Session Type: ePosters
Session Title: ePosters
Authors(s): T.N. Vu (1), E. Jang (2), L.P. Nguyen (1), H.S. Seo (3), J.M. Park (2), D. Yong (2)
Authors Affiliations(s): (1) Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea. Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea - Seoul (Korea, Republic of), Korea, Republic of, (2) Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea - Seoul (Korea, Republic of), Korea, Republic of, (3) Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea, Korea, Republic of
Background:
Research on lytic spectrum of bacteriophage (phages) is important to understand molecular interactions between phages and hosts throughout the infection cycle. However, the mechanisms that enable phages to infect multiple hosts and their evolution remain unstudied for phages targeting carbapenem-resistant Acinetobacter baumannii (CRAB), which has been listed in the critical group of priority pathogens by WHO. In this study, we designed a culture based in vitro evolution accelerating method for evolving therapeutic phage cocktails from well-defined phages. Then, we assessed how the experiment was able to expand phage cocktail host range and the mechanisms of host range expansion were also examined.
Methods:A cocktail of 3 parental phages was grown on a collection of ten separate hosts in a 96-well format. The hosts showing distinct antibiograms were identified using rpoB-based scheme and genotyped by PFGE. The hosts including mostly refractory A. baumannii isolates were maintained from their original stocks and kept separate throughout. The experiments were performed until the cocktail evolved the ability to lyse the phage-resistant strains. Progeny phages from wells in which lysis occured were pooled, and used for the next round. After every 10 rounds, the host range of the pooled lysates were checked using spot tests. The evolved phages generated from the final round were purified and amplified individually for further genetic analysis by WGS and annotation.
Results:The host range of a mixture of phages targeting CRAB could be expanded by the method. The evolved cocktail generated plaque on the strains which were not lysed by the phages in the original mixture. Individual evolved phages isolated from the protocol have expanded their host range. The output cocktail could be able to lyse 80% of the host bacteria after ninety rounds of the experiment.
Conclusions:The method presented an advance for overcoming the limitation of phage’s highly specific host range and can be used in generating a library of expanded host range phages against CRAB. Further investigations of the genetic mechanisms including mutation or recombination, by which one or more phages adapt to a bacterium that was refractory to the original cocktail, are in progress.
Keyword(s): lytic spectrum expansion, Acinetobacter baumannii, carbapenem resistanceSession Type: ePosters
Session Title: ePosters
Authors(s): T.N. Vu (1), E. Jang (2), L.P. Nguyen (1), H.S. Seo (3), J.M. Park (2), D. Yong (2)
Authors Affiliations(s): (1) Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea. Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea - Seoul (Korea, Republic of), Korea, Republic of, (2) Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea - Seoul (Korea, Republic of), Korea, Republic of, (3) Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea, Korea, Republic of
Background:
Research on lytic spectrum of bacteriophage (phages) is important to understand molecular interactions between phages and hosts throughout the infection cycle. However, the mechanisms that enable phages to infect multiple hosts and their evolution remain unstudied for phages targeting carbapenem-resistant Acinetobacter baumannii (CRAB), which has been listed in the critical group of priority pathogens by WHO. In this study, we designed a culture based in vitro evolution accelerating method for evolving therapeutic phage cocktails from well-defined phages. Then, we assessed how the experiment was able to expand phage cocktail host range and the mechanisms of host range expansion were also examined.
Methods:A cocktail of 3 parental phages was grown on a collection of ten separate hosts in a 96-well format. The hosts showing distinct antibiograms were identified using rpoB-based scheme and genotyped by PFGE. The hosts including mostly refractory A. baumannii isolates were maintained from their original stocks and kept separate throughout. The experiments were performed until the cocktail evolved the ability to lyse the phage-resistant strains. Progeny phages from wells in which lysis occured were pooled, and used for the next round. After every 10 rounds, the host range of the pooled lysates were checked using spot tests. The evolved phages generated from the final round were purified and amplified individually for further genetic analysis by WGS and annotation.
Results:The host range of a mixture of phages targeting CRAB could be expanded by the method. The evolved cocktail generated plaque on the strains which were not lysed by the phages in the original mixture. Individual evolved phages isolated from the protocol have expanded their host range. The output cocktail could be able to lyse 80% of the host bacteria after ninety rounds of the experiment.
Conclusions:The method presented an advance for overcoming the limitation of phage’s highly specific host range and can be used in generating a library of expanded host range phages against CRAB. Further investigations of the genetic mechanisms including mutation or recombination, by which one or more phages adapt to a bacterium that was refractory to the original cocktail, are in progress.
Keyword(s): lytic spectrum expansion, Acinetobacter baumannii, carbapenem resistance
1Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, South Korea 2Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, South Korea
Session Type: ePosters
Session Title: ePosters
Authors(s): T.N. Vu (1), E. Jang (2), L.P. Nguyen (1), H.S. Seo (3), J.M. Park (2), D. Yong (2)
Authors Affiliations(s): (1) Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea. Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea - Seoul (Korea, Republic of), Korea, Republic of, (2) Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea - Seoul (Korea, Republic of), Korea, Republic of, (3) Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea, Korea, Republic of
Background:
Research on lytic spectrum of bacteriophage (phages) is important to understand molecular interactions between phages and hosts throughout the infection cycle. However, the mechanisms that enable phages to infect multiple hosts and their evolution remain unstudied for phages targeting carbapenem-resistant Acinetobacter baumannii (CRAB), which has been listed in the critical group of priority pathogens by WHO. In this study, we designed a culture based in vitro evolution accelerating method for evolving therapeutic phage cocktails from well-defined phages. Then, we assessed how the experiment was able to expand phage cocktail host range and the mechanisms of host range expansion were also examined.
Methods:A cocktail of 3 parental phages was grown on a collection of ten separate hosts in a 96-well format. The hosts showing distinct antibiograms were identified using rpoB-based scheme and genotyped by PFGE. The hosts including mostly refractory A. baumannii isolates were maintained from their original stocks and kept separate throughout. The experiments were performed until the cocktail evolved the ability to lyse the phage-resistant strains. Progeny phages from wells in which lysis occured were pooled, and used for the next round. After every 10 rounds, the host range of the pooled lysates were checked using spot tests. The evolved phages generated from the final round were purified and amplified individually for further genetic analysis by WGS and annotation.
Results:The host range of a mixture of phages targeting CRAB could be expanded by the method. The evolved cocktail generated plaque on the strains which were not lysed by the phages in the original mixture. Individual evolved phages isolated from the protocol have expanded their host range. The output cocktail could be able to lyse 80% of the host bacteria after ninety rounds of the experiment.
Conclusions:The method presented an advance for overcoming the limitation of phage’s highly specific host range and can be used in generating a library of expanded host range phages against CRAB. Further investigations of the genetic mechanisms including mutation or recombination, by which one or more phages adapt to a bacterium that was refractory to the original cocktail, are in progress.
Keyword(s): lytic spectrum expansion, Acinetobacter baumannii, carbapenem resistanceSession Type: ePosters
Session Title: ePosters
Authors(s): T.N. Vu (1), E. Jang (2), L.P. Nguyen (1), H.S. Seo (3), J.M. Park (2), D. Yong (2)
Authors Affiliations(s): (1) Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea. Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea - Seoul (Korea, Republic of), Korea, Republic of, (2) Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea - Seoul (Korea, Republic of), Korea, Republic of, (3) Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea, Korea, Republic of
Background:
Research on lytic spectrum of bacteriophage (phages) is important to understand molecular interactions between phages and hosts throughout the infection cycle. However, the mechanisms that enable phages to infect multiple hosts and their evolution remain unstudied for phages targeting carbapenem-resistant Acinetobacter baumannii (CRAB), which has been listed in the critical group of priority pathogens by WHO. In this study, we designed a culture based in vitro evolution accelerating method for evolving therapeutic phage cocktails from well-defined phages. Then, we assessed how the experiment was able to expand phage cocktail host range and the mechanisms of host range expansion were also examined.
Methods:A cocktail of 3 parental phages was grown on a collection of ten separate hosts in a 96-well format. The hosts showing distinct antibiograms were identified using rpoB-based scheme and genotyped by PFGE. The hosts including mostly refractory A. baumannii isolates were maintained from their original stocks and kept separate throughout. The experiments were performed until the cocktail evolved the ability to lyse the phage-resistant strains. Progeny phages from wells in which lysis occured were pooled, and used for the next round. After every 10 rounds, the host range of the pooled lysates were checked using spot tests. The evolved phages generated from the final round were purified and amplified individually for further genetic analysis by WGS and annotation.
Results:The host range of a mixture of phages targeting CRAB could be expanded by the method. The evolved cocktail generated plaque on the strains which were not lysed by the phages in the original mixture. Individual evolved phages isolated from the protocol have expanded their host range. The output cocktail could be able to lyse 80% of the host bacteria after ninety rounds of the experiment.
Conclusions:The method presented an advance for overcoming the limitation of phage’s highly specific host range and can be used in generating a library of expanded host range phages against CRAB. Further investigations of the genetic mechanisms including mutation or recombination, by which one or more phages adapt to a bacterium that was refractory to the original cocktail, are in progress.
Keyword(s): lytic spectrum expansion, Acinetobacter baumannii, carbapenem resistance