Biofouling and Biofilm Processes Section, Water and Steam Chemistry Division, BARC Facilities, Kalpakkam, India.
Homi Bhabha National Institute, Anushaktinagar, Mumbai, India.
Curr Protoc. 2022 Mar;2(3):e395. doi: 10.1002/cpz1.395.
Bacteriophages are bacteria-targeting viruses that may prove useful as therapeutic agents against multidrug-resistant bacterial strains. Though phage therapy is a century-old concept, there is very limited progress on its therapeutic application due to the rapid expansion of antibiotics portfolios in the last few decades. However, the emergence of multidrug-resistant organisms has brought our attention back to bacteriophages. The first step towards developing effective phage therapy against multidrug-resistant bacteria is isolation, amplification, and purification of specific bacteriophages. There are many reported protocols for isolating host-specific bacteriophages from the environment. However, most of them are complex, multistep, low-yielding, resource-intensive protocols, requiring elaborate laboratory setup. We have demonstrated a simple two-step, high-yielding protocol for isolating and amplifying bacteriophages against methicillin-resistant Staphylococcus aureus (MRSA). We have shown that mixing various environmental samples (i.e., sample pooling) and phage amplification at two different temperatures significantly enhance the yield of MRSA phages. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of water sample filtrate for isolation of bacteriophages Basic Protocol 2: Bacterial strain and culture conditions Basic Protocol 3: Native bacteriophage count in water sample filtrate Basic Protocol 4: Isolation and enrichment of MRSA-specific bacteriophages Basic Protocol 5: Quantification of bacteriophages by drop cast method Basic Protocol 6: Effect of incubation temperature and heat shock on bacteriophage yield.
噬菌体是靶向细菌的病毒，有望成为对抗多药耐药菌的治疗药物。尽管噬菌体治疗是一个有百年历史的概念，但由于过去几十年抗生素种类的迅速增加，其治疗应用几乎没有进展。然而，多药耐药菌的出现使我们重新关注噬菌体。开发针对多药耐药菌的有效噬菌体疗法的第一步是分离、扩增和纯化特定的噬菌体。已经有许多报道从环境中分离宿主特异性噬菌体的方案。然而，大多数方案都很复杂，需要多步操作、产量低、资源密集，需要精心的实验室设置。我们已经证明了一种简单的两步法，可用于分离和扩增抗耐甲氧西林金黄色葡萄球菌（MRSA）的噬菌体。我们表明，混合各种环境样本（即样本混合）并在两种不同温度下进行噬菌体扩增，可显著提高 MRSA 噬菌体的产量。©2022 威利父子公司。基本方案 1：用于分离噬菌体的水样过滤液的制备基本方案 2：细菌菌株和培养条件基本方案 3：水样过滤液中天然噬菌体的计数基本方案 4：MRSA 特异性噬菌体的分离和富集基本方案 5：滴铸法定量噬菌体基本方案 6：孵育温度和热休克对噬菌体产量的影响。
