| 摘要: |
| 为探究噬菌体作为新型生物抗菌剂防控肺炎克雷伯菌引起的奶牛乳房炎和乳制品及其加工过程污染，以及对生物被膜清除的能力，以ATCC 700603为宿主菌从某牛场水样中分离得到1株肺炎克雷伯菌噬菌体vB_KpnM-YP11，并通过透射电镜观察噬菌体形态特征；通过双层平板法测定噬菌体宿主谱；通过最佳感染复数、一步生长曲线、温度和pH稳定性测定噬菌体生物学特性；通过全基因组测序分析基因组的结构特征、比较其进化关系；通过结晶紫染色法和平板计数法测定噬菌体对生物被膜的抑制和清除效果；通过牛奶试验分析该噬菌体对牛奶中肺炎克雷伯菌的抑菌效果。结果表明：1）从牛场水样中分离获得肺炎克雷伯菌噬菌体vB_KpnM-YP11，能形成清晰透亮噬菌斑，透射电镜观察vB_KpnM-YP11具有肌尾噬菌体的典型结构。2）测定噬菌体宿主谱，表明vB_KpnM-YP11能够裂解 18/34 株牛源肺炎克雷伯菌。3）生物学特性结果表明vB_KpnM-YP11最佳感染复数为0.1，vB_KpnM-YP11感染宿主菌潜伏期为30 min，爆发期为50~90 min，110 min后进入平台期，平均裂解量为69 PFU/cell，在pH（3~11）和温度（4~60 ℃）范围内活性稳定。4）vB_KpnM-YP11基因组全长166 607 bp，GC含量为39.52%，注释结果显示共有276个ORF和15个tRNA，不含耐药基因及毒力基因。5）vB_KpnM-YP11最佳感染复数为0.1时，具有显著抑制肺炎克雷伯菌生物被膜的形成能力。vB_KpnM-YP11效价为108 PFU/mL，109 PFU/mL能有效清除肺炎克雷伯菌生物被膜。在室温条件下，12 h内vB_KpnM-YP11使牛奶中细菌浓度下降98%。综上，本研究分离获得噬菌体vB_KpnM-YP11潜伏期短、裂解效率高，不仅能抑制和清除生物被膜，同时对牛奶中肺炎克雷伯菌也有显著的抑菌效果，具有作为肺炎克雷伯菌生物抗菌剂的应用潜力。 |
| 关键词: 肺炎克雷伯菌 噬菌体 生物学特性分析 基因组分析 生物被膜 |
| DOI：10.11841/j.issn.1007-4333.2024.09.09 |
| 投稿时间：2024-03-21 |
| 基金项目:国家自然科学基金青年科学基金项目（31802226）；黑龙江省自然科学基金联合引导项目（LH2022C072）；黑龙江省牛病防制重点实验室开放课题（PCBD201710） |
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| Isolation, identification and antibacterial effect study of Klebsiella pneumoniae phage |
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KONG Xiangyu, YANG Wenjing, PANG Shenyu, YUAN Qingxin, PAN Shunyuan, GAO Dongyang, SONG Jun, WANG Jintao
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| (College of Animal Science and Veterinary Medicine/Ministry of Agriculture and Rural Affairs of Key Laboratory of Prevention and Control of Bovine Diseases/Key Laboratory of Bovine Disease Control in Northeast Cold Regions of Heilongjiang Province，Heilongjiang Bayi Agricultural University， Daqing 163319， China) |
| Abstract: |
| The study was to explore the phage as a new type of biological antibacterial agent to control Klebsiella pneumoniae， which cause dairy cow mastitis and contaminate dairy products and their processing， and the ability of biofilm clearance.This study utilized ATCC 700603 as the host bacteria isolated a strain of Klebsiella pneumoniae phage vB_KpnM-YP11 from a water sample of a cattle farm and the morphological characteristics of the phage were observed by transmission electron. The host spectrum of phage was determined by double-layer plate method. The biological characteristics of phages were assessed through an optimal multiplicity of infection， one-step growth curve， as well as temperature and pH stability tests. The structual characteristics and evolutionary relutionary of the genome were analyzed by whole genome sequencing. The inhibitory and scavenging effects of phage on biofilms were determined using crystal violet staining and plate counting methods. The antibacterial effect of the phage on Klebsiella pneumoniae in milk was analyzed. The results showed that： 1） The Klebsiella pneumoniae phage vB_KpnM-YP11 isolated from the water samples of cattle farms could form clear and translucent plaques.The typical structure of tailed phage was observed by TEM. 2） Determination of host spectrum showed that vB_KpnM-YP11 could lyse 18/34 clinical isolates of Klebsiella pneumoniae from cattle. 3） The optimal multiplicity of infection （MOI） of this phage was 0.1. The one-step growth curve showed that the latent period was 30 min， and the average lysis amount was 69 PFU/cell. The phage was stable at 4-60 ℃ or pH 3-11. 4） The full-length genome of vB_KpnM-YP11 was 166 607 bp， and the GC content was 39.52%. The annotation results revealed that the phage contained 276 ORF and 15 tRNA， while lacking drug resistance and virulence genes. 5） When the MOI of vB_KpnM-YP11 was 0.1， it had the ability to inhibit the biofilm formation of Klebsiella pneumoniae. The mature biofilm were cleared by 108 PFU/mL and 109 PFU/mL. At room temperature， the bacterial concentration in milk decreased by 98% within 12 h.In conclusion， Phage vB_KpnM-YP11 with short incubation period and high lysis efficiency not only has a good lytic effect on Klebsiella pneumoniae in milk， but also can effectively remove the biofilm formed by Klebsiella pneumoniae. It has the potential to be used as a biological antibacterial agent for Klebsiella pneumoniae. |
| Key words: Klebsiella pneumoniae phage biological characteristics genomic analysis biofilm |
