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石潆含, 王秀丽, 圣朝军, 王凯飞, 肖坤, 解立新, 张伟华. 噬菌体PCCM_KpP1172的鉴定及其对大蜡螟幼虫碳青霉烯耐药高毒力肺炎克雷伯菌感染的疗效评估[J]. 解放军医学院学报. DOI: 10.12435/j.issn.2095-5227.2023.137
引用本文: 石潆含, 王秀丽, 圣朝军, 王凯飞, 肖坤, 解立新, 张伟华. 噬菌体PCCM_KpP1172的鉴定及其对大蜡螟幼虫碳青霉烯耐药高毒力肺炎克雷伯菌感染的疗效评估[J]. 解放军医学院学报. DOI: 10.12435/j.issn.2095-5227.2023.137
shu
SHI Yinghan, WANG Xiuli, SHENG Zhaojun, WANG Kaifei, XIAO Kun, XIE Lixin, ZHANG Weihua. Identification of bacteriophage PCCM_KpP1172 and its therapeutic efficacy evaluation against CR-hvKP infection in Galleria mellonella larvae[J]. ACADEMIC JOURNAL OF CHINESE PLA MEDICAL SCHOOL. DOI: 10.12435/j.issn.2095-5227.2023.137
Citation: SHI Yinghan, WANG Xiuli, SHENG Zhaojun, WANG Kaifei, XIAO Kun, XIE Lixin, ZHANG Weihua. Identification of bacteriophage PCCM_KpP1172 and its therapeutic efficacy evaluation against CR-hvKP infection in Galleria mellonella larvae[J]. ACADEMIC JOURNAL OF CHINESE PLA MEDICAL SCHOOL. DOI: 10.12435/j.issn.2095-5227.2023.137
shu

噬菌体PCCM_KpP1172的鉴定及其对大蜡螟幼虫碳青霉烯耐药高毒力肺炎克雷伯菌感染的疗效评估

Identification of bacteriophage PCCM_KpP1172 and its therapeutic efficacy evaluation against CR-hvKP infection in Galleria mellonella larvae

    摘要
  • 摘要:
      背景  碳青霉烯耐药的高毒力肺炎克雷伯菌感染发生率逐年增加，临床治疗困难，死亡率高。使用具有高裂解能力的噬菌体治疗细菌感染是具有前景的治疗方法。
      目的  针对碳青霉烯耐药高毒力肺炎克雷伯菌分离一株新型裂解性T7噬菌体，对该分离株进行生物学特性测定和基因组学测序分析，为临床开展噬菌体治疗提供可应用的噬菌体资源。
      方法  从肺部感染患者的痰液中分离出肺炎克雷伯菌，通过细菌鉴定、药敏分析、全基因组测序与PCR检测验证菌株的毒力基因与耐药基因，以这株肺炎克雷伯菌为宿主菌，在污水中分离出一株裂解性噬菌体，命名为PCCM_KpP1172。测定该噬菌体生物学特性，分析其基因组序列，并通过大蜡螟幼虫感染模型检测该噬菌体的治疗效果。
      结果  该株肺炎克雷伯菌基因组分析存在耐药基因与毒力基因rmpA2、rmpA、iroN与icu。以此菌株为宿主菌分离到新型肺炎克雷伯菌噬菌体，命名为PCCM_KpP1172，在宿主菌菌苔上可形成完全透明的噬菌斑并伴晕圈；透射电镜下呈现有尾噬菌体目短尾病毒科病毒的典型形态特征；一步生长曲线显示其潜伏期为15分钟，最佳感染复数(MOI)为0.000 1，对ST11KL64型肺炎克雷伯菌有广泛裂解范围。基因组分析显示，该噬菌体为双链DNA(总长度为40 222 bp)，G + C含量为53%，基因组包含49个开放阅读框(ORF)，无毒力或抗生素耐药性相关基因。基于系统发育分析，该噬菌体可归属于有尾噬菌体目Studiervirinae亚科Przondovirus属的一个新种。此外，噬菌体PCCM_KpP1172能在体外3h内有效抑制碳青霉烯耐药肺炎克雷伯菌的生长，并能提高宿主菌感染的大蜡螟幼虫存活率(P<0.01)。
      结论  本研究针对碳青霉烯耐药的高毒力肺炎克雷伯菌分离并鉴定了一株新的T7噬菌体PCCM_KpP1172，具有高裂解力，无耐药基因及毒力基因，对大蜡螟幼虫感染模型具有良好治疗效果。

     

    Abstract:
      Background  The incidence of carbapenem-resistant and highly virulent Klebsiella pneumoniae infections has been increasing annually, posing significant clinical challenges and high mortality rates. Phage therapy, utilizing highly lytic bacteriophages, holds great promise as a potential therapeutic approach.
      Objective  To isolate a novel lytic T7 phage specifically targeting carbapenem-resistant and highly virulent K. pneumoniae strains and conduct biological characterization and genomic sequencing analysis of the isolated phage, PCCM_KpP1172, so as to provide applicable phage resources for clinical phage therapy.
      Methods  K. pneumoniae was isolated from sputum samples of patients with pulmonary infections. Bacterial identification, antibiotic susceptibility testing, whole-genome sequencing, and PCR detection were used to validate the bacterial strain's virulence and antibiotic resistance genes. A lytic bacteriophage was isolated from sewage using bacterial strain as the host and named PCCM_KpP1172. The biological characteristics of the phage were determined, and its genome sequence was analyzed. The therapeutic efficacy of phage PCCM_KpP1172 was assessed using the Galleria mellonella infection model.
      Results  The genomic analysis of the K. pneumoniae strain revealed the presence of antibiotic resistance genes and virulence genes, including rmpA2, rmpA, iroN, and icu. A novel K. pneumoniae phage, PCCM_KpP1172, was isolated using this bacterial strain as the host. The phage formed clear plaques with a halo on the host lawn, and under transmission electron microscopy, it exhibited typical characteristics of the Siphoviridae family within the Caudovirales order. The one-step growth curve showed a latent period of 15 minutes and an optimal multiplicity of infection (MOI) of 0.0001, indicating a broad lytic range against ST11KL64-type K. pneumoniae. Genome analysis revealed that the phage contained a double-stranded DNA with a total length of 40 222 bp and a G + C content of 53%. It consisted of 49 open reading frames (ORFs) without any genes related to virulence or antibiotic resistance. Based on phylogenetic analysis, the phage was classified as a novel species within the Studiervirinae subfamily of the Podoviridae family. Additionally, phage PCCM_KpP1172 effectively inhibited the growth of carbapenem-resistant Klebsiella pneumoniae in vitro within 3 hours and significantly increased the survival rate of G. mellonella larvae infected with K. pneumoniae (P<0.01).
      Conclusion  This study has successfully isolated and identified a novel T7 phage, PCCM_KpP1172, specifically targeting carbapenem-resistant and highly virulent K. pneumoniae strains. The phage exhibits high lytic activity and lacked genes associated with antibiotic resistance or virulence. Furthermore, it demonstrates significant therapeutic effects in the G. mellonella infection model. These findings provide valuable evidence for the potential clinical application of phage PCCM_KpP1172 in the treatment of hypervirulent carbapenem-resistant K. pneumoniae infections.

     

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