Susceptibility of Vibrio spp. from Viscera Organ and Flesh of Lates calcarifer Against Antibiotics

of Lates calcarifer is widely developed in Indonesia due to its high


Introduction
Cultivation of Seabass (Lates calcarifer) is widely practiced in Indonesia.Cultivation can be carried out in floating net cages or in ponds.Several advantages of Seabass serve as the basis for its high cultivation activity.These advantages include the high economic value of Seabass, with prices ranging from Rp. 75,000 to Rp. 80,000 per kilogram (Santika et al., 2021).Seabass has a wide market coverage both domestically and internationally, including countries like Germany, Spain, England, and Italy (Asdary et al., 2019).Seabass has a broad physiological tolerance range and rapid growth, making the cultivation process easier (Hasibuan et al., 2018).
The biggest challenge in aquaculture is diseases caused by pathogenic bacteria.The presence of pathogenic bacteria can disrupt cultivation activities and even lead to mass mortality in fish (Azhar et al., 2020).This can be detrimental to the aquaculture sector and pose risks to human health in the vicinity.Factors that can potentially contribute to the development of pathogenic bacteria in fish farming include poor water conditions (Lein et al., 2020).Pathogenic bacteria can also originate from contamination of the aquaculture equipment and the use of inappropriate cultivation techniques (Palawe et al., 2018).
Vibrio spp. is one type of pathogenic bacteria that can cause diseases in marine organisms (Azhar and Yudiati, 2023).Vibrio spp.bacteria are commonly found in shallow tropical waters because they thrive in waters with temperatures up to 37°C (Rahmaningsih et al., 2012).The use of antibiotics is one of the measures taken to inhibit the growth rate of Vibrio spp.bacteria (Santi et al., 2017).Antibiotics are widely used in fish farming to control diseases caused by bacteria (Nurhasnawati et al., 2016).However, this has led to cases of antibiotic-resistant bacteria, necessitating resistance testing to determine the level of bacterial resistance to the antibiotics to be used.This research aims to identify the types of antibiotics that can be used to treat diseases in L. calcarifer caused by Vibrio spp.bacteria and to determine the resistance of Vibrio spp.bacteria isolated from the viscera organs and flesh of L. calcarifer affected by the disease to antibiotics.

Collection of Vibrio spp. Isolates.
The Vibrio spp.isolates used in this study were collected from the Laboratory of Biology, Faculty of Fisheries and Marine Sciences, Diponegoro University.The L. calcarifer fish were sourced from aquaculture at the Marine Science Technopark, Diponegoro University, Jepara, Faculty of Fisheries and Marine Science, Diponegoro University, Jepara, Indonesia.

Preparation of Agar Slant Solid Media
The preparation of slant solid media begins by assembling the necessary equipment and materials.The weighed materials are 1.3 grams of Nutrient Broth (Merck) and 1.5 grams of Agar (Merck), which are placed into an Erlenmeyer flask and mixed with 100 ml of distilled water.A magnetic stirring bar is added to the Erlenmeyer flask, and the mixture is homogenized on a hot plate magnetic stirrer.Once the media starts foaming, the Erlenmeyer flask is removed from the hot plate magnetic stirrer and allowed to warm up.When the media is sufficiently warm, it is poured into reaction tubes, filling them to a volume of approximately 5 ml.The reaction tubes are then sealed with cotton and aluminum foil and sterilized using an autoclave.After autoclaving, the tubes containing the media are tilted on a stand on a sterile table surface.Be cautious not to tilt them too much to prevent spillage onto the cotton covering, and allow the media to solidify.

Characterization and purification of Vibrio spp. bacteria
The Vibrio spp.bacteria to be characterized originate from samples of the visceral organs and muscles of snapper fish, which have been previously incubated.The bacteria are observed from petri dishes with the assistance of a flashlight, then marked and identified based on their characteristics following the bacterial characterization method by Ejikeugwu (2017).The characteristics of the bacteria are recorded, and colonies with similar characteristics are counted.Once characterized, the bacteria are purified on prepared solid agar media.Purification is carried out using a sterilized needle that has been heated until it becomes red-hot with a Bunsen burner.After it cools down, the needle is streaked through the bacteria on the petri dish and then streaked onto the solid agar media.Bacteria with different characteristics are placed into separate test tubes and labeled accordingly.The test tubes are sealed with plastic wrap in an aseptic manner and incubated in a sterile container.

Preparation of Liquid Media and Purification of Vibrio spp. Bacteria from Solid Agar to Liquid Media
The preparation of liquid media begins with the preparation of the necessary equipment and materials.Nutrient Broth weighing 1.3 grams and NaCl weighing 1.5 grams are placed into an Erlenmeyer flask, followed by the addition of 100 ml of distilled water.A magnetic stirring bar is placed inside the Erlenmeyer flask, which is then homogenized using a hot plate magnetic stirrer.Once all the ingredients are homogenized, the Erlenmeyer flask is removed from the hot plate magnetic stirrer and left to warm.The liquid media is then transferred into separate vials, each containing 5 ml.The filled vials are sealed with cotton and aluminum foil, followed by sterilization using an autoclave.
Vibrio bacteria grown in the test tubes are scraped with an inoculating needle.The inoculating needle is sterilized using a Bunsen burner until it becomes red hot.After the needle has cooled down, it is used to scrape the bacteria from the solid agar medium.The inoculating needle, now containing a trace of bacteria, is then dissolved in the liquid media inside the vial.The vial is sealed again with cotton in an aseptic manner, sealed with plastic wrap, labeled according to the sample, and incubated in a sterile container.

Preparation of Solid Media
The preparation of solid media begins with the preparation of the necessary equipment and materials.The weighed ingredients, Nutrient Broth (1.3 grams) and Nutrient Agar (1.5 grams), are placed in an Erlenmeyer flask, followed by the addition of 100 ml of distilled water (aquades).A magnetic stirring bar is inserted into the Erlenmeyer flask, and the contents are homogenized on a hot plate magnetic stirrer.Once the media has foamed, the Erlenmeyer flask is removed from the hot plate magnetic stirrer and sealed with cotton and aluminum foil.The media in the Erlenmeyer flask is then sterilized using an autoclave.
After sterilization, the media is allowed to cool slightly and is poured aseptically into petri dishes.The dishes are sealed with plastic wrap and covered with plastic secured with rubber bands.The petri dishes are then stored separately in sterile containers to prevent contamination.The preparation of the McFarland 0.5 standard solution begins with the preparation of the necessary equipment and materials.Sodium chloride (NaCl) weighing 0.85 grams is placed into an Erlenmeyer flask and then 100 ml of distilled water (aquades) is added.A magnetic stirring bar is placed inside the Erlenmeyer flask, and the mixture is homogenized using a hot plate magnetic stirrer.Once all the components are homogenized, the Erlenmeyer flask is removed from the hot plate magnetic stirrer and allowed to cool slightly.The McFarland standard solution is then transferred into individual vials, each containing 5 ml of the solution.These vials are sealed with cotton and aluminum foil, and then sterilized using an autoclave.

Testing the
The standardization of Vibrio spp.bacteria is performed by aseptically transferring the bacteria from the liquid media purification into the vials containing the McFarland standard solution, using a micropipette, until the turbidity level matches the McFarland 0.5 standard.Vials that have reached the McFarland 0.5 standard are sealed with plastic wrap, labeled according to the sample, and stored in a sterile container.2.6.2The preparation of antibiotic stock solutions.
Before starting the preparation of antibiotic stock, 20 ml vials and their caps are sterilized using autoclave.Seven types of antibiotics, namely Ciprofloxacin 500mg, Doxycycline 100mg, Tetracycline 500mg, Chloramphenicol 250mg, Ampicillin 500mg, Co-Amoxiclav 125mg, and Azithromycin 500mg are ground into a fine powder using a mortar and pestle.They are then transferred into separate vials and labeled accordingly.Ethanol 96% is added to the vials containing Ciprofloxacin 500mg, Doxycycline 100mg, Tetracycline 500mg, Chloramphenicol 250mg, Co-Amoxiclav 125mg, and Azithromycin 500mg, each with 10 ml of ethanol.Aquades (sterile water) is added to the vial containing Ampicillin 500mg, also with 10 ml of aquades.The antibiotic solutions are homogenized, capped, and sealed with plastic wrap.The antibiotic stock is stored in the refrigerator.
The antibiotic dilution process begins with the sterilization of aquades in test tubes and 10 ml vials using autoclave.Each vial is labeled for easy identification of the antibiotics.10μL of Ampicillin is added to the vial using a micropipette, followed by the addition of 990μL of sterile aquades.500μL of Genta-100 is added to the vial using a micropipette, followed by the addition of 500μL of sterile aquades.150μL of Doxycycline is added to the vial using a micropipette, followed by the addition of 850μL of sterile aquades.30μL of Tetracycline is added to the vial using a micropipette, followed by the addition of 970μL of sterile aquades.60μL of Chloramphenicol is added to the vial using a micropipette, followed by the addition of 940μL of sterile aquades.5μL of Ciprofloxacin is added to the vial using a micropipette, followed by the addition of 995μL of sterile aquades.80μL of Co-Amoxiclav is added to the vial using a micropipette, followed by the addition of 920μL of sterile aquades.The antibiotic dilution process is carried out aseptically, and the vials are capped and sealed with plastic wrap.

Preparation of Paper Disks and Antibiotic Injection into Paper Disks
The Whatman paper No. 3 is cut using a hole punch to create paper disks.These paper disks are then placed into a glass beaker and covered with aluminum foil.The beaker containing the paper disks is sterilized using an autoclave.
The injection of antibiotics into the paper disks begins with the preparation of sterilized petri dishes using an autoclave.Next, the sterilized paper disks are aseptically arranged in the petri dishes using sterile forceps.The paper disks are placed with some spacing between them to avoid sticking together.The injection of antibiotics is performed with aseptic techniques.Each paper disk is injected with 20μL of the respective antibiotic.The number of paper disks used corresponds to the number of samples in the petri dish.The dishes containing the paper disks are sealed with plastic wrap and left to air dry until the paper disks are partially dry.If the top surface of the dishes with paper disks is still wet, it can be dried using sterile gauze.Each dish containing antibiotic disks is labeled to differentiate its antibiotic content.

The testing of antibiotic potency through paper disk diffusion
The process involves the preparation of agar plates.Bacteria that have been standardized using the MacFarland solution are inoculated into the solid media with 100μL each, using a micropipette.Each type of bacteria is inoculated into two solid media plates.The bacterial inoculation is performed aseptically.The bacteria are then evenly spread across the surface of the solid media using a sterilized spreader heated by a Bunsen burner.Petri dishes are then sealed with plastic wrap, labeled, and placed in an incubator until the bacteria have fully integrated with the solid media.
The next step involves placing antibiotic disks onto the solid media.Four different types of antibiotic disks are placed in a single media plate.Paper labels are used to identify the antibiotic disks placed in each dish.Antibiotic disks are positioned on the solid media using sterilized forceps.They should be spaced apart and not too close to the edge of the dish.Petri dishes containing the media and antibiotic disks are then sealed with plastic wrap and incubated in the incubator for 24 hours.
After the incubation period, zones of inhibition will become visible in the media.These zones are measured using calipers at opposite edges of the inhibition zones, perpendicular to the edge.The diameters of the inhibition zones are measured.The results, including the diameter of the inhibition zones, are recorded.These results are then processed using Microsoft Excel to determine the sensitivity of the bacteria to each type of antibiotic, whether the bacteria are resistant, sensitive, or intermediate, following the criteria for the diameter of the inhibition zone as defined by CLSI (2011) as shown in Tabel 1.

Results
The results of the measurement of inhibition zones and the criteria for the Gentamicin inhibition zone can be seen in Table 2.The measurement results and criteria for Tetracycline HCl inhibition zones can be found in Table 3.The measurement results and criteria for Ciprofloxacin HCl inhibition zones can be seen in Table 4.The measurement results and criteria for Ampicillin inhibition zones can be seen in Table 5.The measurement results and criteria for Chloramphenicol inhibition zones can be seen in Table 6.The measurement results and criteria for Azithromycin inhibition zones can be seen in Table 7.The measurement results and criteria for Doxycycline inhibition zones can be seen in Table 8.The inhibition zones formed on the isolates can be seen in Figure 2. The percentage effectiveness of antibiotics against Vibrio spp.bacteria can be seen in Table 9.

Discussion
Testing the resistance of Vibrio spp.bacteria from the viscera organ and flesh of L. calcarifer begins with the characterization of bacterial colonies.Characterization is done by observing bacterial colonies on agar plates.Colonies are characterized and grouped based on their margin shape, color, and elevation.Bacterial colonies isolated from the JerTR37 plate have a round shape with a convex elevation and are yellow in color.This is consistent with the morphological characteristics of Vibrio spp.colonies as reported by Arisandi et al. (2019), which describes round colonies with a yellow color.In contrast, bacterial colonies isolated from the OTTR36 plate have round and irregular shapes with a convex elevation.The differences in the shape of Vibrio spp.bacterial colonies in one plate are influenced by abiotic environmental factors such as pH, temperature, oxygen levels, nutrient availability in the growth medium, and temperature (Situmeang et al., 2016).The color of Vibrio spp.bacterial colonies is influenced by their ability to utilize sucrose.This is supported by the statement from Ilmiah et al. (2012), which explains that yellow colonies of Vibrio spp.indicate the ability to utilize sucrose, while green colonies indicate the inability to utilize sucrose.
The level of antibiotic resistance is determined by measuring the inhibition zones formed by the reaction of Vibrio spp.bacteria from the viscera organ and flesh of L. calcarifer.Vibrio spp.bacteria from the viscera organ showed resistance to Azithromycin and Ampicillin with percentages above 50%.This is consistent with data from Kusmarwati et al. (2017), which reported that Vibrio spp.bacteria found in shrimp ponds exhibited resistance to Ampicillin, reaching up to 73%.Vibrio spp.bacteria from the white snapper muscle did not show resistance to excessive antibiotics, as indicated by resistance percentages below 50%.The highest resistance percentage was 35.7%, which was observed for Ampicillin and co-Amoxiclav antibiotics.Based on the results obtained, it can be concluded that Vibrio spp.bacteria from the viscera organ and flesh of L. calcarifer have the highest sensitivity to Gentamicin, with a sensitivity percentage of 100%. .

Conclusions
Vibrio spp.bacteria from the viscera organ and flesh of Lates calcarifer exhibit varying levels of resistance and sensitivity to different types of antibiotics.In general, the Vibrio spp.obtained in this study are still sensitive to antibiotics.
Resistance of Vibrio spp.Bacteria to Antibiotics 2.6.1 Preparation of McFarland 0.5 Standard Solution and Standardization of Vibrio spp.Bacteria with McFarland Standard Solution

Table 2 .
Measurement Results and Criteria for Gentamicin Inhibition Zones

Table 9 .
Percentage Effectiveness of Antibiotics against Vibrio spp.