Characterization of the novel broad-spectrum lytic phage Phage_Pae01 and its antibiofilm efficacy against Pseudomonas aeruginosa

Abstract

Introduction: 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.

Methods and results: 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.

Conclusion: 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.

Keywords: Pseudomonas aeruginosa; bacteriophage; biofilm; gentamicin; multidrug resistance; phage therapy.

Figure 1

The plaque and phage morphology of Phage_Pae01. (A) Plaques of Phage_Pae01 to the host strain Pa021, were clear with an expanding halo. (B) Morphology of Phage_Pae01 by transmission electron microscopy. The scale bar represents 100 nm.

Figure 2

One-step growth curve and lysis kinetics of Phage_Pae01. (A) One-step growth curve of Phage_Pae01 on P. aeruginosa strain Pa021. (B) Killing curves of P. aeruginosa strain Pa021 by Phage_Pae01 at various MOIs (10, 1, 0.1, and 0.01).

Figure 3

Biological properties of Phage_Pae01. (A) Thermal stability of Phage_Pae01. Phages were incubated for 1 h at different temperatures. (B) pH stability of Phage_Pae01. Phages were incubated for 2 h at different pHs.

Figure 4

Whole-genome map of Phage_Pae01.

Figure 5

Comparative genome analysis of the P. aeruginosa phages Phage_Pae01, vB_PA45_GUMS, vB_PaeM_C2-10_Ab1 and vB_PaeM_C2-10_Ab02. The direction of transcription is shown by arrows next to the predicted ORFs. According to the key provided at the bottom left of the graphic, the arrows are coloured according to their functions. The corresponding ORFs are presented with functional annotations.

Figure 6

Phylogenetic tree based on the phage terminal large subunit comparisons of selected phages. Amino acid sequence comparison was compared using the ClustalW program, and the phylogenetic trees were generated using the neighbor-joining method with 1,000 bootstrap replications. The numbers at the nodes represent the percent bootstrap values. The numbers in parentheses represent the GenBank accession number.

Figure 7

Effects of the phage on biofilms. (A) Effects of Phage_Pae01 and/or antibiotics on preformed biofilms. The effects of Phage_Pae01 and/or antibiotics on the preformed biofilm at 4, 8, and 24 h are shown. *p < 0.05, **p < 0.01, ***p < 0.0005, ****p < 0.0001. (B) Effects of phage coincubation on biofilm formation. The effects of Phage_Pae01 on biofilm formation after 4, 8, and 24 h of coincubation are shown. **p < 0.01, ****p < 0.0001. (B) Effects of phage co-incubation on biofilm formation. The effects of Phage_Pae01 on biofilm formation after 4, 8, and 24 hours of co-incubation are shown. **p < 0.01, ****p < 0.0001.

Figure 8

Scanning electron microscopy images of P. aeruginosa Pa021 treated with Phage_Pae01 and/or antibiotics: (A, E) P. aeruginosa Pa021; (B, F) P. aeruginosa Pa021 + Gentamicin; (C, G) P. aeruginosa Pa021 + Phage_Pae01; and (D, H) P. aeruginosa Pa021 + Gentamicin + Phage_Pae01.