Department of Clinical Laboratory, The First People's Hospital of Yunnan Province, Kunming, Yunnan, China.
The Affiliated Hospital of Kunming University of Science and Technology, Kunming, Yunnan, China.
Ann Clin Microbiol Antimicrob. 2024 Aug 20;23(1):73. doi: 10.1186/s12941-024-00734-y.
Klebsiella pneumoniae is the most commonly encountered pathogen in clinical practice. Widespread use of broad-spectrum antibiotics has led to the current global dissemination of carbapenem-resistant K. pneumoniae, which poses a significant threat to antibacterial treatment efficacy and public health. Outer membrane vesicles (OMVs) have been identified as carriers capable of facilitating the transfer of virulence and resistance genes. However, the role of OMVs in carbapenem-resistant K. pneumoniae under external pressures such as antibiotic and phage treatments remains unclear.
To isolate and purify OMVs under the pressure of phages and tigecycline, we subjected K. pneumoniae 0692 harboring plasmid-mediated bla and bla genes to density gradient separation. The double-layer plate method was used to isolate MJ1, which efficiently lysed K. pneumoniae 0692 cells. Transmission electron microscopy (TEM) was used to characterize the isolated phages and extract OMV groups for relevant morphological identification. Determination of protein content of each OMV group was conducted through bicinchoninic acid assay (BCA) and proteomic analysis.
K. pneumoniae 0692 released OMVs in response to different environmental stimuli, which were characterized through TEM as having the typical structure and particle size of OMVs. Phage or tigecycline treatment alone resulted in a slight increase in the mean protein concentration of OMVs secreted by K. pneumoniae 0692 compared to that in the untreated group. However, when phage treatment was combined with tigecycline, there was a significant reduction in the average protein concentration of OMVs compared to tigecycline treatment alone. Proteomics showed that OMVs encapsulated numerous functional proteins and that under different external stresses of phages and tigecycline, the proteins carried by K. pneumoniae 0692-derived OMVs were significantly upregulated or downregulated compared with those in the untreated group.
This study confirmed the ability of OMVs to carry abundant proteins and highlighted the important role of OMV-associated proteins in bacterial responses to phages and tigecycline, representing an important advancement in microbial resistance research.
肺炎克雷伯菌是临床实践中最常见的病原体。广谱抗生素的广泛使用导致了目前全球范围内碳青霉烯类耐药肺炎克雷伯菌的传播，这对抗菌治疗效果和公共卫生构成了重大威胁。外膜囊泡（OMVs）已被确定为能够促进毒力和耐药基因转移的载体。然而，在抗生素和噬菌体等外部压力下，OMVs 在碳青霉烯类耐药肺炎克雷伯菌中的作用尚不清楚。
为了在噬菌体和替加环素的压力下分离和纯化 OMVs，我们对携带质粒介导的 bla 和 bla 基因的肺炎克雷伯菌 0692 进行了密度梯度分离。双层平板法分离出能有效裂解肺炎克雷伯菌 0692 细胞的 MJ1。透射电子显微镜（TEM）用于对分离出的噬菌体进行特征描述，并提取 OMV 组进行相关形态学鉴定。通过二喹啉甲酸（BCA）法和蛋白质组学分析测定各 OMV 组的蛋白含量。
肺炎克雷伯菌 0692 对不同环境刺激释放 OMVs，TEM 特征为具有 OMVs 的典型结构和粒径。与未处理组相比，噬菌体或替加环素单独处理仅导致肺炎克雷伯菌 0692 分泌的 OMVs 的平均蛋白浓度略有增加。然而，当噬菌体处理与替加环素联合使用时，与单独使用替加环素相比，OMVs 的平均蛋白浓度显著降低。蛋白质组学表明，OMVs 包裹了大量功能蛋白，并且在噬菌体和替加环素的不同外部应激下，与未处理组相比，肺炎克雷伯菌 0692 衍生的 OMVs 携带的蛋白质显著上调或下调。
本研究证实了 OMVs 携带丰富蛋白质的能力，并强调了 OMV 相关蛋白在细菌对噬菌体和替加环素反应中的重要作用，这是微生物耐药性研究的重要进展。
