铜绿假单胞菌噬菌体的分离筛选和体内外抗菌活性研究

Isolation and Screening of Bacteriophages Infecting Pseudomonas aeruginosa and Evaluation of Their Antibacterial Efficacy in vitro and in vivo

目的:分离筛选针对铜绿假单胞菌的强裂解性噬菌体,并研究其生物学特性和体内外抗菌活性。方法:药敏纸片法筛选铜绿假单胞菌的耐药菌株,以此为宿主菌,采用双层平板法和斑点法分离筛选噬菌体并分析其生物学特性,透射电镜观察噬菌体的微观形态,检测噬菌体和细菌共培养液的OD600值分析体外抗菌活性,建立小鼠全身感染模型评估噬菌体疗效。结果:分离获得3株铜绿假单胞菌噬菌体,从中筛选出一株宽谱的强裂解活性噬菌体PPa24。PPa24属于长尾噬菌体科,其在55℃以下、pH6~10条件下活性稳定,最佳感染复数为0.1,一步生长曲线显示PPa24感染宿主菌的潜伏期为20 min、裂解期为70 min、裂解量为62 PFU·cell^(-1)。噬菌体PPa24可以有效降低细菌培养物的OD_(600)值,用来治疗小鼠全身感染可显著降低血液载菌量(P<0.01),并使存活率提高至75%(P<0.001)。结论:筛选得到的噬菌体PPa24在抗击铜绿假单胞菌感染方面初步显示了良好的体内外活性,可能成为治疗铜绿假单胞菌感染的潜在抗菌剂。

Objective:To isolate and screen lytic phages infecting drug-resistant Pseudomonas aeruginosa and study their biological characteristics and activity of lyticating bacteria in vitro and in vivo.Methods:A disk diffusion method was used to screen drug-resistant strains from clinically isolated P.aeruginosa strains.The phages were isolated and screened by a double-layer agar plating method and a spotting method with the selected drug-resistant strains as host bacteria.The biological characteristics of the phages were detected.The transmission electron microscope was used to observe the microstructure of the phages.OD600 values were determined to analyze phage activities of lyticating bacteria in vitro,and a sepsis model of mice was established to evaluate the efficacy of phages.Results:Three strains of phages were isolated,and the broad-spectrum phage PPa24 with high lytic activity was screened out.Phage PPa 24 belonged to the Siphoviridae family.The results of biological characteristics study showed that the suitable temperature was below 55℃,the suitable pH was pH 6-10,the optimal multiplicity of infection was 0.1.The one-step growth curve showed that the latent period was 20 min,the outbreak period was 70 min,and the burse size was 62 PFU·cell^(-1).In vitro,phage PPa24 significantly reduced the OD_(600) value of bacterial culture.In vivo,phage therapy reduced the blood bacterial load(P<0.01)and increased the survival rate to 75%(P<0.01).Conclusion:Phage PPa24 exhibited good activity in vitro and in vivo and might become a potential antimicrobial agent in fighting against the infection of drug-resistant P.aeruginosa.