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RISS 인기검색어
- 검색키워드
- 저자 : Vu Thao Nguyen (검색결과 2 건)
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Thao Nguyen Vu Graduate School, Yonsei University 2020 국내석사
Acinetobacter baumannii is an oppotunistic pathogen that causes serious nosocomial infection in intensive care units. Especially, carbapenem-resistant A. baumannii (CRAB) strains has emerged as a life-threatening pathogen in hospital settings. In this study, we isolated eight A. baumannii phages, which cause lysis of CRAB strains, from sewage sample at a hospital in South Korea. Among the phages, phage Bϕ-R2919 and Bϕ-R1888, which exhibited a broad host range and showed synergy with colistin in double disk potentiation test (DDPT), respectively, were studied. These two phages belonging to the Myoviridae family showed high absorption rate (> 98% within 5 min) and burst size (112-398 PFU/ cell). Both phages showed strong host cell lytic activities at high-dose (MOI = 10), and exhibited broad stability at various temperatures (4-50°C), but low stability at all pH values (pH 4-10). Complete genomes of these phages were sequenced using the Illumina platform. The phages have a double-stranded circular DNA genome with a length of approximately 44 kbp and 37% G+C content. Bioinformatics analysis showed that most of the contents of the phage proteins associated with morphogenesis, replication, and lysis were similar between phage Bϕ-R2919 and Bϕ-R1888 genomes in regards to protein sequence identity, but ORFs organization and direction were different. This study was successful to establish DDPT for the detection of synergy between phage and antibiotics using homemade phage disks on modified Mueller-Hinton agar (MHA) plates. Besides, the in vitro turbidity assays were designed to evaluate interaction between the purified phages and colistin, and to compare the efficacy between monotherapy and combination therapy. Because of inherent one well dilution variation with this in vitro assay and possibility of reproducibility error, the method was difficult to interpret results via observation. However, it provided obvious information of bacterial growth by recording OD600 values over 72 hr of incubation. To investigate isolated phages as monotherapy or in combination with colistin against A. baumannii, their activity was assessed in vivo in Galleria mellonella model of CRAB infection. The survival rate in the larvae model can be explained by results obtained from the in vitro methods. In summary, two A. baumannii phages, Bϕ-R2919 and Bϕ-R1888, infecting clinical CRAB strains were isolated and studied in detail on physiological characterizations and whole genome sequence analysis. To the best of our knowledge, this is the first report using DDPT to determine synergy between phage and antibiotics. Additionally, the preliminary in vitro testing provided proof of the interaction between lytic phages and antibiotics against A. baumannii. Overall, this study demonstrates that in vitro testing exhibit a linear relationship with in vivo activity. Further standardization for the in vitro methods is important to direct combination therapy for antimicrobial-resistant organisms.
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Vu Thao Nguyen Graduate School, Yonsei University 2024 국내박사
Prior to the discovery and widespread use of antibiotics, bacteriophages (phages) were a potential solution for preventing and treating bacterial infections (1, 2). With the emergence of antimicrobial resistance, there has been a renewed interest in phages (3). However, a major limitation to using phage as therapy is their narrow host range, limiting their effectiveness to specific bacterial strains of the same species (4). To address this limitation, phage training, such as Appelmans protocol, has been proposed as a strategy for expanding the host range of phages (5). Despite its potential, there are currently no reports of the protocol being applied to phages targeting Acinetobacter baumannii, an opportunistic pathogen responsible for a variety of hospitalacquired infections with high mortality rate (6). The aims of my dissertation were to apply the Appelmans protocol for expanding the host range of a phage cocktail targeting A. baumannii and evaluate the therapeutic potential of the expanded host range phages generated from the protocol. Additionally, the genetic mechanism underlying the protocol was also investigated to gain insights into its effectiveness. Chapter I provides a brief overview of Acinetobacter baumannii and highlights the significant global concern surrounding carbapenem-resistant A. baumannii (CRAB) strains. Furthermore, it introduces the fundamental background on bacteriophages and phage therapy, along with their limitations and discusses various approaches to overcome these challenges. Chapter II describes the application of the Appelmans protocol as a host range expansion method to broaden the host range of a phage cocktail targeting CRAB. The assessment of the host range expansion covered both the output cocktail and individual phage clones generated from the method. While the protocol consistently demonstrated the capability to expand the host range of the cocktail, it faced challenges when dealing with CRAB strains. Chapter III delves into the screening and characterization process used to identify potential therapeutic phages. The focus was on evaluating individual phage clones generated from the Appelmans protocol for therapeutic applications. Throughout the evaluation, key factors such as host range stability,lytic growth, transduction potential, and the absence of toxin genes were rigorously examined. However, it is noteworthy that the expanded host range phages obtained from this protocol exhibited limited stability, raising concerns about their suitability for therapeutic purposes. Chapter IV discusses the genetic insights into the Appelmans protocol’s mechanism, not only based on previous studies involving phages targeting different bacterial species but also when applied to the phage cocktail targeting CRAB. A bioinformatics workflow was established to analyze the ancestral of the expanded host range generated from the protocol and the finding revealed that they were recombinant derivatives of prophages induced from encountered bacterial strains. Favorable conditions and explanations for prophage induction during this protocol were proposed to support this genetic mechanism. In conclusion, this dissertation focused on the potential and challenges of a phage training method, especially, the Appelmans protocol in expanding the host range of phages for therapeutic applications. The findings emphasize the importance of understanding the genetic mechanisms underlying phage-host interactions to optimize phage therapy strategies for combating antibioticresistant bacterial infections. Parts of this dissertation are adapted from Vu TN, Clark JR, Jang E, D'Souza R, Nguyen LP, Pinto NA, et al. Appelmans protocol - A directed in vitro evolution enables induction and recombination of prophages with expanded host range. Virus Res. 2023;339:199272.
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