Antibiotic resistance of Aeromonas hydrophila isolated from diseased catfish. Sarakham University, Maha Sarakham, Thailand 44000

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1 Antibiotic resistance of Aeromonas hydrophila isolated from diseased catfish Chutharat Kanchan a,*, Puttachat Imjai a, Nukoon Kanchan b and Leklai Chantabut a a Aquaculture Technology Program, Faculty of Agricultural Technology, Rajabhat Maha Sarakham University, Maha Sarakham, Thailand b Animal Production Technology Program, Faculty of Agro-Industrial Technology, Kalasin University, Kalasin, Thailand * Corresponding author. Tel.: ; fax: address: cmunchan@yahoo.com 1. Abstract The antibiotic susceptibility test of 3 strains of Aeromonas hydrophila isolated from diseased catfish was determined by disk diffusion method. Thirteen antibiotic drugs were investigated namely amoxycillin, ampicillin, cefoxitin, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, kanamycin, novobiocin, oxytetracyline, norfloxacin, sulphamethoxazole-trimethoprim and trimethoprim. All strains of A. hydrophila were resistant to amoxycillin, novobiocin and trimethoprim, however, all bacterial strains were sensitive to cefoxitin and gentamicin. Multi-resistance patterns were observed in other drugs (8 out of 13 drugs) which showed 66.67% of resistance. It can be concluded that gentamicin was a best drug for choosing to treatment of A. hydrophila in diseased catfish. Keywords: Aeromonas hydrophila, antibiotic drugs, catfish

2 2. Introduction Catfish (Clarias spp.) is an economically major species of freshwater fish in Thailand. In the present time, an intensive fish culture system is mainly popular for industrial production. This system is due to high populations of fish densities then fish are risk to becoming a diseases which caused by a various pathogens such as parasite, fungi, virus and bacteria. (Kanchan et al. 2014). The bacterial infection caused by Aeromonas hydrophila has so far been documented in several fish species including catla (Catla catla), rohu (Labeo rohita), mrigal (Cirrhinus mrigala), gold fish (Carassius auratus), anabas (Anabas testidenus) and clarias (Clarias betrachus), catfish and Channa species (Channa punctatus and Channa marulius) (Samal et al. 2014), catfish (Clarias gariepinus) (Laith and Najiah, 2013), tilapia (Tilapia nilotica) and catfish (Clarias betrachus) (Ashiru et al. 2011), carp (Stojanov et al. 2010), ornamental fish (Jongjareanjai et al. 2009) and Piaractus mesopotamicus and Oreochromis niloticus (Belém-Costa1 and Cyrino, 2006). Therefore, A. hydrophila is an important bacterial pathogen in fish species from the previous reports. The damage of fish populations are usually occurred by bacterial diseases in private farm and aquaculture industry. To control and treatment of the bacteria pathogens are mainly used the antibiotic drugs in all period of the fish such as in the fingerling until the adult fish. The present study was aimed to determine the antibiotic resistance against the A. hydrophila which isolated from diseased catfish. 3. Material and methods Sampling of diseased fish Five fish samples were collected from local farm culture Kosumpisai District, Maha Sarakham Province during October to December Catfish were average 2.21 g in body weight and 5.33 cm in body length and then transported to the laboratory of aquaculture technology program for bacterial investigations.

3 Isolation and identification of bacteria Kidneys and liver from diseased fish were aseptically collected into Tryptic Soy agar (TSA, Difco, USA) plates and incubated at 30 C until pure cultures were obtained. Purified cultures were inoculated onto TSA plates and kept at 4 C for stock. For identification of the isolates; the basic tests namely Gram's stain, morphology, oxidase test and catalase were observed. Further identification was performed using the commercial API 20E (biomerieux, France). Finally, A. hydrophila strain was chosen for antibiotic sensitivity test. Antibiotic susceptibility testing Antibiotic susceptibility testing was determined against A. hydrophila which isolated from diseased catfish by the disc diffusion method. Thirteen antibiotic drugs including amoxycillin (AML), 10µg; ampicillin (AMP), 10µg; cefoxitin (FOX), 30 µg; chloramphenicol (C), 30µg; ciprofloxacin (CIP), 5µg; erythromycin (E), 15µg; gentamicin (CN), 10µg; kanamycin (K), 30µg; novobiocin (NV), 30µg; oxytetracyline (OT), 30µg; norfloxacin (NOR), 10µg; sulphamethoxazole-trimethoprim (SXT), 25 µg and trimethoprim (W), 5µg were tested against the bacteria which performed according to the Clinical Laboratory Standards Institute (CLSI, 2008). The inhibition zone diameter of each drug was recorded by vernia caliper measurement. 4. Results The three strains of A. hydrophila were all resistant to amoxicillin, trimethoprim and novobiocin. Furthermore, multi-resistance profile of all strains of A. hydrophila showed 66.67% against ten antibiotics namely ampicillin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, oxytetracycline, norfloxacin and sulphamethoxazole-trimethoprim. However, all strains were susceptible to cefoxitin and gentamicin as shown in Table 1.

4 Table 1 Antibiotic susceptibility and resistance of 13 drugs against A. hydrophila Antibiotic Disk content of A. hydrophila A. hydrophila A. hydrophila antibiotic (µg) strain 1 strain 2 strain 3 AML 10 R R R AMP 10 S R R FOX 30 S S S C 30 S R R CIP 5 S R R E 15 S R R CN 10 S S S K 30 S R R NV 30 R R R OT 30 S R R NOR 10 S R R SXT 25 S R R W 5 R R R Remark: S, susceptible; R, resistant 5. Discussion The bacterial diseases caused by A. hydrophila have been documented in many fish species (Samal et al. 2014; Laith and Najiah, 2013; Ashiru et al. 2011; Stojanov et al. 2010). The antibiotic resistance has also been determined in several authors to control or treatment of the diseased fish in aquaculture and to check the development of resistant bacteria as well. From this study, three strains of A. hydrophila were resistant to amoxicillin, novobiocin and trimethoprim which classified into three groups of antibiotic drugs including penicillin, aminocoumarin and sulphanamides (Orozoca et al.2010). As was aforementioned that all strains showed resistant to amoxycillin which conformed to previous researchers such as Belém-Costa and Cyrio (2006) has been reported of A. hydrophila isolated from pacu and tilapia in Brazil. Moreover, Ho et al. (2008) has been

5 revealed that 2 strains of A. hydrophila isolated from tra catfish in Vietnam showed resistant to amoxycillin. The resistance of all strains to novobiocin in this study was similar to some authors which have been investigated the antibiotic susceptibility against the A. hydrophila isolated from various kinds of fish in many countries for instance in Bulgaria (Orozoca et al.2010), in Brazil (Belém-Costa and Cyrio, 2006) and in China (Xia et al. 2004). In the other hand, all bacterial strains in this study showed high sensitive to gentamicin (100%) which corresponded with Belém-Costa and Cyrio (2006), (Orozoca et al.2010) and Ashiru et al. (2011). So far, many researchers were concerned to study the development of antibiotic resistance in fish and environments. The major antimicrobial drugs have so far been used in aquaculture for example sulphonamides, penicillins, macrolides, quinolones, phenicols and tetracyclines (Sapkota et al. 2008). In this study, the antibiotic drugs as mentioned above were overall investigated against A. hydrophila isolated from catfish. The multiresistance pattern of A. hydrophila was observed in several drugs. The inappropriate use of antibiotics is leading to increase of incidence of high bacterial resistance in fish or environment (Asari et al. 2011). For alternation to reduce the antibiotic resistance of bacterial pathogens in fish or environments have to play concern in a good management of aquaculture and to change a rotation of antibiotic drugs in fish treatment (Miranda et al. 2013). 6. Conclusions Three strains of A. hydrophila were isolated from catfish. Their antibiotic resistance profiles were resistant to 3 out of 13 drugs. Furthermore, 2 out of 13 drugs were sensitive and 8 out of 13 drugs showed multi-resistance patterns. From these results can be suggested that gentamicin was an effective drug for curing the bacterial pathogens namely A. hydrophila in diseased catfish.

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