Isolation and characterization of φkm18p, a novel lytic phage with therapeutic potential against extensively drug resistant Acinetobacter baumannii

Abstract

Aims: To isolate phages against extensively drug resistant Acinetobacter baumannii (XDRAB) and characterize the highest lytic capability phage as a model to evaluate the potential on phage therapy.

Methods and results: Eight phages were isolated from hospital sewage and showed narrow host spectrum. Phage φkm18p was able to effectively lyse the most XDRAB. It has a dsDNA genome of 45 kb in size and hexagonal head of about 59 nm in diameter and no tail. Bacterial population decreased quickly from 10(8) CFU ml(-1) to 10(3) CFU ml(-1) in 30 min by φkm18p. The 185 kDa lysis protein encoded by φkm18p genome was detected when the extracted protein did not boil before SDS-PAGE; it showed that the lysis protein is a complex rather than a monomer. Phage φkm18p improved human lung epithelial cells survival rates when they were incubated with A. baumannii. Combination of phages (φkm18p, φTZ1 and φ314) as a cocktail could lyse all genotype-varying XDRAB isolates.

Conclusion: Infections with XDRAB are extremely difficult to treat and development of a phage cocktails therapy could be a therapeutic alternative in the future. Phage φkm18p is a good candidate for inclusion in phage cocktails.

Figure 1. Relationship between phage titers and inhibition efficiency.

Phages φkm18p were used at different titers to infect host cells in order to determine the best titer for lysis of the host during 2 hr (A) and 24 h (B) periods. Phage concentration: (•) no phage added, (○) MOI = 1, (▾) MOI = 0.1 and (▵) MOI = 0.01.

Figure 2. Electron micrographs of A. baumannii phage φkm18p.

Phage suspension was loaded onto a copper grid, stained with 2% uranyl acetate and observed with transmission electron microscopy. The phage particles (the arrows indicated) showed a hexagonal head about 59 nm in diameter that lacks a neck and tail.

Figure 3. Phage φkm18p genomic DNA restriction patterns and size determination.

(A) Agarose gel electrophoresis of HincII fragments. (B) NheI fragments separated by PFGE. The total size of genomic DNA was estimated at approximately 45 kb.

Figure 4. The phage particle virion proteins were separated by SDS-PAGE.

The phage particles (1.5×10¹⁰ PFU) were boiled with cracking buffer and subjected to SDS-PAGE using 12% gels. The most abundant protein was 39 kDa.

Figure 5. Phage endolysin activity and size were determined by an overlay assay.

The protein suspension extract (about 13 mg) from the phage lysate was mixed with sample buffer without β-mercaptoethanol. Unboiled samples were analyzed by SDS-PAGE. The control set used a sample buffer containing β-mercaptoethanol and the sample was boiled for 10 min. The SDS-PAGE gel was poured onto a plate and soft agar mixed with Ac. baumannii KM18 was overlaid. The protein with endolysin activity produced a clear region on the overlay.

Figure 6. Protection efficacy test of φkm18p on A549 human lung epithelial cells.

A549 cells were distributed in a 24-well ELISA plate (10⁵ cells well⁻¹) and incubated for 12 h. The cells were infected with 10⁶ CFU Ac. baumannii KM18. Phage at different titers (MOI = 0, 1000, 1, 0.1, 0.01) was added to the cells. The “+” indicates the addition of phage and “−” means phage were not added. The cell counts of surviving A549 cells suspended in trypsin solution were determined by examination under the microscope (Nikon, E2000, Japan). Results were confirmed by repeating the experiments three times.