The Isolation And Identification Of Salmonella Bacteriophages And Their Preliminary Application On Poultry Meats

In recent years,foodborne diseases caused by pathogenic bacteria have been emerged frequently in the world,especially Salmonella.Poultry are considered one of the most important reservoirs,the contaminated poultry carcasses and parts with Salmonella organisms can infect humans through food chain,and cause acute gastroenteritis,septicaemia,food poisoning.It represents a major threat to public health worldwide.The conventional strategies of pathogenic bacteria control are based on antibiotics,but the repeated misuse and overase of antibiotics have accelerated the emergence of antibiotic-resistant bacteria,leading to serious clinical infection,simultaneously the therapeutic limitation of current antibiotics has added to the difculty in treating multidrug-resistant bacterial infections.Hence,the development of alternative therapeutic treatments over antibiotics is essential.Bacteriophages are bacterial viruses that infect and lysis bacterial cells,it has the advantages of strong specificity,significant effect,wide distribution as compared with antibiotics,bacteriophages have been recognized as a possible alternative to antibiotics,and it has provided a new route for the control of foodborne pathogens.In this study,a set of bacteriophages were isolated and purified used different serotypes of Salmonella as the host.Two of them,named LP31 and SP55 were selected having the best lysis efficiency.Their characteristics were identified including host range determination,transmission electron microscopy,optimal multiplicity of infection assay,one step growth curve,thermal stability,pH tolerance capacity.Then through whole genome sequencing and bioinformatics analysis,we had better understand the genetic background of bacteriophages.Finally,we explored the optimum conditions of their application and evaluated their ability to reduce Salmonella at surface of chicken skin and those of biofilm-forming strains of Salmonella spp.1 Isolation,identification of Salmonella bacteriophages1100 samples(sewage water,poultry feces)were collected from six places in Jiangsu province,and bacteriophages were isolated used S.Pullorum C79-13,S.Enteritidis C50041 and S.Indiana 218 as the host,325 positive samples(29.5%)were detected from the samples,then 175 bacteriophages were purified by successive single-plaque isolation until homogenous plaques were obtained,and saved.The host ranges of the 175 bacteriophages were determined using 27 strains including different strains of Salmonella,Escherichia coli and Listeria monocytogenes.And the results indicated that bacteriophages had a good lytic effect on S.Pullorum,S.Enteritidis,S.Typhimurium,cracking rate exceeds 90%,then S.Typhi(86.9%)S.Derby(60%),S.Indiana(27.4%),S.Paratyphi A(24%);S.Rissen,S.Anatum,S.Meleagridis,S.London,S.Braenderup,Escherichia coli and Listeria monocytogenes are not lysed.2 Characterization and whole genome sequence of two bacteriophagesBacteriophages LP31 were purified and formed clear plaques of approximately 4 mm in diameter on Salmonella bacterial lawns,bacteriophages had a good lytic effect on S.Pullorum(59/61,97%)and S.Enteritidis(100/104,96%).Its optimal MOI is 0.01.Bacteriophages LP31 were able to survive in a pH range between 4-11,and it was able to survive in a treatment of 40??60? for 60 min.One-step growth kinetics showed that the latent periods were 10 min,burst period 90 min and burst size 23 PFU/cell.It revealed that bacteriophages LP31 is belonged to family Siphoviridae using transmission electron microscopy.This phage possesses an icosahedral head(diameter,60nm)and a noncontractile tail(length,110nm).Bacteriophages SP55 were purified and can form clear plaques of approximately 3 mm in diameter on Salmonella bacterial lawns.It had a good lytic effect on S.Pullorum(59/61,97%)and S.Enteritidis(97/104,93%).Its optimal MOI is 0.01.Bacteriophages SP55 were able to survive in a pH range between 4-11 and temperature of 40??60? for 60 min.One-step growth kinetics showed that the latent periods were 10 min,burst period 80 min and burst size 40 PFU/cell.It revealed that bacteriophages SP55 is belonged to family Siphoviridae using transmission electron microscopy.This phage possesses an icosahedral head(diameter,55 nm)and a noncontractile tail(length,120nm).According to sequence analysis,the LP31 genome is composed of 42,766 bp,with a total GC content of 50.33%.There are a total of 57 open reading frames(ORFs)in the LP31 genome,25 ORFs were predicted to encode proteins with known functions.The SP55 genome is composed of 42,781 bp,with a total GC content of 50.28%,its genome contains a total of 58 ORFs,27 ORFs were predicted to encode proteins with known functions.These proteins with known functions belong to the bacteriophage replication/transcription module,structure and packaging module and the host lysis module.No putative virulence factors and antibiotic resistance genes were identified according to the whole genome sequences of bacteriophages.The results suggested that there is a potential use of bacteriophages LP31 and SP55 as a prophylactic agent for the control of the desired serotypes of Salmonella.3 Preliminary application of Salmonella bacteriophages on poultry meatTwo lytic bacteriophages LP31 and SP55 were evaluated their capacity to reduce Salmonella spp,including S.Pullorum or S.Enteritidis on poultry carcasses and poultry products.The optimum conditions for the interaction between bacteriophages and host bacteria was explored.The results revealed optimal time was 2 h,and the concentration of host bacteria and bacteriophages was 104 CFU/mL,108 PFU/mL,respectively.According to the optimal conditions,chicken skin samples were inoculated with Salmonella and then surface treated with bacteriophages,untreated skin samples were used as negative controls.After the test samples stored at 4? and 25? for 2 h,Salmonella counts were performeded.The S.Pullorum counts in phage-treated samples stored at 4? were significantly(P<0.05)reduced by 2.16 log CFU/mL in LP31 group,1.64 log CFU/mL in SP55 group,and S.Enteritidis 1.56 log CFU/mL in LP31group,1.36 log CFU/mL in SP55 group.At 25?,LP31 and SP55 caused a significant reductions(P<0.05)of S,Pullorum at the level of 1.52 log CFU/mL,1.46 log CFU/mL,respectively,and reduced S.Enteritidis at the level of 1.09 log CFU/mL,0.99 log CFU/mL,respectively,compared to the negative controls.The results showed that two bacteriophages had antibacterial activity at different temperatures(4?,25 ?)against S.Pullorum or S.Enteritidis,and the antibacterial effect at 4? was better than 25?.5 strains of Salmonella spp(3 for S.Pullorum and 2 for S.Enteritidis)were screened and proved able to form biofilms at 24 h of incubation(P<0.05).Then treatment with bacteriophages LP31,their biofilm-forming were significantly reduced(P<0.05).It was shown that these two phages have the great potential for controlling Salmonella biofilms.