Screening And Genomic Analysis Of Salmonella Lytic Phages And Their Antibacterial Application On Broiler Products

The foodborne pathogen Salmonella is a threat to global public health and causes considerable global economic losses.Salmonellosis,caused by nontyphoidal Salmonella spp.,is a common foodborne communicable disease that affects humans and broiler industries.S.enteritidis can induce intestinal diseases in animals.A common cause of salmonellosis is the consumption of Salmonella spp.-contaminated chicken meat or eggs.An alternative approach to the use of antibiotics is urgently needed to control Salmonella contamination in food,especially in chicken meat and eggs.Lytic bacteriophages（referred to as phages）are promising green biocontrol agents for targeting and controlling foodborne pathogenic bacteria.Phages can reduce pathogenic bacteria in chicken product-producing environments and can be used as biocontrol agents against chicken meat and egg contamination.However,before phages are widely used,their safety should be ensured priority.The safety of phage genomes is an important prerequisite for the basic application of phages.Lytic phages,which are not capable of transduction,are considered to be a powerful tool for lysing pathogenic bacteria.Given this,this paper aims to address chicken meat and egg contamination problems of Salmonella from slaughter to sale in broiler industries,looking for effective lytic phages as an effective green biocontrol agent to assist traditional antibacterial measures.This study proves the safety of the phage at the genomic level and evaluates the application effects of phages to reduce Salmonella contamination,and also provides a theoretical basis to improve the safety of chicken meat and eggs and ensure public health.First,a total of 20 Salmonella strains were obtained from different institutions or samples in this study.Among them,the wild-type Salmonella strain ZC-S1 was isolated and identified from the intestine of broilers.The 16S r RNA gene comparison results showed that this strain was similar to Salmonella enterica ser.Newlands.The results of drug susceptibility experiments showed that S.ZC-S1 was resistant to 10 kinds of antibiotics（14 types）or intermediate antibiotics（12 types）and susceptible to 6 kinds（13 types）.S.ZC-S1 belongs to multidrug resistant bacteria.Combined with the third-generation sequencing（TGS）technology of Nanopore,the complete genome map of the S.ZC-S1 genome was constructed.In the next-generation sequencing（NGS）rawdata of S.ZC-S1,a complete prophage with inducible activity was found.Using the genome and 16S r RNA gene to construct evolutionary trees,S.ZC-S1 was inferred to belong to the serotype of S.enterica subsp.enterica serotype Enteritidis.S.ZC-S1 has a strong sensitivity to all phages isolated in this study and is used as a general-purpose amplification host and test strain for phages.Second,a total of 13 isolates of phages were screened and isolated from different samples.All of these phages are ds DNA-tailed phages,with the morphology of a long-flexible tail,a contractile tail,or a short tail.Their taxonomic status belongs to 4 families,including the families Demerecviridae,Siphoviridae,Myoviridae,and Podoviridae;their status specifically belongs to 7 genera,including the genera Epseptimavirus,Tequintavirus,Chivirus,Jerseyvirus,Rosemountvirus,Dhakavirus,and Lederbergvirus.In this study,6long-tailed phages were used as research objects for comparative genomics analysis to clarify their species taxonomic status and the type of genetic relationship and to predict the safety of their genome.The host profile test results show that the host ranges of these 6 phages are different,but they can only lyse bacteria from the Salmonella genus and cannot infect other species across genera.The genomes of these 6 phages are similar to T5-like phages.The genomes of these 6 phages were analyzed and compared with 3 type species from their genera in subfamily Markadamsvirinae,as well as other similar or homologous phages.This confirmed the phylogenetic status of these 6 phages belonging to the genus Epseptimavirus,subfamily Markadamsvirinae,and family Demerecviridae.The genomes of these 6 phage strains did not contain lysogenic genes,drug resistance genes,virulence transfer genes,integrase,repressor proteins,pathogenic genes,or any other unwanted genes.Genome analysis results show that they are all lytic phages.According to the host profile results and lysis ability of these 6 Epseptimavirus phages,two phages（1-23 and 3-29）with a broad host spectrum and strong lytic properties were selected from these phages as the representative phages.In this study,the antibacterial effectiveness was evaluated,and the ability of the lysate to control S.ZC-S1 on chicken meat and eggs was detected.The results show that the representative phages are all insensitive to chloroform;their OMOIs are 0.01;their latent period is short（approximately 10 min）;and the outbreak period is 20 and 30 min,respectively.They could survive within an environmental p H of 3-11,and the temperature tolerance range（-20-60℃）was wider.In this study,the survival cell count of S.ZC-S1 in the treatment groups using representative phages and the phage-free control group were compared,and the in vitro antibacterial effect of the two phages was determined.The results show that at 37°C,the representative phages were able to reduce the viable bacteria count of S.ZC-S1 to below the detection limit（0 CFU/m L）within4 h and the average reduction was 7.80×10⁵CFU/m L.Subsequently,phages 1-23 and 3-29 were prepared into a phage mixture to examine the feasibility and control effect on the surface of chicken meat and eggs contaminated with S.ZC-S1 at 4°C.Compared with the initial value of the experiment,the phage mixture with MOI=100 could reduce the viable bacteria count of S.ZC-S1 from 5.53×10⁵CFU/m L to under 20 CFU/m L in all 4 sample groups within 7 hours(the reduction is 4.47-5.67 log₁₀CFU/m L).Compared with MOI=1,the phage mixture with MOI=100 more quickly reduced the viable bacteria count of S.ZC-S1 to fall below the detection limit（0 CFU/m L）on chicken breast slices,ground chicken meat,and egg groups within 1-2 hours.The phage mixture with MOI=1 could reduce the viable bacteria count of S.ZC-S1 from 1.19×10⁶CFU/m L to231,1.10×10⁴,and 178 CFU/m L in chicken breast slices,chicken skin,and eggs groups within 7 hours(the reduction is 2.00-4.07 log₁₀CFU/m L).In the ground chicken meat group,the phage mixture with MOI=1 failed to inhibit the growth of S.ZC-S1.The results show that the high-concentration phage mixture with an MOI=100 displayed rapid and powerful biocontrol efficacy in vitro antibacterial applications.Combined with the results of genomics analysis,this study proves that phages 1-23 and 3-29 are two safe,stable,and lytic phages.They can be used as green biocontrol agents for the prevention of foodborne Salmonella spp.At the same time,it is inferred that the other 4 Epseptimavirus phages isolated in this study could also be used as phage biocontrol agents.In the future,this research needs to be combined with other detection measures for multifaceted verification to ensure the effectiveness and safety of biocontrol agents.This study provides a technical basis for research directions such as phages to ensure the food safety of chicken meat and eggs and to develop green and pollution-free biocontrol agents.