CONTACT : ANDI MUHAMMAD MUNAWIR U nawier_uh@yahoo.co.id 26 Abstract Milkfish is a seafood commodity that is very popular with the community. However, milkfish can also be a source of spreading bacteria if it is not managed hygienically and safely. The objective of this experiment was to activite Salmonella typhii in milkfish using the invitro test. This research was called out in Microbiology Laboratory at the Faculty of Medical, Hasanuddin University, Makassar, Indonesia. The result of this experiment indicated that Salmonella typhii gave a positive result for the Biochemical test with TSIA media, SIM media, MRVP media, citrate, urea, Glucose, Sucrose, Lactose, Mannitol at three samples of milkfish out of ten samples taken randomly at the fish market and fish auction in Makassar, the contamination level of Salmonella typhii bacteria in all observed samples was moderate with a percentage level of 30%. Next test antibacterial and antibiotic tests were carried out with MHA medium and antibiotics, and the antibiotic Ciprofloxacin has a high degree of sensitivity test. ISSN : 2580-2410 eISSN : 2580-2119 The Activity of Salmonella Typhii in Milkfish (Chanos chanos) with Invitro. Andi Muhammad Munawir U1, Anastasia A Basir2 1Department of Health Analyst, Stikes Yapika Makassar, Indonesia 2Department of Nursing, Stikes Yapika Makassar, Indonesia. Introduction Salmonellosis is a disease caused by Salmonella bacterial infection in humans and animals that attacks the gastrointestinal tract, which includes the stomach, small intestine and large intestine or colon (Zelpina and Noor, 2020). Bacteria that are often found in food and drink contaminated one of which is Salmonella sp. Usually, infection of Salmonella sp causes salmonellosis (Lestari et al, 2017). Salmonellosis is zoonotic, meaning that this disease can be transmitted from animals to humans (Dharmojono, 2001). Salmonella that plays the most role is S.typhi because it is still a major health problem causing typhoid fever in developing countries. Bacteria with a level of danger that can cause death (Ariyanti and Supar, 2017). Typhoid fever is generally found in developing and low - income countries, as well as countries with tropical climates, WHO estimates that there are around 16 million cases annually worldwide and around 600,000 people die (WHO, 2018). Data information from GBD for Indonesia itself according to gender in 2019, the value OPEN ACCESS International Journal of Applied Biology Keyword Salmonella typhii, Invitro, TSIA, MRVP, Ciprofloxacin Article History Received February 22, 2023 Accepted June 14, 2023 International Journal of Applied Biology is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. International Journal of Applied Biology, 7(1), 2023 27 obtained for men is 187.06 DALYs per 100,000 while for women it is 122.99 DALYs per 100,000 (Global Burden of Disease, 2020). Specifically for South Sulawesi Province, a value of 147.88 DALYs per 100,000 was obtained in 2019 (Ainil, M. 2021). Transmission of bacteria in f oods, there are 39% that are positively contaminated by Salmonella typhi. This can be influenced by several factors, namely food processing, selling environmental hygiene, contaminated water, and flies as disease vectors (Putri et al, 2019) Foodborne disease is a disease caused by microorganisms and is a poison found in food that then enters through the mouth and is digested in the stomach inside the body (Muna et al, 2020). Fish is foodstuff with a high protein source, containing essential amino acids needed by the body. Fish contains high nutrients but is easily decomposed , caused by the presence of microbes found in fish (Melawati, et al, 2019). Milkfish can also be a source of spreading bacteria if not managed hygienically and safely. The multiplication of bacteria in the body of fish is not only caused by bacteria naturally found in fish (in the stomach contents, gills, and skin) but also by bacteria originating from other sources that contaminate the fish, including pathogenic bacteria, such as Salmonella typhii (Samiadi, 2021). Salmonella species encompasses a group of common rod shape, gram-negative rods that are motile, 1.0 to 3.0 µm long, and 0.8 to 1.0 µm wide (Darmawan et al, 2020). Salmonella will produce pink bars on Gram stain on microscopic examination. Salmonella can ferment glucose, produce gas, but do not ferment lactose and sucrose. Generally , Salmonella sp. produces H2S, is resistant to certain chemicals, and grows at 15-41°C with a pH of 6-8. The formation of gas comes from the reaction of acids which are retained based on media (Ulya, 2020). On media containing high protein, Salmonella sp. Grow quickly and can even grow as many as 90,000 cells in just 6 hours (Yunus, et al. 2017). In a culture so that the colonies are large, 2-8 millimeters in diameter, slightly convex round, clear, smooth, on BAP media do not cause hemolysis, on Mac Concey media Salmonella sp. Does not ferment lactose (NLF), the consistency is smooth Salmonella sp. survive in water that is frozen for a long time, these bacteria are resistant to certain chemicals (eg brilliant green, sodium tetrathionate, sodium deoxycholate) that inhibit the growth of other enteric bacteria, but these compounds are useful to be added to the isolation medium Salmonella sp. on the stool sample (Irianto, 2018). Typhoid fever is one of the endemic diseases in Indonesia, so it must be given serious attention because it can be a threat to public health. Moreover, there has been an increase in relapse cases and there is resistance to the drugs used, which will make it more difficult for various efforts to be made related to this disease (Depkes, 2006). Salmonella incubation time lasts 7-14 days depending on the number of bacteria, virulence, and immune response (Hardianto, 2019) One way to prevent bacterial contamination is to wash your hands (Taliya, 2021). Contaminated microbes in food are the result of direct or indirect contamination with pollutant sources (Amiruddin et al, 2017). Cross contamination can also occur if the International Journal of Applied Biology, 7(1), 2023 28 processing and preparation process does not meet the requirements, such as food that is not clean and undercooked (Yuniatun, et al. 2017). The objective of this research was to look at the activity of contaminated Salmonella typhii in milkfish sold in unclean fish markets, besides that the length of the process from cutting until the milkfish is ready for consumption, is very possible for contamination of milkfish, including contamination at the time of slaughter, namely with the market or unhygienic fish auction places, tools used for cutting, water used for washing, workers' hands, contact with parts that are often contaminated such as scales, internal organs, gill covers, and others. Materials and Methods Experimental Location and Samples This experiment was conducted in Microbiology Laboratory at the Faculty of Medical Hasanuddin University, Makassar, Indonesia, in June 2021. The population is all samples of milkfish scattered in markets and fish auctions in Makassar City. Samples were taken as ten samples with areas selected by Probability Sampling. Isolation and Identification with Specific Medium Isolation and identification with McConkey Agar (MCA) or BHIB medium, SSA medium, and NA medium were incubated at 35oC for 24 hours. Biochemical Test for Salmonella Typhii For the identification of S.typhii, specific biochemical tests were conducted according TSIA media, SIM media, MRVP media, citrate, glucose, sucrose, urea, Lactose, mannitol were incubated at 35oC for 24 hours. Gram Strain Gram stain with crystal violet, iodine, safranin, and alcohol 70%. Antibacterial and Antibiotic Tests Antibacterial test with MHA media (Mueller Hinton Agar) and antibiotics used such as Ceftriaxone, Sulfamethazole, Ampicillin, Ciprofloxacin, Chloramphenicol (Andi Munawir, 2021) Results and Discussion Milkfish samples totaled ten samples by taking milkfish (Chanos chanos) samples at different locations in the Fish Market and Auction in Makassar City by providing sample codes A-J. The sample criteria examined were scales, gill covers, gills, fluids in the fish's mouth, and parts of the fish's intestines figure 1. International Journal of Applied Biology, 7(1), 2023 29 Figure 1. Samples Milkfish (Chanos Chanos) Milkfish samples are put into BHIB (Brain Heart Infusion Broth) Medium , as seen in figure 2, where BHIB is a microorganism growth medium which is rich in nutrients which is suitable for various types of microorganisms including bacteria. Figure 2. Identification Test with BHIB Medium Test Selective medium SSA (Salmonella Shigella Agar) and NA (Nutrient Agar) is a selective medium for detecting and identifying Salmonella and Shigella bacteria at figure 3. Figure 3. Culture on SSA and NA Selective Medium International Journal of Applied Biology, 7(1), 2023 30 Salmonella typhii belongs to the gram-negative bacteria in the form of rods as shown in the gram stain with Figure 4. Figure 4. Gram Strain at S.typhii Biochemical test with TSIA media, SIM, MR-VP, Citrate, Glucose, Lactose, Sucrose, and Mannitol in Figure 5. Specific characteristics of Salmonella typhii in that the TSIA medium gives a reddish color change with a red slant which includes alkali and a red butt forming H2S gas, then the SIM medium changes color, and there is movement of motile bacteria characterized by the spread of bacteria with the spread of growth in the area around the ose puncture, then on the MR-VP medium there is a color change from yellow to reddish, then citrate test, urea test, lactose test, and the sucrose test did not change color, while the glucose and mannitol test gave a color change from red to yellow. Figure 5. Biochemistry Test with TSIA, MR-VP, Citrate, Urea, Glucose, Lactose, Mannitol, Sucrose In Table 1, samples coded C, G, and J were suspected of Salmonella typhii, then sample A was suspected of Providencia stuartii bacteria, then samples B, D, E, H , and I were suspected of Proteus mirabilis bacteria, with reference to Great Wohls (1980). International Journal of Applied Biology, 7(1), 2023 31 Table 1. Table of Observation of Biochemical Test of Bacterial Samples In Figure 6, the measurement of inhibition using the antibiotics ceftriaxone, sulfamethoxazole, ampicillin, ciprofloxacin, chloramphenicol, and tetracycline, can be seen in Table 2 the most sensitive antibiotic against Salmonella typhii is ciprofloxacin (CIP) with a high inhibition value compared to with other antibiotics which refer to the measurement and inhibition of antibiotics with reference to Great Wohls (1980) or (Andre B, and Novita, 2022). The level of contamination of Salmonella typhii bacteria in milkfish shows a percentage of 30%, which is included in the moderate level, this indicates that the level of contamination with this bacteria is still high. Figure 6. Antibacterial Test with Antibiotics Cefriaxone, Sulfamethazole, Ampicillin, Ciprofloxacin, Chloramphenicol No Code BHIB Media Mac Conkey Agar (MCA) Media SSA and NA Media Gram Stain TSIA SIM MRVP Cit Ur ea G L S M Interpretation Slant Butt H2S Gas Indol Motil H2S MR VP 1. A Cloudy Colourless, Concave Round, Big Basil gram (-) Alkali Acid - - + + - + - + - + - - - Providencia stuartii 2. B Cloudy Cloud red Round, Big Basil gram (-) Acid Acid + + - + + + - + - + + + + Proteus mirabilis 3. C Cloudy Colourless, Convex Round, Small Basil gram (-) Alkali Acid + - - + + + - - - + - - + Salmonella typhii 4. D Cloudy Cloud red Round, Big Basil gram (-) Acid Acid + + - + + + - + - + + + + Proteus mirabilis 5. E Cloudy Cloud red Round, Big Basil gram (-) Acid Acid + + - + + + - + - + + + + Proteus mirabilis 6. F Cloudy Convex red Round, Small, Smooth Basil gram (-) Acid Acid - - + + - + - - - - + + + Escherechia coli 7. G Cloudy Colourless, Convex Round, Small Basil gram (-) Alkali Acid + - - + + + - - - + - - + Salmonella typhii 8. H Cloudy Cloud red Round, Big Basil gram (-) Acid Acid + + - + + + - + - + + + + Proteus mirabilis 9. I Cloudy Cloud red Round, Big Basil gram (-) Acid Acid + + - + + + - + - + + + + Proteus mirabilis 10. J Cloudy Colourless, Convex Round, Small Basil gram (-) Alkali Acid + - - + + + - - - + - - + Salmonella typhii International Journal of Applied Biology, 7(1), 2023 32 Table 2. Antibacterial Test Using Certain Antibiotics AMP TE SXT C CIP 1. Salmonella typhii 17 19 16 18 21 2. Proteus mirabilis 8,2 24,4 17,4 16,6 25,5 3. Providentia stuartii 6,7 6,3 19,5 22 21 4. Escherechia coli 6,9 20,4 22,3 25,5 11,9 Description : AMP : Ampicilin TE : Tetracyclin SXT : Sulfamethazole C : Chloromfenikol Conclusion Samples of Salmonella typhii were obtained in 3 samples of the ten samples studied. The level of contamination of Salmonella typhii bacteria in all samples observed was moderate, with a percentage level of 30% of samples taken at fish markets and auctions in Makassar City. The antibiotic Ciprofloxacin has a high sensitiv ity test rate compared to the four antibiotics, namely Tetracycline (TE), Chloramphenicol (C), Ampicillin (AMP), and Sulfamethoxazole (SXT). International Journal of Applied Biology, 7(1), 2023 33 References Ainil, M. 2021. Angka kejadian demam tifoid berdasarkan kajian serologis di RS Unhas Makassar, Puskesmas Tamalanrea tahun 2019-2020. Fakultas Kedokteran, UNHAS, Makassar. Amiruddin, R. R., Darniati., dan Ismail, 2017. Isolasi dan identifikasi Salmonella sp. pada ayam bakar di rumah makan kecamatan Syiah Kuala kota Banda Aceh. Jimvet, 01(3): 265- 274. Andi Munawir, Anastasia A, 2021. Uji Aktivitas Bakteri Salmonella typhii pada sayuran Lalapan Kemangi (Ocimum sanctum L) secara invitro. UMI Medical Journal Vo 6, No 2, 31 Desember 2021, e-ISSN: 2685-7581. Andre Budi, Novita L. 2022. Pola Resistensi Bakteri Salmonella typhii terhadap antibiotik Ceftriaxone dan Ciprofloxacin. Journal Health and Science, Gorontalo, Vol 6 Nomer 1, April 2022, e-ISSN: 2656-9248. Ariyanti, T., dan Supar. 2017. Problematika salmonellosis pada manusia. Lokakarya Nasional Penyakit Zoonosis, Jakarta. Departemen Kesehatan Republik Indonesia, 2006, Pedoman Pengendalian Demam Tifoid. Menteri Kesehatan Repubik Indonesia, Jakarta. Darmawan, A., Lucia, M., Sitti, A., dan Hapsari, M. (2020). Kontaminasi Salmonella sp. pada daging ayam broiler yang dijual di beberapa Pasar Tradisional di Makassar. Indonesia Medicus Veterinus, 9(2) : 168-176. Dharmojono, 2001, Limabelas Penyakit Menular dari Binatang ke Manusia. Milenia Populer, Jakarta. Global Burned of Disesase, 2020. Global burden of 369 diseases and injuries in 204 countries and territories. The Lancet, 396(10258), pp. 1204–1222. Doi : 10.1016/S0140-6736(20) 30925-9. Hardianto, D. 2019. Telaah metode diagnosis cepat dan pengobatan infeksi Salmonella thypi. Jurnal bioteknologi dan biosains Indonesia, 6(1): 149-158. Irianto, K., 2018, Epidemiologi Penyakit Menular dan Tidak Menular Panduan Klinis, Alfabeta, Bandung, Jawa Barat. Lestari, I Dewa A.M.D Dan Hendrayana, Made Agus, 2017. Identifikasi Dan Diagnos is Infeksi Bakteri Salmonella typhi. Skripsi. Fakultas Kedokteran : Universitas Udayana Denpasar. Melawati, B., Fakhrurrazi, dan Abrar, M., 2019. Deteksi bakteri Salmonella sp pada ikan asin talang-talang (Scomberoides tala) di kecamatan Leupung kabupaten Aceh Besar. Jimvet, 3(3): 175-180 Muna, Fauzul dan Khariri. 2020. Bakteri Patogen Penyebab Foodborne Disease. Jurnal UIN Alaudin. Makassar. Putri, Meiwa R., Tri Umiana S., Syazili Mustofa dan Ety Apriliana. 2019. Identifikasi Bakteri Salmonella typhi Pada Makanan Jajanan Gorengan Yang Dijual di Depan Sekolah Dasar Negeri Kecamatan Kedaton Kota Bandar Lampung. Universitas Lampung : Jurnal Agromedicine,Vol 6, No. 2. Samiadi, L.A. 2021. Salmonellosis. Artikel Kesehatan Pencernaan. http://hellosehat.com. Salmonellosis.id Diakses 15 Januari 2022 Pukul 11.00 WIT Taliya Putri Wardani, 2021. Identifikasi Salmonella typhii pada swab telapak tangan pedagang makanan di SD Kanisus Semanggi II Surakarta. Stikes Nasional, Surakarta International Journal of Applied Biology, 7(1), 2023 34 Ulya, N. N., Fitri, I. dan Widyawati, I. D. 2020. Gambaran makroskopis dan mikroskopis bakteri Salmonella typhi dan Salmonella paratyphi pada penderita demam tifoid. Jurnal Sintesis, 1(2): 40-46. World Health Organization. 2018. Thypoid Fever. http://www.WHO.int. diakses pada tanggal 28 July 2021. Yuniatun, T., Purwantisari, S., dan Yuliawati, S. 2017. Hubungan hygiene sanitasi dengan kualitas mikrobiologis pada makanan gado -gado di Kecamatan Tembalan Kota Semarang. Jurnal Kesehatan Masyarakat, 5(4) : 491-499. Yunus, R., Mongan, R., dan Rosnani. 2017. Cemaran bakteri Gram negatif pada jajanan siomay di Kota Kendari. Medical Laboratory Tecnology Journal, 3(1) : 87-92. Zelpina, E., dan Noor, SM. 2020. Non-Typhoid Salmonella penyebab Foodborne disease: pencegahan dan penanggulangannya. Wartazoa, 30(4):221-229.