Applications Of Phage Endolysin In The Detection And Treatment Of Methicillin-resistant Staphylococcus Aureus

The abuse of antibiotics has led to frequent emergence of multidrug-resistant superbugs,which brings huge challenge to clinical treatment of infectious diseases.Also,because of slowed development of new antibiotics,drug-resistant bacteria have become one of the greatest threats to global public health.Staphylococcus aureus（S.aureus）is a common pathogen causing various human infectious diseases.The emergence of methicillin-resistant Staphylococcus aureus（MRSA）and its rapid growth of drug resistance have brought huge challenge to the clinical treatment of S.aureus infections.Therefore,besides of developing new antibiotics,it is also necessary to establish rapid and sensitive methods for drug-resistant bacteria detection and antibiotic susceptibility test（AST）for achieving efficient diagnosis of bacterial infections and accurate decision for subsequent treatment.In our works,endolysin LysP108 and its cell wall binding domain（CBD）of MRSA phages were recombinantly expressed and their activities were verified.Based on the recognition effect of CBD toward MRSA,novel methods for MRSA detection and AST were established in combination with dual-site fluorescent analysis.Based on the lytic effect of endolysin toward MRSA,it was applied to the treatment of bacterial infection.（1）Expression and purification of phage endolysin and CBDPhage endolysin is a kind of cell wall hydrolase synthesized by phage gene in the late stage of phage infection.In general,endolysin targeting Gram-positive bacteria are composed of N-terminal enzymatic activity domains with bacterial cell-lysing capability and C-terminal CBD for adhering to the bacterial cell wall.Previously we obtained MRSA phage P108（NC025426）and stored it in our laboratory.DNA sequences of phage endolysin LysP108 and its CBD were obtained from National Center for Biotechnology Information（NCBI）retrieval and local sequence alignment tool BlastP analysis.Primers were designed based on the relevant DNA sequences.The gene fragments encoding CBD and LysP108 were amplified by PCR,digested with restriction endonuclease Nde I and NotI,and seamlessly ligated into pET21a plasmid to construct recombinant vectors pET-21a-CBD and pET-21a-LysP108.Meanwhile,the green fluorescent protein gfp gene and the CBD gene were fused and expressed to construct the recombinant vector pET-21a-CBD-GFP.After CBD,CBD-GFP and LysP108 recombinant proteins were expressed and purified in Escherichia coli（E.coli）,it was found that a large amount of the target proteins were present in E.coli cells in soluble form after IPTG-induced expression.After purification,proteins with molecular weight of 13 kDa,35 kDa and 34 kDa were obtained,and their molecular weight were consistent with expected.（2）Detection and AST of MRSA by utilizing the molecular recognition capability of phage CBDDistinct from the strain-specific MRSA phage,CBD proteins displayed broad-spectrum recognition capability toward all five staphylococcal cassette chromosome mec（SCCmec）types of MRSA.Furthermore,they did not display any lytic activity toward the host bacteria,and were able to distinguish live bacteria from dead ones,which facilitated the capture of whole MRSA live cells with ideal flexibility for downstream manipulation and tracing.A magnetic separation based flow cytometry was established for broad-spectrum detection of MRSA strains by employing CBD and CBD-GFP as the recognition agent and the signal probe,respectively.The linear range for the detection of MRSA was 1.5×10²-1.5×10⁶ CFU mL^-1,and the limit of detection was 40 CFU mL^-1.Gram-negative bacteria and other Gram-positive bacteria showed minor interference to the detection of MRSA,indicating ideal selectivity and specificity.The method was successfully applied to detect MRSA in physiological saline injection,lake water and human urine,with recovery values between 81.0%and 104.8%.We further applied this dual-site fluorescent method to AST of MRSA.The whole procedure can be completed within 2.5 h by using a microplate reader,which realized rapid acquisition of drug susceptibility results.（3）Study on antibacterial effect of phage endolysin LysP108As a potential antibacterial agent,endolysin can directly lyse Gram-positive bacteria from the outside and is not prone to result in drug resistance.Therefore,endolysin is regarded as an important weapon for the treatment of multidrug-resistant pathogen infections.Standard plate counting method showed that the phage endolysin LysP108could directly lyse S.aureus and Pseudomonas aeruginosa（P.aeruginosa）with damaged outer membrane,resulting in a significant reduction in the number of live bacteria.Enzyme activity experiments indicated that the optimal working concentration of the endolysin was 250μg mL^-1,the optimal pH was 7.0,and the optimal temperature was37℃.Live/dead bacteria staining results indicated that endolysin possesses strong bactericidal ability,with anti-bacterial rate of about 90%.Crystal violet staining results indicated that endolysin could also inhibit and destroy bacterial biofilms.In vivo animal experiments demonstrated that the area of subcutaneous abscess of mice infected with MRSA was significantly reduced after the combined injection of endolysin LysP108 and vancomycin in comparison with monotherapy.The results demonstrated the synergistic antibacterial effect of endolysin LysP108 and vancomycin.Therefore,endolysin LysP108is expected to be combined with various antibiotics to play an important role in the treatment of multiple drug-resistant bacterial infections.