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Isolation and Phylogenic Analysis of Emerging New Antibiotic Resistant Bacteria, Acinetobacter Lwoffii, Associated with Mortality in Farmed Rainbow Trout

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Isolation and Phylogenic Analysis of Emerging New Antibiotic Resistant Bacteria, Acinetobacter Lwoffii, Associated with Mortality in Farmed Rainbow Trout Iranian Journal of Fisheries Sciences 15(4) 1279-1292 2016 Isolation and phylogenic analysis of emerging new antibiotic resistant bacteria, Acinetobacter lwoffii, associated with mortality in farmed rainbow trout Dadar M.1; Adel M.2*; Zorriehzahra M.J.2 Received: May 2016 Accepted: September 2016 Abstract Whereas it is well documented that the genus Acinetobacter is associated with antibiotic resistant bacteria and human clinical infections, there are very few studies to date that report it as an emerging new pathogen for fish. In the present study, Acinetobacter lwoffii was isolated from diseased rainbow trout (Oncorhynchus mykiss) from Iranian farms, in the Khuzestan Province by both biochemical tests and polymerase chain reaction (PCR)-based methods and further confirmed as the causative agent by infectivity experiment. Uni or bilateral exophthalmia, blackening of the skin, abdominal distension, hemorrhages around the mouth cavity and eyes, basal fine, skin, gills and in internal organs were the main clinical signs in the affected fish. Phylogenetic analysis of the 16S rRNA sequence of MD77 revealed a 99% homology with that of A. lwoffii (GenBank accession no.KR856323.1) form Iran. Histopathological changes of challenged rainbow trout including focal necrosis of liver cells (hepatocyte), pyknotic nucleus and karyolysis of hepatocyte, hyperemia, fatty acid composition change of liver, infiltration of inflammatory cells, sinusoidal dilatation and congestion. The drug resistance of isolates and minimal inhibitory concentration (MIC) was examined and showed that A. lwoffii was multiresistant to 8 of the antimicrobial agents tested. Keywords: Acinetobacter lwoffii, Phylogenic analysis, Pathogenic effects, Drug resistance, Oncorhynchus mykiss, Iran 1- Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran. 2-Department of Aquatic Animal Health & Disease, Iranian Fisheries Science Research Institute (IFSRI), Agricultural Research Education and Extension Organization (AREEO), P.O.Box: 13185-116, Tehran, Iran. *Corresponding author's email: [email protected]; [email protected] 1280 Dadar et al., Isolation and phylogenic analysis of emerging new antibiotic resistant bacteria… Introduction charecteristics. Other species of Acinetobacter, belonging to family Acinetobacter were isolated form Moraxellaceae, class γ-Proteobacteria Mandarin fish, Siniperca chuatsi (Gu et and order Pseudomonadales, is a genus al., 1996) and channel catfish (Ictalurus of gram-negative, non-motile, non- punctatus) in China (Xia et al., 2008). fermentative rods, oxidase-negative, During the last decade, antibiotic aerobic bacteria which are widely resistant bacteria became an important distributed in the different environment environmental contamination issue, and including water, soil, human, plant, there is a growing concern about the aquatic organisms, the polar region, persistence and epidemic spread of hydrocarbon-contaminated sites and these organisms in the environment sediments (Baumann, 1968; Berlau et (Harnisz et al., 2011; Maravić et al., al., 1999; Joly‐Guillou, 2005; Yonar et 2015). The genes of resistance to al., 2010; Kostka et al., 2011; Naviner antibiotics can be transferred between et al., 2011; Jung and Park, 2015). The bacteria in the environment through genus comprises both nonpathogenic plasmids, integrons and transposons and pathogenic species (de Berardinis (Dijkshoorn et al., 2007). Infections et al., 2009). Most strains of these caused by resistant strains of organisms grow at a wide range of Acinetobacter spp. are costly to treat in temperatures (El-Sayyad et al., 2010; fish and humans. These infections Doughari et al., 2011). Within the prolong the pathological situations and Acinetobacter genus, Acinetobacter if they are not treated with the right lwoffii, a pathogen resistant to a range antibiotics they can increase mortality, of antibiotics, is the causative agent of a morbidity and risk of antibiotic- nosocomial infections, including associated adverse events (Peleg et al., bacteraemia, secondary meningitis, 2008). urinary tract infection, surgical sites The main aim of our research was the infection, gastritis and pneumonia, identification, as well as the especially in intensive care unit (ICU) determination of genotype relations and patients (Zavros et al., 2002). While clinical features of Acinetobacter sp. this pathogen is well documented to be resistant to antibiotic treatment in associated with multi-drug resistant rainbow trout farms. bacteria and human clinical infections, to the best of our knowledge, to date Material and methods there was only one report that described Fish sampling it as a pathogen for fish. A. lwoffii was During the incidence of antibiotic for the first time isolated by Kozińska resistant bacteria in 2015, et al. (2014) from common carp in Acinetobacter sp. was isolated from Poland and identified in terms of its rainbow trout of Iranian farms. A total biochemical and molecular of 100 moribund rainbow trout Iranian Journal of Fisheries Sciences 15(4) 2016 1281 weighing 70±30 g (mean±SD), Pure cultures of isolate were stored at 4 originating from two farms, in ±1°C for further examination. Khuzestan Province, (Southwest of Iran) were investigated during May and Phenotypic characterisation of the September 2015. Clinical signs of isolate diseased fish included exophthalmia, Morphology of colonies was blackening of the skin, as well as investigated and recorded. Then, pure hemorrhages around the eyes, base of culture of MD77 isolate was dorsal and caudal fins, skin, gills and characterized with Gram-staining and internal organs. The farms were run on biochemically tested as described a flow-through system of fresh water at previously (Gu et al., 1996; Kozińska et a temperature of 17±3ºC. The moribund al., 2014). fish were selected and transferred to the Central Veterinary Laboratory, Ahvaz, PCR and sequencing Iran. Mortality rate among the fish was Molecular identification of isolates was 28-30% especially in the trout conducted by sequencing of 16s rDNA population on warm days. The disease gene fragment as described in different was noted in different seasons, but was works elsewhere (Marchesi et al., most common in May and September. 1998). Briefly, genomic DNA was extracted from a pure culture using the Isolation of bacteria genomic DNA purification kit The body surface and gills were (GeneAll) according to the carefully swabbed using 70% ethyl manufactures’ instructions. The alcohol to remove normal external extracted DNA was amplified using a bacterial flora. After opening the body polymerase chain reaction (PCR) kit in cavity, the brain, eyes, liver, kidney, accordance with the manufacturer’s and spleen were aseptically removed. protocol (Qiagen, Germany). Nearly Bacterial subculturing was done under full length 16S rDNA genes were the conditions described in Kozińska et amplified using the 1492r primer (5′- al. (2014). Briefly, the samples were GGTTACCTTGTTACGACTT--3′) and diluted (1:1) in PBS and homogenized. one of the following three 27f primer Then they were inoculated onto formulations: twofold-degenerate tripticase soya agar (TSA, Sigma, USA) primer 27f (5′- and nutrient agar (Sigma, USA) and AGAGTTTGATCMTGGCTCAG-3′, supplemented with 5% horse blood where M is A or C). The thermal PCR (BA). After incubating at 28 °C for 24 steps were 1 cycle 95ºC for 2 min, h, bacteria isolated from rainbow trout followed by 35 cycles of denaturation at were subcultured onto TSA to check the 95ºC for 30 sec, annealing temperature purity of the isolation. The isolated at 50ºC for 30 sec and extension at 72ºC bacterial strain was named as MD77. for 1 min. This was followed by a final extension of 10 min at 72ºC. The PCR 1282 Dadar et al., Isolation and phylogenic analysis of emerging new antibiotic resistant bacteria… products were separated by agarose gel Enrofloxacin (5 μg/mL), flumequine electrophoresis, purified with gel (30μg/mL), ampicillin (10 μg/mL), extraction kit (Qiagen, Germany), and gentamicin (10μg/mL), subjected to nucleotide sequence chlorotetracycline (20μg/mL) and analysis by dideoxy chain termination oxolinic acid (10μg/mL) were method. The 16S rDNA gene sequence purchased from Sigma-Aldrich France. of four isolates and the representatives After 24 h incubation at 30 °C, isolates of the other species from GenBank were defined as resistant using database were analyzed using MEGA 6 inhibition zones around the discs, software. Similarity between the breakpoint tables for interpretation of sequences of the tested isolates and Minimum inhibitory concentrations sequences available at GenBank was (MICs) and zone diameters established defined by the use of MEGA 6 by European Committee on software. The amplified fragments were Antimicrobial Susceptibility Testing sequenced and deposited in the NCBI (EUCAST) (2006) was used. database with the accession number Psedumonase aeruginosa ATCC 27853 KU695542. was used as quality control strain. Phylogenetic analysis Ethics statement The sequence data were analyzed using For the protection of animals used for Blast (NCBI), Mega 6 software. scientific purposes the experiments Finally, the results were compared with described comply with the Guidelines other existing sequences in the data of the European Union Council bank and a phylogenetic tree was http://ec.europa.eu/environment/chemic produced by the maximum-likelihood als/lab_animals/legislation_en.htm analysis with bootstrap
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