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Phenotypic and genomic characterization of two novel lytic bacteriophages targeting multidrug-resistant Pseudomonas
Rafwana Ibrahim1, Vipin Kalikot Valapil2, Shaila Angela Lewis3
1Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, India.
Background:
The increasing prevalence of multidrug-resistant (MDR) Pseudomonas aeruginosa highlights the urgent need for innovative therapeutics beyond conventional antibiotics.
Method:
In this study, two novel lytic bacteriophages, Pseudomonas phage_SW_PA2862_14_24 (N2) and Pseudomonas phage_SW_PA2862_11_24 (N3) of the myovirus morphology, were isolated and comprehensively characterized for activity against P. aeruginosa isolates. Transmission electron microscopy was used to determine morphotypes, while adsorption assays, one-step growth curves, and time-kill assays assessed infection dynamics. Genomic characterization included whole-genome sequencing and bioinformatic analysis for safety profiling and comparative genomics.
Results:
Electron microscopy revealed distinct morphologies: N2 exhibited a contractile tail, and N3 displayed a flexible, non-contractile tail. Adsorption assays showed that N3 achieved rapid host binding (∼95% within 5 min), while N2 demonstrated a higher burst size of 231 PFU/cell. Genomic analysis identified genome sizes of ∼92.8 kb with 202-203 coding sequences, 15 tRNA genes, and a high proportion (∼73%) of hypothetical proteins. Both phages contained complete lytic modules and conserved genes related to replication and DNA metabolism. Comparative analysis demonstrated high genomic homology with established therapeutic phages (SPA01, EPA1, OMKO1). Time-kill assays confirmed dose-dependent bactericidal efficacy, with N2 showing superior and sustained lytic activity at moderate to high MOIs. Importantly, both genomes lacked lysogeny, antimicrobial resistance, and virulence genes.
Conclusion:
These novel phages constitute biosafe, genetically defined lytic agents with distinct infection kinetics and potent activity against MDR P. aeruginosa. Their complementary features and favorable genomic profiles support their potential use in precision phage therapy to combat antibiotic-resistant infections.
Insights
Two novel bacteriophages, N2 and N3, show potent lytic activity against multidrug-resistant Pseudomonas aeruginosa. These genetically defined phages are biosafe and demonstrate potential for precision phage therapy against antibiotic resistance.
Area of Science:
- Microbiology
- Virology
- Genomics
Background:
- Rising multidrug-resistant (MDR) Pseudomonas aeruginosa necessitates novel therapeutic strategies.
- Conventional antibiotics are becoming less effective against MDR P. aeruginosa infections.
Purpose of the Study:
- To isolate and characterize novel lytic bacteriophages for activity against MDR P. aeruginosa.
- To assess the safety and efficacy of these bacteriophages for potential therapeutic applications.
Main Methods:
- Isolation and comprehensive characterization of two novel myovirus bacteriophages (N2 and N3).
- Morphological analysis via transmission electron microscopy.
- Infection dynamics assessed through adsorption, one-step growth, and time-kill assays.
- Genomic analysis including whole-genome sequencing and bioinformatic profiling.
Main Results:
- Phages N2 and N3 exhibited distinct morphologies and infection kinetics, with N3 showing rapid adsorption and N2 a high burst size.
- Genomic analysis revealed complete lytic modules, conserved genes, and a high percentage of hypothetical proteins, with no identified lysogeny, antimicrobial resistance, or virulence genes.
- Time-kill assays confirmed dose-dependent bactericidal efficacy, with N2 demonstrating superior sustained activity against MDR P. aeruginosa.
Conclusions:
- Novel bacteriophages N2 and N3 are biosafe, genetically defined lytic agents with potent activity against MDR P. aeruginosa.
- Distinct infection kinetics and favorable genomic profiles support their potential use in precision phage therapy.
- These phages offer a promising alternative to combat antibiotic-resistant infections.
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