Aquaculture Research, 2009, 40, 13^17

doi:10.1111/j.1365-2109.2008.02101.x

Isolation and identication of sh pathogen

Edwardsiella tarda from mariculture in China

Jingfan Xiao, Qiyao Wang, Qin Liu, Xin Wang, Huan Liu & Yuanxing Zhang
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
Correspondence: Y Zhang, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China. E-mail: yxzhang@ecust.edu.cn

Abstract
The causative agent was isolated from diseased turbots (Scophthalmus maximus) stricken by a highmortality outbreak of bacterial septicaemia occurring in a mariculture farm in Yantai, a northern coastal city of China. Seven pure isolates, namely EH-15, EH103, EH-107, EH-202, EH-203, EH-305 and EH-306, belonged to Edwardsiella tarda. The phenotypic features of the cultures were analysed extensively. Three of the isolates showed high 16S rDNA sequence similarities with E. tarda sequence (GenBank accession no. EF467289). However, unlike the E. tarda ATCC 15947, all the isolates, except EH-15, contained a novel large plasmid sized about 23.7 kb. Furthermore, pathogenicity of the isolates was addressed by experimental challenges with sh models. The isolates exhibited strong virulence to swordtail sh with LD50 ranging between 3.8 103 and 3.8 105 CFU g 1, and EH202 displaying the lowest LD50 value among them. Antibiotic susceptibilities of E. tarda isolates were assayed. Compared with E. tarda ATCC 15947, the isolates displayed strong resistance to chloramphenicol, and the probable dominant chloramphenicol resistance determinant was cat III. Depicting the main biological properties of turbot-borne E. tarda strains in China, the study provided useful information for further unveiling their pathogenic mechanisms.

Keywords: antibiotics susceptibility, aquaculture, chloramphenicol resistance, Edwardsiella tarda, pathogenicity

portant mariculture species in the coastal areas of northern China. In recent years, increasing frequency has been assumed in outbreaks of epizootic diseases, especially bacterial enteric septicaemia, a curse for farmed turbot industry in China. Edwardsiella tarda was rst reported in Japan by Sakazaki and Murata (1962) and further described by Ewing, McWhorter, Escobar and Lubin (1965). Edwardsiella tarda infections resulting in important economical losses have been reported from a variety of cultured sh in Asia, especially Japan and India, and channel catsh in the United States (Herman & Bullock 1986). The organism was rst isolated from diseased turbot by Oei, Hindley and Berry (1992), standing as one of the major causative agents of such diseases in brackish or freshwater sh species around Asian countries (Herman & Bullock1986). Fish aected by E. tarda displayed extensive skin lesions and necrosis in internal organs such as the liver, kidney, spleen and muscle, and developed into mass-mortality outbreaks of edwardsiellosis (Rao, Lim & Leung 2001). In this study, E. tarda strains were isolated from moribund S. maximum in a recent outbreak of edwardsiellosis in 2006 in a mariculture farm inYantai, a northern coastal city of China. Examinations of the biological properties, antibiotic susceptibilities, plasmid proles and virulence of these turbot-borne E. tarda strains were performed to provide useful information for further studies on their pathogenesis.

Materials and methods Isolation and identication of E. tarda strains

Introduction Turbot (Scophthalmus maximus), rst introduced in China in 1992 from Europe, was an economically im-

Edwardsiella tarda isolates were obtained from moribund S. maximum in a mariculture farm in Yantai. The isolates were grown on Salmonella^Shigella (SS)

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agar at 37 1C. Small colonies with black centres were picked up and re-streaked onto SS agar to ensure purity. Well-dierentiated single bacterial colony was streaked onto tryptone soy agar (TSA; Difco, Detroit, MI, USA) to obtain pure culture. The colonies were initially characterized by Gram staining and catalase tests. Colonies primarily conrmed as Edwardsiella species (Gram negative, catalase positive and lactose non-fermenting) were subjected to further characterization by biochemical tests (indole, methyl red,Voges^Proskauer test, fermentation of xylose and mannitol, production of H2S and motility test). The strains were grown at 37 1C in Luria^Bertani (LB) broth and preserved in 20% glycerol at 70 1C. Edwardsiella tarda ATCC 15947 was purchased and used as reference strain for this study. Edwardsiella tarda strains ET-M and ET-W were kindly provided by Dr Zhao Lan Mo and Mr Xiu HuaWang.

Antibiotics susceptibility test Antibiotics susceptibilities of the E. tarda isolates and ATCC 15947 were assayed using standard Kirby^ Bauer disc diusion method performed in MuellerHinton medium (Kangrun Biotech, Shanghai, China). Staphylococcus aureus ATCC 25923 was used as the reference strain. Discs containing the following antibiotics were spotted with an interval of 3 cm: sulfamethoxazole, oxacillin sodium, cefotaxime, cefuroxime, cefoperazone, tetracycline, tobramycin, piperacillin, nitrofurantoin, ceftriaxone, rifampicin, gentamicin, ceftazidime, cefazolin, cefradine, chloramphenicol, amikacin, erythromycin, noroxacin, penicillin G, ooxacin, ampicillin, streptomycin, kanamycin, ciprooxacin hydrochloride and vancomycin. The plates were incubated at 37 1C for 18 h and zones of growth inhibition were evaluated.

16S rDNA analysis The 16S rDNA of the isolates and E. tarda ATCC 15947 was amplied using the polymerase chain reaction (PCR) with the universal primers (forward primer, 5 0 -AGAGTTTGATC(A/C)TGGCTCAG-3 0; reverse primer, 5 0 -TACGG(C/T)TACCTTGTTACGACTT-3 0). The PCR reactions were performed with Taq DNA polymerase (TaKaRa, Dalian, China), with initial denaturation at 94 1C for 5 min followed by 30 cycles of denaturation at 94 1C for 1min, annealing at 50 1C for 2 min, extension at 72 1C for 2 min and a nal extension at 72 1C for 7 min. Amplied DNA fragments were examined using horizontal electrophoresis in 1.0% agarose gel (Genebase Gene-Tech, Shanghai, China) with 5 mL aliquots of PCR products. The gel images were visualized through UV gel image acquisition camera (FR-200A, Shanghai Furi Science & Technology, Shanghai, China). Automated DNA sequencing and primer synthesis were carried out by Invitrogen (Shanghai, China) and DNA analysis was completed using BLAST network services. LD50 determination The LD50 values of E. tarda ATCC 15947 and isolates were determined. Healthy swordtail sh (Xiphophorus helleri) of about 2 g body weight was obtained from a commercial sh farm, kept at 20 1C and infected intramuscularly (i.m.) with the E. tarda strains. The mortality of the sh was recorded over a period of 90 h after infection. The LD50 values were calculated using the method of Reed and Muench (1938).

Polymerase chain reaction determinations of the cat and oR genes All the isolates and reference strains were screened for the cat genes using a multiplex PCR method (Yoo, Huh, Kim, Lee & Jeong 2003). Representative PCR products were sequenced for conrming the identities of cat genes. The anking region upstream the multiplex PCR product was obtained using genome walking kit (TaKaRa, Tokyo, Japan). The oR gene was also screened as its product also mediates chloramphenicol resistance. Primers o-FW (5 0 -TGGCT CCTTTCGA CATCC-3 0) and o-RV (5 0 -A(C/T) CCACAT CGGTAGG ATGA-3 0) were synthesized to amplify the 889 bp oR homologous region (Dang, Zhang, Song, Chang & Yang 2007).

Growth of E. tarda isolates The growth of three out of seven isolated strains as well as ATCC 15947 was examined in LB medium. The growth of a fast-growing EH-202 strain in LB medium with dierent sodium chloride concentrations was also investigated. Sodium chloride concentration of sterile LB broth was adjusted to 0.5%, 1%, 1.5%, 2% and 3% (w/v) respectively. The broth (10 mL) in an Erlenmeyer ask was inoculated with 0.1mL of an overnight culture. The asks were incubated on a shaker (200 r min 1) at 37 1C for 36 h. Bacterial densities were spectrophotometrically determined at 600 nm at 5 h intervals. Samples were assayed in duplicates.

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Isolation and identication of Edwardsiella tarda J Xiao et al.

Plasmid proles The plasmids of E. tarda were extracted using Plasmid Extraction Kit (Tiangen, Shanghai, China). Plasmid purity was determined using agarose gel electrophoresis. All strains were examined for the presence of extra-chromosomal elements with horizontal electrophoresis on 0.7% agarose gels as well as UV visualization.
23730 bp 9416 bp 6557 bp 4361 bp 2322 bp 2027 bp

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Results Isolation and identication of strains In the present study, seven E. tarda strains were successfully isolated from spleen (EH-202 and EH-203), liver (EH-103 and EH-107) and ascites (EH-15, EH-305 and EH-306) of the diseased S. maximum. The isolates were tentatively conrmed using morphological and biochemical tests. On SS agar, the E. tarda colonies were featured with black centre as described previously (Wyatt, Nickelson II & Vanderzant 1979). Corresponding to reference strain ATCC15947, the isolates showed typical characteristics of E. tarda in biochemical reaction including Gram negative, motile and catalase positive, indole and H2S gas producing, methyl red reducing, and xylose and mannitol non-fermentive.

Figure 1 Plasmid proles of isolates and ATCC 15947. Lane M, Lambda DNA/Hind III Markers; lane 1, ATCC 15947; lanes 2^9, EH-202, EH-103, EH-306, EH-15, EH-305, EH-107, EH-203, and E. coli DH5a (negative control); lane 10, genome of Edwardsiella tarda.

necrotic lesions in skin and muscle with suppurative abscesses. Bacteria with the same characteristics as E. tarda were isolated from all dead and moribund X. helleri. The 50% lethal dose values for EH-15, EH-103, EH-107, EH-202, EH-203, EH-305 and EH-306 were 9.5 104, 2.2 105, 3.8 105, 3.8 103, 1.5 105, 2.3 105 and 3.2 105 CFU g 1 respectively. The 50% lethal dose value for ATCC 15947 was 6.0 106 CFU g 1.

Antibiotics susceptibility test The antibiotic susceptibilities of E. tarda EH-202 and ATCC 15947 were investigated. EH-202 and ATCC 15947 had similar antibiotic-resistant patterns for aminoglycosides (gentamycin, amikacin, kanamycin and tobramycin), quinlolones (ciprooxacin hydrochloride, ooxacin and noroxacin) and cephalosporins among a set of 26 antibiotics (Table 1). EH-202 and ATCC 15947 also displayed dierent resistances to penicillins, erythromycin, tetracycline, vancomycin and chloramphenicol. As contrasted with strain ATCC 15947, strain EH-202 was resistant to tetracycline and chloramphenicol and sensitive to erythromycin. As previously described (Stock & Wiedemann 2001), the E. tarda strains shared native resistance to rifampin.

16rS DNA analysis The 16S rDNA sequences of the isolates were analysed via BLAST network services. The 16S rDNA genes of the E. tarda EH-107, EH-202 and EH-306 isolates were sequenced, which showed a high similarity (99% identity) to that of E. tarda (EF467289), and the sequence of EH-202 was deposited in GenBank under accession no. EU121410.

Plasmid proles Plasmids patterns of each isolate were shown in Fig.1. ATCC 15947 harboured two plasmids sized about 6.5 and 4.3 kb (lane 1), respectively, while all the E. tarda isolates, except EH-15 (lane 5), presented a novel and larger plasmid sized about 23.7 kb (lanes 2^8).

The PCR determinations of cat and oR genes All the isolated strains gave single and specic PCR amplication products of 275 bp (Fig. 2). The sequencing and BLAST analysis of the fragment suggested that the chloramphenicol resistance determinant was cat III. The cat genes were not detected in the reference strains ATCC 15947, ET-M and ET-W. The oR gene could not be detected in all the isolated and

LD50 determination in sh model The E. tarda isolates were pathogenic to X. helleri when i.m.-challenged. Typical symptoms of edwadsiellosis were observed, including haemorrhage,

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Table 1 Antibiotic susceptibilities of Edwardsiella tarda strains

Antibiotic agents
Penicillins Penicillin G Ampicillin Oxacillin sodium Piperacillin Tetracyclines Tetracycline Quinolones Noroxacin Ciprooxacin hydrochloride Ooxacin Aminoglycosides Amikacin Tobramycin Kanamycin Gentamycin Cephalosporins Cefotaxime Cefoperazone Cefazolin Cefuroxime Ceftazidime Cefradine Ceftriaxone Other antibiotics Sulfamethoxazole Streptomycin Chloramphenicol Nitrofurantoin Rifampicin Erythromycin Vancomycin

M 1200 bp 800 bp 500 bp 200 bp

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ATCC 15947

EH-202

R I R S S S S S S S S S S S S S S S S R R S S R R R

S I S S R S S S S S S S S S S S S S S S R R S R S S

Figure 2 Electrophoretic analyses of the polymerase chain reaction screening results of cat genes from the seven selected isolated strains from turbot, ATCC 15947, ETM and ET-W respectively. Lane M, Marker III; lane 1, ATCC 15947; lanes 2^8, EH-202, EH-103, EH-306, EH-15, EH-305, EH-107 and EH-203; lane 9, ET-M; lane 10, ET-W.

grew well in medium supplemented with 0.5^2% NaCl and the optimal NaCl concentration was 2%. The NaCl concentration that appeared growth inhibition to the strains was above 3%. However, EH-202 could still survive in medium supplemented with 5% NaCl (data not shown), demonstrating its high halo-tolerating ability.

Discussion In this work, virulent E. tarda strains were isolated from liver, spleen and ascites of diseased turbots. Conventional biochemical tests as well as analysis of 16S rDNA were performed to identify the E. tarda isolates from dierent tissues of the sh. The spleenderived E. tarda EH-202 was found to be more virulent than other isolates to swordtail sh (X. helleri). EH-202 also displayed strong resistance to chloramphenicol, tetracycline, streptomycin and rifampicin. Except EH-15, all the isolates harboured a novel and large plasmid sized about 23.7 kb. Reger, Mockler and Miller (1993) reported that ve of ten E. tarda isolates gave an identical plasmid pattern of four plasmids ranging in size from 76 to 5 kb; one exhibited a 54 kb plasmid and the other four strains did not contain plasmid DNA. Compared to their results, all the turbot isolates in this work, except EH15, harboured a similar plasmid sized above 23.7 kb. Since 2003, X. helleri was approved as laboratory animal by State Evaluation Committee of Fisheries Stock (GS01003-2003, China) and thus extensively used as model sh for many sh pathogens. Xiphophorus helleri infected by E. tarda displayed septicaemia and extensive skin lesions in our experiments, demonstrating that the turbot-borne E. tarda was also pathogenic to X. helleri. The LD50 values of X. helleri were ranged between 3.8 103 and 3.8 105 CFUg 1, similar to an earlier report by Pan, Wu, Li, Huang and Si (2000).

reference strains. Furthermore, the1800 bp fragment containing the cat III ORF region (642 bp) was obtained using genome walking strategy. In the obtained fragment, another two hypothetical proteins were found upstream the cat III ORF region, and all the identied ORFs displayed 99% identity to that of the previously isolated gene fragment from E. tarda (EF 467364).

Growth of E. tarda isolates Four E. tarda strains, ATCC 15947, EH-107, EH-202 and EH-306, displayed typical sigmoidal growth kinetics with identiable lag and logarithmic growth phases. EH-202 showed signicant faster growth rate than other strains during logarithmic growth phases. The other isolates displayed similar growth rate to that of ATCC 15947. In addition, the isolates

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Compared with ATCC 15947 and six other isolates, the spleen-derived EH-202 had the lowest LD50 value, correlating with its high growth rate, suggesting that the fast proliferating ability of EH-202 might be relevant to its strong virulence. In the investigation of antibiotic susceptibility of the strains, the reference strains ATCC 15947, ET-M and ET-W were susceptible to chloramphenicol, the same as the E. tarda strains documented previously by Stock and Wiedemann (2001). However, E. tarda isolates in our study showed strong resistance to chloramphenicol, and the minimal inhibitory concentration (MIC) value was 100 mg mL 1. The dominant chloramphenicol resistance determinant was cat III in our isolated strains rather than cat II or cat IV reported by Yoo et al. (2003) and Dang et al. (2007) via multiplex PCR and gene sequence analysis. Furthermore, the 1035 bp fragment upstream the cat III gene ORF region was analysed via BLAST network services. Two hypothetical proteins were found which shared 99% identity on the nucleotide level to the homologues (GenBank accession no. EF 467364) from the E. tarda strain TX1, and this suggested that the two strains might share the same origin of chloramphenicol resistance. The sequenced 1672 bp ragment containing the cat III ORF genes and two hypothetical proteins showed an average G1C content of 41.98%, which is much lower than the 53^59% of Edwardsiella, suggesting that a probable lateral gene transfer event occurred at the locus. These data indicate that chloramphenicol resistance is not an inherent characteristic of E. tarda. Dang et al. (2007) assumed that this situation was related to the drug application regime in mariculture. Additionally, the resistance of E. tarda might be obtained by horizontal genetic material exchange with other micro-organisms. The dierence between antibiotic-resistant genes detected from isolates in China and the reported isolates in Korea, indicates that antibiotic resistance was acquired through dierent mechanisms. In this work, evaluation of antibiotic resistances of the bacteria inhabiting turbot could be useful for further studies in demonstrating the origin and evolution of the drug-resistant genes. Furthermore, it would help farmers and veterinarians setting a more ecient and appropriate farm management.

Wang (Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences) for sending us the reference strains. The work was supported by grants from National High Technology Research and Development Program of China (2006AA100310), the Ministry of Agriculture (nyhyzx07-046) and Shanghai Leading Academic Discipline Project (B505).

References
Dang H., Zhang X., Song L., ChangY. & Yang G. (2007) Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China. Journal of Applied Microbiology 103, 2580^2592. Ewing W.H., McWhorter A.C., Escobar M.R. & Lubin A.H. (1965) Edwardsiella, a new genus of Enterobacteriaceae based on a new species, E tarda. International Bulletin of Bacteriological Nomenclature andTaxonomy 15, 33^38. Herman R.L. & Bullock G.L. (1986) Pathology caused by the bacterium Edwardsiella tarda in striped bass. Transaction of theAmerican Fisheries Society 115, 232^235. Oei C., Hindley J. & Berry C. (1992) First isolation of Edwardsiella tarda from diseased turbot (Scophthalmus maximus) reared in a sea farm in the Bay of Biscay. Bulletin of the European Association of Fish Pathologists 14, 28^129. Pan H.J.,Wu S.Q., Li K.B., Huang Z.B. & Si C.B. (2000) Application of Xiphophorus helleri. Journal of Fisheries of China 24, 467^471. Rao P .S., Lim T.M. & Leung K.Y. (2001) Opsonized virulent Edwardsiella tarda strains are able to adhere to and survive and replicate within sh phagocytes but fail to stimulate reactive oxygen intermediates. Infection and Immunity 69, 5689^5697. Reed L.J. & Muench H. (1938) A simple method of estimating fty percent end points. American Journal of Hygiene 27, 493^497. Reger P.J., Mockler D.F. & Miller M.A. (1993) Comparison of antimicrobial susceptibility, b-lactamase production, plasmid analysis and serum bactericidal activity in Edwardsiella tarda, E. ictaluri and E. hoshinae. Journal of Medical Microbiology 39, 273^281. Sakazaki R. & Murata Y. (1962) The new group of Enterobacteriaceae, the Asakusa group. Japanese Journal of Bacteriology 17, 616^617. Stock I. & Wiedemann B. (2001) Natural antibiotic susceptibilities of Edwardsiella tarda, E. ictaluri, and E. hoshinae. AntimicrobiologyAgents and Chemotherapy 45, 2245^2255. Wyatt L.E., Nickelson R. II & Vanderzant C. (1979) Edwardsiella tarda in freshwater catsh and their environment. Applied and Environmental Microbiology 38,710^714. Yoo M.H., Huh M.D., Kim E.H., Lee H.H. & Jeong H.D. (2003) Characterization of chloramphenicol acetyltransferase gene by multiplex polymerase chain reaction in multidrug-resistant strains isolated from aquatic environments. Aquaculture 217,11^21.

Acknowledgments We wish to thank Dr Zhao-Lan Mo (Institute of Oceanology, Chinese Academy of Sciences) and Mr Xiu-Hua

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