American Journal of Biochemistry and Biotechnology Original Research Paper Isolation, Identification and Genomic Analysis of Plesiomonas shigelloides Isolated from Diseased Percocypris pingi (Tchang, 1930) 1, 2, 3 Lei Pan, 4Shuiyi Liu, 1Xuwei Cheng, 1Yiting Tao, 5Tao Yang, 5Peipei Li, 1,2 Zhengxiang Wang, 3Dongguo Shao and 6Defeng Zhang 1School of Resources and Environmental Science, Hubei University, Wuhan 430062, People's Republic of China 2Hubei Key Laboratory of Regional Development and Environmental Response (Hubei University), Wuhan 430062, People's Republic of China 3State key Laboratory of Water Resources and Hydropower Engineering Science (Wuhan University), Wuhan, 430072, People's Republic of China 4Department of Medical Laboratory, the Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, People's Republic of China 5Wuhan Heyuan Green Biological Co., Ltd., Wuhan 430206, People's Republic of China 6Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, People's Republic of China Article history Abstract: Recently, the outbreak of a serious infectious disease of Received: 25-10-2017 unknown etiology was noted in Percocypris pingi (Tchang, 1930) farms in Revised: 11-12-2017 Yunnan province. Due to currently limited information, we aimed to Accepted: 20-12-2017 identify the pathogen isolates, determine the susceptibility of the isolates, evaluate the pathogenicity and analyze the genome of the representative Corresponding Author: Defeng Zhang strain. Ten strains of Gram-negative rods were isolated from diseased P. Key Laboratory of Fishery Drug pingi and the isolates were identified as Plesiomonas shigelloides based Development, Ministry of on biochemical characteristics, 16S rRNA gene sequencing and species- Agriculture, Pearl River Fisheries specific PCR detection. The results of susceptibility analysis showed that Research Institute, Chinese two selected strains LS1 and LL2 were resistant to ampicillin, penicillin Academy of Fishery Sciences, G, trimethoprim/sulfamethoxazole, tetracycline, enrofloxacin, nalidixic Guangzhou, People's Republic of acid and enoxacin. A virulence assay indicated that the pathogen was China virulent to zebrafish. Genomic analysis revealed that the LS1 isolate was Email: [email protected] closely related to strain GN7, which was isolated from animal farms. To the best of our knowledge, this is the first report of P. shigelloides as a pathogen of P. pingi . This study will provide a rational framework for exploration of epidemiological analysis of P. shigelloides in fish diseases and would further benefit conservation of the species. Keywords: Plesiomonas shigelloides , Percocypris pingi (Tchang, 1930), Identification, Genomic Analysis Introduction emerging enteric pathogen that is linked to intestinal and extra-intestinal infections in humans (González-Rey et al ., Plesiomonas shigelloides is a Gram-negative, motile, 2011). It is recognized as a potential fish pathogen and has non-spore-forming, oxidase-positive and rod-shaped been isolated from red hybrid tilapia (Nadirah et al ., bacterium belonging to the Enterobacteriaceae family 2012). In China, P. shigelloides is the pathogen of grass (Brenner et al ., 2005). P. shigelloides is widely distributed carp ( Ctenopharyngodon idella ), sturgeon ( Huso in the aquatic environment even under cold climatic huso ×Acipenser ruthenus ), gibel carp ( Carassius auratus conditions (Salerno et al ., 2010). Thus far, P. shigelloides gibelio ), black carp ( Mylopharyngodon piceus ) and tilapia has been isolated from streams, lakes, estuarine waters, (Oreochromis niloticus ). mammals, crustaceans, mollusks, reptiles, amphibians and Percocypris pingi (Tchang, 1930), an endemic and fish (Alexander et al ., 2016). P. shigelloides is an commercially important species only found in the upper © 2017 Lei Pan, Shuiyi Liu, Xuwei Cheng, Yiting Tao, Tao Yang, Peipei Li, Zhengxiang Wang, Dongguo Shao and Defeng Zhang. This open access article is distributed under a Creative Commons Attribution (CC-BY) 3.0 license. Lei Pan et al . / American Journal of Biochemistry and Biotechnology 2017, 13 (4): 226.232 DOI: 10.3844/ajbbsp.2017.226.232 reaches of the Yangtze River in China, is a benthic fish Molecular Characterization that is typically found in rivers with torrential flow. The population of this fish decreased intensely due to Genomic DNA from ten isolates was extracted using overfishing, dam construction, habitat deterioration and the EasyPure Genomic DNA Kit (Transgen Biotech, water pollution, becoming endangered in the past Beijing, China) in accordance with the manufacturer’s decade. In May 2015, this species has been listed as instructions. A partial 16S rRNA gene sequence was Endangered (EN) in the Red List of China’s Vertebrates amplified as described previously (Zhang et al ., 2011). (Li et al ., 2016). Aquaculture may counteract this decline Briefly, universal PCR primers 27F (5′- and improve biodiversity by sustaining healthy fish AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′- populations (Pan et al ., 2016). Therefore, further TACGGMTACCTTGTTACGACTT-3′) were used for research is essential to maintain sufficient production of amplification of the 16S rRNA gene. In addition, primers P. pingi to meet fishing demands for conservation. PS23FW3 (5′-CTCCGAATACCGTAGAGTGCTATC Recently, artificial propagation of P. pingi has been C-3′) and PS23RV3 (5′- conducted in Sichuan and Yunnan provinces in China to CTCCCCTACCCAATAACACCTAAA-3′) were used restore their population and the hatchery-reared fish have for detection of the 23S rRNA gene of P. shigelloides been released into the river by several institutions (Li et al ., (González-Rey et al ., 2000). 2016). Unfortunately, bacterial diseases are one of the Biochemical Characteristics major obstacles to the expansion of P. pingi farming. In 2016, an outbreak caused by P. shigelloides occurred in Of the ten isolates, two (strains LS1 and LL2) were farmed P. pingi in China. However, the information identified based on biochemical characteristics, which were examined by API 20E system (bioMèrieux, Marcy about P. pingi disease is limited. In this study, we have l’Etoile, France) as reported previously (Krovacek et al ., described the isolation, identification and genomic 2000) and by API strips (Chen et al ., 2013). Hemolytic analysis of P. shigelloides from diseased P. pingi . The activity was assayed by spot inoculation of these strains aim of this study is to provide a theoretical foundation on 5% sheep erythrocytes in BHI agar and erythrocyte for further understanding of the epidemiology of P. lysis was recorded after 24 h of incubation at 37°C. shigelloides strains and ensure effective prevention and control measures. Pathogenicity Test In this study, zebrafish ( Danio rerio ) was used as the Materials and Methods target fish to evaluate the pathogenicity of strain LS1 (Saralahti and Rämet, 2015). The isolate LS1 was used Sample Collection for the experimental infection of healthy zebrafish to In August 2016, a disease outbreak in P. pingi (average assess their pathogenic potential. A total of 300 healthy body weight 45 gram) were obtained from a commercial zebrafish were randomly divided into seven groups (20 source, which were cultured in a recirculating system in per group) and then allowed to acclimatize at 28°C for 7 Lijiang city, Yunnan province, China. The culture water d prior to challenge. Challenge assays were performed in temperature was maintained at 24±1°C. In the early stage triplicate. Isolate LS1 was cultured in BHI broth of the disease, several fish began to show clinical signs overnight, centrifuged and collected. The bacteria were including anorexia, caudal fin and skin ulceration and resuspended in sterile PBS buffer at six different doses. ascites (accumulation of abdominal fluid). Four days later, Experimental groups were intraperitoneally inoculated mortalities started to occur in this farm. Moribund fish with 20 µL of cultured cells at doses of 3 ×10 8, 3 ×10 7, were sampled and kidney tissues were incubated on Brain 3×10 6 and 3 ×10 5 CFU/mL, respectively. The zebrafish in Heart Infusion (BHI, Difco Laboratories, Detroit, MI, the control group were injected with the same volume of USA) agar for bacterial isolation. PBS buffer. Clinical signs and mortality were recorded daily for 14 d. In addition, bacteria were isolated from Bacterial Isolation the moribund zebrafish and were detected by PCR assay Tissue samples were aseptically collected from the based on the 23S rRNA gene of P. shigelloides . The kidney of moribund P. pingi (N=10) and incubated on statistical analysis was calculated using SPSS software BHI agar. Plates were incubated at 28°C for 24 h. The (version 22.0) and the survival curve was performed by bacterial colonies were re-streaked on BHI agar to obtain Origin software (version 9.0). a pure culture. For long-term preservation the bacterial Antimicrobial Susceptibility Testing isolates were stored at -80°C in sterile BHI containing 15% glycerol. In this study, a total of ten strains were In order to select the antimicrobial agents for treatment isolated and two of them, were selected for further study. and define the antibiotic pattern of the isolates, antibiotic 227 Lei Pan et al . / American Journal of Biochemistry and Biotechnology 2017, 13 (4): 226.232 DOI: 10.3844/ajbbsp.2017.226.232 susceptibility of bacterial