[논문]어류병원성 Edwardsiella spp.의 2010년 이전 분리균의 재분류와 특성

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doi:10.7847/jfp.2023.36.1.095

어류병원성 Edwardsiella spp.의 2010년 이전 분리균의 재분류와 특성
Reclassification of fish pathogenic Edwardsiella spp. isolated before 2010 and their characteristics 원문보기

韓國魚病學會誌= Journal of fish pathology, v.36 no.1, 2023년, pp.95 - 105

초록
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이전에 Edwardsiella tarda로 분류되었던 많은 어류 병원세균들이 다양한 연구에서 E. piscicida 또는 E. anguillarum으로 재분류되었다. 본 연구에서는 국내에서 1996년부터 2010년까지 양식 넙치와 뱀장어의 감염어에서 분리하여 E. tarda로 동정하였던 17균주의 재분류를 실시하였다. 이를 위해 species-specific primers를 이용한 특정 PCR amplicon 검출과 gyrB 염기서열에 기반한 계통발생학적 분석을 수행하였다. 또한 숙주에 따른 분리주 특성의 차이를 알아보기 위하여 항균제 감수성 패턴과 random amplified polymorphic DNA (RAPD) PCR의 amplicons에 대한 염기서열을 분석하였다. E. piscicida 특이 프라이머를 사용한 PCR 결과 17개의 분리주만 amplicon을 형성하였고, E. tarda 프라이머를 사용했을 때는 E. tarda type strain (ATCC 15947)에서만 amplicon이 나타났다. 17개 분리주의 gyrB 염기 서열에 따른 계통수에서 모든 분리주는 E. piscicida의 참조 균주들과 함께 E. tarda type strain 과는 분리되는 clade를 형성하였으며 넙치와 뱀장어에서 분리된 균들은 각각의 진화 그룹으로 구분되었다. 항균제 감수성 시험 결과 넙치에서 분리된 균들은 doxycycline, oxytetracycline, tetracycline에서 non-wild type (NWT)이 나타났고 oxolinic acid, flumequine에 대해서는 뱀장어에서 분리된 균에서만 NWT이 나타났다. E. piscicida 분리주들의 RAPD 패턴에서 차이나는 3개 amplicons의 뉴클레오티드 서열을 분석한 결과 Edwardsiella phage GF-2, E. piscicida MS-18-199의 플라스미드 pEPMS-18199 및 E. anguilllarum ET080813의 플라스미드 1에서 일치하는 영역이 확인되었다. 이러한 결과에 근거하여, 본 연구의 17 분리주는 모두 E. piscicida로 분류될 수 있으며, 분리주의 특성은 숙주의존적인 차이를 보이는 것으로 판단되었다.

Abstract ▼ AI-Helper

Edwardsiella piscicida previously identified as E. tarda is an important pathogen of numerous fish species. In this study, reclassification was conducted on 17 strains isolated from diseased cultured olive flounder or eel and identified as E. tarda between 1996 and 2010 in Korea. For this, the detection of a polymerase chain reaction (PCR) amplicon by species-specific primers and phylogenetic analysis based on gyrB sequences were performed. And, nucleotide sequences of three amplicons detected in RAPD profile of the isolates originated from eel and the antimicrobial susceptibility were compared. Only 17 isolates formed an amplicon in PCR using E. piscicida primer. The gyrB of the 17 isolates diverged with those of the E. piscicida reference strains in a phylogenetic tree constructed with MEGA11 software. Regions matching the nucleotide sequences of the RAPD amplicons were identified in Edwardsiella phage GF-2, E. piscicida plasmid pEPMS-18199 and E. anguilllarum ET080813 plasmid 1. The antimicrobial susceptibility of the isolates originated from flounder showed non-wild type (NWT) for doxycycline, oxytetracycline, and tetracycline, and only isolates derived from eel were NWT for oxolinic acid and flumequine. These results confirmed that all 17 isolates of this study classified as E. piscicida, and that the characteristics of the isolates showed differences depending on the isolate's host.

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표/그림 (6)

표 Table 1. Primers used for the detection of species-specific amplicons (Griffin et al., 2014)
표 Table 2. Origin of Edwardsiella reference strains used for comparisons of gyrB sequences and their GenBank accession numbers
표 Table 3. Results of species-specific PCR
그림 Fig. 1. Phylogenetic tree constructed using the Neighbor-Joining method with DNA gyrase subunit B gene (gyrB) sequences of reference strains obtained from GeneBank and those of Edwardsiella isolates from Paralichthys olivaceus and common eel. Klebsiella pneumoniae KAM260 was used as outgroup. The evolutionary distances were computed using the Kimura 2-parameter method. Boostrap values based on 1,000 replications are shown next to the branches. Evolutionary analysis were conducted in MEGA11 (Tamura et al., 2021). The picture inside the square box is the topology of the subtree.
표 Table 4. Reference sequences showing high homology in BLAST searching of RAPD amplicon of R1 in E09-2, and R2 and R3 in E97-1
표 Table 5. Non-wild type(NWT) profiles determined according to wild type cut-off (COwt) value of inhibition zone in disc diffusion test of various antibacterial agents against Edwardsiella isolates

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용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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