Study Of Lysin P26ly On Biofilm Clearance Of Staphylococcus Aureus And Escherichia Coli

Biofilms are aggregated structural communities of bacteria wrapped in a matrix composed of proteins,DNA and polysaccharides.Compared with plankton,biofilms are 10 to 1000 times more resistant to various antibiotics,and almost all microorganisms are highly resistant to most antibiotics after biofilm formation.Phage lysin,a class of cell wall hydrolases expressed by phages at the late stage of bacterial infection,are bactericidal and resistant to a variety of bacteria,and can inhibit biofilm formation and degrade and remove them,making them promising new antibacterial substances.In this paper,based on the sequence of lysin gene P26ly obtained by macrovirulence sequencing,we used PCR amplification to identify the phage strains containing this gene,and studied the biological properties of the phage,and cloned and expressed the lysin gene P26ly to study the inhibition effect of lysin on Escherichia coli and Staphylococcus aureus,and to explore its effect on biofilm removal.The results showed that the lysin gene P26ly corresponds to the phage SP26,which belongs to the order Arthrophage,family Long-tailed Phage,and has higher activity at pH 5?8 and temperature 4?40?.The molecular weight of lysin P26ly is17 k Da,and the optimum enzyme activity temperature and pH value are 37?and7.5,respectively.Mg²⁺and K⁺ions have activating effects on the enzyme,and the concentration of lysin at 75?g/mL can lyse the cell wall of host bacteria,and has good bactericidal effects on a variety of Gram-negative and Gram-positive bacteria.After co-incubation with E.coli and S.aureus strains at a concentration of 75?g/mL,the lysin P26ly did not form an obvious biofilm shape,and the bacteria were dispersed and of low density,which could effectively inhibit the formation of biofilms of S.aureus and E.coli.After its co-incubation with the formed biofilm E.coli and S.aureus respectively,it could be observed by XTT method combined with fluorescence staining,and it could be found that the number of viable bacteria in the biofilm was significantly reduced,the scanning electron microscope results showed that the cells of biofilm were disrupted after the action of lysin P26ly,causing cytoplasmic contents to lyse out and leading to cell deformation.It indicates that lysin has a good scavenging effect on biofilm.Further,after combining lysin P26ly with antibiotic amoxicillin,it was found that the biofilm was cleared better,indicating that the bactericidal effect of the combination of the two was more significant than that of using them alone.In conclusion,lysin P26ly can effectively inhibit the formation of biofilm of S.aureus and E.coli,and also has a degrading effect on the already formed biofilm.The lysin P26ly was able to reduce the use of antibiotics when combined with the antibiotic amoxicillin.The results indicate that lysin P26ly is expected to be a novel antibacterial substance,and this study provides a new idea for the alternative technology of antibiotics.