Prevalence, antibiotic susceptibility, and presence of drug resistance genes in Aeromonas spp. isolated from freshwater fish in Kelantan and Terengganu states, Malaysia

Abstract Background and Aim: The emergence of antibiotic-resistant bacterial pathogens has been increasingly reported, which has resulted in a decreasing ability to treat bacterial infections. Therefore, this study investigated the presence of Aeromonas spp., including its antibiotic resistance in various fish samples, Oreochromis spp., Clarias gariepinus, and Pangasius hypophthalmus, obtained from Kelantan and Terengganu, Malaysia. Materials and Methods: In this study, 221 fish samples, of which 108 (Oreochromis spp., n=38; C. gariepinus, n=35; and P. hypophthalmus, n=35) were from Kelantan and 113 (Oreochromis spp., n=38; C. gariepinus, n=35; and P. hypophthalmus, n=40) were from Terengganu, were caught using cast nets. Then, samples from their kidneys were cultured on a Rimler Shott agar to isolate Aeromonas spp. Polymerase chain reaction (PCR) was used to confirm this isolation using specific gene primers for species identification. Subsequently, the isolates were tested for their sensitivity to 14 antibiotics using the Kirby–Bauer method, after which the PCR was conducted again to detect resistance genes: sul1, strA-strB, aadA, blaTEM, blaSHV, tetA-tetE, and tetM. Results: From the results, 61 isolates were identified as being from the genus Aeromonas using PCR, of which 28 were Aeromonas jandaei, 19 were Aeromonas veronii, seven were Aeromonas hydrophila, and seven were Aeromonas sobria. Moreover, 8, 12, and 8 of A. jandaei; 4, 3, and 12 of A. veronii; 6, 0, and 1 of A. hydrophila; and 3, 3, and 1 of A. sobria were obtained from Oreochromis spp., C. gariepinus, and P. hypophthalmus, respectively. In addition, the isolates showed the highest level of resistance to ampicillin (100%), followed by streptomycin (59.0%), each kanamycin and nalidixic acid (41.0%), neomycin (36.1%), tetracycline (19.7%), sulfamethoxazole (14.8%), and oxytetracycline (13.1%). Resistance to gentamicin and ciprofloxacin both had the same percentage (9.8%), whereas isolates showed the lowest resistance to norfloxacin (8.2%) and doxycycline (1.6%). Notably, all Aeromonas isolates were susceptible to chloramphenicol and nitrofurantoin. Results also revealed that the multiple antibiotic resistances index of the isolates ranged from 0.07 to 0.64, suggesting that the farmed fish in these areas were introduced to the logged antibiotics indiscriminately and constantly during their cultivation stages. Results also revealed that the sul1 gene was detected in 19.7% of the Aeromonas isolates, whereas the tetracycline resistance genes, tetA and tetE, were detected in 27.9% and 4.9% of the isolates, respectively. However, β-lactam resistance genes, blaTEM and blaSHV, were found in 44.3% and 13.1% of Aeromonas isolates, respectively, whereas strA-strB and aadA genes were found in 3.3% and 13.1% of the isolates, respectively. Conclusion: This study, therefore, calls for continuous surveillance of antibiotic-resistant Aeromonas spp. in cultured freshwater fish to aid disease management and better understand their implications to public health.


Introduction
Aquaculture plays an important role in the food supply of Malaysia. Under the Economic Transformation Program, the Malaysian government established aquaculture as one of the key thrust areas for the agro-food industry [1]. In 2014, Malaysia's annual per capita fish intake was one of the highest in Asia at 56.5 kg, with tilapia (Oreochromis spp.) and African catfish (Clarias gariepinus) being the favored farmed fish. Interestingly, in freshwater aquaculture, the African (C. gariepinus) and Pangasius (Pangasius hypophthalmus) catfishes being produced are leading because of a higher local demand, followed by tilapia (Oreochromis spp.), which is small in terms of production and was valued at RM223,000 (USD 58,000) [1].
Antimicrobials have progressively been used in animal farming for disease prevention and treatment over the past few years, including as growth promoters [18]. However, their usage is based on modern medicine; the misuse of these antibiotics has increased the risk of emerging antimicrobial resistance cases in pathogenic and nonpathogenic bacteria. This has resulted in the lower treatment potency of commonly used antimicrobials in treating diseases, such as tuberculosis, pneumonia, and gastrointestinal infections, in humans [19]. In addition, during animal farming, antimicrobial deposits have been discovered in terrestrial, freshwater, and marine habitats close to agriculture and aquaculture facilities [20,21]. Antimicrobials are also applied in the feed or directly to water in aquaculture systems. Thus, they are proposed to subsequently be disposed into the environment by run-off water, sedimentation of feces, or uneaten feed pellets that can then be eaten by local fish or invertebrates [21][22][23][24][25]. The unconstrained use of antimicrobials in aquaculture can therefore transmit antibiotic-resistant bacteria, which are commonly transferred through R plasmids, with fish bacteria acting as intermediates [18,20,22,[26][27][28][29][30][31].
Therefore, this study investigated the presence of Aeromonas spp., including its antibiotic resistance in various fish samples, Oreochromis spp., Clarias gariepinus, and Pangasius hypophthalmus, obtained from Kelantan and Terengganu, Malaysia.

Ethical approval
The study was approved by the Institutional Animal Care and Use Committee (IACUC), Faculty of Veterinary Medicine, University Malaysia Kelantan (UMK/FPV/ACUE/PG/4/2019).

Study period and location
This study was conducted from February 2019 to December 2019. Samples were taken from three freshwater fish farms, each in state of Kelantan and Terengganu. In Kelantan, the farms located in Tumpat, Kota Bharu and Bachok. In Terengganu, two farms located in Kuala Terengganu and one farm in Hulu Terengganu. All the samples were processed in situ with an aseptic technique.

Sample collection
Here, 221 freshwater fish were collected, with 108 samples from Kelantan and 113 samples from Terengganu. Of the 108 fish samples from Kelantan, 38 were red hybrid tilapia (Oreochromis spp.), 35 were African catfish (C. gariepinus), and the remaining 35 were Pangasius catfish (P. hypophthalmus). However, of the 113 fish samples collected from Terengganu, 38 were Oreochromis spp., 35 were C. gariepinus, and 40 were P. hypophthalmus. Next, a specimen of the kidneys was collected from these fish.

Bacterial isolation and identification
The specimen was inoculated on Rimler Shott agar (RSA) (HiMedia, India) supplemented with novobiocin antibiotics and incubated at 30°C for 24 h. Next, yellow colonies on RSA were chosen and further sub-cultured on Trypticase soy agar (TSA) (Oxoid, Hampshire, UK) for purity. Subsequently, morphological and biochemical tests were used to identify all isolates, such as Gram staining, oxidase, catalase, and motility tests, after which the biochemical characteristics of Aeromonas spp. were examined using the analytical profile index 20E kit (bioMerieux, France) according to the manufacturer's instructions. Finally, the strip was incubated at 30°C for 24 h.

Confirmation of Aeromonas spp. using polymerase chain reaction (PCR) assay
Genomic DNA was extracted using the Bacterial Genomic DNA kit (Geneaid, USA) following the manufacturer's instructions. To determine the presence of Aeromonas spp., a PCR assay was then conducted using 16S rRNA and a specific gene [32]. Next, PCR amplification was conducted using a Mastercycler gradient (Bio-Rad, USA). A final PCR volume of 25 μL containing 12.5 μL Go Taq ® Green Master Mix (Promega, USA), 1 μL of each 10 ρmol forward and reverse primers, and 2 μL DNA template were used. The conditions for thermocycling were set as follows: 94°C for 3 min, 35 cycles of 94°C for 60 s, 58°C for 60 s, 72°C for 1.5 min, and a final extension at 72°C for 3 min. Finally, amplified products were electrophoresed on 2.0 % agarose gels, after which the gels were visualized and captured using GelDoc (Bio-Rad).
MAR index was calculated using the formula provided by Sarter et al. [34]: Where, X=Total cases of antibiotic resistance; Y=Total number of isolates; Z=Total number of isolates The MAR index value of equal to, or less than, 0.2 was defined as antibiotics that were seldom or never used.

Detection of associated drug resistance genes
Resistance genes were detected using PCR amplification with the different primers as described in Table-1 [35][36][37][38][39][40][41]. Assays were then conducted in 25 μL volume mixtures, according to the manufacturer's protocol (Promega, USA). Next, all PCR reactions were subjected to amplification according to the cycling parameter suggested by a previous researcher (Table-1). Finally, PCR products were run on 2.0% agarose, after which the gel was visualized and captured using Gel Doc (Bio-Rad).

Results
From the results, 61 isolates obtained from freshwater fish samples were identified as genus Aeromonas using PCR. Table-2 shows that from the 61 Aeromonas spp. isolated, 22 isolates were from P. hypophthalmus, 19 from Oreochromis spp., and 20 from C. gariepinus. Furthermore, Aeromonas species isolated from freshwater fish in Kelantan were higher (43 isolates) than those from Terengganu (18 isolates). Figure-1 shows the confirmed identification using the PCR assay of Aeromonas spp. The positive isolates for the 16S rRNA gene were then sent for sequencing. Figure-2 shows the distribution of Aeromonas species     Figure-3 shows Aeromonas spp. colonies formed on TSA, which were creamy in color, round, and convex, whereas Aeromonas colonies on RSA were yellow-green in color, round, and convex. The biochemical test results from Aeromonas spp. isolates revealed Gram-negative staining, rod-shaped, motile, fermentative, oxidase-positive, catalase-positive, and indole negative characteristics.
In addition, all Aeromonas isolates displayed varying trends of resistance, where all isolates were ampicillin-resistant (100%), followed by streptomycin (59.0%), kanamycin and nalidixic acid with the same percentage (41.0%), neomycin (36.1%), tetracycline (19.7%), sulfamethoxazole (14.8%), and oxytetracycline (13.1%). Gentamicin and ciprofloxacin both had the same percentage resistance (9.8%), whereas norfloxacin (8.2%) and doxycycline (1.6%) had the lowest (Figure-4). However, all Aeromonas isolates were sensitive to chloramphenicol and nitrofurantoin. Figure-5 shows the presence of antibiotic resistance genes in Aeromonas isolates. Results showed that the sul1 gene (related to sulfonamide resistance) was detected in 19.7% of the Aeromonas isolates. However, for tetracycline resistance genes, only tetA and tetE were detected in 27.9% and 4.9% of isolates, respectively. In addition, the β-lactam resistance genes, bla TEM and bla SHV , were found in 44.3% and 13.1% of Aeromonas isolates, respectively, whereas the strA-strB gene (related to streptomycin resistance) was found in 3.3% of the isolates, and the aadA gene (related to streptomycin and spectinomycin resistance) in 13.1% of the isolates. Table-3 shows the resistance phenotype and antibiotic resistance genes of all Aeromonas spp. isolates.

Discussion
H 2 S production is one of the features of Aeromonas spp. pathogenic piscine strains [42]. Shotts and Rimler [43] indicated that Aeromonas spp. grown on an RSA medium formed yellow colonies; however, the colonies with black centers had to be tested for oxidase production to exclude the probability of Citrobacter spp. or other species of bacteria. MAS is broad, which includes A. hydrophila and several species of Aeromonas that have been reported to be risks to freshwater fish in aquaculture [13,14,44,45]. Motile aeromonad infections are possibly the most significant bacterial infection in freshwater fish. They are also discovered regularly in the intestines and gills of freshwater fish. Therefore, in this study, bacteria of the genus Aeromonas were isolated from the kidneys of red hybrid tilapia (Oreochromis spp.), including African (C. gariepinus) and Pangasius (P. hypophthalmus) catfishes obtained from the states of Kelantan and Terengganu in Malaysia.   Available at www.veterinaryworld.org/Vol.14/August-2021/11.pdf   Furthermore, among the 61 isolates from the genus Aeromonas isolated, 28 isolates were A. jandaei, 19 were A. veronii, and seven isolates were A. hydrophila and A. sobria, respectively. These results are in agreement with those observed in earlier studies by Radu et al. [46] that found A. veronii, A. sobria, A. hydrophila, and A. caviae in the market fish samples from Selangor state in Malaysia. In India, A. hydrophila has also been isolated from fish obtained from retail shops [47]. In addition, Ashiru et al. [48] isolated A. hydrophila, A. caviae, and A. sobria in catfish (Clarias betrachus) and tilapia fish (Tilapia nilotica) obtained from the Makoko market in Nigeria. The authors reported that A. caviae was predominant in tilapia fish, whereas A. hydrophila and A. sobria were predominant in catfish. Other studies have also reported that A. jandaei is pathogenic to aquaculture fish, such as European eels (Anguilla anguilla) [49] and Pangasius catfish (P. hypophthalmus) [50]. Besides, other studies have shown that A. veronii infected high numbers of fish, such as Chinese long snout catfish (Leiocassis longirostris) [51], loach (Misgurnus anguillicaudatus) [45], Oscar (Astronotus ocellatus) [52], and tilapia (Oreochromis spp.) [13,14,44]. This bacterial genus attacks catfish, which is among the main freshwater resources, and infects all sizes of fish as well, which can lead to death and result in big losses of freshwater fish production [53].
Aeromonas genus generates single or MARs rapidly, indicating that this genus is an effective marker of antimicrobial resistance in the freshwater aquaculture system [54]. The MAR index varying from 0.07 to 0.64 with 60% (37/61) of the isolates have MAR indices of more than 0.2 (high-risk source of contamination), suggesting that the Aeromonas spp. in these farmed fish have been exposed to a high level of antibiotics during the cultivation processes, which can result in the development of antibiotic resistance among the bacteria isolates. Results from this study prove this fact, which revealed a high level of multidrug resistance (MDR) among the isolates tested (Table-3). However, the percentage of MAR index of more than 0.2 in this study (60%) was much lower than that obtained from the study by Odeyemi and Ahmad [55] in Aeromonas spp., isolated from 53 aquatic samples in Melaka, Malaysia (100%). This result indicates that the indiscriminate use of antibiotics in West Coast Malaysia (Melaka) is higher than in East Coast Malaysia (Kelantan and Terengganu). High resistance of MDR due to Aeromonas spp. has also been reported as serious public health pathogens that cause gastroenteritis, septicemia, and skin infections in humans, which enter the human body through contaminated food and water consumption, including skin lesions [56].
In this study, all Aeromonas isolates were highly ampicillin-resistant. A previous study reported that these Aeromonas species acquired β-lactams resistance through the expression of chromosomal lactamases [57]. This finding is also proposed to be due to a high intrinsic β-lactam resistance, which is enhanced by an active efflux mechanism or cooperation through external membrane impermeability or secondary resistance mechanisms known as β-lactamases or antibiotic efflux pumps [54,57,58]. Furthermore, resistance rates to tetracycline, oxytetracycline, streptomycin, kanamycin, nalidixic acid, neomycin, sulfamethoxazole, ciprofloxacin, and gentamicin have also been recorded, which is suggested to be due to the extensive consumption of such antimicrobials in the ornamental fishery [59,60]. All isolates were also susceptible to chloramphenicol and nitrofurantoin. This observation is due to that these antibiotics were banned in Malaysia for use in treating aquatic animal diseases [61]. Several antibiotics were completely banned as well for food animals and aquaculture in Malaysia because of serious toxicity and the development of antibiotic-resistant bacterial strains, such as avoparcin, chloramphenicol, nitrofurans (i.e., nitrofurantoin, nitrofurazone, furazolidone, and furaltadone), teicoplanin, vancomycin, and norfloxacin [61,62].
Furthermore, in this study, no trends of significant antibiotic resistance specific to the fish species were observed. The current findings follow other research on MDR occurrence from aquatic habitats and seafood samples in Aeromonas spp. [63,64]. These classes of antibiotics are broadly used worldwide as Available at www.veterinaryworld.org/Vol.14/August-2021/11.pdf well, particularly in developing countries in Asia, because of their low cost and diverse-spectrum antimicrobial activity, which increases the chances for any bacterial species to develop resistance to these antibiotics [65,66].
The presence of resistance genes was also detected in several of the isolates during this study, including those encoding resistance to ampicillin (bla TEM and bla SHV ), streptomycin (aadA and strA-strB), and tetracyclines (tetA and tetE). The present findings agree with earlier studies where tetA genes were the most significant determinants of tetracycline resistance and have commonly been observed in Aeromonas spp. [39,67]. Increased resistance to β-lactam antimicrobials (penicillins and derivatives, cephalosporins, carbapenems, and monobactams) through the existence of genes that code for the formation of β-lactamase has also been reported [68]. In addition, Jones-Dias et al. [69] mentioned that in Aeromonas spp., three main β-lactamases were identified: Metallo-β-lactamase Class B, cephalosporinase Class C, and penicillinase Class D. Fosse et al. [70] have also categorized the β-lactamases into five (i*v) groups of Aeromonas species: (i) The A. hydrophila complex strains exhibiting Classes B, C, and D β-lactamases; (ii) the A. caviae strains exhibiting Classes C and D β-lactamases; (iii) the A. veronii strains identifying Classes B and D lactamases; (iv) the Allium schubertii strains recognizing Class D lactamases; and (v) the Aeromonas trota strains containing Class C β-lactamases. It is also suggested that several isolates of A. veronii biovar sobria do contain a class C cephalosporinase [58]. Therefore, because of the presence of β-lactamase genes, increased resistance to β-lactam antibiotics was reported in the Aeromonas genus [4,68,71,72].

Conclusion
This study has identified several Aeromonas spp. that are resistant to several types of antibiotics in freshwater fish from Kelantan and Terengganu states, with 60% (37/61) of the isolates having a MAR index of more than 0.2. The result suggests that aquaculture waste deposited into the aquatic ecosystems is one of the factors that enhance the incidence of aeromonad MDR in the river water. The presence of Aeromonas species in freshwater fish can thus be a major problem if the fish is not cooked properly. This drug resistance has become a major public health concern since these fish species (Oreochromis spp., C. gariepinus, and P. hypophthalmus) are important sources of aquatic proteins consumed in Malaysia. Therefore, regular surveillance for antibiotic resistance of Aeromonas spp. should be conducted among freshwater fish. Finally, more intensive studies should discover the specific existence of antibiotic resistance in Aeromonas spp., including the levels of antibiotics that affect their resistance profile.

Authors' Contributions
NNFNMF: Designed the study and drafted the manuscript. MM, RHH, AI, AAMZ, and NFAM: Data analysis. RHH and MM: Direction and supervision of the study. All authors read and approved the final manuscript.