Isolation and characterization of bacteriophages from wastewater as potential biocontrol agents for Escherichia coli.

Abstract

Host-specific lytic bacteriophages have regained momentum as an alternative treatment option to control and eliminate pathogenic bacteria. This study aimed to isolate, characterize and evaluate the potential application of Escherichia coli phages as a biocontrol agent in wastewater. In this study, four lytic Escherichia coli phages were isolated from wastewater for biocontrol purposes, using the double-layer method with E. coli (ATCC-25922) as a host. The phage morphology was characterized using transmission electron microscopy, with further parameters such as host range, phage stability at different temperatures, and pH values analyzed additionally. The genome of two selected phages (NPS and NPM) was sequenced, and the capacity of the phage isolate NPM to eliminate E. coli from artificial wastewater was evaluated and compared to conventional chlorination. All the four phage isolates showed typical T4 phage appearance with isometric capsids and contractile tails of different sizes, matching the family Myoviridae in the order Caudovirales. They exhibited a narrow host range limited to E. coli isolates, with two exceptions: phage NPS and NPM additionally lysed Salmonella Typhimurium (ATCC-14028). The four phage isolates were even able to lyse MDR (multidrug-resistant) E. coli isolates, such as the strain FP29. The four phages had burst sizes ranging from 70-115 per host cell and a latency period of 10-20 minutes. All the four bacteriophages were stable at pH 5-9 but completely inactivated at pH 12. Exposure to 60°C for 10 minutes reduced phage titers by 1.5log, while exposure to 80°C for 10 minutes completely inactivated all four phage isolates. The two genomes (NPS and NPM) were 99% identical and had similar sizes (169 536 bp), but phage NPS differed from phage NPM in view of its host range and plaque morphology. Another difference observed at the genome level was a shift of coding sequences between phage NPS and NPM. Phage isolate NPM achieved a 3.5log reduction of E. coli cells present in artificial wastewater at an MOI of 0.1 in 120 minutes. A 90-minute chlorine treatment achieved a log reduction in the same range, highlighting that phages have the potential as environmentally friendly biocontrol agents in wastewater treatment.iii Acknowledgements I would like to acknowledge God first for the blessing of life and the strength to do this Master's degree. It has been a rough but rewarding journey, surviving a car accident was one of the most remarkable experiences in my life during the cause of this Master's degree and for that I am forever grateful.• I would like to express my sincere gratitude to my supervisor Professor Stefan Schmidt for his mentorship, guidance, support, and opportunities he offered me for this Master's degree to be an amazing journey.• The MMU staff at the UKZN Pietermaritzburg campus for providing the electron microscopy facilities that were essential for this project.• National Research Fund for believing in this project and funding it. I am grateful.• Colleagues from Jiangsu Academy of Ag...