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Isolation and characterization of a lytic bacteriophage against Pseudomonas aeruginosa.

ABSTRACT: In recent years, the use of bacteriophages (or 'phages') against multidrug-resistant (MDR) bacteria including Pseudomonas aeruginosa has drawn considerable attention, globally. In this work, we report the isolation and detailed characterization of a highly lytic Pseudomonasphage DRL-P1 isolated from wastewater. Under TEM, DRL-P1 appeared as a member of the phage family Myoviridae. DRL-P1 featured rapid adsorption (~ 5 min), short-latency (~ 30 min), and large burst size (~ 100 PFU per infected cell). DRL-P1 can withstand a wide temperature range (4 °C to 40 °C) and pH (5.0 to 10.0) conditions. The 66,243 bp DRL-P1 genome (MN564818) encodes at least 93 ORFs, of which 36 were functionally annotated based on homology with similar phage proteins available in the databases. Comparative analyses of related genomes suggest an independent evolutionary history and discrete taxonomic position of DRL-P1 within genus Pbunavirus. No toxin or antibiotic resistance genes was identified. DRL-P1 is tolerant to lyophilization and encapsulation techniques and retained lytic activity even after 18 months of storage. We also demonstrated decontaminating potentials of DRL-P1 in vitro, on an artificially contaminated cover-slip model. To the best of our knowledge, this is the first Pbunavirus to be reported from India. Our study suggests DRL-P1 as a potential candidate for various applications.

SUBMITTER: Sharma S

PROVIDER: S-EPMC8481504 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Pseudomonas aeruginosa is present throughout nature and is a common opportunistic pathogen in the human body. Carbapenem antibiotics are typically utilized as a last resort in the clinical treatment of multidrug-resistant infections caused by P. aeruginosa. The increase in carbapenem-resistant P. aeruginosa poses an immense challenge for the treatment of these infections. Bacteriophages have the potential to be used as antimicrobial agents for treating antibiotic-resistant bacteria. In this study, a new virulent P. aeruginosa phage, Phage_Pae01, was isolated from hospital sewage and shown to have broad-spectrum antibacterial activity against clinical P. aeruginosa isolates (83.6%). These clinical strains included multidrug-resistant P. aeruginosa and carbapenem-resistant P. aeruginosa. Transmission electron microscopy revealed that the phage possessed an icosahedral head of approximately 80 nm and a long tail about 110 m, indicating that it belongs to the Myoviridae family of the order Caudovirales. Biological characteristic analysis revealed that Phage_Pae01 could maintain stable activity in the temperature range of 4~ 60°C and pH range of 4 ~ 10. According to the in vitro lysis kinetics of the phage, Phage_Pae01 demonstrated strong antibacterial activity. The optimal multiplicity of infection was 0.01. The genome of Phage_Pae01 has a total length of 93,182 bp and contains 176 open reading frames (ORFs). The phage genome does not contain genes related to virulence or antibiotic resistance. In addition, Phage_Pae01 effectively prevented the formation of biofilms and eliminated established biofilms. When Phage_Pae01 was combined with gentamicin, it significantly disrupted established P. aeruginosa biofilms. We identified a novel P. aeruginosa phage and demonstrated its effective antimicrobial properties against P. aeruginosa in both the floating and biofilm states. These findings offer a promising approach for the treatment of drug-resistant bacterial infections in clinical settings.

Project description:The ESKAPEE pathogen Pseudomonas aeruginosa is a common cause of chronic wound and cystic fibrosis lung infections, as well as acute burn and nosocomial infections. Many of these infections are recalcitrant to conventional antibiotic therapies due to both traditional antibiotic resistance mechanisms and antimicrobial tolerance. Recent successes with bacteriophage (phage) therapy to treat chronic human P. aeruginosa infections have led to a renewed interest in isolating and characterizing new P. aeruginosa phages. Here, we isolated and characterized a new lytic phage (termed PIP, pili-infecting phage) capable of infecting P. aeruginosa PA14. PIP is a tailed phage with an icosahedral head and flexible tail containing a genome that is 57,462 bp in length. Phylogenetic analysis reveals that PIP belongs to the subfamily Queuovirinae and genus Nipunavirus but is highly divergent in gene content from known Nipunaviruses. By isolating and characterizing a P. aeruginosa strain that spontaneously evolved resistance to PIP, we show that the receptor for PIP is Type IV pili. In summary, we isolated a new P. aeruginosa phage species with a unique genome, thus increasing the diversity of phages known to infect this important human pathogen.IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa causes both acute and chronic human infections. These infections are notoriously difficult to treat due to both antibiotic resistance and antibiotic tolerance. The increasing frequency of antibiotic failure in P. aeruginosa infections has led scientists to explore other treatment options, including bacteriophage (phage) therapy. To this end, there has been a significant effort to identify new Pseudomonas phages. Here, we isolated and characterized a bacteriophage (termed PIP, pili-infecting phage) that infects P. aeruginosa PA14. Examination of the PIP genome revealed that this phage represents a new species in the subclass Queuovirinae. The isolation and characterization of spontaneous PA14 mutants that are resistant to PIP infection revealed Type IV pili as the PIP receptor. Ultimately, this study characterizes a new species of Pseudomonas phage, thus enhancing the known diversity of phages that infect this important pathogen.

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Isolation and characterization of a lytic bacteriophage against Pseudomonas aeruginosa.

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