Isolation and Characterization of a Novel Lytic Phage, vB_PseuP-SA22, and Its Efficacy against Carbapenem-Resistant Pseudomonas aeruginosa

Abstract

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses a serious public health threat in multiple clinical settings. In this study, we detail the isolation of a lytic bacteriophage, vB_PseuP-SA22, from wastewater using a clinical strain of CRPA. Transmission electron microscopy (TEM) analysis identified that the phage had a podovirus morphology, which agreed with the results of whole genome sequencing. BLASTn search allowed us to classify vB_PseuP-SA22 into the genus Bruynoghevirus. The genome of vB_PseuP-SA22 consisted of 45,458 bp of double-stranded DNA, with a GC content of 52.5%. Of all the open reading frames (ORFs), only 26 (44.8%) were predicted to encode certain functional proteins, whereas the remaining 32 (55.2%) ORFs were annotated as sequences coding functionally uncharacterized hypothetical proteins. The genome lacked genes coding for toxins or markers of lysogenic phages, including integrases, repressors, recombinases, or excisionases. The phage produced round, halo plaques with a diameter of 1.5 ± 2.5 mm on the bacterial lawn. The TEM revealed that vB_PseuP-SA22 has an icosahedral head of 57.5 ± 4.5 nm in length and a short, non-contractile tail (19.5 ± 1.4 nm). The phage showed a latent period of 30 min, a burst size of 300 PFU/infected cells, and a broad host range. vB_PseuP-SA22 was found to be stable between 4-60 °C for 1 h, while the viability of the virus was reduced at temperatures above 60 °C. The phage showed stability at pH levels between 5 and 11. vB_PauP-SA22 reduced the number of live bacteria in P. aeruginosa biofilm by almost five logs. The overall results indicated that the isolated phage could be a candidate to control CRPA infections. However, experimental in vivo studies are essential to ensure the safety and efficacy of vB_PauP-SA22 before its use in humans.

Keywords: Bruynoghevirus; Podovirus; Pseudomonas aeruginosa; anti-biofilm effect; bacteriophage; broad host range; carbapenem resistance.

Figure 1

Radar plot showing the antibiotic resistance profile of P. aeruginosa strain B10. R = resistant, S = sensitive. The size of the inhibition zone is presented in mm; 0 mm indicates no inhibition zone detected (i.e., the tested bacterium was not sensitive to the respective antibiotic).

Figure 2

(A) Spot assay (lytic zone on the lawn of P. aeruginosa strain B10); (B) typical vB_PseuP-SA22 plaques; and (C) transmission electron micrograph of vB_PseuP-SA22 under negative staining with 2% uranyl acetate. The size of petri plates = 90 mm; bar = 100 nm.

Figure 3

(A) Phage adsorption plots of vB_PseuP-SA22. The X-axis represents the time of exposure and Y-axis indicates the percentage of free phage particles found at different time points. (B) Three phases of one round lytic cycle of vB_PseuP-SA22. All values represent the mean + SD of triplicated experiments.

Figure 4

(A) Stability of vB_PseuP-SA22 under thermal treatment; (B) pH stability of vB_PseuP-SA22; (C) Determination of optimal MOI; and (D) Lysis kinetics. Error bars represent mean value ± SD. Two-way ANOVA and the Bonferroni post-hoc test were conducted using the GraphPad Prism software. Significant statistical differences were noted between the control and the respective phage-treated samples at different MOIs (p < 0.05).

Figure 4

(A) Stability of vB_PseuP-SA22 under thermal treatment; (B) pH stability of vB_PseuP-SA22; (C) Determination of optimal MOI; and (D) Lysis kinetics. Error bars represent mean value ± SD. Two-way ANOVA and the Bonferroni post-hoc test were conducted using the GraphPad Prism software. Significant statistical differences were noted between the control and the respective phage-treated samples at different MOIs (p < 0.05).

Figure 5

Circular genome map of vB_PseuP-SA22. The outer circle designates the ORFs of vB_PseuP-SA22. The red inner landscape shows the GC content, while the second inner circle with the purple and green landscape shows the GC skew. The CDSs with identified functions are labeled in black color, along with their location, while other unlabeled CDSs (blue) represent hypothetical proteins. The direction of the functional CDCs is represented as (+) for the main strand and (-) for the complementary strand. Scale units are base pairs.

Figure 6

Neighbor-joining phylogenetic trees based on (A) whole-genome sequence and (B) terminase large sub-unit of vB_PseuP-SA22. The red color indicates the position of vB_PseuP-SA22. Reference phage sequences were obtained from the above-mentioned databases.

Figure 7

Circos plot showing the whole-genomic comparison of five Pseudomonas phages (including vB_Pseu-SAS22) in five different quadrants. The colored ribbons found inside the circle denote the local BLAST alignments, representing the different levels of the maximum score, as indicated in the legend.

Figure 8

(A) Anti-biofilm activity of vB_PseuP-SA22 on biofilms of CRPA at an MOI of 0.1. (B) Viable bacterial cell counts at different ages of biofilm at an MOI of 0.1. All values designate mean ± SD of triplicate experiments. Two-way ANOVA and the Bonferroni post-hoc test were carried out using the GraphPad Prism software. Significant differences are illustrated using asterisks (p < 0.05; **, p < 0.01; ***, p < 0.001); and (C1–C4) Scanning electron microscopic images showing the biofilm at different time intervals (C1, 0 h; C2, 4 h; C3, 8 h; C4, 12 h). Scale bar = 10 μm at a magnification of 5000×.

Figure 8

(A) Anti-biofilm activity of vB_PseuP-SA22 on biofilms of CRPA at an MOI of 0.1. (B) Viable bacterial cell counts at different ages of biofilm at an MOI of 0.1. All values designate mean ± SD of triplicate experiments. Two-way ANOVA and the Bonferroni post-hoc test were carried out using the GraphPad Prism software. Significant differences are illustrated using asterisks (p < 0.05; **, p < 0.01; ***, p < 0.001); and (C1–C4) Scanning electron microscopic images showing the biofilm at different time intervals (C1, 0 h; C2, 4 h; C3, 8 h; C4, 12 h). Scale bar = 10 μm at a magnification of 5000×.