2500), the mixture was then shaken for one minute.
Saponin compounds were tested for using 10 mg heterotrophic bacteria extract added to 5 mL distilled water which was then shaken for 1 minute. 150 µL 1N HCl (Merck 1.00317. 2500) was then added, and shaken for another minute.
A positive alkaloid test was indicated by the formation of a white precipitate after adding Mayer regent. A positive terpenoid test was indicated by the formation of a red colour. A positive flavonoid test was indicated by a red colour change. Phenolic compounds were indicated by a blue colour change. Saponin compounds were indicated by a foam forming.
Meanwhile, to determine the functional groups in secondary metabolite extracts, FT-IR (Shimadzu, IR prestige-21, IR solution software ver. 1.1) spectroscopy analysis was performed. This was conducted by crushing 1 mg of each extract, added to KBr (Merck-1.04950.0500), and mixed vigorously until homogenized. This mixture was then measured for infrared absorbance at 4500-450 cm wavelength.

Inhibitory activity of heterotrophic bacterial extract
The secondary metabolite extracts of heterotrophic bacteria obtained were tested on pathogenic bacteria namely, V. algynolyticus, A. hydrophila, and P. aeruginosa using agar diffusion method, and 6 mm disc paper (Macherey-nagel, MN827 ATD) 55 . The procedure is as follows, 1 ml of pathogenic inoculants (OD 600nm = 0.08-0.1) (OD measured with Thermo scientific, Genesys 10S UV-Vis) added to 15 ml liquid nutrient agar media at 50°C, then homogenized, and poured into a petri dish to solidify. Furthermore, Oxytetracycline antibiotic disc paper (Oxoid, CT0041B, OT30 mcg) was used as the positive control, while methanol disc paper was the negative control. The metabolite extracts were then dissolved in 1 mg / mL methanol (P.a) and incubated at 30°C for 24 hours. The inhibitory power of heterotrophic bacterial extracts was measured from the diameter of clear zone formed around the disc.

Data analysis
The data were subjected to one-way analysis of variance followed by the Post Hoc Tukey multiple range test using R 4.0 software (GNU General Public License), p<0.05 is considered to indicate a statistically significant difference.

Phytochemical test and functional groups
Phytochemical test results of the metabolite extracts when added to Lieberman-Burchard reagents produced a red colour indicating the presence of terpenoids in the seven isolates. Meanwhile, the test for alkaloid, flavonoid, phenolic, and saponin compounds gave negative results.

Inhibitory activity
The results showed that the seven heterotrophic bacterial isolates inhibited the growth of pathogenic bacteria. The extracts inhibitory activity against pathogenic bacteria are shown in Table 2 56 . The average inhibition zone diameter of the extracts against pathogenic bacteria namely, V. alginolyticus, A. hydrophila, and P. aeruginosa ranges from 9.3 to 17.5 mm, 9.3 to 16.8 mm, and 8.5 to 17.3 mm, respectively. This inhibitory zone activity was indicated by the presence of clear zones formed around the disc paper. The largest inhibition zone diameter of the extracts against the growth of pathogenic bacteria was derived from isolates of Bacillus sp. strain JS04 (17.5 mm) on

Discussion
Phytochemical test results showed that the seven heterotrophic bacterial isolates produced terpenoids, which consist 5 carbon atoms or isoprene (C5) units. Microbes carry out biosynthesis by producing isopentyl pyrophosphate and dimethyl allyl pyrophosphate for terpenoid formation 57 . A significant relationship between terpenoids gene expression and isoprene production in Bacillus subtilis has previously been reported 58 .
Infrared spectrum analysis provided information about the detected compounds in the mixture 59 . Metabolite extracts showed the presence of hydroxyl, aldehyde, carboxylic acid, alkene, alkane, carbonyl, and amine functional groups in these extracts. This indicated that the seven bacterial isolates produced terpenoids, while the functional groups contained in the terpenoids were namely, O-H hydroxyl, C-H aliphatic, carbonyl, C-H cyclic, and carboxylic acid 60 .
The result of inhibitory activity in the secondary metabolite extracts of Bacillus sp. strain JS04 showed the largest inhibition zone against the growth of pathogenic bacteria. The formation of clear zones on culture media indicated that heterotrophic bacteria produced terpenoid compounds for antibacterial purposes.
The terpenoid compounds contained several phytochemicals that possess antimicrobial activity 61 . For example, Terpenes and terpenoids have been reported to exert antimicrobial activity against a wide variety of bacteria, both Gram-positive and Gram-negative 62 . Terpenes cause membrane disruption through acting on lipophilic compound in the membrane 63 . Therefore, terpenoid compounds were able to prevent the forming of biofilm cell in the bacterium Streptococcus mutans 60

Conclusion
The secondary metabolite extracts produced by the seven isolates of haterotrophic bacteria can inhibit the growth of pathogenic bacteria, namely V. alginolyticus, A. hydrophila, and P. aeruginosa. The secondary metabolite extracts of Bacillus sp. strain JS04 has the highest inhibitory activity against the growth of these three pathogenic bacteria.