Isolation, identification, and analysis of virulence gene and drug resistance gene in pathogenic Edwardsiella piscicida from cultured turbot Scophthalmus maximus
HUANG Hua, YU Guanglei, WANG Liyong, GAO Yan, ZHANG Lan, SUN Baiming, ZHUANG Yanwen1, WANG He1*
1.Yantai Marine Economic Research Institute, Yantai 264006, China; 2.Shandong Provincial Key Laboratory of Restoration for Marine Ecology, Shandong Marine Resource and Environment Research Institute, Yantai 264006, China
Abstract: In order to explore the causes of diseases characterized by ascites and sepsis in turbot Scophthalmusmaximus with body weight of (350±5)g farmed in Yantai, Shandong Province in 2019, predominant bacterial strains were isolated from moribund individual tissues including liver, kidney, spleen, and heart, and identified by morphological observation, 16S rRNA gene sequence analysis, gyrB gene sequence analysis, rpoB gene sequence analysis, and specific PCR amplification. Genetic characteristics of the isolated strain were analyzed by virulence genes and drug resistance gene, and the susceptibility of the isolated strain to 10 categories of drugs including 23 antimicrobial dugs was determined using Kirby-Bauer diffusion method. The results showed that the strain isolated from the visceral tissues as Gram-negative bacillus named as H4-S18 was identified as Edwardsiella by the physiological features of positive lysine, ornithine and glucose acid production, and negative citrate, arginine, sucrose, mannitol, amygdalin, urea, nitrate reduction and oxidase. The strains H4-S18 was identified as Edwardsiellapiscicida by 16S rRNA, gyrB and rpoB gene sequence alignment and specific amplification of Edwardsiella. The virulence genotype was found to be fimA+-fimB+-fimC+-fimD+-esrB+-mukF+-katB+-sodB+-citC+-gadB+. The drug resistance genotype was found to be TEM+-ant(3″)-Ⅰ+-tet(A)+-mcr-1+-Sul2+-Sul3--oqxA-. The strain H4-S18 had median lethal concentration( LD50 )of 1.2×108 CFU/mL determined in the healthy turbot injected with the bacterial solution intraperitoneally at a dose of 7.8×1010, 7.8×109, 7.8×108 and 7.8×107 CFU/mL. Antibiotic sensitivity test showed that among 23 antibiotics tested, H4-S18 was resistant to 5 antibiotics such as sulfonamides, polypeptides, and rifamycins, but was sensitive to 18 antibiotics such as β-lactams, aminoglycosides, quinolones, tetracyclines, macrolides, chloramphenicols, and furan. It is considered that H4-S18, E.piscicida bacterium is the first pathogenic strain isolated from turbot with five resistant genes. The finding provides basic support for the research of fish-sourced E.piscicida disease surveillance, precision prevention, and control.
2022, Vol. 37 
