一株克氏原螯虾雷氏普罗威登斯菌的分离鉴定与联合药敏试验

Identification and synergistic susceptibility of pathogenic Providencia rettgeri isolated from diseased Procambarus clarkii

为了探明克氏原螯虾(Procambarus clarkii)5—6月死亡的病因并制定相应的治疗方案,本试验从患病克氏原螯虾肝胰腺中分离到1株病原菌(L20190509),通过生理生化特征、16S rRNA序列分析和进化树进行鉴定,通过琼脂扩散法测定了该菌株的药物敏感性,进一步研究了抗菌药物与天然化合物的联合抑菌作用。结果发现,该菌株对克氏原螯虾的半数致死浓度为9.27×10^(4)CFU·mL^(-1),其生理生化特征与雷氏普罗威登斯菌(Providencia rettgeri)一致,16S rRNA测序和进化树分析发现,分离株与雷氏普罗威登斯菌的亲缘关系最近,综合以上结果将该菌株鉴定为雷氏普罗威登斯菌。进一步通过药敏试验发现该菌株对20种受试药物中的亚胺培南、头孢噻肟、恩诺沙星3种药物敏感,对氟苯尼考和多西环素2种药物中度敏感,对其他受试药物均不敏感。此外,联合抑菌试验发现氟苯尼考与厚朴酚联用具有协同抑菌作用。以上研究发现,分离株L20190509是一株能导致克氏原螯虾发病的病原菌,具有多重耐药性,研究结果有助于进一步了解克氏原螯虾“五月瘟”的病因,并制定科学有效的治疗方案。

To clarify the cause of death of Procambarus clarkii(P.clarkii)during May to June and bring out treatment protocols,a bacterial pathogen(L20190509)was isolated from P.clarkia with typical symptom.The isolated pathogen was characterized by physiological and biochemical assay,16S rRNA sequencing and phylogenetic analysis.Then the sensitivity of the isolated pathogen was tested by agar diffusion method,the synergistic effect of antibiotics and natural compounds were determined by checkerboard assays.The results showed that the 50%lethal concentration of the isolate to healthy P.clarkii was 9.27×10^(4)CFU·mL^(-1),which exhibited similar physiological and biochemical characteristics to Providencia rettgeri(P.rettgeri)and shared over 99%homologies with P.rettgeri.Thus,the isolate was determined to be a P.rettgeri strain.The sensitivity study showed that the isolate was susceptible to imipenem,cefotaxime and enrofloxacin;intermediate to florfenicol and doxycycline;resistant to other tested antibiotics.Moreover,florfenicol combined with magnolol had synergistic effect against L20190509.Taken together,L20190509 is a bacterial pathogen of farmed P.clarkii which exhibited multi-resistance to antibiotics,these findings help to further acknowledge the pathogens of P.clarkii in May and formulate scientific and effective treatments.