Effect Of Phage On Eradication Of Salmonella Biofilm

Salmonella spp.is a common foodborne pathogen that causes food contamination and food safety incidents frequently worldwide each year.The biofilm increases the spread and pollution of the bacteria,which can easily accumulate on the surface of animal slaughter factories and meat processing equipment,increasing the resistance to fungicides and the possibility of cross-contamination.As the natural enemy of bacteria,phages take an advantage in killing bacteria and lysing biofilms.However,a single bacteriophage has a narrow host spectrum,which limits the application advantage to the complex microbial flora environment in food factories.Therefore,phage cocktail,which include the phages with a wide host spectrum and high lytic activity,are not only easy to prepare,but also have a clear and safe genetic background,which has become a hotspot in food safety research.The purpose of this experiment is to isolate phage with broad-host spectrum and high-efficiency lysis taking Salmonella Enteritidis as the host,and make the mixture of those phages to prepare the phage cocktail.By exploring the biological characteristics and genomics of phage,the applicability and safety of phages to food factories were revealed.The degradation effect of the phage cocktail to Salmonella biofilm were investigated,which provide theoretical basis for the development of technology on biofilm control on food processing equipments.1.Isolation and biological characteristics of broad-host spectrum Salmonella phageIn this study,Salmonella phages were isolated from the fecal and carcass water samples of the chicken farm by double-layer plate culture using the S.Enteritidis,and their host spectrum was determined.The morphological,environmental stability,optimal infection number and reproduction curve were studied for one of the broad-host spectrum phages.Results were as follows:21 Salmonella phages were isolated in total.The broadest-host spectrum phage CW1 can lyse 43 Salmonella strains of 10 serotypes（70.49%（43/61））,and the comprehensive lysis rate is as high as 60.56%（43/71）.The results showed that the phage CW1 is amember of the Siphoviridae family.The optimum growth p H was 6-9.The optimum growth temperature was 30°C-50°C.The optimal infection number was 0.1,and the titer was 1.413?10¹¹ PFU/ml.Its latency time was 15 min,the outbreak time was 45 min,the adsorption capacity of the phage was 62.33%,and the average lysed capacity was 253.2.The genomic analysis of new-isolated Salmonella phagesThe structural genomics and functional genomics analysis of 21 Salmonella phages were performed on the Illumina sequencing platform.Genomic analysis indicated that 11phages are in common with gene and their sources include fecal and carcass water.The rest of 10 phages have homology in collinearity and they all belong to the Siphoviridae family.Representative phage CW1 is double-stranded linear DNA and total gene length is 41763bp,with a G+C content of 50.20%and a gene density of 1.412,only 28 functional genes are predicted as known function genes among total 59 open reading frames,they were divided into 17 structural genes including head protein and tail protein,7 metabolic genes related to DNA replication,recombination and modification,and 4 host lysis genes.There is a complete set of holin-endolysin system for lysing host and its biofilm.Integrase,virulence factors and antibiotic resistance were not found in phage CW1 genome.3.The effect of the phage cocktail on Salmonella biofilmThe crystal violet staining method was used to determine the effect of 10 phages on Salmonella biofilms in different states（formation and maturity stage）and different incubation time（12 h,24 h,48 h）.Combined with the results of host spectrum in chapter 1,four phages,CW1,M10（high-efficiency,broad-spectrum）and CW11,M4（high-efficiency）were selected and made into the phage cocktail.The effect of the cocktail to the biofilm on the stainless steel was explored by the viable cell counting,and the space structure and the extracellular polymer（EPS）composition of the biofilm by cocktail treatment were observed using CLSM,SEM and Roman technology.The results showed that the phage cocktail effectively reduced the number of viable cells of the developing biofilm（24 h,48 h）and mature biofilm by about 0.87 Log（CFU/cm²）,0.79 Log（CFU/cm²）and 0.4 Log（CFU/cm²）respectively,and reduced the thickness of the developing biofilm（48 h）and mature biofilm by 3.41μm and 2.56μm respectively.The phage destroyed the morphology of the cell and the structure of EPS,reducing the secretion and adhesion of the attached-cells.The cocktail mainly destroyed carbohydrates and proteins of EPS,affected the stability of the three-dimensional structure of the biofilm.