鸭源多杀性巴氏杆菌的分离鉴定及药敏试验

Isolation,identification and antimicrobial susceptibility test of Pasteurella multocida from duck

目的:从疑似禽霍乱的4只病死鸭的病料中分离并鉴定多杀性巴氏杆菌(Pasteurella multocida,Pm),并通过药敏试验筛选敏感药物用于治疗。方法:无菌采集肝脏组织病料,接种至普通营养琼脂、麦康凯及血琼脂培养基上进行病原菌的培养、分离纯化;对纯化后的分离菌进行革兰染色、镜检,提取其核酸作为模板,进行16S rRNA基因的PCR鉴定;圆纸片扩散试验测定分离菌株对18种抗菌药物的敏感程度。结果:在血琼脂培养基上分离菌株表现为稍微隆起、表面光滑、湿润、灰白色、露珠样、半透明的圆形菌落,但在普通营养琼脂和麦康凯培养基上不生长。革兰染色镜检显示分离菌为一种革兰染色阴性、菌体两端着色深、中间着色较浅的球状短小杆菌。PCR扩增16S rRNA出现1 500 bp的目的条带,其序列分析与Pm菌株Q的16S rRNA基因的相似性为99.86%;药敏试验表明,分离菌株对米诺环素敏感性最高,抑菌圈直径达19 mm,对四环素、强力霉素、庆大霉素、氨苄西林、头孢哌酮表现中度敏感,其他药物均为耐药。结论:本研究从一例疑似禽霍乱的病死鸭中分离鉴定出1株多杀性巴氏杆菌,分离菌株对米诺环素较为敏感,建议用于临床治疗。

Objective:To isolate and identify Pasteurella multocida from 4 dead ducks suspected to be suffered from cholera,and the sensitive drugs were selected for treatment by antimicrobial susceptibility test.Methods:Liver tissue samples were collected aseptically and pathogenic bacteria were isolated on common nutrient medium,MacConkey medium,blood agar medium,then Gram staining and microscopic examination were conducted for the single colony of the isolated strain.The nucleic acid was extracted from a single purified colony as a template for PCR identification of 16S rRNA.At last,the sensitivity test of the isolates to 18 antimicrobial agents was determined by disk diffusion method.Results:The isolated strain on blood agar showed round colony with slightly raised,smooth,moist,gray-white,dew-like,translucent,but those did not grow on common nutrient medium and MacConkey medium.Gram staining microscopy showed micrococcus globularis with negative gram staining,deep coloring at both ends and light coloring in the middle.The 16S rRNA was amplified by PCR with a target band of 1500 bp,and the similarity of 16S rRNA sequencing results was 99.86%compared with the strain Q of Pasteurella multocida.The results of antimicrobial susceptibility test showed that,the isolates were most sensitive to minocycline,with an inhibition zone of 19 mm in diameter,and moderately sensitive to tetracycline,doxycycline,gentamicin,ampicillin and cefoperazone,while resistant to other drugs.Conclusion:In this study,a strain of Pasteurella multocida was isolated from the dead ducks suspected to be avian cholera.The strain was most sensitive to minocycline and could be used for clinical treatment.