Stability of spoilage- Pseudomonas- specific phages to selected physicochemical conditions

Abstract

Bacteriophages are viruses that specifically require bacterial hosts’ metabolic activity to replicate their genomic material and produce proteins necessary for their survival. They are showing promising results as antimicrobial agents against quiet a number of pathogenic bacteria. In the present study, that was carried out at Makerere University, in Kampala, Uganda; Pseudomonas specific bacteriophages, that were coded as Psa1, Psa2, Psa3 and Psa4, were isolated and characterized for their potential to control and manage fish spoilage mainly caused by Pseudomonas spp. Phages were sourced from fish Gastrointestinal tract and those specific against Pseudomonas were isolated using an enrichment procedure with a single strain of P. aeruginosa originally isolated from fish samples. A spot assay was used to detect presence and determine the lytic spectra of Pseudomonas-specific phages. Phage purification and concentration (titer) were determined using the agar overlay method. Phage bactericidal activity was compared at different Multiplicity of Infection (MOI). The study aimed at using Pseudomonas specific bacteriophages to control fish spoilage by Pseudomonas spp. A One-step growth curve was constructed by determining the phage titer over time. Phage titers before and after exposure to various temperature and pH for up to 60 minutes allowed determination of stability under the specific physicochemical factors. The titers for the purified phage lysates were recorded as 1010 PFU/ml for Psa1, Psa2, and Psa4 and 108 PFU/ml for Psa3. One step growth curve analysis revealed that the isolated bacteriophages have a short latent period and a large burst size. All the selected phages were stable at temperature ranges of 30 – 50˚C with a titer of 1010 PFU/ml for Psa1, Psa2 and Psa4 and 108 PFU/ml for Psa3 with a slight decrease at 60˚C; but were sensitive to higher temperatures. Phage viability was affected at higher temperature of 70˚C and no plaques were detected after 20 minutes of incubation. The pH stability studies showed that Psa1, Psa2 and Psa4 were stable at pHs of 6, 7, 8 and 9 whereas Psa3 infectivity was slightly reduced at pH 8. The optimum MOI was 100 for Psa1 and Psa2, while MOI of 0.1 was the best for Psa4 and Psa3 was 10.