Biological Characteristics Of Phages Against Enterobacter Hormaechei And Its Application In Food Decontamination

Enterobacter hormaechei is a zoonotic pathogen belonging to the family Enterobacteriaceae formerly known as enteric group 75.In recent years,many neonatal septicemia cases caused by E.hormaechei have been reported.The harmfulness of E.hormaechei has gradually attracted people's attention.In addition,the abuse of antibiotics led to the increasing number of drug-resistant pathogenic bacteria.E.hormaechei isolates also showed multi-drug resistance,including their resistance to ?lactams,aminoglycosides,and quinolones.Therefore,it is an urgent need to establish rapid detection and bio-controlling technologies to control Enterobacter hormaechei pollution and transmission in the food chain.In the present study,a strain of E.hormaechei isolated from the spoiled meat was used as the host bacterium to isolate lytic phages from sewage in Yangzhou,Jiangsu Province,and 6 E.hormaechei phages were obtained.The biological properties,genomic characteristics,and decontamination abilities of 6 phages against E.hormaechei were investigated,and their potential use as bio-control agents were evaluated.This work provided phage tools and related information for further E.hormaechei identification,bio-control,and ecological studies.Details of this study are presented as the following:(1)Identification,genomic and drug resistance analysis of E.hormaechei EhYZU05 isolated from vacuum-packed meat products were performed.This strain was identified as E.hormaechei(named Eh-YZU05)by 16S rRNA sequencing.The wholegenome sequencing results showed that the genome of Eh-YZU05 is 4,701,521 bp,with 4,189,419 bp coding gene length and 56.8%GC content.The genome encoded 4358 protein genes,including 955 cell process and signaling-related genes,713 information storage processing-related genes and 1890 metabolism-related genes.Among them,57 antibiotic resistance genes and 142 bacterial virulence factor genes were included.The results of drug-resistance analysis showed that Eh-YZU05 was resistant to 10 antibiotics,including penicillin G,amoxicillin,benzocillin,vancomycin,lincomycin,neomycin,furantoin,erythromycin and azithromycin.(2)Isolation and biological characterization of highly efficient lytic phage of E.hormaechei were performed.Six E.hormaechei virulent phages were isolated from sewage,named Ehp-YZU05,Ehp-YZU08,Ehp-YZU10,Ehp-YZU9-1,Ehp-YZU9-2 and Ehp-YZU9-3.Transmission electron microscopy results showed that Ehp-YZU05 and Ehp-YZU9-1 belong to the Myoviridae family,and Ehp-YZU08,Ehp-YZU10,Ehp-YZU9-2 and Ehp-YZU9-3 belong to the Podoviridae family.The burst sizes of the six phages are 70-283 after a latent period of 5-40 min.Phage Ehp-YZU08 and EhpYZU10 showed the shortest latent period and Ehp-YZU9-2 showed the most amount of burst size.The test of thermal and pH stability showed that 6 phages are able to remain active in the pH range of 5-10 and resist temperatures up to 60? for 60 minutes.(3)Genomic composition and comparative genomic analysis of efficiently lytic phages of E.hormaechei were performed.The full genome sequences of the six phages ranged from 39,502-173,418 bp in length.The proportion of coding genes to the total genes ranged from 86.9%-94.6%.The known functional gene annotations of the six phage strains can be divided into two major categories:phage structural protein genes(e.g.,head protein genes,tail protein genes,capsid protein genes,etc.)and genes related to phage nucleic acid metabolism and expression(e.g.,endolysin genes,holin genes,etc.).Clustering analysis of the genome sequences of the eight known E.hormaechei phage strains revealed low sequence similarity to each other,indicating that the E.hormaechei phage genes as a whole exhibit a high degree of genetic diversity.Three phage Ehp-YZU08,Ehp-YZU10 and Ehp-YZU9-1 were compared to the GenBank database respectively.The whole genome sequences of Ehp-YZU08,Ehp-YZU10 and Ehp-YZU9-1 were analyzed by comparative genomics with those of known phages with the highest homology in the GenBank database,and the results showed that these four phages had 118,13,13 and 257 gene fragments with?90%homology,and 82,32,32 and 36 gene fragments with no homology or<90%,respectively.No genes associated with bacterial virulence or drug resistance were found in the genomes of the 6 phages.In addition,the genomes of all 6 phage strains contained 23?110 genes associated with phage replication.(4)A gravy culture model was constructed to determine the maximum reduction rate(Rmax)and lysis kinetics of the six phages on the growth of E.hormaechei.The inhibition test showed that all six phages had strong lysis abilities against E.hormaechei.Phage Ehp-YZU08,Ehp-YZU10,Ehp-YZU9-1 and Ehp-YZU9-2 showed a corresponding increase in Rmax and a corresponding decrease in the time to reach maximum reduction rate(Tmax)with increasing phage treatment concentration,while phage Ehp-YZU05 and Ehp-YZU9-3 had the highest Rmax and Rfinal at the concentration of MOI=0.1.The Rmax of phage Ehp-YZU9-3 at MOI=0.1 was 5.48 Log and Rfinal was 4.50 Log,which showed the best inhibitory effect on E.hormaechei.In the gravy model,all six phages showed a small increase in potency within 2 h.The increase was accelerated after 2 h,and the maximum potency was reached after 4-6 h.The smaller the initial dose of phage,the more effective the phage could be to proliferate and produce the maximum number of zygotic phages.(5)The frequency of phage-insensitive mutations and the characteristics of resistant strains were performed.The resistance mutation frequency of E.hormaechei to Ehp-YZU10 was the lowest(2×l0-6),and the resistance mutation frequencies of the other three phages were around 10-5.The four phages determined in this study could cause a low frequency of phage resistance mutations in E.hormaechei,which is beneficial for the development and application of biocontrol agents.The heat resistance of the phage-resistant mutant strain of E.hormaechei and the sensitive strain were analyzed,and the results showed that the sensitive strain reached undetectable level in 35 min at 55?,while the resistant strain reached undetectable level in 20-30 min,indicating that the heat resistance of the strain decreased in different degrees after the resistance mutation.The resistant mutant strains formed small colonies during constant temperature incubation at 37?,and the growth rate and maximum population density decreased significantly,but the degree of decrease was different for different strains,indicating that the pasteurization conditions after phage treatment were more favorable to kill the formed resistant strains.