Background: Microbial keratitis (infection of the cornea, the clear membrane overlying the pupil and coloured iris in the eye) is often caused by Pseudomonas aeruginosa, especially in contact lens wearers. Commonly used antibiotics are failing against this bacterium as it is becoming increasingly resistant to antimicrobials. This study was conducted to isolate and characterize bacteriophages (phages) that could be used to design a phage cocktail that is active against multi-drug resistant (MDR) ocular P. aeruginosa isolates.
Methods: Raw untreated sewage water was used for isolation and purification of phages following the Phage-on-Tap protocol. Phage confirmation, enumeration and identification were performed by spot assay, double layer plaque assay and transmission electron microscopy (TEM), respectively. Lytic activity of phages was determined by host range evaluation against thirteen P. aeruginosa isolates that had previously been characterised as MDR and resistant to another older set of phages. Further characterization of new phages used one step growth curves and thermal stability.
Results: Four phages were isolated and purified. These phages produced high titres of phage, approximately 1010 plaqe forming untis (pfu)/ml. TEM analysis revealed that the phages belong to two families, Podoviridae and Siphoviridae. All phages were stable at temperatures from 4°C to 50°C. Host range via spot assays showed that Sullo-1, Sullo-2, Sullo-3 and Sullo-4 had lytic activity against 77%, 69%, 55% and 55% of P. aeruginosa strains. The latent period was around ~ 20 minutes for Sullo-1, Sullo-2 and Sullo-3 and 40 minutes for Sullo-4. The burst size of Sullo-1, Sullo-2, Sullo-3 and Sullo-4 was 38, 140, 246 and 73 pfu/infected cell, respectively.
Conclusions: The newly isolated phages had efficient lytic activity against ocular P. aeruginosa isolates and could be distinguished in terms of morphology, host range, thermal stability, burst size and latent period.
