Pathogenicity test bacteria from Oreochromis niloticus and Clarias gariepinus aquaculture ponds Uji patogenisitas bakteri yang diisolasi dari media budidaya ikan nila Oreochromis niloticus dan ikan lele Clarias gariepinus

Present research aimed to examine the pathogenicity of some bacteria which were isolated from ponds aquaculture to some freshwater fish. A number of novel pathogenic bacterial strains that infect fish have been identified within the previous five years. Isolating and testing bacteria from culture media for pathogenicity is one method used to stop disease outbreaks in fish farming. Previously, the bacteria were isolated from the water and feces of Oreochromis niloticus and Clarias gariepinus aquaculture in Samarinda, which were identified as Escherichia coli , Enterobacter cloacae , and Enterobacter amnigenus . An 0.1 mL of each bacterium with 1.23-5.46 (×10 6 CFU/mL) was intramuscular injection and evaluated the clinical signs, external pathology, and fish mortality. The freshwater fish using in this research were O . niloticus , C . gariepinus , and Pangasius pangasius with size 10-15 g from Loa Kulu, Kutai Kartanegara Regency, East Kalimantan, Indonesia. The result showed that each bacteria caused mortality in fish: E . coli bacteria caused fish mortality 23.33-66.67%; E . cloacae 10.00 to 90.00%; and E . amnigenus by 3.33-56.67%. The average death time of E . coli and E . amnigenus in all three fish were under 48 hours, while E . cloacae caused mortality less than 48 hours in C . gariepinus and P . pangasius . In conclusion, bacteria Koch postulate test showed that E . coli , E . cloacae , and E . amnigenus are putative pathogenic bacterium in O . niloticus , C . gariepinus , and P . pangasius .


INTRODUCTION
In recent decades, bacterial infections affect aquaculture productivity, causing high mortality (Karvonen et al., 2019;Thongkao & Sudjaroen, 2019;Nugroho et al., 2017). Bacteria of Escherichia coli, Enterobacter cloacae, and E. amnigenus are commonly found in humans, animals, water, soils, plants, insects, and processed products, and can even be used as an indicator of pollutant waste in waters (Dong et al., 2020;Onmaz et al., 2020;Heiman et al., 2015). Many types of Enterobacter sp. bacteria have been identified and there are several species that are pathogenic not only in humans (Davin-Regli & Pagès, 2015), but also in fish. However, some of them are not causing mortality in cultured fish. In Aquaculture, almost every years founded the new bacteria which are pathogenic in fish (Rajasekaran, 2008;Schubiger et al., 2015).
Recent research showed that some bacteria exist in fish farming ponds and have been found that infected O. niloticus i.e Escherichia coli, Salmonella arizonae, Citrobacter braakii, C. freundii, Enterobacter sakazakii, E. cloacae, Raoultella ornithinolytica, Klebsiella ozaenae (Oliveira et al., 2017). In addition, isolated the E. cloacae which caused mortality in Mugil cephalus in Muttukadu lagoon (Sekar et al., 2008), while  successfully found 13 isolates of bacteria from O. niloticus and C. gariepinus ponds in Samarinda which caused mortality up to 90%, in O. niloticus. Past research also determined that LD50 of Enterobacter cloacae, and E. amnigenus bacteria in O. niloticus fish ranged from 10 6.4 CFU/mL. Tilapia fish injected with Escherichia coli, Enterobacter cloacae, and E. amnigenus bacteria showed pale signs of the liver and kidney, anemia, intestinal congestion, ulceration of the anus accompanied by mucus and bleeding in their infected external organs (Monir et al., 2020). This research evaluated characteristics and pathogenicity Escherichia coli, Enterobacter cloacae, and E. amnigenus bacteria in some freshwater fish O. niloticus, C. gariepinus, and P. pangasius.

Aquarium test preparation
The study was performed using an aquarium (41×27×30 cm, length × width × height). Before being used, the aquarium was washed using chlorine, dried, and filled with 30 L of water.
Aeration for each aquarium was provided for 24 hours before the fish were distributed into the aquarium. Water pH for cultivation was maintained in a range 7-8, while temperature about 28 o C, and DO 6-8 mg/L.

Fish preparation
The fish that were used in the pathogenicity test were tilapia (O. niloticus), African catfish (C. gariepinus), and pangasius fish (P. pangasius). All fish had average initial body weight about 10-15 g which was obtained from Loa Kulu Village, Loa Kulu, Kutai Kartanegara, East Kalimantan, Indonesia. Ten fish was used in each aquarium with three replications for each bacterial test. Firstly, fish were acclimatized in the laboratory for seven days and followed by immersion in 3% formalin solution for five minutes for sterilization from bacteria in fish body (Francis-Floyd, 1996). The immersion was repeated if bacteria growth was found. The examination was continued by isolating the gills and kidneys in the BHIA media (OXOID) at a temperature of 35 o C for 24-48 hours.

Enterobacter bacteria preparation
The three species bacteria (Escherichia coli, Enterobacter cloacae, and E. amnigenus) for the pathogenicity test were isolated from the water and feces in O. niloticus and C. gariepinus aquaculture ponds in Samarinda City . In total of eight isolates were grown in tryptic soy agar (TSA) media at a temperature of 30 o C for 24-48 hours, followed by the reidentification and sensitivity test to antibiotics using API 20E. The obtained isolates were cultured in tryptic soy broth (TSB) media and incubated at 30 o C for 24 hours. The isolates bacteria obtained and density (CFU/mL) at LD50-96 for fish injection were list in Table 1 based on the previous research by .

Identification bacteria using API 20 E
This bacterium is prone to contamination; therefore, a new identification is required to confirm that the bacteria utilized in this investigation are the right bacteria. The reidentify and characterize bacteria, API 20 E kit was used (BioMerieux, Inc). Respectively, each isolate was stabbed into tubes, containing semi-solid nutrients and incubated at 35-37 °C for 24 h. The incubated tubes were examined for detecting motility of inoculated isolates followed by preservation in the refrigerator at 4 °C.

Sensitivity test to antibiotic
The fact that certain pathogenic bacteria in fish are currently resistant to antibiotics is a grave warning that improper chemical drug use hastens the pathogenicity of microorganisms. As part of bacterial identification and timely warning against disease outbreaks, bacterial sensitivity testing is necessary. Antibiotics bacterial sensitivity test (Inhibition zone method) was completed using seven commercial antibiotics, namely: nitrofurantoin (Nitro), ciprofloxacin (Cipro), oxytetracycline (Oxy), chloramphenicol (Chlo), nalidixic acid (Na), gentamicin (Gent), and norfloxacin (Nor). Each bacteria was cultured in BHIA and after 5 min, the antibiotic disc was placed on the medium and incubated at 35 o C for 24 h. The diameter of inhibition zone (mm) was measured to determine antibiotic resistance.

Pathogenicity test of bacteria in freshwater fish
Each fish was intramuscularly injected using each isolate bacteria (0.1 mL) with densities, as in Table 1. The density of bacteria was calculated using the TPC method as previously performed by  and Hardi et al. (2016). After being injected, the fish were kept for 96 h. Observation of macroscopic anatomic pathology of external organs, the number of mortalities, and the average main time death, were measured at 96 h after injection.

Water Quality Control
Water quality measurement during the research was carried out to control the condition of aquaculture water. Temperature, dissolved oxygen, and pH were checked every week using the U-50 Series Multi-Parameter Water Quality Checker Tool.

Observed Parameters
Characteristic of bacteria was observed, bacterial identification using API 20E and sensitivity to various antibiotics nitrofurantoin; ciprofloxacin; oxytetracycline; chloramphenicol; nalidixic acid; gentamicin; and norfloxacin.

Pathology of external organs anatomy
Observations were done to detect any abnormalities in the external organs anatomy of fish at 96 h after injection with bacteria. The observation of pathology was focused in the eye of fish (opacity/O, exophthalmia/ E, lysis/L); skin (slimy/B, pale/P, loose scales/Sl); and fin (fin rot/ Sg).

Mortality and mean time death
The cumulative mortality of O. niloticus, C. gariepinus, and P. pangasius were calculated every day or 24, 48, 72, and 96 h after injection. Mean time death (MTD) was measured by calculating the average time needed by each isolate bacteria to cause the mortality in O. niloticus, C. gariepinus, and P. pangasius, using the MTD calculation method (Fitri et al., 2019).
Tests with antibiotic sensitivity showed that each bacteria have different sensitivity to commercial antibiotics (Nitro, Cipro, Oxy, Chlor, Na, Gent, and Nor). All bacteria except E. amnigenus-02 was only sensitive to antibiotics with inhibitory zones of >15 mm ie Cipro, Chlor, Na, and Nor (Table 3).

Pathogenicity of isolate bacteria in tilapia, catfish, and pangasius
Injection with bacteria was carried out with densities ranging from 10 5 -10 6 CFU/mL (Table  1). After the injection, O. niloticus, C. gariepinus,   and P. pangasius showed pathology signs in the organs of the eye, skin and fins (Table 4). Until the 96 hours of post-injection in O. niloticus, the fish appeared to have opacity and eye lysis ( Figure  1A); the changes in the eyes of P. pangasius were also found to have opacity and exophthalmia ( Figure 1B), whereas C. gariepinus showed that no changes were seen in the eye. Meanwhile, in skin organs, the most common symptom was the pale of fish body both O. niloticus, C. gariepinus ( Figure 1C), and P. pangasius. In addition, loose scales was also found in O. niloticus and the fin organ of all fish also showed the presence of fin rot.
The mortality of fish after injected was shown in Table 5. The highest mortality, occurred in

Discussion
The Oreochromis niloticus, C. gariepinus, and P. pangasius are freshwater fish species that are widely cultivated by farmers. Many cultivation technologies developed can be a source of problems if it is not accompanied by good environmental management (Austin & Austin, 2016). The emergence of pathogenic bacteria from the cultivation environment needs to be considered as a step to maintain the safety of aquaculture. There are many studies on zoonotic bacteria from fish that are reported to be potentially pathogenic not only in humans but also in cultured fish (Austin & Austin, 2016;Sekar et al., 2008).
The E. cloacae bacteria even caused mortality up to 90% in P. pangasius, while E. coli reached 66.67% in P. pangasius. Meanwhile, the E. amnigenus bacteria caused mortality of up to 56.67% in C. gariepinus. From the three of fish, P. pangasius was susceptive to E. coli, E. cloacae, and E. amnigenus. The fish mortality post-injection with bacteria was caused by the presence of bacterial virulence factors such Siderophores like anguibactin or piscibactin, have been described in Vibrio and Photobacterium pathogens (Lemos & Balado, 2020).
The previous result findings that the Streptococcus agalactiae bacteria which produces hemolysin causes death to O. niloticus faster and more than the S. agacatiae bacteria that does not produce the hemolysin protein . Li et al. (2020); Mahendra (2016); Moreira et al. (2021), Abalaka et al. (2015) explained that enteric bacteria produce extracellular proteins that are virulent in fish in the form of hemolysin and leukotoxins protein, which cause damage to the fish red blood cells, resulting hemorrhage, hyperplasia, and necrosis in cells and tissues. In addition, hemolysin is a protein that cause virulence factor in bacteria. Austin and Austin (2016), Park et al. (2012) and Susanti et al. (2016) stated that fish infected with Edwardsiella tarda shows some clinical signs of hemorrhage in organs, exophthalmia (protruding eyes), opacity of the eyes, distended abdomen, bloody ascites, and enlarged spleen and kidneys. Similar to current findings, fish injected with S. agactiae bacteria  the Vibrio alginolyticus bacteria (Yanuhar et al., 2022); and E. cloacae (Sekar et al., 2008) revealed the same results that the same damages also occurred in fish kidneys.
Meanwhile, (Witeskaa et al., 2022) found that fish infected with E. vulneris bacteria at 10 6 cells /L density has some clinical signs such as hemorrhagic lesions on the skin, exophthalmia, bloody exudate in the intestinal tract or empty digestive tract and abnormal liver. In addition, E. coli bacterial infection which has a high level of pathogenicity in fish and humans also has the same pathogenicity homologous with Edwardsiella tarda (Nakamura et al., 2013). The presence of bacteria in the brain organs will cause fish to swim abnormally like gasping swimming, sideways swimming and even whirling . Whereas the presence of bacteria in fish kidneys usually causes discolouration in the body of both fish to turn pale or blackened, this is seen histopathologically in fish kidneys that appear to have an increase in the amount of blood in the vessels, which is indicated by dilation of blood capillaries full of erythrocytes in cranial vessels (Dalum et al., 2022).
The pathogenicity of bacteria in fish is determined by bacterial strains, fish species, bacterial entrances, and environmental conditions. Based on the different type of bacteria from E. coli, E. cloacae, and E. amnigenus, all of those bacteria can cause abnormality and mortality in fish. However, based on the strains of each type of E. coli, only E. coli-02; Ent. cloacae-01 and 02 which cause death in tilapia, in fact, E. coli-03 did not cause mortality in catfish and pangasius. This finding revealed that each strain has different characteristics that affected the level of pathogenicity. Moreover, according to API 20 E test results, bacteria which has the ability to hydrolyze more sugar has higher pathogenicity compared to enterobacteria that hydrolyze less sugar. This ability may relate to bacterial adhesion properties and colonize in cells of host tissues.

CONCLUSION
The E. coli, E. cloacae, E. amnigenus which come from the results of the postulat Koch test indicated the possibility of pathogens in freshwater aquaculture (O. niloticus, C. gariepinus and P. pangasius), which may be seen from the presence of macroscopic anatomical pathologies in eye, skin and fins organs; and cause the death of aquaculture fish, as well as the fish meantime death (MTD) after bacterial infection which is less than 48 hours.