Study On The Inhibitory Effect Of Bacteriophage Lysin On Biofilm Of Two Pathogenic Bacteria

The formation of Bacterial biofilm（BBF）and the massive use of antibiotics have led to the emergence of drug-resistant bacteria,which has threatened global public health.BBF refers to a microbial community with a certain structure formed by bacteria producing a polymer complex matrix,which wraps itself and adheres to the surface of an object or active tissue.Phage lysin has good bactericidal specificity and is not easy to produce drug resistance.It has the potential to develop as an antibacterial drug against drug-resistant bacteria,providing a new safe and efficient method for inhibiting the formation and degradation of biofilm.At present,bacteriophage lysin has very little biofilm inhibition effect.In this study,we used high-temperature bacteriophage lysin TSPphg and MMPphg as materials to explore the inhibitory effect of lysin on the biological formation of Staphylococcus aureus and Pseudomonas aeruginosa.Firstly,the TSPphg vector with EGFP tag was constructed by molecular biology technology,and the fusion protein expression and enzyme activity verification were carried out.Secondly,the biofilm models of Staphylococcus aureus and Pseudomonas aeruginosa were constructed,and TSB medium containing 5%glucose was used for the formation of their biofilms.The crystal violet staining method,XTT reduction method,and fluorescent staining were used for legal quantitative analysis Effects of lysin TSPphg and MMPphg on the biofilm of Staphylococcus aureus and Pseudomonas aeruginosa.Determine the minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of the lytic enzymes TSPphg and MMPphg against Staphylococcus aureus and Pseudomonas aeruginosa by double dilution method,and use the double dilution method The MIC values of lysin TSPphg,antibiotics alone and combined use were measured to study the effect of lysin TSPphg combined with antibiotics.The research results are as follows:（1）The results showed that p ET28a-EGFP-TSPphg（PGTphg）was successfully constructed,and the lysin PGTphg was expressed in a soluble manner.The optimal induction conditions were 20℃,150 rpm,and induction for 12 h（induction concentration 0.5 m M）.The expression was stable in shake flasks,The expression level can reach 189 mg/m L,and the lysin PGTphg has lysin activity and fluorescent activity.（2）Studies with crystal violet staining method and XTT reduction method show that TSPphg（concentration 50μg/ml）can reduce the percentage of Staphylococcus aureus biofilm formation by 49.3%,and its OD₆₀₀ value is reduced from 2.8 to 1.4（P<0.05）;Pseudomonas aeruginosa decreased by 39%,and its OD₆₀₀ value decreased from 2.3 to 1.3（P<0.05）.MMPphg（concentration 50μg/ml）can reduce the percentage of Staphylococcus aureus biofilm formation by 22.7%,and its OD₆₀₀ value decreased from 2.8 to 2.1（P<0.05）;Pseudomonas aeruginosa decreased by 49.3%,its OD600 The value decreased from 2.3 to 1.2（P<0.05）.These results indicate that the lysin TSPphg and MMPphg can effectively inhibit the formation of biofilms on Gram-positive bacteria（Staphylococcus aureus）and Gram-negative bacteria（Pseudomonas aeruginosa）.XTT reduction method results showed that TSPphg（concentration 50μg/m L）can reduce the percentage of S.aureus enveloped bacteria proliferation activity by 41.7%（P<0.05）,Pseudomonas aeruginosa decreased by 42.7%（P<0.05）;MMPphg（Concentration 50μg/m L）can reduce the percentage of the Staphylococcus aureus enveloped bacteria proliferation activity by 22%,Pseudomonas aeruginosa by 52.3%.This indicates that the lysin TSPphg and MMPphg can effectively inhibit the formation of biofilms of Staphylococcus aureus and Pseudomonas aeruginosa.（3）Fluorescence microscopy observed that the lysin PGTphg acted on the Staphylococcus aureus biofilm 1h,the Staphylococcus aureus enveloped bacteria began to dissociate,the lysin PGTphg has the effect of destroying the extracellular polysaccharide of the biofilm,which can dissociate the enveloped bacteria.At a concentration of 100μg/m L,the lysin TSPphg and MMPphg acted on the Staphylococcus aureus biofilm for 12 h,and the number of coated bacteria treated with the lysin was small and the colonies were sparse.The role of cocci biofilm.（4）The results of the ratio dilution method showed that the bacteriophage lysin TSPphg had a MIC value of 12.5μg/m L for Staphylococcus aureus and an MBC value of 25μg/m L;the MIC value for Pseudomonas aeruginosa was 25μg/m L,MBC The value is 50μg/m L.The MIC value of bacteriophage lysin MMPphg for Staphylococcus aureus was 25μg/m L,MBC value was 50μg/m L;for P.aeruginosa,the MIC value was 25μg/m L,and MBC value was 50μg/m L.The results showed that the combined use of lysin TSPphg and antibiotics reduced the amount of antibiotics used,and the combined MIC value was smaller than that when antibiotics were used alone.In summary,the lysin TSPphg and MMPphg can effectively inhibit the formation of S.aureus and P.aeruginosa biofilms,and have a degrading effect on the biofilms that have been formed.The lysin TSPphg and MMPphg are expected to become new antibacterial substances.This study provides a new way of thinking about alternative technologies for antibiotics.