Isolation, characterization, molecular analysis and application of bacteriophage DW-EC to control Enterotoxigenic Escherichia coli on various foods

Abstract

Among food preservation methods, bacteriophage treatment can be a viable alternative method to overcome the drawbacks of traditional approaches. Bacteriophages are naturally occurring viruses that are highly specific to their hosts and have the capability to lyse bacterial cells, making them useful as biopreservation agents. This study aims to characterize and determine the application of bacteriophage isolated from Indonesian traditional Ready-to-Eat (RTE) food to control Enterotoxigenic Escherichia coli (ETEC) population in various foods. Phage DW-EC isolated from Indonesian traditional RTE food called dawet with ETEC as its host showed a positive result by the formation of plaques (clear zone) in the bacterial host lawn. Transmission electron microscopy (TEM) results also showed that DW-EC can be suspected to belong to the Myoviridae family. Molecular characterization and bioinformatic analysis showed that DW-EC exhibited characteristics as promising biocontrol agents in food samples. Genes related to the lytic cycle, such as lysozyme and tail fiber assembly protein, were annotated. There were also no signs of lysogenic genes among the annotation results. The resulting PHACTS data also indicated that DW-EC was leaning toward being exclusively lytic. DW-EC significantly reduced the ETEC population (P ≤ 0.05) in various food samples after two different incubation times (1 day and 6 days) in chicken meat (80.93%; 87.29%), fish meat (63.78%; 87.89%), cucumber (61.42%; 71.88%), tomato (56.24%; 74.51%), and lettuce (46.88%; 43.38%).

Bacteriophage DW-EC with the capability to destruct and inhibit biofilm formed by several pathogenic bacteria

“…In addition, bacteriophage DW-EC was found to lyse the bacterial cell wall, seen from the irregular shape of the cell. This observation indicates that adding DW-EC can decrease the number of bacterial cells and degrade their biofilm matrix 4,21 .…”

“…Phylogenetic tree analysis was used in a previous study to determine the relationship between bacteriophage DW-EC phage tail fiber to other phages. The DW-EC putative phage tail fiber protein was found to be closely related to Escherichia phage ukendt putative tail fiber protein 4 . Escherichia phage ukendt recognizes the glycan surface and ECA as the first receptor and the first glucose of the outer membrane LPS as the second receptor.…”

“…DW-EC can inhibit biofilm formation by lysing cells from within using lysine and putative T4-like lysozyme, attacking each cell individually, lyses cells, and inhibits proliferation, thereby preventing bacteria from reaching the required density to produce EPS. Moreover, mature biofilms are destroyed when depolymerase enzymes degrades bacterial polysaccharides 4,13 .…”

“…In our previous molecular analysis study, we found that bacteriophage DW-EC had a putative T4-like lysozyme associated with cell lysis. A putative T4-like lysozyme is a hydrolytic enzyme that can break the 1,4-glycosidic bond with N-acetylglucosamine, resulting in cell wall perforations and eventual host cell lysis 4 . In addition, this phage produces endolysin enzymes that can damage the peptidoglycan in the bacterial cell wall, also resulting in lysis 3 .…”

“…In addition, this phage produces endolysin enzymes that can damage the peptidoglycan in the bacterial cell wall, also resulting in lysis 3 . Bacteriophage DW-EC lacks integrase and excisionase genes associated with the lysogenic phage stage, indicating that DW-EC phages are lytic 4 , avoiding the disadvantage of gene transfer which can prevent the transmission of antibiotic resistance genes and other virulence-associated genes that make bacterial pathogens more difficult to eradicate 22 .…”