Guo Ai, Zuo Dianbao, Shi Li, Guo Ming, Li Jinquan, Li Caili, Wang Puqing, Sun Xiaodong, Sang Ming
Research Center for Translational Medicine, Department of Neurology, Hubei Provincial Clinical Research Center for Parkinson's Disease at Xiangyang No.1 People's Hospital, Hubei Key Laboratory of Wudang Local Chinese Medicine Research, Hubei University of Medicine, 15 Jiefang Road, Xiangyang, 441000, Hubei, China.
College of Biomedicine and Health, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
Ann Clin Microbiol Antimicrob. 2025 Aug 20;24(1):46. doi: 10.1186/s12941-025-00812-9.
The emergence of MDR K. pneumoniae poses a critical challenge in treating respiratory-associated pneumonia. Bacteriophages are promising antibiotic alternatives with unique features. This study aimed to isolate new bacteriophages from the hospital environment and investigate their therapeutic potential and mechanisms.
We employed plaque assays, transmission electron microscopy, and whole-genome sequencing to systematically characterize the biological properties, morphology, and genomic profiles of the phages in parallel. The bacteriostatic curve, biofilm staining quantification, and biofilm inhibition rate assay were employed to evaluate the in vitro lytic efficacy of the phage. More importantly, we established the murine pneumonia infection models through nasal instillation, assessed the therapeutic potential of the phage in vivo by observing pathological morphology via HE staining, detecting pro-inflammatory cytokine levels via qPCR and ELISA, and monitoring bacterial load changes in lung tissue through PCR analysis.
Phages vB_KpnP_XY3 and vB_KpnP_XY4, taxonomically classified as Siphoviridae, demonstrated broad temperature (4-60 °C), pH (4-11) tolerance, chloroform resistance, latent periods of 40/35 min, and burst sizes of 340/126 PFU/cell. Both genomes contained circular dsDNA genomes (47,466 bp/50,036 bp) without virulence or antibiotic resistance genes. The bacterial concentration markedly decreased at 2 h post-treatment, reaching its biological nadir by 6 h. Concurrent biofilm assays demonstrated 80% biofilm inhibition and rapid bacterial clearance. In murine pneumonia models, both phage monotherapy and phage-antibiotic combinations significantly reduced bacterial loads compared with antibiotics alone (P < 0.05), concurrently attenuating inflammation (IL-1β/IL-6/TNF-a. P < 0.0001) and restoring alveolar architecture with reduced necrosis.
The phages vB_KpnP_XY3 and vB_KpnP_XY4 demonstrated robust environmental adaptability. Its antibacterial effect is related to its specific biofilm dissolution performance in vivo and in vitro. These findings provide strong evidence for the precise phage treatment of MDR K. pneumoniae infections.
耐多药肺炎克雷伯菌的出现给呼吸道相关性肺炎的治疗带来了严峻挑战。噬菌体作为具有独特特性的抗生素替代品，具有广阔前景。本研究旨在从医院环境中分离新的噬菌体，并研究其治疗潜力及机制。
我们采用噬菌斑测定、透射电子显微镜和全基因组测序，并行系统地表征噬菌体的生物学特性、形态和基因组概况。采用抑菌曲线、生物膜染色定量和生物膜抑制率测定来评估噬菌体的体外裂解效力。更重要的是，我们通过滴鼻建立小鼠肺炎感染模型，通过苏木精-伊红（HE）染色观察病理形态、通过实时定量聚合酶链反应（qPCR）和酶联免疫吸附测定（ELISA）检测促炎细胞因子水平以及通过PCR分析监测肺组织中的细菌载量变化，来评估噬菌体在体内的治疗潜力。
噬菌体vB_KpnP_XY3和vB_KpnP_XY4，分类上属于长尾噬菌体科，表现出广泛的温度（4 - 60°C）、pH（4 - 11）耐受性、氯仿抗性、潜伏期为40/35分钟以及裂解量为340/126噬菌斑形成单位/细胞。两个基因组均包含环状双链DNA基因组（47,466碱基对/50,036碱基对），无毒性或抗生素抗性基因。治疗后2小时细菌浓度显著下降，6小时达到最低点。同时进行的生物膜测定显示生物膜抑制率为80%且细菌快速清除。在小鼠肺炎模型中，与单独使用抗生素相比，噬菌体单一疗法和噬菌体 - 抗生素联合疗法均显著降低了细菌载量（P < 0.05），同时减轻了炎症（白细胞介素-1β/白细胞介素-6/肿瘤坏死因子-α。P < 0.0001）并恢复了肺泡结构，坏死减少。
噬菌体vB_KpnP_XY3和vB_KpnP_XY4表现出强大的环境适应性。其抗菌作用与其在体内和体外的特定生物膜溶解性能有关。这些发现为耐多药肺炎克雷伯菌感染的精准噬菌体治疗提供了有力证据。
