Phage endolysin activity was shown by bacterium
overlay of a PAGE gel
Upon release, the phages burst the bacteria, causing endolysin
to flow outside into the medium. Proteins of supernatant were
precipitated by 30% ammonia sulfate and separated by SDSPAGE,
as described in the materials and methods. Bacterium
overlay on SDS-PAGE showed a clear band at 185 kDa, but clear
band disappeared when the sample was boiling treatment before
electrophoresis (Fig. 5). From this result, we suggest that the
endolysin functions in a complex rather than as a monomer.
Phage ensures A549 cell survival under bacterial
infection
To ensure the safety of phage therapy, A549 human lung
epithelial cells were used as an indicator for phage toxicity. The
phage wkm18p was found to provide the highest protection against
A. baumannii KM18 infection of cells (105 cells) (Fig. 6). Phage at an
MOI of 1, 0.1 or 0.01 enabled cells inoculated with A. baumannii
KM18 (106 CFU) to survive as well as uninoculated controls. Cells
treated with phages at an MOI of 1000 (109 PFU), but not
inoculated with bacteria, survived as well as the control cells. Cells
inoculated with the bacterial strain A. baumannii KM18 only
without any phage treatment were completely killed. The results
showed that wkm18p eliminated bacteria and protected A549 cells
from immediate killing by KM18 bacteria. They also suggested
that a high dose of phages did not impact A549 survival. therapy. In our study, clinical A. baumannii isolates were used to
survey lytic phages. We chose the most active phages for further
analysis of their plaque morphology, effect on bacterial growth,
genome size, endolysin activity and protection of cells. We
examined the antibiotic sensitivity of these 34 strains of A.
baumannii, and classified them as CS, CRAB, MDRAB and
XDRAB. All of the bacterial strains were sensitive to the phages