Isolation and Characterization of Pseudomonas aeruginosa Phages with a Broad Host Spectrum from Hospital Sewage Systems and Their Therapeutic Effect in a Mouse Model

Abstract

This study aimed to isolate and characterize phages as an alternative treatment of multidrug- or pan-drug-resistant Pseudomonas aeruginosa. Phage titers and bacterial densities correlated, with the phages disappearing after bacteria were eliminated. We isolated phages in filtered sewage water by a double-layered agar spot test. Fifty-eight P. aeruginosa strains were used to screen the host spectrum of the 14 phages isolated. Random amplification of polymorphic DNA-typing polymerase chain reaction was used to analyze the genomic homologies of the 58 host bacteria strains and four phages with a broad host spectrum. Transmission electron microscopy was used to observe the morphology of the four phages with a broad host spectrum. Mice with intraabdominal P. aeruginosa infection were used as an in vivo animal model to investigate the therapeutic effect of the selected phage. Four virulent phages with a broad host spectrum specific to P. aeruginosa strains were isolated. They were all double-stranded DNA viruses and belonged to four different genotypes. The test curve showed that phage I had the highest adsorption rate, the shortest latent period, and the largest burst size. The infected mouse model indicated that small doses of phage I could prevent the death of infected mice. Phage titers and bacterial densities correlated, with phages disappearing after bacteria were eliminated. Phage I was the most effective and promising treatment of drug-resistant P. aeruginosa.

Figure 1.

Homologous cluster analysis of the 46 Pseudomonas aeruginosa strains that could be lysed by the phages. This indicates that bacteria numbers 5, 8, 16, and 20 belonged to the same genotype, whereas the other host bacterial genotypes were different. RFLP = restriction fragment length polymorphism.

Figure 2.

Morphology of the four phages with a broad host spectrum. (A) Phage I belongs to the Siphoviridae family; the size of its head was approximately 100 nm and the length of its tail was approximately 405 nm. (B) Phage II belongs to the Myoviridae family; the size of its head was approximately 80 nm and the length of its tail was approximately 100 nm. (C) Phage III belongs to the Myoviridae family; the size of its head was approximately 78 nm and the length of its tail was approximately 100 nm. (D) Phage IV belongs to the Siphoviridae family; the size of its head was approximately 400 nm and the length of its tail was approximately 450 nm. Magnification, 150,000×. Scale bars, 100 nm.

Figure 3.

Adsorption efficiency of the four phages. The adsorption efficiency of the four phages was compared using the χ² test (P = 0.001). Data are mean ± SD (error bars) from triplicate experiments.

Figure 4.

One-step growth curve of the four phages. Data are mean ± SD (error bars) from triplicate experiments. PFU = plaque-forming unit.

Figure 5.

Phage particles and Pseudomonas aeruginosa distribution in vivo. “Only bacteria” indicates mice injected with P. aeruginosa only. “Only phage” indicates mice injected with the phage only. “Bacteria (coexist)” indicates in vivo bacterial distribution in mice injected with both the bacteria and the phage. “Phage (coexist)” indicates in vivo distribution of the phages in mice injected with both the bacteria and the phage. The line for the “only bacteria” group stops at 6 hours because no treatment was given and all mice in this group died before the 24-hour time point. Data are mean ± SD (error bars) from three independent experiments with five mice per group. CFU = colony-forming unit; PFU = plaque-forming unit.