Antimicrobial Resistance in Bacteria Isolated from Aquaculture Environments in the Philippines E.A. Tendencia and C.R. Lavilla-Pitogo Aquaculture Department Southeast Asian Fisheries Development Center, Tigbauan 5021 Iloilo, Philippines Abstract. Antibiotics have been used in aquaculture as feed additives to promote growth and added in water to prevent disease occurrence. Therapeutic doses are used to treat diseases. Long-term use and misuse of antibiotics may result to drug resistant bacterial strains and accumulation of unwanted residues in the cultured commodity upon slaughter and harvest. Some of the antibiotics that have been used in shrimp hatcheries and grow-out ponds are tetracycline, rifampicin, chloramphenicol, nitrofurans, erythromycin, oxolinic acid and furazolidone. Oxytetracycline, furanace, terramycin, Ektecin, chloramphenicol and sulfa drugs have been used to treat fish bacterial infections. Antibiotic resistant bacteria have been isolated from shrimp rearing water, natural bodies of water that received effluents from aquaculture, and from cultured apecies in the Philippines. Resistance to oxytetracycline and furaltadon were reported in bacteria isolated from crab and nearshore sediments. Bacteria from shrimp ponds have been reported to be resistant to oxytetracycline, furazolidone, oxolinic acid and chloramphenicol. Luminous vibrios from shrimp larvae and ponds were resistant to erythromycin, kanamycin, oxytetracycline, Penicillin, streptomycin, sulfadiazine and triple sulfa. Aeromonas sp. from fish, shrimp, and their rearing water were resistant to streptomycin, oxytetracycline and trimethoprim/sulphamethoxazole. Multiple antibiotic resistance has also been reported in bacteria isolated from shrimp, shrimp ponds, luminous bacteria from shrimp post larvae, and Aeromonas hydrophila from fish. Despite the recorded usage, antibiotic residues in cultured food fish have not yet been a problem of the Philippine aquaculture industry as far as trade is concerned. However, chloramphenicol and nitrofurazolidone residues have been detected in shrimp for export using the enzyme link immunoassay (ELISA) technique. To minimize the use of antibiotics, different alternative methods to prevent diseases in aquaculture have evolved. Government agencies also issued regulations on the use of antibiotics. Keywords- Antibiotic, resistance, Philippines, aquaculture INTRODUCTION In the Philippines, aquaculture has been a major source of livelihood and food. Farming practices have intensified to increase production and maximize profits. This intensification is accompanied by disease problems. Fish disease problems have been dealt usually by using chemicals or drugs [1,2]. The indiscriminate use of antimicrobials could lead to problems such as drug resistance [3,4,5,6] and presence of antibiotic residues in food fishes [7], which could affect therapy of human diseases. This report reviews the use of antibiotics in the Philippines, and includes the reasons why chemotherapeutants are used, the problems and constraints for their use, the drugs currently being used, alternative methods to control disease and some recommendations. REASONS FOR USAGE Antibiotics have been used in finfish, crustaceans and mollusks hatcheries as a prophylaxis during larval rearing [8.9] wherein low levels of antibiotics are incorporated into the rearing water to prevent disease occurrence. Antibiotics are used in aquaculture as feed additives to promote growth and added in water to prevent disease occurrence[10]. Therapeutic doses are used to treat diseases [11]. Growth promoters are drugs that destroy or inhibit bacteria and are administered at a low, sub-therapeutic dose. Antibiotic growth promoters are used to help growing animals digest their food more efficiently, get maximum benefit from it and allow them to develop into strong and healthy individuals. Administration of growth promoters is based on the principle that the antibiotic suppresses sensitive populations of bacteria in the intestines and if the microbial population is better controlled, then energy could be diverted to growth. In Philippine aquaculture, growth promoters are indirectly administered during integrated fish-livestock farming. Animal manure, which serves as fertilizers and supports growth of photosynthetic organisms, is shed directly into the fishpond. Livestock are often fed with feed containing growth promoters that are excreted with the manure. Antibiotics are also used to treat diseases. The rationale for antibiotic therapy of infectious disease is that if a disease is caused by a certain pathogen, eradicating the pathogen will end or stop the disease. Pathogens are treated with antibiotics that are effective against them. PROBLEMS The indiscriminate use of antibiotics in the Philippines resulted to the isolation of antibiotic resistant bacterial strains and the detection of antibiotic residues in shrimp for export [7]. Antibiotic resistant bacteria have been isolated from shrimp rearing water, natural bodies of water that received effluents from aquaculture, and from cultured crab, shrimp, and fish in the Philippines. Table 1 shows the resistance of different bacterial isolates to different antibiotics as well as the source of the isolates. Table 1. Antibiotic resistance of bacteria isolated from aquaculture environments Source Bacteria Antibiotics Tilapia13 Streptococcus OTC, Fx, E Shrimp6 luminous Vibrio C, E, Fx, K, NB, P, OTC, P, Pb, S, SD, SSS Shrimp zoea12 FD, OTC Catfish, grouper Aeromonas OTC, SXT,S Seabass 5 Crab4 A Shrimp ponds3 OTC, OXA, Fx, C OTC=oxytetracycline, 30 µg/ml; Fx=furazolidone, 100µg/ml, OXA=oxolinic acid 30µg/ml; C=chloramphenicol, 30µg/ml; E=erythromycin, 15 µg/ml; K=kanamycin, 30 µg/ml; NB=novobiocin, 30 µg/ml; P= penicillin, 10 units; Pb= polymyxin B, 300 units; S=streptomycin, 10 µg/ml; SD=sulfadiazin, 25 µg/ml; SSS=triple sulfa, 250 µg/ml; SXT= Trimethoprim/sulphamethoxazole, 25 µg/ml; A=ampicillin, 30 µg/ml. Reference [12] reported that 1.6% of the Vibrio bacteria isolated from crab Scylla sp. and nearshore sediments are resistant to oxytetracycline and 57% to furaltadon. Reference [3 ]reported that bacteria isolated from shrimp ponds are resistant to oxytetracycline, furazolidone, oxolinic acid and chloramphenicol. Luminous vibrios from shrimp larvae and ponds were resistant to erythromycin, kanamycin, oxytetracycline, Penicillin, streptomycin, sulfadiazine and triple sulfa [6]. Reference [4] also observed resistance to ampicillin in Vibrio isolated from crab hemolymph, pond/creek/reservoir water and P. monodon. Aeromonas sp. from fish (milkfish Chanos chanos, grouper Epinephelus sp., seabass Lates calcarifer), shrimp, and their rearing water were resistant to streptomycin, oxytetracycline and trimethoprim/sulphamethoxazole [5]. Streptococcus sp. from tilapia has been reported to be resistant to oxytetracycline, furazolidone, and erythromycin [13]. Multiple antibiotic resistance has been reported in bacteria isolated from shrimp and shrimp ponds [3]. Reference [5] also reported multiple antibiotic resistance in luminous bacteria from shrimp post larvae, and Aeromonas hydrophila from fish. Table 2 shows the antibiotic resistance profile of bacteria isolated from shrimp ponds, while, Table 3 shows the multiple antibiotic resistance profile observed in Vibrio sp. from the rearing water, fish and shrimp; and Aeromonas spp. from fish. Table 2. Antibiotic resistance profile of bacteria isolated from shrimp ponds. Resistance profile Occurrence (%) OTC, OXA, Fx, C 6 OTC, OXA, FX 5 OTC, OXA, C 1 OXA, C, FX 2 OTC, C, Fx 4 OTC, OXA 2 OTC, FX 12 OTC, C 12 OXA, Fx 15 Oxa, C 1 C, Fx 3 OTC=oxytetracycline, 30 µg/ml; Fx=furazolidone, 100 µg/ml, OXA=oxolinic acid 30 µg/ml, 2; C=chloramphenicol, 30 µg/ml. Table 3. Antibiotic resistance profile of Vibrio spp and Aeromonas spp. isolated from fish/shrimp and their rearing water. Bacteria Antibioticprofile Occurrence(%) Vibrio spp. OTC, SXT, S 0.9 OTC, OXA, S 0.9 SXT, S 0.9 OXA, S 0.9 C, S 1.8 OTC, S 10.1 Aeromonas spp. OTC, SXT, C, S 42.9 OTC, SXT, S 57.1 OTC=oxytetracycline, 30 µg/ml; S=streptomycin, 10 µg/ml; SXT=Trimethoprim/sulphamethoxazole, 25 µg/ml; OXA=oxolinic acid 30 µg/ml,; C=chloramphenicol, 30 µg/ml. Despite the recorded usage, antibiotic residues in cultured food fish have not yet been a problem of the Philippine aquaculture industry as far as trade is concerned. However, chloramphenicol and nitrofurazolidone residues have been detected in shrimp for export using the enzyme link immunoassay (ELISA) technique (Regidor, pers. comm.). CURRENT STATUS OF USAGE IN AQUACULTURE Table 4 summarizes the different antibiotics used in shrimp hatcheries and grow-out ponds and in milkfish grow-out ponds [9]. Erythromycin, nitrofurans, oxolinic acid, tetracycline, chloramphenicol, and the sulfa drugs are some of the antibiotics that are available in the market. The chemicals recommended for use in aquatic animals are listed in Table 5. Table 4. List of antibiotics used in shrimp hatcheries/ grow-out ponds. Antibiotics Usage Tetracycline disease control Rifampicin disease control Chloramphenicol disease control Nitrofuran disease control Erythromycin disease control Table 5. List of antibiotics recommended for use in aquatic animals. Antibiotics Target species Dihydrostreptomycin sulfate ornamental FW fish Erythromycin phosphate ornamental fish Furazolidone ornamental fish Gentamycin sulfate ornamental fish Isoniazid ornamental fish Kanamycin ornamental fish Neomycin sulfate ornamental fish Nyfurpyrinol ornamental fish Oxolinic acid food/ornamental F/C ormetoprim food/ornamental F/C Sulfadimethoxine food/ornamental F/C Sulfamerazine food/ornamental F/C Sulfisoxazole food/ornamental F/C Sulfisoxazole food/ornamental F/C Trimethoprim/sulfadiazine food/ornamental F/C FW= freshwater; F=food; C=crustacean ALTERNATIVE APPROACHES To minimize the use of antibiotics, different alternative methods to prevent diseases in aquaculture have evolved. Some of the culture techniques currently implemented by shrimp farmers in the Philippines are the low salinity technique, use of ozone, crop rotation, the finfish-shrimp integrated culture system or more commonly known as the greenwater culture system, the low-discharge system, and the use of probiotics. In the low salinity culture technique, 90 days old shrimp are cultured in ponds with water salinity of 4-15 ppt. Water from a deep well is used to achieve the desired salinity. Reference [14] reported higher survival rate (65- 99%)in shrimp cultured in low salinity compared to those in conventional method (15-25%) during disease outbreak. Furthermore, it has been reported that shrimp grown in low salinity have faster growth rate and are of good quality [14]. Ozone is widely used in shrimp hatcheries and processing plants to destroy harmful organisms such as virus and bacteria. However, the use of ozone may also destroy beneficial bacteria that may create an imbalance in the pond ecosystem. Thus, ozone is used only to disinfect water in the reservoir and not in the culture pond [15]. Low-discharge system makes use of a close re- circulating system [16]. Sludge collectors are installed at the center of each grow-out pond. The pond system also includes a sedimentation pond and a reservoir, which is at least 25% of the shrimp grow-out pond area, wherein fish cages are installed. Tilapia or milkfish are stocked in the net cages. To prevent disease shrimp farmers also use probiotics. Probiotics are mono- or mixed culture of live microorganisms that, when applied to animals, beneficially affect the host by improving the properties of the indigenous microflora [17]. In Negros Island, Philippines, 80- 100% shrimp survivals were observed in ponds where probiotics were used even in the presence of luminous Vibrio [17]. Crop rotation is the culture of phylogenetically different species for a definite culture period. It is based on the principle that the microbiota of the environment is altered if two phylogenetically different species are cultured alternately. Reference [18] observed that the greater the phylogenetic differences between two cultured organisms, the better sanitary effect. Shrimp and finfish belong to different orders in the animal kingdom, thus are considered good candidates for crop rotation. Shrimp monoculture may cause the accumulation of harmful bacteria in the environment due to biofilm formation. Beneficial bacteria associated with finfish culture may replace these harmful bacteria. Reference [19] reported an increase in shrimp survival after using the shrimp pond for tilapia culture. Another technique used by shrimp farmers to prevent disease is the green water culture system [20] or the finfish shrimp integrated culture system [14]. The greenwater culture system is a technique wherein shrimp are cultured in water collected from a pond where finfishes are grown. The fish may also be cultured in an isolated net pen inside the shrimp culture pond. The most commonly used species is the Tilapia hornorum. Reference [21] reported that stocking of tilapia a biomass not lower than 300 g/m3 efficiently inhibits the growth of luminous bacteria in shrimp (biomass=60 g/m3) rearing water, Government agencies issued regulations on the use of antibiotics. Under the Food, Drug, and Cosmetic Act, the Food and Drug Administration (FDA) has the authority to restrict the use of antibiotics in animals based upon the potential risk to human health [22]. In the United Kingdom, antibiotics are used only as therapeutants and not for prophylaxis or as growth promoter [23]. In the Philippines, olaquindox and carbadox were banned and withdrawn from the market after studies revealed that both drugs possess genotoxic potential [24]. In 1992, some 71 Filipino scientists issued a statement that urged that antibiotics, non-biodegradable pesticides, and disease- control chemicals be banned from aquaculture use [7]. The Food and Agriculture Organization also published the “Code of Conduct for Responsible Fisheries” [25] which calls for the implementation of the following: 1) improved selection and use of appropriate feeds, feed additives, and fertilizers, including manures; 2) minimal use of chemicals such as hormones, antibiotics and other chemotherapeutants; 3) regulate the use of chemicals which are hazardous to public health and the environment; and 4) disposal of excess veterinary drugs and other hazardous chemicals should not pose hazard to public health and the environmenmt. CONCLUSIONS Antibiotic resistant bacteria have been found in shrimp rearing water and pond soil. Multiple antibiotic resistance has been associated with antimicrobial use [3]. 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