Characterization of integrons and antibiotic resistance determinants in Aeromonas spp. isolated from freshwater fish culture in east coast Malaysia

Aquaculture has become the main source of protein for the 30.75 million people of Malaysia. The major concern in fish farms industries is bacterial infections including Aeromonas spp. Aeromonas contains class 1 integrons associated with plasmid that facilitate the emergence and dissemination of antibiotic resistance among bacteria population in aquaculture environments. Antimicrobials have been used for disease prevention and treatment, as well as growth promoters in animal farming, including aquaculture. The aim of this study is to isolate Aeromonas bacteria from freshwater fish species including tilapia and catfish which are the main industries in Malaysia, to determine the presence of drug resistance genes, integrons, its associated genes and possible horizontal transfer of these integron genes among the bacteria species in the aquaculture environments. A total of 334 freshwater fish samples consisting of red hybrid tilapia (Oreochromis spp.), African catfish (Clarias gariepinus) and Pangasius catfish (Pangasius hypopthalmus) were collected from nine farms in East Coast Malaysia (Kelantan, Terengganu and Pahang). Aeromonas spp. were identified by biochemical test and confirmed by polymerase chain reaction (PCR) using 16SrRNA gene. Ninety-two isolates of Aeromonas spp. were obtained by direct streaking of the kidney samples onto Rimler Shotts agar and trypticase soy agar. The isolates were then subjected to antibiotic sensitivity test against 14 antibiotics using disc diffusion method and multiple antibiotic resistance (MAR) index was calculated. Among the Aeromonas bacteria isolated, 38 isolates (41.3%) were Aeromonas veronii, 28 isolates (30.4%) were Aeromonas jandaei, 14 isolates (15.2%) were Aeromonas sobria and 12 isolates (13.0%) were Aeromonas hydrophila. Aeromonas isolates showed the highest resistance to ampicillin (96.7%) followed by streptomycin (62.0%), neomycin (47.8%), nalidixic acid (45.7%), kanamycin (34.8%), oxytetracycline (30.4%), tetracycline (29.3%), sulphamethoxazole (14.1%), gentamycin (8.7%), ciprofloxacin (6.5%), norfloxacin (5.4%), chloramphenicol (3.3%), nitrofurantoin (3.3%) and doxycycline (2.2%). Furthermore, MAR index value ranging from 0.07 to 0.64 with 70.7% of isolates were higher than 0.2. These results suggest that Aeromonas isolates have been continually exposed to the antibiotics. The presence of integrons, integron associated genes, and antibiotic resistance genes were also examined by PCR. An intI1 gene was detected in six of the isolates (6.5%) and none of the isolate has intI2 and intI3 genes. Two isolates harboured the inserted resistance gene cassettes in variable regions including aac(6’)II-blaOXA-21-catB3 in A. veronii (T1K6) and dfrA1-orfC in A. jandaei (K2K11) isolates. Tetracycline resistance genes, tetA and tetE were detected in 30 (32.6%) and 7 (7.6%) isolates, respectively. The blaTEM and blaSHV genes were found in 54 (58.7%) and 14 (15.2%) isolates, correspondingly. The strA-strB gene was found in 3 (3.3%) isolates, aadA gene in 8 (8.7%) isolates and sul1 gene in 11 (12.0%) isolates. One isolate contains complete structure of integrons with gene cassettes, T1K6 (A. veronii) was tested for their transferability into E. coli DH5α and E. coli ATCC 25922 recipients. The transformation experiment was done using heat shock method and natural conjugation using filter paper method. It was found that the class 1 integrons can be transferred horizontally via the natural conjugation in E. coli ATCC 25922 and transformation in E. coli DH5α. The findings of this study indicate that freshwater fish culture could be a significant reservoir for Aeromonas spp. Multidrug resistance (MDR) Aeromonas and antibiotic resistance integron gene can be transferred horizontally from one bacterium to other bacteria species. Thus, this study has proved that an environmental pool of antibiotic resistance genes in which some of the genes can be transferred horizontally among bacteria species can pose a potential risk to public health. The data provided from this study is important for the effective control or treatment of bacterial infection and could act as a wake-up call to certain authorities to change existing policies.