中华绒螯蟹病原弗氏柠檬酸杆菌的鉴定

目的对从一起自然发病的病(死)中华绒螯蟹(Eriocheir sinensisH.Milne-Edwards)肝胰腺中,所分离做纯培养的6株(HQ010516B-1至HQ010516B-6)菌,进行在主要生物学性状、感染试验的致病作用、对抗菌类药物的敏感性等方面的检验,旨在明确分离菌的种类、病原学意义及耐药特征等。方法在发病情况及主要病变检验的基础上,以6只病(死)蟹的肝胰腺为材料做细菌分离与纯培养,对纯培养菌做形态特征、理化特性、16S rRNA基因序列测定及系统发育学分析等方面较系统的鉴定,主要依据《Bergey’s Manual of Determinative Bacteriology.9th ed》(1994)并结合系统发育特征做菌种归类判定;以3×108CFU/ml的菌悬液,对健康蟹做感染试验,测定相应的致病作用;用琼脂扩散(K-B)法做对常用抗菌类药物的敏感性测定,以抑菌圈直径作为敏感与耐药的判定指标。结果分离做纯培养的6株菌为同种的弗氏柠檬酸杆菌(Citrobacter freundi-i),对供试健康蟹有致病性,对供试37种抗菌类药物中的头孢噻肟等22种药物呈敏感或高度敏感、对四环素等5种呈低度敏感、对青霉素G等10种耐药。结论所检出的弗氏柠檬酸杆菌,构成了所检中华绒螯蟹病例的病原菌之一,对供试抗菌类药物未表现出敏感与耐药的明显株间差异性。

Identification of Citrobacter freundii isolated from crab (Eriocheir sinensis)

Six strains(HQ010516B-1 to HQ010516B-6) isolated from liver of naturally diseased(or dead) crab(Eriocheir sinensis) were examined by examine the main biological properties,pathogenicity,and sensitivity to antimicrobial agents,so as to clarify classification of isolates,their pathogenicity and traits of resistance to antimicrobial agents.Liver of 6 diseased crab were used as samples for isolation and cultivation of pathogenic bacteria.Identification of pure cultures were conducted systematically including morphological characteristics,physiological and biochemical characteristics,detection of 16S rRNA gene sequence and phylogenetic analysis.The isolates were classified based on the criteria described in the《Bergey's Manual of Determinative Bacteriology》and the results of phylogenetic analysis;and pathogenicity of the isolates were determinded by inoculation of the healthy crab with bacteria suspension of 3×10~8CFU/ml.Antimicrobial susceptibility tests of isolates were detected by the routine method of K-B,susceptibility determination to antimicrobial agents as demonstrated by the diameter of the zones of inhibition.Six pure cultures were identified as Citrobacter freundii;and isolates have strong pathogenicity to healthy crab.Antibiotic sensitivity using 37 antimicrobial agents showed that all strains were sensitive or high sensitive to 22 agents including cefotaxime.And were slight sensitive to 5 agents including tetrecycline,but were resistant to 10 agents including penicillin G.The examined Citrobacter freundii were one of the pathogen of crab disease,and there is no difference in sensitivity and resistance to the tested antimicrobial agents among different strains.