Isolation and characterization of lytic bacteriophages from sewage at an egyptian tertiary care hospital against methicillin-resistant clinical isolates.

BACKGROUND

Methicillin resistant (MRSA) is a pathogen to humans causing life-threatening infections. MRSA have the capability to grow resistance to many antibiotics, and phage therapy is one treatment option for this infection.

OBJECTIVES

The aim of the present study was to isolate and characterize the lytic bacteriophages specific to MRSA from domestic sewage water at a tertiary care hospital in Egypt.

METHODS

Thirty MRSA strains were isolated from different clinical samples admitted to the microbiology lab at Theodor Bilharz Research institute (TBRI) hospital, Giza, Egypt. They were confirmed to be MRSA through phenotypic detection and conventional PCR for gene. They were used for the isolation of phages from sewage water of TBRI hospital. Plaque assay was applied to purify and quantify the titer of the isolated phages. The host range of the isolated phages was detected using the spot test assay. The morphology of phages was confirmed using transmission electron microscope (TEM). Digestion of DNA extracted from phages with endonuclease enzymes including and was performed. SDS-PAGE was performed to analyze MRSA specific phage proteins. As a positive control prophages were isolated from a mitomycin C (MitC) treated culture of . strain ATCC25923. Further characterization using conventional polymerase chain reaction (PCR) was used to select three known Staphylophages by detecting the endolysin gene of phage K, the polymerase gene of phage 44AHJD, and the minor tail gene of phage P68.

RESULTS

Isolated phages in this research displayed a wide host range against MRSA using the spot test, out of thirty tested MRSA isolates 24 were sensitive and got lysed (80%). The titer of the phages was estimated to be 1.04 × 10 pfu/ml using plaque test. Identification of head and tail morphology of the phages was achieved using TEM and they were designated to tailed phages of order , they composed an icosahedral capsid. Prophages were isolated through MitC induction. DNA of phages was digested by endonuclease enzymes. Conventional PCR yielded 341 bp of phage K endolysin gene and phage P68 minor tail protein gene 501 bp. Protein analysis using SDS-PAGE showed 4 proteins of sizes between 42 kDa and 140 kDa.

CONCLUSION

Phages isolated here are alike to others mentioned in previous studies. The high broad host range of the isolated phages is promising to control MRSA and can be in the future commercially suitable for treatment as lysate preparations. Animal models of phage-bacterial interaction will be our next step that may help in resolving the multidrug resistant crisis of MRSA in Egypt.