Antibiotic-resistant threads of Aeromonas hydrophila as a major pathogen in Indonesia freshwater aquaculture

. Aeromonas hydrophila is a zoonotic, important primary fish pathogen in many economic fish species. Aeromonas acquires and exchanges antimicrobial resistance genes (ARG) and has been widely studied as a potential reservoir of ARGs. This research aims to observe the impact of antibiotics used through the role of Aeromonas hydrophila as a pathogenic bacteria in aquaculture. The tested antibiotics are based on the Minister of Marine Affairs and Fisheries No. 1/2019 Decree, namely tetracycline, oxytetracycline, and enrofloxacin, which the Indonesian government allows to be used in aquaculture. Random sampling was conducted using four to five fishes per farm in West Java, namely Bogor (17 farms) and Sukabumi (10 farms), Central Java at Banyumas (38 farms), and Magelang (25 farms). The test method used is CLSI-2020, an Antimicrobial Sensitivity Test disk diffusion with standard bacteria ATCC Escherichia coli 25922. The results show that A. hydrophila is resistant to tetracycline by 15,06% and oxytetracycline by 54,54%. However, A. hydrophila is sensitive to enrofloxacin by 84%. The aquatic environment acts as a reservoir for antibiotic-resistant A. hydrophila and emphasizes the importance of prudent antimicrobial use and timely AMR surveillance in aquaculture.


Introduction
Aquaculture is the future hope for a portion of sustainable world food.Asia, including Indonesia, accounts for 90% of global production and is predicted to continue to increase until 2050.The fish villages organized by the Indonesian government are one way to increase small-scale home-based production to support food security in rural areas.This is applied by low to middle-income countries.Medium-scale aquaculture is also geared towards contributing to the export and industrial-scale production of high-tech products that are traded globally.
Sustainability in aquaculture faces challenges that must be overcome.Environmental challenges, especially in shrimp farming, include environmental degradation due to aquaculture exploitation, high carbon footprint, and disproportionate and excessive use of antibiotics in aquaculture production.Food-producing animals, especially aquaculture, contribute to the potential risk of resistant bacteria and residues in the food chain that are harmful to human health [19].Toxic metabolites can be formed from several antibiotics, which are excreted unchanged due to imperfect absorption or metabolism in the digestive tract [24,35].Antibiotics are in the environment due to uncontrolled application, without pauses, and inappropriate doses [33].About 90% of antibiotics are not entirely absorbed by the gastrointestinal tract and are excreted in the urine, and 75% of them remain unchanged in the feces [35,30].
One Health is a collaborative, multisectoral, and transdisciplinary approach to achieving health and well-being outputs that benefit people, animals, and the environment [28], how to serve protein derived from aquatic animals that do not hurt human health and the environment.The genetic element of the Antibiotic Resistance Gene (ARG) in aquaculture systems is of major concern due to the intensive and often inappropriate use of antibiotics to treat disease.Antimicrobial Resistance (AMR) is a global public health issue and food security risk that results in ineffective prevention and treatment measures for infections in humans and animals and risks to food safety due to the uncontrolled and harmful use of antibiotics [7,25].
Antimicrobial resistance should be prevented in the future by designing a One Healthbased aquaculture system.This research aims to observe the impact of antibiotics used through the role of Aeromonas hydrophila as a pathogenic bacteria in aquaculture.

Material and Method
Bacterial isolate samples were isolated from farmed fish (Oreochromis niloticus, Clarias gariepinus, Osphronemus goramy, and Cyprinus carpio) in West Java and Central Java, Indonesia.The sampling location can be seen in Figure 1.The samples were Oreochromis niloticus, Clarias gariepinus, Osphronemus goramy, and Cyprinus carpio from cultivators with a 50-200 g size range.Ninety samples (pooling of spleen, kidney, and liver) were taken from several fish farmers, namely Banyumas with 38 samples, Bogor Regency with 17 samples, Sukabumi with 10 samples, and Magelang with 25 samples.One farm is represented by 4 to 5 fish, which are pooled into 1 sample, which represents the farm.The fish were taken from ponds randomly and in healthy condition.The fish were dissected using surgical instruments and under sterile conditions.
Fish target organs isolated from the spleen, kidney, and liver on Nutrient Broth (NB) medium and incubated at 28°C for 6-8 hours and then isolated onto Aeromonas selective media, Rimler-Shotts (RS) agar (Himedia) at 37°C for 24 hours.Typical yellow colonies were re-inoculated until pure colonies were obtained.
The pure bacteria of A. hydrophila will then be identified using PCR with the specific primer according to [35] design, namely F-5' GCC GAG CGC CCA GAA GGT GAG TT 3'; R-5' GAG CGG CTG GAT GCG GTT GT 3' with amplification result target was 130bp.
The Kirby-Bauer disk diffusion method on Mueller-Hinton agar (Sigma®) was used to examine antibiotic susceptibility patterns for A. hydrophila in vitro, according to Hudzicki [16], with slight modifications.The antibiotics used were tetracycline (TE; 30μg), enrofloxacin (ENR; 5μg), and oxytetracycline (OT; 30μg) from Oxoid.Before use, the bacterial isolate and the reference strain (Escherichia coli 25922, from the American Type Culture Collection, ATCC) were propagated in nutrient broth and incubated at 37°C for 24 hours.Then, the bacterial suspension was adjusted to a turbidity equivalent to 0.5 McFarland standard and spread onto the surface of Mueller-Hinton agar (Sigma) for even growth.Three antibiotic discs were then placed on the surface of the agar plate and incubated again at 35°C for 16 hours.The zone of inhibition formed by the isolates was measured and interpreted as sensitive, intermediate, or resistant isolates based on the Clinical Laboratory Standards Institute [10] (Table 1).*Ciprofloxacin (5 ì g) [9] The data obtained was then collected in a Microsoft Excel® spreadsheet to calculate averages and proportions and presented descriptively.Meanwhile, inferential statistics were processed using the statistical package for Social Sciences (SPSS®), version 22.0.

Result and Discussion
Low and Middle-Income Countries (LMICs) face severe problems in the use of antibiotics that contribute to the increase in global antimicrobial resistance (AMR).Southeast Asia, including Indonesia, is a global region with the greatest risk of spreading AMR.Aquaculture in Southeast Asia is a major food production industrial center supplying both domestic and international markets [32,6,9].
Abundance and antibiotic resistance of bacteria of Aeromonas hydrophila isolated from various types of freshwater fish were found in this study (Figure 2).The majority of A. hydrophila is generally resistant to tetracycline and oxytetracycline.However, almost all Aeromonas isolates were susceptible to enrofloxacin.In Sukabumi Regency in 2019, A. hydrophila showed resistance to OT, 55%, and to TE, 48%.Meanwhile, A. hydrophila against ENR is still very sensitive, namely 84%.However, in 2022 A. hydrophila, its sensitivity to ENR decreased to 22% from the previous 84% (Figure 3.A1.and A2.).The freshwater fish cultivated in Sukabumi include catfish, carp, tilapia, and ornamental koi fish.Sukabumi Regency is directly adjacent to the south by the Indonesian Ocean, with a coastline length of ± 117 km, which extends from the sub-districts of Cisolok, Cikakak, Palabuhanratu, Simpenan, Ciemas, Ciracap, Surade, Cibitung, and Tegalbuleud.Potential coastal and marine resources include marine fish, coral reefs, mangrove forests, seaweed, turtles, mining materials, and minerals.
Banyumas is known as a center for Gourami cultivation, which can contribute about 20% of Gourami production in Central Java Province.In Banyumas Regency in 2020, A. hydrophila in fish samples was still sensitive to OT and ENR of 60%, respectively.The condition of the Banyumas district in 2021, A. hydrophila, sensitive to OT and ENR decreased to 21% respectively (Figure 3.B1 and B2).
In 2021, A. hydrophila bacteria in fish samples in the Bogor district appeared to show a low sensitivity, only around 9%, to the three types of antibiotics, namely TE, OT, and ENR (Figure 3.C.).The working area of Bogor Regency covers six areas consisting of Region I to VI, namely Cibinong, Babakan Madang, Jonggol, Ciomas, Cigudeg, and its surroundings.During the last five years (2010-2014), catfish production in Bogor Regency has continued to increase with an average increase of 30% per year, followed by carp, tilapia, and catfish.
Magelang Regency, with its superior commodity of tilapia, is one of the locations designated by the Ministry of Marine Affairs and Fisheries (MMAF) to be developed into a center for aquaculture villages especially for tilapia.In 2021, A. hydrophila in fish samples from the Magelang district also showed low sensitivity to TE and ENR and resistance to OT (Figure 3 Risk management begins with preliminary activities in the form of an AMR survey by FAO (FAOTCP/RAS/3702 Support mitigation of AMR risk associated with aquaculture in Asia) cooperating with the Indonesian government.At that time, sampling was carried out at four locations: one location in West Java (Subang), two locations in Central Java (Banyumas and Sleman), and one location in East Java (Blitar).Aeromonas hydrophila dominates in the four areas, respectively, 48%, 31%, 60%, and 46% [1].From the results of this study, it can be seen that there are additional resistant areas, namely Sukabumi, Bogor, and Magelang.In addition, Aeromonas hydrophila in Banyumas, which FAO surveyed, seems to have decreased sensitivity to OT and ENR.
Fish from aquaculture centers sampled in this study, namely A1, B1, and D, were resistant to two types of antibiotics: tetracycline and oxytetracycline.Tetracycline (TE) and oxytetracycline (OT) are often used for therapy in fish farming, livestock, and humans because of their low cost and productivity for controlling pathogens [33].Antibiotics in aquaculture are usually given through feed or immersion, according to [14].TE and OT are most commonly used in cultivation for prevention and treatment.WHO includes TE and OT in the criteria for antibiotics, which are very important for human health, and the use of these antibiotics is limited in the veterinary and aquaculture sectors [31].
The decrease in A. hydrophila sensitivity to three types of antibiotics found in fish samples obtained at observation sites in West and Central Java was thought to be due to the use of doses of the three antibiotics that were not by the recommended doses.This is the opinion expressed by [24], who found that antimicrobial agents in disproportionate doses are given to fish through mixed feed.
Due to unconsumed antimicrobials, fish products, undigested feed, and fish waste can contain antimicrobial residues.Because of their concentration and biodegradability, the secreted antimicrobial metabolites can remain in water and sediments in fish ponds for long periods [14].The antibiotics that are given to fish, 70-80% are excreted back into the water, affecting the microbial community in ponds and causing major changes in sediment and water biodiversity near open aquaculture systems, even at small doses [23,4,5].Changes in biodiversity in the surrounding sediments also increased the resistome of mobile genetic elements and antibiotic resistance genes [3,12,37].
OT was banned by the EU in 2006 if it is used as a growth promoter [8].[27] reported that OT is a commonly used antibiotic in aquaculture and is frequently detected in culture media in various countries.The rest of the use of OT from aquaculture is often disposed of into the environment.Moreover, its absorption in the fish body is less effective and requires high doses [11].
Risk assessment can be traced from hazard identification and characterization.In this research, A. hydrophila is a pathogen determined as a hazard.Aeromonas hydrophila is a pathogen that is always the cause of sick fish, acting as a primary or secondary infection.The following risk assessment step is risk characterization and exposure assessment, as shown in Figure 4 below.According to the China Food and Drug Administration, humans and animals use more than 100 types of antibiotics, and 70 can be found in water and sediments [10,13].Moreover, [39] said that antibiotics were found in wastewater treatment plants in South China and sewage drainage, livestock farms in China and Japan at levels of ng L -1 to 1g L -1 [29,20], river water, and sediment at the Yellow River, Hai River and Liao River in Northern China [28], municipal sewage sludge digesters [26], land irrigated by wastewater in Hebei, China [15], and also found in various types of waters both on land and at sea such as groundwater in Northern China [22], surface water [20], seawater in Liaodong Bay and the adjacent Liao River basin, North China [16], and drinking water [39].
Antibiotic-resistant A. hydrophila is a problem that requires attention because it involves medical, social, and environmental problems.The main threat from resistant A. hydrophila in water can cause antibiotic resistance in fish.Fish and its processed products can be consumed by humans through the food chain, thus causing human disease to be no longer sensitive to antibiotics.

Conclusion
Due to the uncontrolled use of antibiotics in West and Central Java fish farming, the sensitivity of A. hydrophila has decreased to OT, TE, and ENR antibiotics.Therefore, in the future, monitoring these three types of antibiotics needs to be carried out carefully.Antimicrobial resistance (AMR) surveillance activities must be carried out to identify, control, and take early action against bacteria in the culture environment.The results of this study can be used as a policy recommendation for the type of antibiotic used for the treatment of fish.Planning for future surveillance activities is essential to expand the area of sampling point locations in fish farming production areas in Indonesia.

Fig. 1 .
Fig. 1.Research sampling locations were sample location taken.(A) Sukabumi Regency of West Java; (B) Bogor Regency of West Java; (C) Banyumas Regency of Central Java; (D) Magelang Regency of Central Java.

Table 1 .
Breakpoints for three antibiotics according to the Clinical and Laboratory Standard Institute Standards.