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Malaysian Journal of Microbiology, Vol 12(3) 2016, pp. 191-198 http://dx.doi.org/10.21161/mjm.77915 Malaysian Journal of Microbiology Published by Malaysian Society for Microbiology (In since 2011) Evaluation of identification techniques for the fish pathogen, Aeromonas hydrophila, from Indonesia Diah Kusumawaty 1,2, Adi Pancoro1, I. Nyoman P. Aryantha1 and Sony Suhandono1* 1School of Life Sciences and Technology, Bandung Institute of Technology, Indonesia. Jl. Ganesha No. 10, Bandung 40132 Indonesia. 2Dept. of Biology Education, Indonesia University of Education, Jl. Dr. Setiabudi No 229 Bandung 40154 Indonesia. Email: [email protected] Received 30 September 2015; Received in revised form 9 December 2015; Accepted 9 December 2015 ABSTRACT Aims: This study evaluated the accuracy of three methods used in the identification of Aeromonas hydrophila, a Gram- negative bacterium found in warm aquatic environments. A. hydrophila samples from Indonesia were tested using (a) SNI 7303, developed by the Indonesian government, (b) the method of Dorsch and (c) the method of Cascón. The results obtained were compared to that of the gold standard method, which used 16S rDNA sequences. Methodology and results: Based on the Indonesian government standard identification method SNI7303, we identified 56 out of 95 samples as A. hydrophila. The samples were then screened using the PCR amplification approach developed by Dorsch and Cascón. Of the 56 samples, only 20 samples were found to be positive by either the Dorsch or Cascón methods. DNA from these 20 samples was amplified using common 16S rDNA primers and the sequences compared with available 16S rDNA sequences from the GenBank. Phylogenetic analyses on the data were performed using Clustal X and MEGA 5 software. Conclusion, significance and impact of study: Of the 56 samples positively identified as A. hydrophila using the BSN method, identity in only five samples were positively confirmed using the16S rDNA method, giving an accuracy of only 8.9%. In this connection, the Dorsch method was 31.3% accurate while the Cascón method provided 45.5% correct identification. When all three methods were used in combination, 71.4% of the samples were correctly identified. The results of the study show that methods used to identify A. hydrophila cannot be used with confidence to identify A. hydrophila from Indonesia and probably from other tropical regions as well. The genetic diversity of Aeromonas bacteria in Indonesia appears to be considerably higher than that encountered by Dorsch or Cascón. Therefore, there is a need to develop a new simple method to identify A. hydrophila from tropical regions. Keywords: Aeromonas hydrophila, identification, phylogenetic, 16S rDNA INTRODUCTION Aeromonas hydrophila is a pathogenic bacterium found in USD235,000 and 2.7 million juvenile fish worth various aquatic environments, such as fish ponds, rivers, USD200,000 died in three days (Diraja, 2007 in Eka, lakes, even in sparkling chlorinated drinking water 2010). Laboratory experiments have shown that 105–1010 reservoirs. It is pathogenic to shrimps, frogs, and fishes CFU/mL A. hydrophila can kill the fish in only three days (Martin-Carnahan and Joseph, 2005; EPA, 2006). The after infection (Triyaningsih et al., 2014). Moreover, A. infection caused by this bacterium can lead to significant hydrophila is frequently associated with human diarrhea. mortality rates within a short period, causing losses to fish Alberts et al. (1990) found that 12.2% of toddlers with and shrimp breeders (Illanchezian et al., 2010; acute diarrhea in Dhaka, Bangladesh, tested positive for Mangunwardoyo et al., 2010). The disease symptoms A. hydrophila. Hence, a rapid method to determine A. vary, but infected fish generally show skin ulcers and hydrophila concentration in fish ponds and to ensure the haemorrhaging in the skin, gills, and oral cavity (Gardenia supply of safe drinking water is urgently needed. et al., 2010). The difficulty in distinguishing between A. hydrophila High mortality in gourami fish occurring over short and other species within the Aeromonas genus stems periods has caused great losses to fish breeders. For from the complexity of its identifying characters, example, in Lubuk Pandan, a city in the West Sumatra sometimes varying even within the species (Soler et al., Province of Indonesia, 47 tons of gourami worth 2004; Ottaviani et al., 2011). Identification using classical *Corresponding author 191 ISSN (print): 1823-8262, ISSN (online): 2231-7538 Mal. J. Microbiol. Vol 12(3) 2016, pp. 191-198 phenospecific methods is hence error-prone as shown by Tasikmalaya, Garut, Bandung, Indramayu, and Beaz-Hidalgo et al. (2010) in the recent Bergey’s Manual Sukabumi. When initially cultured on Rimler-Shotts of Systematic Bacteriology. For example, a positive ADH medium + novobiocin, the colonies of suspected A. (arginine dihydrolase) test and hydrolysis of glutamine hydrophila appeared white with a bright yellow zone in the cannot differentiate between A. hydrophila and A. sobria. middle of the colony. The isolates were sub-cultured on Moreover, conventional methods that do not employ Trypticase Soy Agar (TSA) slants and incubated at 28 °C genetic constitution tend to be tedious and complex for 24 h before being subjected to an array of tests and (Abbott et al., 2003). analyses, including Gram staining, motility test, oxidase The gold standard method in the identification of test, blood agar test, oxidative-fermentative test and bacteria involves analysis of 16S rDNA. However, this Rimler-Shotts+novobiocin test (Rimler and Shotts, 1973; method is dependent on DNA sequencing, facilities for BSN, 2009). During this procedure, the morphology of the which are not available in many laboratories. Therefore, isolate under investigation was compared with that of the there is a need to develop alternative practical and reference A. hydrophila ATCC 7966 obtained from accurate identification methods for A. hydrophila. A Microbiologic Co. standard biochemical method to identify A. hydrophila SNI 7303 (BSN, 2009) has been developed by Indonesian Bacterial DNA isolation and analysis Marine and Fisheries Department. This official protocol is frequently used to detect A. hydrophila in Indonesia To isolate bacterial DNA, the bacterial colony was re- (Tanjung et al., 2013; Hardi et al., 2014; Tulung et al., suspended in one milliliter PBS (Phosphate Buffer Saline) 2014). and homogenized using a vortex mixer. The tube was Other DNA-based methods for the identification of A. centrifuged at 1680 rcf for 3 min. The supernatant was hydrophila have been reported by Dorsch et al. (1994) discarded and the pellet was mixed with 100 μL 1× TE pH and Cascón et al. (1996). Dorsch et al. (1994) designed 8.0, and homogenized using a vortex mixer. The sample primers to amplify specific regions of the 16S rDNA of A. was incubated in boiling water for 10 min, and then hydrophila. The 685 bp amplicon that distinguishes A. centrifuged for 1 min at 1680 rcf. A total of 100 µL of the hydrophila from other Aeromonas species has been used lysate was transferred to a new 1.5 mL sterile tube and for identification of the former by Chu and Lu (2008) and dissolved in 900 μL cold 1× TE. Several sample aliquots Pandove et al. (2013). Adopting a different approach, of 200 µL were stored at −20 °C for use as template DNA Cascón et al. (1996) designed PCR primers from the in conjunction with specific primers designed by Dorsch et lipase gene of A. hydrophila H3. This method is rapid and al. (1994), Cascón et al. (1996), and Jiang et al. (2006) specific in identifying A. hydrophila isolated from aquatic for PCR. Following electrophoresis on 1% agarose gel, environments (Hiney and Smith, 1998; Abdullah et al., DNA sequencing was performed at Macrogen Korea. 2003; Salimi et al., 2013; Afsari et al., 2014). The PCR The PCR solution mix and primers used are shown in product, a 760 bp amplicon, has also been employed by Table 1. The sequencing results were subjected to Lee et al. (2000) to identify A. hydrophila in diseased BLAST analysis, with sequences aligned and compared fishes in Korea, while Swaminathan et al. (2004) with the other Aeromonas 16S rDNA from the GenBank successfully detected four out of nine A. hydrophila database NCBI (National Center for Biotechnology isolates from fish and water using the method of Cascón Information;http://www.ncbi.nml.nih.gov/BLAST/Blast.cgi). et al. Eleven sequences of Aeromonas 16S rDNA were chosen Nevertheless, both the methods of Dorsch et al. and from BLAST analysis for comparison with 16S rDNA Cascón et al. have not been validated against the gold sequence from 20 samples which were identified as standard established using complete sequence of 16S positive using the Cascón and Dorch methods. The rDNA (Jiang et al., 2006). This is the first research, to our comparison was made using phylogenetic analysis knowledge, that evaluates the accuracy (sensitivity) of the based on program of Clustal X and MEGA 5 software methods of Dorsch et al. (1994) and Cascón et al. (1996) (Tamura et al., 2011). To construct a phylogenetic tree, for the identification of A. hydrophila from a population of we used three 16S rDNA sequences from A. hydrophila Aeromonas in Indonesia. as references, viz. (1) 16S rDNA generated from living A. hydrophila (ATCC 7966) from Microbiologic Co, (2) 16S MATERIALS AND METHODS rDNA sequence of A. hydrophila (ATCC 7966) available at GenBank NCBI, and (3) 16S rDNA sequence of A. The isolation of Aeromonas hydrophila hydrophila (ATCC 4910) available at GenBank NCBI. Other reference 16S rDNA sequences were later In order to sample Aeromonas species from diverse identified from the NCBI database based on the BLAST environments, we collected bacteria from different results. We also used the 16S rDNA sequence of sources. Bacteria samples were isolated from the Tolumonas auensis strain DSM 9187 from GenBank as intestines of healthy and diseased fish obtained from an out-group.