| Staphylococcus aureus is one of notorious food-borne pathogens,which can produce various toxins,including thermostable enterotoxins,form biofilms,and cause many food safety problems.At the same time,with the abuse of antibiotics,S.aureus has evolved into multidrug-resistant strains such as methicillin-resistant S.aureus(MRSA).MRSA is a new problem in the fields of food safety,animal health,and modern medical care.It is necessary to develop new antibiotics to replace overused traditional antibiotics.In recent years,there was an urgent need for research and development of new sterilization and control technologies for foodborne pathogens such as MRSA.Phage lysin was considered to be a promising antimicrobial agent.In this paper,a virulent MRSA bacteriophage was screened,and the lysin and its functional domains were constructed according to its genome sequence.Based on the enzymatic properties of each functional domain,the application of lysin in different fields was carried out,which provided a theoretical basis for the application of lysin and its functional domains in food and medicine.The main research contents and results are as follows:1.Screening and identification of S.aureus bacteriophage:A virulent phage Z was screened from food-borne waste,which could lyse MRSA with a titer of about 105.The phage belongs to siphoviridae,with a tail length of about 200 nm and a head diameter of about 80 nm by transmissio electron microscopy(TEM).The growth of S.aureus can be completely inhibited on the plate by the phage Z.Through the determination and analysis of the genome sequence of the phage Z,it was shown that it contains a gene lysz encoding lysin LysZ.The gene was fully synthesized subcloned to a vector p ET15b to construct a recombinant plasmid p ET15b-LysZ.The formed plasmid was transformed into E.coli BL21(DE3)and the LysZ was obtained by induction and purification.The seven strains containing five MRSA strains(2101,2102,2107,2403,and 2701)can be lysed despite different lysis levels,indicating that LysZ can be used for the control of S.aureus and MRSA.The optimum temperature and p H of LysZ were 35oC and 6.0,respectively.LysZ was a metal-dependent enzyme.The activity of LysZ gradually increased with the increasing ionic strength.The lytic activity of LysZ in milk was related to the component NaCl that accounts for?0.049%.The low-salt environment of milk caused the loss of its lytic activity.However,after NaCl was added into milk,the lytic activity of LysZ was recovered.2.The correlation between the effect of milk components on lytic activity of LysZ and its fuctional domains(lytic domain and binding domain)was also studied.The fusion protein EGFP-CBDz was obtained and it can specifically bind to S.aureus.The optimum temperature and p H for the specific binding were 30oC and 4.0,respectively.The binding ability increased with the increase of ionic strength in the solution.In milk,binding activity of EGFP-CBDz increased with the increasing NaCl content.Therefore,the low activity of LysZ at low ionic strength may be related to the weak binding ability of EGFP-CBDz to cell wall under this condition.Similar to LysZ,CHAPLysZ could rapidly cleave seven S.aureus strains,indicating that CHAPLysZ can also be used for the control of S.aureus and MRSA,with the optimum p H of 9.0and the optimum temperature of 40oC.CHAPLysZ was also a metal-dependent enzyme.CHAPLysZwas highly sensitive to ionic strength and its activity decreaseed with the increase of ionic strength.However,the activity of CHAPLysZ was not affected by NaCl and milk fat.CHAPLysZcould play its lytic activity in whole milk and skim milk without adding NaCl,which has a potential to reduce the pollution of MRSA.3.Selection of application strategy of S.aureus phage lysin in non-acid foods.The bactericidal ability of phage lysin in meat products with different salt contents was studied.LysZ and CHAPLysZ presented a promising biocontrol effect in high-salt food and low-salt food,respectively.The 0.4 nmol/cm2 of LysZ and CHAPLysZ were applied in Chinese bacon and fresh pork,where they could efficiently decrease MRSA from 104 to 0 CFU/cm2 after 2 h(LysZ to bacon,and CHAPLysZ to fresh pork).Based on the investigation of LysZ and CHAPLysZ,a strategy using the two enzymes in foods was proposed to fight against S.aureus.LysZ and CHAPLysZ can be chosen according to the salt content of food.For instance,CHAPLysZ was chosen for low-salt and non-acidic foods.Given the high efficiency of LysZ at high-salt concentration(200?700 m M),LysZ could be chosen for high-salt and weak-acid foods.In order to avoid pollution in food processing,biofilm removal was also essential.Different strains of S.aureus have different ability to generate biofilm.Among the seven strains used in this paper,MRSA 2102 had the strongest ability to generate biofilm,which was the most difficult to be removed by LysZ and CHAPLysZ,and its MIC to methicillin was 128?g/m L.LysZ and CHAPLysZ were used to remove biofilms produced by S.aureus.The ability of CHAPLysZ alone or in combination with methicillin to remove biofilms produced by MRSA was better than that of LysZ.The synergistic effect of CHAPLysZ(0.05 n M)combined with methicillin(32?g/m L)can remove more than 97%of the biofilm formed by MRSA 2102,which may be related to the mechanism and size of the lytic domain.CHAPLysZ can enter the biofilm through small pores in the biofilm,and it does not need to bind to the bacterial cell wall to play a role.4.LysZ and CHAPLysZ can be used to remove MRSA infection on skin wounds,promote wound healing,and reduce food safety problems caused by pollution caused by trauma.LysZ(1 nmol/cm2)could promote wound healing in mice.LysZ could reduce about 2.5 Log10 CFU of bacteria one day,while CHAPLysZ only reduced 0.4 Log10 CFU.Probably because of the high ionic strength of cytochylema,LysZ was more suitable for application in vivo to reduce MRSA contamination.5.A two-dimensional gold nanoparticle Au@PEG@EGFP-CBDz was synthesized,which can specifically bind to the cell wall of MRSA.The material was triangular,with an average hydration particle size of 72.32 nm and a Zeta potential of-5.08 m V.It had a good photothermal conversion effect under 808 nm laser irradiation.The heating rate and temperature were proportional to the material concentration and laser power.This material had good biocompatibility and does not affect cell viability.In the aqueous solution containing 1×104MRSA,50?g/m L Au@PEG@EGFP-CBDz were added and irradiated by NIR laser(808 nm,1 W/cm2)for 300 s,360 s,and 420 s,respectively.The bacteria were reduced by about 1.2Log10CFU,1.6 Log10CFU,and 3.1 Log10CFU,respectively,and the MRSA was specifically killed.Au@PEG@EGFP-CBDz can be used as a protein to specifically bind to MRSA infected in the lungs of mice.The targeted MRSA can be killed by photothermal irradiation to reduce the pulmonary bacterial infection caused by MRSA,reduce the inflammatory response caused by MRSA infection,and promote the improvement of the body. |
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