Screening Of Potential Genes Affecting Phage Replication In Enterobacter Cloacae

Species of the Enterobacter cloacae are widely spread in nature,but they can be virolent.Although the Enterobacter cloacae strains are among the most common Enterobacter spp.causing nosocomial bloodstream infections in the last decade,little is known about their virulence-associated properties.In contrast,much has been published on the antibiotic-resistance features of these microorganisms.In fact,they are capable of overproducing AmpC b-lactamases by derepression of a chromosomal gene or by the acquisition of a transferable ampC gene on plasmids conferring the antibiotic resistance.Many other resistance determinants that are able to render ineffective almost all antibiotic families have been recently acquired.Phage,viruses of bacteria,were considered as “bacteria eaters” with potential for use in combating antimicrobial resistance.In order to enrich the theoretical basis of the interaction between Enterobacter cloacae and phage,our aim is to screen the candidate genes affecting the replication of Enterobacter cloacae phage.We hope this reserch can promote the further study of the interaction mechanism between Enterobacter cloacae and phage,thus promoting the steps of phage therapy.In this study,we isolated and identified a strain of Enterobacter cloacae E20,via gram stain microscopy,biochemical reaction identification and PCR identification.Genotyping shows that E20 belonged to ST122.As a multi-drug resistant strain,E20 showed resistance to ?-lactams(amoxicillin,cefotaxime,ampicillin,cefoxitin),aminoglycosides(gentamicin,kanamycin,streptomycin),tetracyclines(tetracycline and doxycycline)and was also resistant to sulfa drugs,sulfamethoxazole,and the amide alcohol,chloramphenicol.In order to isolate Enterobacter cloacae lytic phage,this study used a double-layer agar plate separation method to isolate and purify a strain of Enterobacter cloacae phage named vB_Ecop_S523(S523)from a dairy farm environment sample.The phage has a head of 58-59 nm in diameter and a tail of 10 nm in length,which forms round,transparent and well-defined plaques in the double-layered agar plate.The genome of S523 consisted of double-stranded DNA(39,825 bp,48.8% G+C)and was morphologically classified under Podoviridae(T7-like phage).To explore the biological characteristics of S523,the physicochemical and biological characteristics of it were analyzed.The host we found of S523 include E19,E20(Enterobacter cloacae)and MC1061,DH5?,BL21(K12,Escherichia coli).As a potential bacteriostatic agent,S523 has good thermal stability and has a wide tolerance range for pH.Furthermore,S523 was resistant to chloroform,but sensitive to ultraviolet light.When the optimal multiplicity of infection(MOI)of S523 was 0.1,the one-step growth curve shows that the incubation period of S523 was 5 min,and the amount of lysis was about 90 PFU per cell.In order to screen related candidate genes,a random insertion mutant library was constructed by using the Enterobacter cloacae as a parent strain,and a total of 2473 mutant strains were constructed by using the pUTmini-Tn5 transposon plasmid.Two rounds of screening were carried out based on the change of bacterial turbidity.A total of 13 mutant strains showing phage resistance were screened,and the inactivated genes were identified by Walking PCR.At the same time,the phage sensitivity of the five mutant strains was verified by double-layer agar plate method.The results showed that the inactivation of Succinate dehydrogenase flavoprotein subunit(SDHA)significantly reduced the phage sensitivity to Enterobacter cloacae.To further verify our finding,detailed investigation and in-depth research are needed.