Maharjan Archana, Nepal Roshan, Dhungana Gunaraj, Parajuli Apshara, Regmi Madhav, Upadhyaya Elisha, Mandal Dipendra, Shrestha Mitesh, Pradhan Pragati, Manandhar Krishna Das, Malla Rajani
Central Department of Biotechnology, Institute of Science and Technology, Tribhuvan University, Kirtipur, Nepal.
J Nepal Health Res Counc. 2022 Mar 13;19(4):717-724. doi: 10.33314/jnhrc.v19i04.3837.
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen frequently causing healthcare-associated infections. The apocalyptic rise of antimicrobial resistance has rekindled interest in age-old phage therapy that uses phages (viruses that infect bacteria) to kill the targeted pathogenic bacteria. Because of its specificity, phages are often considered as potential personalized therapeutic candidate for treating bacterial infections.
In this study, we isolated and purified lytic phages against multi-drug resistant P. aeruginosa using soft agar overlay technique. Phage characteristics like thermal and pH stability, latent period and burst size were determined using one-step growth assay while multiple host range spectrum was determined by spot assay. The phages were further characterized using protein profiling.
Three Pseudomonas phages (øCDBT-PA31, øCDBT-PA56 and øCDBT-PA58) were isolated from the holy rivers of Kathmandu valley. Among 3 phages, øCDBT-PA31 demonstrated multiple host range and could lyse multi-drug resistant strain of P. aeruginosa. Further, øCDBT-PA31 showed latent period of 30 minutes with corresponding burst sizes of 423-525 PFU/cell. Interestingly, øCDBT-PA31 also tolerated a wide range of adverse conditions, such as high temperature (50°C) and pH 3-11. Further, protein profiling revealed that øCDBT-PA31 has 4 and øCDBT-PA11 had 3 distinct bands in the gradient gel ranging from approximately 3.5-29 kilodaltons (kDa) suggesting them to be morphologically distinct from each other.
As multi-drug resistant bacteria are emerging as a global problem, lytic phages can be an alternative treatment strategy when all available antibiotics fail.
铜绿假单胞菌是一种革兰氏阴性机会致病菌，常引起医疗保健相关感染。抗菌药物耐药性的急剧上升重新激发了人们对古老的噬菌体疗法的兴趣，该疗法利用噬菌体（感染细菌的病毒）杀死目标病原菌。由于其特异性，噬菌体常被视为治疗细菌感染的潜在个性化治疗候选物。
在本研究中，我们使用软琼脂覆盖技术分离并纯化了针对多重耐药铜绿假单胞菌的裂解性噬菌体。使用一步生长试验确定噬菌体的热稳定性和pH稳定性、潜伏期和裂解量等特性，同时通过点滴试验确定多个宿主范围谱。通过蛋白质谱对噬菌体进行进一步表征。
从加德满都谷地的圣河中分离出三种假单胞菌噬菌体（øCDBT-PA31、øCDBT-PA56和øCDBT-PA58）。在这三种噬菌体中，øCDBT-PA31表现出多个宿主范围，能够裂解铜绿假单胞菌的多重耐药菌株。此外，øCDBT-PA31的潜伏期为30分钟，相应的裂解量为423-525 PFU/细胞。有趣的是，øCDBT-PA31还能耐受广泛的不利条件，如高温（50°C）和pH 3-11。此外，蛋白质谱显示，øCDBT-PA31在梯度凝胶中有4条不同的条带，øCDBT-PA11有3条不同的条带，范围约为3.5-29千道尔顿（kDa），表明它们在形态上彼此不同。
由于多重耐药细菌正在成为一个全球性问题，当所有可用抗生素都失效时，裂解性噬菌体可以作为一种替代治疗策略。
