Context: Rare report of bacterial isolation and drug resistance from farmed tilapia fishes in Thailand where high yield of tilapia fish products and export. Aims: 1) To isolated and identified of human pathogens in Tilapia fishes (Oreochromis niloticus) from seven aquacultures at Surat Thani, Thailand, including 4 of well-typed fish ponds, 2 of floating baskets and 1 of nursery fish pond 2) To determine water quality of each fish farm. Materials and Methods: The internal organs from 210 of fish samples were collected for pathogen isolation and identification, and then, tested for antibiotic susceptibility. All microbiological laboratory techniques were performed by Clinical and laboratory standards institute (CLSI) criteria. Water quality was evaluated and compared with standard of water quality criteria. Results and Discussion: The three of most bacterial isolations in Tilapia fish from well-typed fish ponds were Klebsiella pneumoniae, Edwardsiella tarda and coagulase negative Staphylococci. While, K. pneumoniae, Proteus mirabilis, and Streptococcus group D non enterococci were isolated from Tilapia fish, which feeding on river floating baskets. Therefore, only K. pneumoniae and C. albicans were isolated from fry in nursery fish pond. Penicillin and ampicillin resistance were occurred in K. pneumoniae and P. mirabilis. Water qualities of fish water farms were evaluated and almost of water parameters were to water standard quality, except ammonia and alkaline values were higher and lower than reference values, respectively. Conclusions: We were deduced that growing of pathogens in fishes, especially K. pneumoniae may relate to water environment. However, antibiotic resistance of isolated bacteria may concern as zoonotic pathogens.
Keywords: Antibiotic susceptibility test, bacterial isolation, Oreochromis niloticus, tilapia fish
The aquaculture is one of the fastest food-producing sectors, which worldwide contributed for approximately half of all aquatic products for human consumption. Among the farmed aquaculture species, tilapia (Nile tilapia, Oreochromis niloticus, Linn.) is considered as the second of most-popular farmed fish worldwide. The large-scale tilapia production in commercial systems is a purpose for domestic consumption and export. The proportion of Thai tilapia is export to the United States of America, middle-east country, and Europe, which are 35%, 26%, and 18%, respectively. Thai tilapia products are 15,496.1 tons, which gaining values are approximately 31.34 million US dollar., The Southern of Thailand is most fish farming area, which commonly conduct as both growth-out pond (well-typed fish ponds) and cage (floating baskets) culture. The tilapia fish infections included by bacteria, virus and parasites are become major problem for aquaculturists, and their effects may vary depended on environmental and/or biological factors.
Particularly focused on bacterial infections are most commonly occurring and antibiotics are often using in fish farm, which caused to rising of antibiotic resistance rate especially in fishes and water environment of aquatic farms. In addition, mode of “fish-to-fish” bacterial transmission in nursery ponds is usually occur and spread in different environments of fish breeding area from one area to another. Major bacterial infections in tilapia fishes are Aeromonas species, Streptoccoccus agalactiae, Streptococcus iniae, Flavobacterium columnare, and Francisella species., However, the World Organization for Animal Health is reported the zoonotic potential of topically acquired bacterial infections caused by S. iniae in aquatic species and cause invasive diseases in humans. The most of the affected individuals S. iniae infections have been reported from patients in Asia (85%) and 58% of infected patients had history concerning with handled or been exposed to fresh fish. The clinical signs of S. iniae infection are including cellulitis, septicemia, endocarditis, arthritis, meningitis, osteomyelitis, fever and abdominal distension and pneumonia.,,
Tilapia fishes can be growth and survive in different temperature ranges, poor quality water, and low dissolved oxygen where most of the other fish fail to survive.,,,, Manmade activities, such as urban waste, antibiotic production waste, and animal farming are increase the numbers of antibiotic resistance bacteria and antibiotic resistance genes (ARGs) and transfer with water and sediment., Thus, Tilapia fishes can become zoonotic reservoir by environmental exposed it to many potential bacterial pathogens carrying ARGs and serve as reservoirs, which an alternative route for human exposure to clinically important ARG-carrying bacteria. At this point, consumption and handling of tilapia fish may pose a potential health risk. The ARGs bacteria associated with farmed Tilapia had reported in Malaysia, Trinidad, and India.,, Hence, the preliminary bacterial isolation and screening for antibiotic resistances in Thai Tilapia fishes, especially from different types of farming was a need to evaluation and follow-up, because of its rare report of bacterial isolation and drug resistance from farmed tilapia fished in Thailand where high yield of tilapia fish products and export.
This study was aimed to isolated and identified of human pathogens in Tilapia fishes (O. niloticus) from three types of aquacultures including 4 of well-typed fish ponds, 2 of floating baskets and 1 of nursery fish pond (totally seven fish farms) at Surat Thani, Thailand during the breeding season (September–December 2016), which have many tilapia juvenile species and had high hatching rates. The isolated bacteria were determined for antibiotic or drug susceptibility. However, this study period was rainy season in Southern part of Thailand and flooding may cause affect, thus, water quality of fish farms was also continuing monitored and interpreted with compared to standard of water quality recommended by Thai government. The ineffective antibiotics using may become the problem of aquaculture farms especially tilapia fish farms and our results from this study may provide the information to suggest and awareness for an appropriate antibiotic used in types and in doses, which was prevent the raising of antibiotic residues to cause resistance in bacteria by genetically genes transfer. A good aquaculture practices (GAP) is become important for Thai aquaculturist by Thai government promotion, and our results were also supported one of GAP recommendation, which was good practice for minimize disease outbreaks and along with proper treatment for reduce pathogens and animal (fish) stress.
Fish sample collection and preparation
Adult and fry (only from nursery fish farm) tilapia fishes were collected from seven aquacultures, Surat Thani, Thailand, including three of well-typed fish ponds (farm 1-3) at Thachana district and one of well-typed fish pond (farm 4) at Thachana district; two of floating baskets (farm 5 and 6) at Punpin district; and one of nursery fish pond at Thachana district (farm 7). Thirty of adult tilapia fishes were collected from each farm (farm 1–6) and swabbing of internal organs included (liver, kidney, and brain) were done and took on Stuart transport medium (Oxoid ™, UK), and thirty of whole fry fishes were grinded and then swabbing same as adult fish samples. All sample preparations (n = 210) were done by sterile technique and icepack-chill transport medium tubes were sent back to laboratories at Bangkok within 24–48 h.
Bacterial isolation and identification
Bacterial isolation and identification from the fish swabbed samples were carried out at microbiological laboratory unit, Faculty of Science and Technology, Suan Sunandha Rajbhat University. Briefly, swab samples were streaked and cultured with blood, MacConkey, and chocolate agars at 37°C, 24 h (for yeast or Candida specie was used potato dextrose agar, PDA agar). Bacterial isolation and identification were performed by colony morphology, Gram staining, and biochemical tests, which were explained by Bergey’s Manual of Systematic Bacteriology. The use of biochemical tests, such as, catalase, oxidase, coagulase, TSI, citrate, lysine indole motility, ornithine decarboxylase, Methyl red-Vogeprokauer, Mannitol, Growth 0% NaCl, Growth 6.5% NaCl, and Bile esculin test, which were interpretation for bacterial genus and species identification according by Clinical and Laboratory Standards Institute (CLSI) guideline.
Antimicrobial susceptibility testing
Antibiotic susceptibility testing was also performed by CLSI criteria, which used agar disc diffusion method (Kirby–Bauer test). Each isolated bacteria was inoculated and cultured in TSB broth at 37°C for 3–4 h and bacterial cell concentration was approximately 108 CFU/mL (0.5 McFarland turbidity standard or OD = 0.08-0.05 from spectrophotometer). 0.1 ml of each broth culture was spread on the Mueller-Hinton agar plate and allows the plate to dry for approximately 5 min. Use an antibiotic disc dispenser to dispense discs containing specific antibiotics onto the plate and incubated at 37°C for 18–24 h. Ten of antibiotic discs (Difco, USA) including amikacin, ampicillin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, penicillin, sulfamethoxazole, tetracycline, and norfloxacin were used in susceptibility testing for each isolated bacteria. Interpretation of this test was depended on diameter (mm) of transparent (clear) zone around testing disc and compared to the CLSI standard, which were interpreted to sensitive or susceptible (S), intermediate (I), and resistance (R).
Fish pond water collection and water quality assessment
The each water samples from fish farms were collected from three points (top, middle, and bottom) in triplicate and mixed of water samples (1000 mL) evaluated for surface water standard quality, including dissolve oxygen, temperatures, pH, alkalinity, hardness, ammonia, total suspended solids, nitrite, nitrate, and phosphate according by Standard Method of the Examination of Water and Wastewater.
According to bacterial isolation and results of biochemical tests [Table 1] were isolated and identified eight of bacteria and one of yeast. The numbers of isolated bacteria in tilapia fishes from well-typed fish ponds (farm 1–4) were 25, 8, 5, 2, 2, and 1 isolated for Klebsiella pneumonia, Edwardsiella tarda, coagulase-negative Staphylococci, Staphylococcus aureus, E. coli, and Candida albicans, respectively. Bacteria isolated from Tilapia fishes, which feeding on river floating baskets, were K. pneumoniae, Proteus mirabilis, Bacillus sp. and Streptococcus Group D nonenterococci. Therefore, only K. pneumonia and C. albicans were isolated from fry samples in nursery fish pond [Table 2]. Thus, K. pneumonia was the most of isolated bacteria from all types of fish farms. Interestingly, isolated bacteria were commonly positive from liver and only few of them were isolated from kidney and brain (data not shown). K. pneumoniae and P. mirabilis were resisted to penicillin and ampicillin by 4–8 and 5–8 mm of clear zone diameters [Table 3], which may concerned as antibiotic resistance bacteria and/or ARGs reservoir. The water qualities of seven fish farms were represented as mean ± standard deviation and compared with standard criteria [Table 4], which were almost acceptable. However, ammonia values were higher than reference values (farm 1, 2, 4 and 7), which may cause by accumulation of food remaining precipitate and fish excretes; and alkaline values were lower than reference values, (farm 1–6), which may affected from heavy flooding in rainy season.
This surveillance on microbial infections (or colonization) in farmed tilapia fishes was revealed eight of bacteria including for K. pneumonia, P. mirabilis, E. tarda, coagulase-negative Staphylococci, S. aureus, E. coli, Bacillus species and Streptococcus Group D nonenterococci, and one of yeast (C. albicans). Some of them, we can ignore as contamination, such as coagulase-negative Staphylococci and S. aureus and as commonly finding in water environment, such as, E. coli, Bacillus species and Streptococcus Group D nonenterococci. However, K. pneumoniae and P. mirabilis were need to concerned as fish and also human pathogens, which can be antibiotic resistance bacteria and/or ARGs reservoir as corresponded to results from previous studies.,,K. pneumonia and other pathogens were better growth in tilapia fishes that living in fish ponds rather than in river floating baskets. However, there is no significance different of all kinds of fish farms in this study, which may due to rainy season and flooding effects.
K. pneumoniae being ubiquitous in nature encounters wide differences in environmental condition, which abundance in natural water reservoirs exposed to temperature variation forms the basis of its persistence and spread in the soil and other farm produce. Significant up-regulation of genes encoding ribosomal proteins at 20 and 50°C possibly suggest their role in the survival of K. pneumoniae cells under low- and high-temperature stress. Hence, changing of temperature and water conditions in Thai fish farms may induce gene up-regulation of K. pneumoniae. K. pneumoniae is a common cause of serious Gram-negative infections in humans, including pneumonia, urinary tract infections; wound infections, bacteremia in humans. Also in aquatic animals, such as hemorrhage and red spottiness along the body of Cyprinus carpi; skin discoloration with ulcer and exopthalmia in Nemipterus japonicus,O. niloticus, skin hemorrhages in Amphiprion nigripes.
Due to our results were found antibiotic resistance K. pneumonia and P. mirabilis, which may prove and confirmed that isolates of K. pneumonia are becoming increasingly resistant to more kinds of antibiotics and subsequently may become even more difficult to treat or antibiotic resistance. Clinically important genes, such as extended-spectrum beta-lactamases (ESBLs) and integrons, are increase in abundance in the receiving rivers, downstream of the water treatment.,, ESBLs confer resistance is a wide variety of beta-lactam antibiotics in pathogens commonly member in Enterobacteriaceae and there are difficult to treat by normal medication , and low immune person, i.e., cancer patients. Integrons are mobile genetic elements responsible for integration and expression of gene cassettes. ESBLs and integrons relating genes are often associated to drug resistance and considered as markers of horizontal gene transfer potential of a bacterial strain., ESBLs  and integron  are reported in bacterial strains isolated from cultured fish. They may transfer from fish to humans and other predators serve as a route for the spread of ARGs. The presence of antibiotic resistance markers such as class 1 and class 2 integrons; ESBLs– bla CTX-M, bla SHV, bla OXA, and aac (6′)-Ib–cr genes had been reported from Indian natural tilapia fishes  and farmed Tilapia.,
We were deduced that growing of pathogens in fishes, especially K. pneumoniae may relate to water environment. Antibiotic resistance of isolated K. pneumoniae and P. mirabilis may concern as zoonotic pathogens and further molecular analysis for drug resistance genes was interesting topic. We were interested to focus on horizontal gene transfer K. pneumonia may carry ARGs and transfer to P. mirabilis by multiplex-PCR technique as we done on the study of gene transfer in different Vibrio species of aquatic animals.
The authors express their sincere appreciation to Research and development institute, Suan Sunandha Rajabhat University, Bangkok, Thailand for the grant support of this work. We would like to sincerely thank the Research Division, Faculty of Science and Technology, Suan Sunandha Rajabhat University for partial research facility support.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]