| Due to the more widespread use of antibiotics,the clinical multi-drug res:istance bacteria are gradually increasing,and the research and development of anti-biotics is far slower than the speed of bacteria to obtain the corresponding antibiotic resistance,resulting in the production of multidrug-resistant bacteria,and causing great challenges to clinical anti-infection treatment.Phages are a special group of viruses that infect and kill microbial hosts.Therefore,using corresponding phage to treat the infection by multidrug-resistant bacterial has re-aroused people’s attention as a potential treatment.However,there are still many problems to be solved in the treatment of drug-resistant bacterial infections by phage,including whether bacteria can evolve resistance to phages and which components of phages are involved in the cracking of bacteria.Therefore,it is urgent to find alternatives to antibiotics for the prevention,control and treatment of drug-resistant bacteria.In this study,Vibrio alginolyticus,Vibrio parahaemolyticus,Vibrio vulnificus,Vibrio fluvialis,Vibrio furnissii was selected as the host bacterium,and the corresponding lytic phage vB_Va1P_IME271 of Vibrio alginolyticus was isolated from seawater of the Qing Dao Yellow Sea.The biological characteristics of the phage are as follows:the incubation period is 90 minutes,the cracking stage is about 60min,and the outbreak volume is 40 pfu.IME271 is sensitive to ultraviolet radiation and high temperatures,and insensitive to chloroform treatment.Phage IME271 has good cracking activity in the range of pH 7.0-9.0.Electron microscopy analysis showed that phage IME271 belonged to the genus Sephvirinae of the Podoviridae.Genome analysis showed that 87%of the IME271 genome query cover with phage Vibrio phage VPp1,and 95.93%Ident(VPp1 was directly submitted to NCBI and had not yet been classified),indicating that IME271 is a new discovered phage.Genome-wide functional annotation and evolutionary analysis of phage IME271 showed that there were 67 open reading frames(ORFs)in the IME271 genome,of which 45 ORFs are hypothetical protein sequences and the remaining 22 ORFs are coding sequences of proteins with known functions.By using mass spectrometer to identify its proteome,a total of 17 proteins were identified,and no bacterial virulence factors or related proteins were found.In order to further study the lytic activity of phage IME271 against its host Vibrio alginolyticus,pET28a was used as vector for expression of IME271 endolysin Lys271.After conditional screening,the optimal expression temperature was 37℃,the induction time was 4 h,and the optimal concentration of IPTG was 1 mmot/L,which produced soluble protein.After protein purification,the protein concentration after desalting and concentration was 4.74 mg/mL.Further experiments using turbidity method and plate method showed that solubly expressed Lys271 had lytic activity against Vibrio alginolyticu.s.This is the first report of successful expression of active lysin against Vibrio alginolyticus.Compared with the original phage IME271,the lysin can not only lyse Vibrio alginolyticus of different serotypes,but also can lyse Vibrio parahaemolyticus and Vibrio vulnificus,with a lysis spectrum much broader than that of the original phage IME271.Lys271 can retain high activity under the conditions of pH 6.0～9.0 and 55 C,and still exhibits 80%activity after 16 month storage under-80℃ conditions.The above study not only provides abundant data resources for the diversity of Vibrio alginolyticus phage genome,but also provides theoretical basis and technical guidance for the prevention and control of Vibrio resistance by using phage lysins,and provides scientific basis for the implementation ofphage therapy at the same time.The results show that Lys271 fills the gap in the study of Vibrio alginolyticus phage therapy,which has a very good application prospect as an antimicrobial strategy of Gram-negative bacteria infection. |
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