Use of bacteriophages to control salmonella in crocodiles and optimisation of a light induced expression system for endolysin.

Abstract

The aims of this study were to isolate bacteriophages from crocodile feeding pen wastewater and to evaluate their effectiveness against clinical Salmonella species, Escherichia coli and Enterobacter cloacae. Also, the recombinant expression of a synthesised endolysin (Lysin ECD7) using a light inducible plasmid vector pDAWN was optimised. Materials and Methods: Crocodile wastewater from Bally Vaughan Bird and Game Sanctuary (Mwanga Lodge) was collected and used to isolate Salmonella species and bacteriophages. Salmonella spp. DNA was isolated and characterised using RISA-PCR, 16S rRNA-PCR, ERIC-PCR and M13-RAPDs-PCR. The isolated bacteriophages were tested against clinical samples of Salmonella species, Escherichia coli and Enterobacter cloacae. Bacteriophage DNA was also isolated and subjected to M13 RAPDs –PCR and 16S rRNA- PCR molecular fingerprinting techniques. Chemically synthesised endolysin gene was cloned and expressed under the induction of both IPTG and light. The molecular weight of the isolated protein was determined using SDS-PAGE analysis and the concentration was determined using BSA standard curve. Result: The presence of Salmonella spp. in crocodile wastewater was confirmed by both physiological and molecular techniques. The isolated bacteriophages were potent against all the tested bacterial strains with very high titre volumes of up to 3.1 x 109 PFU/ml. Protein expression of endolysin was induced by Isopropyl β-D-thiogalactopyranoside (IPTG). The protein sample was analysed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis which showed that the protein was successfully expressed and was approximately 15 kDa. Light- induction was optimised and endolysin protein concentration of approximately 1.007mg/ml was obtained. Conclusion: Salmonella spp. specific bacteriophages were successfully isolated and characterised. A synthetic endolysin gene was successfully expressed under cost effective light-induction. The combination of endolysins and bacteriophages will serve as a cost effective and potent alternative in the treatment of Salmonella associated diseases and other diseases that arise due to antimicrobial resistance in Enterobacteriacae species.