Isolation And Identification Of Salmonella Bacteriophage And Modification Of Host Spectrum Of T4 Bacteriophage

Salmonella is a very important pathogenic bacterium that can cause infection in a variety of animals.At the same time,the bacterium is also a foodborne pathogen that harms human health.Antibacterial drugs have been used as the main means to prevent and control salmonellosis,but with the general emergence of drug-resistant and multi-drug resistant strains,it is necessary to find other effective control methods.Bacteriophages are virus that can infect bacteria and kill bacterial cells.This feature makes phage became one of the alternatives to antibiotics.which limits further clinical use of phages.On the other hand,how to quickly screen out phages that target specific bacteria is also a challenge.Therefore,how to change or broaden the phage host spectrum has become a research hotspot for scientists.In T4 bacteriophage,the tail fiber protein of the bacteriophage is a key region that determines the specificity of the bacteriophage host.This study was conducted in three parts.The first part was Isolation,identification and biological characteristics analysis of Salmonella phage.The second part is the genetic modification of gp37 of T4 phage.The third part is the in vitro antibacterial test of virulent phage1.Isolation,identification and biological characteristics analysis of Salmonella phageIn clinical applications,phages are mainly restricted by the narrow host spectrum.Therefore,the isolation and preservation of a large number of phages with different host spectrum is the basis for phage host-specific research and related research on phage therapy.In this test,samples of sewage or feces were collected from different regions of Jiangsu Province.A large number of Salmonella virulent phages were isolated from the samples by processing the samples and enriching the phages,and the biological characteristics of some phages were determined.A total of 36 virulent phages were isolated in this test.Based on the phage host spectrum results and the lytic characteristics of each phage,phages SP32 and SP34 were selected for morphological and biological analysis.The test results show that the potent bacteriophages SP32 and SP34 belong to the family Myoviridae,with a short incubation period,strong lytic ability,and certain acid,alkali and high temperature resistance.This study provided sufficient experimental materials and basic data for the preparation of phage preparations to prevent and control Salmonella infection.2.Genetic modification of gp37 of T4 phageThe tail fiber protein of phage can recognize the host bacteria surface receptor protein,which can cause the phage to specifically adsorb on the host cell.In this test,the homologous recombination method was used first to replace the gene of the tail filament protein of the parent phage WG01 with a broad host spectrum to the parental phage QL01 with a narrow host spectrum,and then the constructed chimeric phage was screened with the host phage WG01 and The biological characteristics and genetic stability of parental phage and chimeric phage were compared and analyzed.The test results showed that a total of 5 chimeric phages were screened,and the tail fiber protein of phages could be modified to infect more Salmonella.The results of the pH stability test show that the parent phage and chimeric phage QWG have strong acid and alkali resistance,and the optimum pH is 7.0.The temperature stability test results showed that the optimal temperatures of the parent phage and the chimeric phage QWG were different,which were 37,37,and 28℃,respectively.The three phages had higher survival rates in the range of 28～45℃.One-step growth curve and multiple infection test results show that the parent phage and chimeric phage QWG have strong lytic ability,and their incubation period is relatively short.When the multiplicity of infection is 0.001,the multiplication factor is the most.In order to verify the genetic stability of the chimeric phage,we first purified the chimeric phage,then subcultured it,and finally sequenced and compared the gp37 gene of the 1st and 20th generation of chimeric phage.The alignment results showed that the sequencing results of the two generations of chimeric phage gp37 were consistent.In this experiment,a series of phages with a wide host spectrum were obtained by modifying the gp37 of T4 phages,which laid the foundation for the widespread application of phages in the treatment of bacterial diseases.3.Vitro antibacterial test of virulent phageWhen in an adverse environment,bacteria live as a bacterial biofilm in order to increase their resistance.Bacterial biofilm-related infections often cause chronic or recurrent infections.Bacterial biofilm-related infections often cause chronic or repeated infections in the body.As a natural bactericide,phage is used to fight drug-resistant bacteria infection and bacterial biofilm infection.In this test,the virulent phages WG01 and QL01 were cultured together with their common host bacteria to determine the antibacterial effect of the two strains of phages.The phage infects the bacterial biofilm under different conditions to determine the bacterial biofilm’s inhibitory effect,degradation effect and the optimal time for the phage to infect the host bacteria.The test results show that the potent phage WG01 and QL01 can inhibit the formation of bacterial biofilms and the growth of bacteria in vitro,and degrade the mature bacterial biofilms.Compared with the phage alone preparation,the phage cocktail preparation has a better inhibition and degradation effect on the biofilm,and the inhibition effect is better than the degradation effect.In addition,when phages are added when bacteria start to form a biofilm,phages have the best effect on inhibiting biofilm formation.