Isolation and identification of fish pathogen Edwardsiella tarda from mariculture in China

Authors


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 high-mortality outbreak of bacterial septicaemia occurring in a mariculture farm in Yantai, a northern coastal city of China. Seven pure isolates, namely EH-15, EH-103, 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 fish models. The isolates exhibited strong virulence to swordtail fish with LD50 ranging between 3.8 × 103 and 3.8 × 105 CFU g−1, and EH-202 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.

Introduction

Turbot (Scophthalmus maximus), first introduced in China in 1992 from Europe, was an economically important 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 first 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 fish in Asia, especially Japan and India, and channel catfish in the United States (Herman & Bullock 1986). The organism was first 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 fish species around Asian countries (Herman & Bullock 1986). Fish affected 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 in Yantai, a northern coastal city of China. Examinations of the biological properties, antibiotic susceptibilities, plasmid profiles 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 identification of E. tarda strains

Edwardsiella tarda isolates were obtained from moribund S. maximum in a mariculture farm in Yantai. The isolates were grown on Salmonella–Shigella (SS) agar at 37 °C. Small colonies with black centres were picked up and re-streaked onto SS agar to ensure purity.  Well-differentiated 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 confirmed 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 °C in Luria–Bertani (LB) broth and preserved in 20% glycerol at −70 °C. 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 Hua Wang.

16S rDNA analysis

The 16S rDNA of the isolates and E. tarda ATCC 15947 was amplified using the polymerase chain reaction (PCR) with the universal primers (forward primer, 5′-AGAGTTTGATC(A/C)TGGCTCAG-3′; reverse primer, 5′-TACGG(C/T)TACCTTGTTACGACTT-3′). The PCR reactions were performed with Taq DNA polymerase (TaKaRa, Dalian, China), with initial denaturation at 94 °C for 5 min followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 2 min, extension at 72 °C for 2 min and a final extension at 72 °C for 7 min. Amplified DNA fragments were examined using horizontal electrophoresis in 1.0% agarose gel (Genebase Gene-Tech, Shanghai, China) with 5 μL 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 fish (Xiphophorus helleri) of about 2 g body weight was obtained from a commercial fish farm, kept at 20 °C and infected intramuscularly (i.m.) with the E. tarda strains. The mortality of the fish was recorded over a period of 90 h after infection. The LD50 values were calculated using the method of Reed and Muench (1938).

Antibiotics susceptibility test

Antibiotics susceptibilities of the E. tarda isolates and ATCC 15947 were assayed using standard Kirby–Bauer disc diffusion method performed in Mueller-Hinton 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, norfloxacin, penicillin G, ofloxacin, ampicillin, streptomycin, kanamycin, ciprofloxacin hydrochloride and vancomycin. The plates were incubated at 37 °C for 18 h and zones of growth inhibition were evaluated.

Polymerase chain reaction determinations of the cat and floR 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 confirming the identities of cat genes. The flanking region upstream the multiplex PCR product was obtained using genome walking kit (TaKaRa, Tokyo, Japan). The floR gene was also screened as its product also mediates chloramphenicol resistance. Primers flo-FW (5′-TGGCTCCTTTCGA CATCC-3′) and flo-RV (5′-A(C/T) CCACAT CGGTAGG ATGA-3′) were synthesized to amplify the 889 bp floR 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 different 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 flask was inoculated with 0.1 mL of an overnight culture. The flasks were incubated on a shaker (200 r min−1) at 37 °C for 36 h. Bacterial densities were spectrophotometrically determined at 600 nm at 5 h intervals. Samples were assayed in duplicates.

Plasmid profiles

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.

Results

Isolation and identification 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 confirmed 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 ATCC 15947, 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.

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 profiles

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).

Figure 1.

 Plasmid profiles 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 DH5α (negative control); lane 10, genome of Edwardsiella tarda.

LD50 determination in fish model

The E. tarda isolates were pathogenic to X. helleri when i.m.-challenged. Typical symptoms of edwadsiellosis were observed, including haemorrhage, 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 (ciprofloxacin hydrochloride, ofloxacin and norfloxacin) and cephalosporins among a set of 26 antibiotics (Table 1). EH-202 and ATCC 15947 also displayed different 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.

Table 1.   Antibiotic susceptibilities of Edwardsiella tarda strains
Antibiotic agentsATCC 15947EH-202
Penicillins
 Penicillin GRS
 AmpicillinII
 Oxacillin sodiumRS
 PiperacillinSS
Tetracyclines
 TetracyclineSR
Quinolones
 NorfloxacinSS
 Ciprofloxacin hydrochlorideSS
 OfloxacinSS
Aminoglycosides
 AmikacinSS
 TobramycinSS
 KanamycinSS
 GentamycinSS
Cephalosporins
 CefotaximeSS
 CefoperazoneSS
 CefazolinSS
 CefuroximeSS
 CeftazidimeSS
 CefradineSS
 CeftriaxoneSS
Other antibiotics
 SulfamethoxazoleRS
 StreptomycinRR
 ChloramphenicolSR
 NitrofurantoinSS
 RifampicinRR
 ErythromycinRS
 VancomycinRS

The PCR determinations of cat and floR genes

All the isolated strains gave single and specific PCR amplification 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 floR gene could not be detected in all the isolated and reference strains. Furthermore, the 1800 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 identified ORFs displayed 99% identity to that of the previously isolated gene fragment from E. tarda (EF 467364).

Figure 2.

 Electrophoretic analyses of the polymerase chain reaction screening results of cat genes from the seven selected isolated strains from turbot, ATCC 15947, ET-M 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.

Growth of E. tarda isolates

Four E. tarda strains, ATCC 15947, EH-107, EH-202 and EH-306, displayed typical sigmoidal growth kinetics with identifiable lag and logarithmic growth phases. EH-202 showed significant 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 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 different tissues of the fish. The spleen-derived E. tarda EH-202 was found to be more virulent than other isolates to swordtail fish (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 five 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 EH-15, 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 fish for many fish 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 CFU g−1, similar to an earlier report by Pan, Wu, Li, Huang and Si (2000). 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 μg 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 G+C 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 difference between antibiotic-resistant genes detected from isolates in China and the reported isolates in Korea, indicates that antibiotic resistance was acquired through different 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 efficient and appropriate farm management.

Acknowledgments

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

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