56 The Antibacterial Activity of Thermoactinomyces sp. (H24) Extract Against Escherichia coli and Staphylococcus aureus Julia Nanda Puspita1, Rikhsan Kurniatuhadi1, Rahmawati 1 1Department of Biology, Faculty of Mathematics and Natural Sciences, Tanjungpura University, West Kalimantan, lndonesia Correspondence: Rahmawati, Jl. Prof. Dr. H. Hadari Nawawi, Pontianak, West Kalimantan, Indonesia Zip Code: 78124 Email: rahmawati@fmipa.untan.ac.id Received: August 15th, 2020 Revised: February 13th, 2021 Accepted: March 31th, 2021 Published: April 28th, 2021 DOI: 10.33086/ijmlst.v3i1.1700 Abstract Bacteria of the genus Thermoactinomyces have the ability to produce antibacterial bioactive compounds. This bioactive compound can be used for combating diarrheal agents such as Escherichia coli and Staphylococcus aureus. This study aims to determine the antibacterial activity of the metabolite extract from Thermoactinomyces sp. (H24) against E. coli and S. aureus. Methanol was used as a solvent for the extraction of bacterial bioactive compounds. Antibacterial activity was analyzed by the diffusion method with several extract concentrations (0.75 mL, 1.5 mL, 2.25 mL, and 3 mL), 10% DMSO (Dimethyl sulfoxide) as the negative control, and ciprofloxacin as the positive control. Our result shows that Termoactinomyces sp. (H24) extract has an inhibitory effect on the growth of E. coli and S. aureus with an effective concentration of 2.25 mL (inhibition strength: very strong). Keywords Antibacterial, Escherichia coli, Staphylococcus aureus, Thermoactinomyces sp. (H24). This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author. INTRODUCTION Eschericia coli and Staphylococcus aureus are two common causative agents of serious infections in humans (1,2). E. coli can infect the respiratory and digestive tracts and cause several diseases like pneumonia, endocarditis, wound infection, meningitis (3), and diarrhea by producing enterotoxins (4). S. aureus can cause several diseases such as pneumonia, osteomyelitis, arthritis, and inflammation of the brain. S. aureus cause hemolysis of blood, and have the ability to rahmawati@fmipa.untan.ac.id https://journal2.unusa.ac.id/index.php/IJMLST/article/view/1700/version/2183 Ina. J. Med. Lab. Sci. Tech. 2021; 3(1): 56–63 Julia Nanda Puspita, et al. 5 7 produce enterotoxins that led to diarrhea, seizures, and fever (5). The emergence of various kinds of diseases caused by pathogenic bacteria encourages the researcher to find alternative antibacterial compounds. Thermoactinomyces sp. (H24) is one of the bacteria with the ability to produce the antibacterial compound (6) . Jawetz et al. (7) find that Thermoactinomyces sp. (H24) have antifungal activity. Thermoactinomyces sp., in addition to antifungal compound, also produce antibacterial aromatic compound namely Cyclic Hexapeptides thermoactinoamide A-F with ability to inhibit lipophilic bacteria (8). Currently, there is no reported research on the antibacterial analysis of Thermoactinomyces sp. (H24) for inhibiting the growth of several pathogenic bacteria such as E.coli and S. aureus. Therefore, this research was conducted to analyze the antibacterial activity of Thermoactinomyces sp. (H24) for inhibiting the growth of S. aureus and E. coli. MATERIALS AND METHODS Preparation and Sterilization The media used were Nutrient Agar (NA), Mueller Hinton Agar (MHA), and Nutrient Broth (NB) dissolved in 1,000 mL of distilled water. All media, Petri dishes, and test tubes were sterilized using an autoclave at 121°C, 2 atm for 15 minutes (9). Extraction of Termoactinomyces sp. (H24) Metabolites Thermoactinomyces sp. (H24) was cultivated by the streak plate method on Nutrient Agar, then incubated at 50°C for 24 – 72 hours. A single colony of Thermoactinomyces sp. (H24) was inoculated on 50 mL of Nutrient Broth, then incubated in an incubator shaker at 110 rpm, 50°C for 84 hours until the culture reached a stationary phase. Thermoactinomyces sp. (H24) metabolite was extracted by centrifuging the culture at 3,000 rpm for 15 minutes to obtain supernatant. The supernatant obtained was a source of secondary metabolites (9). The supernatant was filtered using Whatman paper no. 1. The filtrate was immersed in 100% methanol for 24 hours in different bottles with a ratio of v/v (1:1). The solvent was removed using a rotary evaporator and then allow to stand for 24 hours (9). Qualitative Test of Thermoactinomyces sp. (H24) Extract A qualitative test was done to analyze the presence of phenols, alkaloids, flavonoids, saponins, tannins, steroids, and terpenoids in Thermoactinomyces sp. (H24) extract. Preparation of Escherichia coli and Staphylococcus aureus culture The colony of E. coli and S. aureus was transferred into 9 mL of sterile distilled water until the Optical Density (OD) value similar to 0.5 Mc Farland Standard. McFarland Ina. J. Med. Lab. Sci. Tech. 2021; 3(1): 56–63 Julia Nanda Puspita, et al. 5 8 Standard was prepared by mixing H2SO4 with 0.05 ml of BaCl 1% in a test tube (10). Antibacterial Activity Test of Thermoactinomyces sp. (H24) Extract Various concentration of Thermoactinomyces sp. (H24) extract (0.75 mL, 1.5 mL, 2.25 mL, and 3 mL) was transferred into 3 ml sterile distilled water. Antibacterial activity test was performed by well diffusion method. E. coli and S. aureus culture were swabbed evenly on the all surface of the MHA medium using a sterile cotton swab. The well was made on a medium with a diameter of 6 mm, then filled with 25 µL of each extract with several concentrations, DMSO 10% as a negative control, and ciprofloxacin 0.25 µg/mL as a positive control. The medium was incubated at 37° C for 24 – 48 hours (11). A clear zone formed after incubation was measured using a caliper. The clear zone categorized into very strong (diameters >20 mm); strong (diameter 11 – 20 mm); moderate (diameter 6 – 10 mm); and weak (<5 mm) (12). RESULTS Qualitative Test of Thermoactinomyces sp. (H24) Extract The qualitative test was revealed the presence or absence of secondary metabolites from Thermoactinomyces sp. (H24) extract (Table 1). Table 1. Qualitative test results of Thermoactinomyces sp. (H24) extract No. Secondary Metabolites Reactor Change Result 1. Alkaloids Wagner Brown deposits are formed + 2. Flavonoids Magnesium (Mg)andamyl- alkohol No yellow discoloration occurs - 3. Saponins HCl No foam - 4. Tannins FeCl3 No dark blue discoloration - 5. Phenol Etanoland FeCl3 No change into green color - 6. SteroidsandTerpenoids CH3COOH glacial, H2SO4 No change into red or green - Note: + : contains secondary metabolites; – : not contains secondary metabolites Ina. J. Med. Lab. Sci. Tech. 2021; 3(1): 56–63 Julia Nanda Puspita, et al. 5 9 A qualitative test of the Thermoactinomyces sp. (H24) extract showed the presence of the alkaloid compounds in the extract, indicated by the formation of brown deposits when Wagner's reagent was added to the extract. Antibacterial Activity of Thermoactinomyces sp. (H24) The formation of the clear zone indicated the ability of Thermoactinomyces sp. (H24) extract to inhibit the growth of E. coli and S. aureus. The diameter of the clear zone showed that the extract had various bacterial growth inhibition level against E. coli from moderate to very strong depending on extract concentration (Table 2). Statistics analysis showed that the different concentrations give significantly different results. Table 2. Average diameter of clear zone against Escherichia coli Concentration of extract (mL) Diameter (mm) Category 24 hrs 48 hrs DMSO 10% 0.00 0.00 - 0.75 9.44 9.62 Moderate 1.5 16.18 16.71 Strong 2.25 21.78 28.49 Very strong 3 26.85 32.94 Very strong Ciprofloaxcin 37.57 41.20 Very strong Figure 1 showed the clear zone formed after incubation 24 hours and 48 hours, indicated the ability of Thermoactinomyces sp. (H24) extract against E. coli. Figure 2 showed the clear zone formed after incubation 24 hours and 48 hours, indicated the ability of Thermoactinomyces sp. (H24) extract against S. aureus. Figure 1. The clear zone of Thermoactinomyces sp. (H24) extract against Escherichia coli: (a) 24 hours, (b) 48 hours of incubation a b Ina. J. Med. Lab. Sci. Tech. 2021; 3(1): 56–63 Julia Nanda Puspita, et al. 6 0 Table 3. Average diameter of clear zone against Staphylococcus aureus Concentrationof Extract (mL) Diameter (mm) Category 24 hrs 48 hrs DMSO 10% 0.00 0.00 - 0.75 7.41 7.43 Moderate 1.5 13.52 13.71 Strong 2.25 22.31 22.68 Very strong 3 26.12 36.48 Very strong Ciprofloaxcin 36.16 36.48 Very strong Figure 2. The clear zone of Thermoactinomyces sp. (H24) extract against Staphylococcus aureus: (a) 24 hours, (b) 48 hours of incubation DISCUSSION Thermoactinomyces sp. (H24) extract has the ability to inhibit the growth of E. coli and S. aureus, indicated by the formation of a clear zone around the well. The effective concentration of Thermoactinomyces sp. (H24) extract against E. coli and S. aureus was 0.75 mL, 1.5 mL, 2.25 mL, and 3 mL with inhibition strength from moderate to very strong. Thermoactinomyces sp. (H24) extract concentration of 0.75 mL could inhibit E. coli and S. aureus at a moderate level (Table 2 and Table 3). Teta et al. (8) was reported that Thermoactinomyces vulgaris isolated from a hydrothermal vent on the coast of Iceland possessed moderate level antibacterial activity with a concentration of at least 0.35 mL against S. aureus and 0.14 ml against Escherichia coli. Bratchkova et al. (13) showed that Thermoactinomyces sp. isolated from penguin droppings on Livingston Island, Antarctica had a strong level of antibacterial activity at the volume of 0.10 mL against S. aureus and E. coli. This high antibacterial activity at a low concentration from Thermoactinomyces sp. extract reported by Teta et al. (8) and Bratchkova et al. (13) because of the different extraction solvents a b Ina. J. Med. Lab. Sci. Tech. 2021; 3(1): 56–63 Julia Nanda Puspita, et al. 6 1 and the use of chromatography result in the more pure extract. The concentration of extract positively correlated with the diameter of the clear zone. The clear zone formed is produced from the antibacterial active substance in the extract. The most effective concentration of extract for inhibiting the growth of E. coli and S. aureus is 2.25 mL. E. coli cell walls have three layers (multilayer) with high-fat content (11 – 22%) and peptidoglycan layer inside stiff layer (10% dry weight) that cause E. coli less absorb antibacterial compound (14). The effective concentration of antibacterial extract produces a biological response. The effective concentration is defined as the lowest concentration that provides a significant response (15). The diameter of the inhibition zone at the 48-hours incubation time was greater than 24-hours incubation, indicated that the antibacterial activity is bactericidal or has the ability to kill bacteria (16). The average diameter of the inhibition zone resulted from the Thermoactinomyces sp. (H24) extract against E. coli was greater than S. aureus. This difference indicates that Gram-negative and Gram-positive bacteria have different responses to antibacterial compounds. E. coli, a Gram-negative bacteria, is more sensitive to non-polar (hydrophobic) antibacterial compounds such as alkaloid compounds than Gram-positive bacteria such as S. aureus (20). The difference in response between Gram- positive and Gram-negative bacteria to alkaloid compounds is due to the cell walls of Gram-negative bacteria contain 11–22% lipids which will more easily react with nonpolar alkaloid compounds, resulting in cell lysis. In contrast, Gram-positive bacteria, such as S. aureus, contains 4% lipids which make it difficult to react with alkaloid compounds. Bratchkova et al. (13) reported that E. coli (Gram-negative) was more sensitive than S. aureus (Gram-positive) bacteria in response to Thermoactinomyces sp. Impact-14 extract. The antibacterial activity of Thermoactinomyces sp. (H24) extract against E. coli and S. aureus is due to the content of metabolite compounds produced by Thermoactinomyces sp. (H24). The qualitative test showed the presence of alkaloid compounds in Thermoactinomyces sp. (H24) extract (Table 1). Several studies also show that many Thermoactinomyces strains extract contained alkaloid compounds as antibacterial agents. Bratchkova et al. (13) explained that Thermoactinomyces sp. impact-14 isolated from penguin droppings on Livingston Island, Antarctica contains β- carboline alkaloid compounds which have antibacterial activity against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Bacillus mycoides, Streptomyces viridoch, Proteus vulgaris, Candida Ina. J. Med. Lab. Sci. Tech. 2021; 3(1): 56–63 Julia Nanda Puspita, et al. 6 2 tropicalis, Candida albicans, Mucor miehei, and Penicillium notatum. Alkaloid compounds found in the Thermoactinomyces sp. (H24) extract play a role in inhibiting the growth of E. coli and S. aureus. The nitrogen group in the alkaloid will react with the amino acids that cause the change in the structure of the amino acid in the bacterial cell wall. Changes in the structure of amino acids trigger lysis in bacterial cells led to bacterial death (17). CONCLUSIONS Thermoactinomyces sp. (H24) extract contained an alkaloid compound and has antibacterial activity against E. coli and S. aureus. Strong inhibition activity against E. coli and S. aureus was achieved by the concentration of 50% Thermoactinomyces sp. (H24) extract. AUTHOR CONTRIBUTIONS Julia Nanda Puspita: conceptualization, methodology, formal analysis, investigation, data curation. Rahmawati: validation, writing-review & editing, project administration, funding acquisition. Rikhsan Kurniatuhadi: validation, writing-original draft, project administration. AUTHOR CONTRIBUTIONS Julia Nanda Puspita: Conceptualization, methodology, formal analysis, investigation, data curation. Rahmawati: validation, writing-review & editing, project administration, funding acquisition. Rikhsan Kurniatuhadi: validation, writing-original draft, project administration. ACKNOWLADGEMENTS The autors are grateful to Jerliman et al. which has permitted to using Thermoactinomyces sp. (H24) and Microbiology Labotarory, Faculty of Mathematics and Natural Science, Tanjungpura University for the facilities and assistance in completing the research work. CONFLICT OF INTEREST There are no conflicts of interest. REFERENCES 1. Arenz S, Wilson DN. Blast from the past: reassessing forgotten translation inhibitors, antibiotic selectivity, and resistance mechanisms to aid drug development. Mol Cell. 2016;61(1):1- 12. 2. Amalia S, Wahdaningsih S, Untari EK. Activity testdragon fruit skin n-hexane fraction antibacterial red (Hylocereus polyrhizus Britton & Rose) against Staphylococcusaureus ATCC 25923. Pontianak: Faculty Tanjungpura University Medicine. J Phy Ina. 2016; 61-64. 3. Entjang I. Microbiology and parasitology for the nursing academy and health power school of the equals. PT. Citra Aditya Bakti; 2018: 56-58 4. Brooks GF, Karen CC, Butel JS, Stephen AM. Jawetz, Melnick & Adelberg’s, Book 1 Medical Microbiology. Jakarta: EGC; 2015. 5. Plata FX, Ebergeny X, Resendiz JL, Villarreal O, Barcena R, Viccon JA, Mendoza GD. Palatability and chemical composition of feeds ingested in captivity by Yucatan white-tailed deer (Odocoileus virginianus yucatanensis). Arc Med Vet. 2019; 123-129. Ina. J. Med. Lab. Sci. Tech. 2021; 3(1): 56–63 Julia Nanda Puspita, et al. 6 3 6. Manalu J, Rahmawati, Hidayat N. Antifungal activity of Actinomycetes isolates from hot springs Ai Sipatn Lotup Sanggau against isolate Hortaea werneckii (T1). Protobiont. 2019; 69-77. 7. Jawetz E, Melnick JL, Adelberg EA. Medical microbiology. Translated by Mudihardi E, Kuntaman, Wasito EB, Mertaniasih NM, Harsono S, Alimsardjono L, Edition XXII, 327-335, 362- 363, Publisher Salemba Medika, Jakarta; 2015. 8. Teta R, Marteinsson VT, Longeon A, Klonowski AM, Groben R, Bourguet KM, CostantinoV, MangoniA. Thermoactinoamide A, an antibiotic lipophilic cyclopeptide from the icelandic thermophilic bacterium Thermoactinomyces vulgaris. J Nat Prod. 2017; 2530-2535. 9. Mulyadi, Sulistyani N. Activity of 12 Actinomycetes isolates against bacteria resistance: Public Health. 2013;7(2): 55-112. 10. Zheng L, Chen H, Han X, Yan X. Antimicrobial screening and active compound isolation from marine bacterium NJ6-3-1 associated with the sponge Hymeniacidon parleve. World J Microb Biotech. 2015; 201-206. 11. Moningka KS, Kojong NS, Sudewi S. Antibacterial activity test of leaf extract of cats (Acalypha hispida Burm. F.) against Staphylococcus aureus and Escherichia coli in vitro. J Pharm Sci. 2015; 2302-2493. 12. Warbung YY, Wowor VNS, Posangi J. Inhibition of sea sponge extract Callyspongia sp. against the growth of Staphylococcus aureus bacteria. EGiGi Journal (eG). 2013; 1 (2): 2. 13. Bratchkova A, Ivanova V, Gousterova A, Laatsch L. β-Carboline alkaloid constituents from a Thermoactinomyces sp. strain isolated from Livingston Island. Biotechnology & Biotechnological Equipment: Antarctica. 2012: 3005-3009. 14. Todar, K. Pathogenic E. coli, Todar’s Online Textbook and Bacteriology, http://textbookofbacteriology.net/e.coli.html., 2018. 15. Liu J. Minimum Effective Dose. Encyclopedia of biopharmaceutical statistics. Taylor & Francis. p. 1493. doi:10.1081/E-EBS3-130001128. 2019. ISBN 978-1-4398-2246-3. 16. Limaye L, Patil R, Ranadive P, Kamath G. Application of potent actinomycete strains for bio-degradation of domestic agro-waste by composting and treatment of pulp-paper mill effluent. Adv Micro. 2017; 94-108. 17. Gunawan I. Preliminary extraction of bioactive compounds as antibacterials as well as toxicity test and Minimum Inhibitor Concentration (MIC) test of soft rocks of origin perairan pulau pulau, kepulauan seribu. Description: Faculty of Fisheries and Marine Science, Bogor. 2019. 18. Bagg J, Mcfarlane, Wallace T, Poxton, Ian R, Smith, Andrew J. Essentials of microbiology for dental students, 2nd Edition. Oxford University, 2017. 19. Chaudhary HS, Yadav J, Shrivastava AR, Singh S, Singh KA, Gopalan N. Antibacterial activity of Actinomycetes isolated from different soil samples of Sheopur. J Adv Pharm Technol. 2017; 118–123. 20. Levinson W. Review of medical microbiology, The McGraw-Hill Companies, 25-26, 78-79, 2018. 21. Sharma SK, Gupta VK. In vitro antioxidant studies of Ficus racemosa linn root, Pharmacognosy Magazine, India. 2008.