Genome Sequencing Of An Escherichia Coli Bacteriophage With Novel Lysin And Activity Detection Of Its Lysin

In recent years, the problem of bacterial resistance has caused a great threat tohuman health and animal husbandry. Faced with resistant bacteria increasing andresistance genes disseminating broader, phage and lysin have caught researchers’attention again. In antibacterial treatment, bacteriophages and lysin have moreadvantages than antibiotics. We must analyze not only their biological characteristics,but also their evolution and harmful genes from genetic level for the safety ofbacteriophage and lysin therapy. When it comes to the use of gram-negative bacteriaphage lysin, we need to consider the extra cellular bacterial lysis.vB_EcoM-ep3was isolated from domestic sewage and then study its biologicalcharacteristics. Its optimal MOI is between0.1and0.001. This phage has goodthermal stability. It can substantially maintain its activity after a cultured at50℃for2h. The incubation period of this phage is20min and the lyse period is40min.Tested E.coli O78and eight clinical isolates of Escherichia coli from chicken aresensitive to this phage. TEM observation showed that it belongs to Myoviridae.These findings suggest that we can draw a conclusion that vB_EcoM-ep3is a lyticphage with strong lytic ability and widerange.Therefore, we sequenced the phage genome, predicted and analyzed thefunction of the each open reading frame and study the evolutionary relationshipswith other phages with software. There are42,351nucleotides in its genome, GCcontent is53.35%,52(48.1%) of ORF have assumed function. It has a highhomology with phage vB_EcoM_ECO1230-10. There is no lysin gene homologouswith Lysep3, but it has a high homology with Pseudomonas PPpW-3lysin at theprotein level. Combined with EDTA, citric acid and malic acid, Lysep3can lyse P.aeruginosa ATCC27853and Escherichia coli DH5α. Lysep3can reduce the number of bacteria by5-fold,4.7-fold and2.1-fold respectively after treated2h at the proteinconcentration of3mg/mL,1.5mg/mL and0.75mg/mL. At the protein concentrationof3mg/mL and1.5mg/mL, Lysep3can reduce the amount of bacteria by twomagnitudes after treated24h. In order to lyse bacteria directly, we constructedLysep3-D8, which can obviously lyse E.coli O78.In conclusion, vB_EcoM-ep3has excellent lytic activity and biological stability.Lysep3has high homology with Pseudomonas PPpW-3lysin. It can effectively lysebacteria combined with EDTA, citric acid and malic acid. Recombinant lysinLysep3-D8can directly lyse bacteria and has medicinal potential.