Mohamed Amira A, El-Zayat Emad M, El-Shibiny Ayman
Center for Microbiology and Phage Therapy, Zewail City of Science and Technology, Giza, 12578, Egypt.
Department of Biotechnology, Faculty of Science, Cairo University, Giza, Egypt.
Virol J. 2025 Jul 12;22(1):236. doi: 10.1186/s12985-025-02848-x.
Pan-drug resistance (PDR) is a ticking time bomb, as it causes high human hospitalizations and mortality rates. For instance, Pseudomonas aeruginosa is associated with significant rates of urinary tract infections (UTIs) due to several reasons including antibiotic resistance, biofilm formation and the presence of various virulence factors. Consequently, there is an urgent need for safe and effective alternative antibacterials. Phage therapy is a promising alternative that uses naturally occurring bacteriophages (phages). Therefore, our present study investigated the isolation and characterization of a novel virulent phage (vB_Ps_ZCPS13) against the PDR Pseudomonas aeruginosa strain (Ps13).
Phage vB_Ps_ZCPS13 was isolated from raw sewage water in Egypt during the springtime. The isolated phage was purified and amplified, followed by estimating its purity and genome size using pulsed-field gel electrophoresis (PFGE), morphology using transmission electron microscopy (TEM), antibacterial activity against other P. aeruginosa hosts, physiochemical stability studies, whole genome sequencing, antibiofilm activity on urinary catheters using scanning electron microscopy (SEM), and cytotoxicity assays against normal human skin fibroblast (HSF) cell lines.
Based on vB_Ps_ZCPS13 morphology under TEM, the phage has been classified as a myovirus. In consistent with the PFGE results, DNA sequencing revealed a phage genome size of 92,443 bp, with lytic-associated genes and no antimicrobial resistance or virulence factors. Phage vB_Ps_ZCPS13 presented a wide host range of over 93% of tested clinical isolates having different multiple antibiotic resistance (MAR) indices. Furthermore, phage vB_Ps_ZCPS13 exhibited high efficiency in plaque formation (EOP ≥ 1) against 13% of the strains and exhibited low frequencies of bacteriophage insensitive mutants (BIM). The physical stability test against harsh environmental conditions revealed phage stability within a pH range of 3.0-11.0 and stable at temperatures below 70 °C. Phage vB_Ps_ZCPS13 also exposed a significant antibacterial activity in vitro across different MOIs, with the highest reduction in bacterial growth observed at lower MOIs. Furthermore, vB_Ps_ZCPS13 demonstrated potent biofilm inhibition and clearance capabilities, effectively eradicating P. aeruginosa from the urinary catheter surface. Moreover, the phage presented no cytotoxicity against normal human skin fibroblast (HSF) cell lines at high titer.
Our study offers an effective phage as a therapeutic candidate against PDR Gram-negative P. aeruginosa infections, including catheter-associated urinary tract infections.
泛耐药性（PDR）是一颗定时炸弹，因为它导致人类住院率和死亡率居高不下。例如，铜绿假单胞菌与显著比例的尿路感染（UTIs）相关，原因包括抗生素耐药性、生物膜形成以及各种毒力因子的存在。因此，迫切需要安全有效的替代抗菌药物。噬菌体疗法是一种有前景的替代方法，它使用天然存在的噬菌体。因此，我们目前的研究调查了一种针对泛耐药铜绿假单胞菌菌株（Ps13）的新型烈性噬菌体（vB_Ps_ZCPS13）的分离和特性。
噬菌体vB_Ps_ZCPS13于春季从埃及的未经处理的污水中分离出来。对分离出的噬菌体进行纯化和扩增，然后使用脉冲场凝胶电泳（PFGE）估计其纯度和基因组大小，使用透射电子显微镜（TEM）观察其形态，检测其对其他铜绿假单胞菌宿主的抗菌活性，进行理化稳定性研究，进行全基因组测序，使用扫描电子显微镜（SEM）检测其对导尿管的抗生物膜活性，以及针对正常人皮肤成纤维细胞（HSF）细胞系进行细胞毒性测定。
基于TEM下vB_Ps_ZCPS13的形态，该噬菌体被归类为肌病毒。与PFGE结果一致，DNA测序显示噬菌体基因组大小为92,443 bp，具有溶菌相关基因，无抗菌耐药性或毒力因子。噬菌体vB_Ps_ZCPS13呈现出广泛的宿主范围，超过93%的测试临床分离株具有不同的多重抗生素耐药（MAR）指数。此外，噬菌体vB_Ps_ZCPS13在针对13%的菌株形成噬菌斑方面表现出高效率（EOP≥1），并且噬菌体不敏感突变体（BIM）的频率较低。针对恶劣环境条件的物理稳定性测试表明，噬菌体在pH值3.0 - 11.0范围内稳定，在70°C以下的温度下也稳定。噬菌体vB_Ps_ZCPS13在不同的感染复数（MOI）下也表现出显著的体外抗菌活性，在较低的MOI下观察到细菌生长的最大减少。此外，vB_Ps_ZCPS13表现出强大的生物膜抑制和清除能力，有效地从导尿管表面根除铜绿假单胞菌。此外，该噬菌体在高滴度下对正常人皮肤成纤维细胞（HSF）细胞系没有细胞毒性。
我们的研究提供了一种有效的噬菌体作为治疗泛耐药革兰氏阴性铜绿假单胞菌感染（包括导管相关尿路感染）的候选药物。
