Isolation And Identification Of Enterococcus Faecalis Phage ZXL-01 And Preliminary Investigation Of Its Therapeutic Effect On Periapical Inflammation

Enterococcus faecalis is widely present in nature and in the human and animal intestines and is a common causative agent of nosocomial infections,causing urinary tract infections,oral infections and other fatal forms of local and systemic infections.In oral infections,it is the main causative agent of refractory periapical infections.The increasing detection rate of multi-drug resistant Enterococcus faecalis and the difficulty of complete removal of the Enterococcus faecalis biofilm pose great difficulties for clinical treatment.Since phages have efficient bactericidal ability and host specificity,phage therapy is expected to be a new way to maintain oral health.Therefore,it is important to isolate the phage of Enterococcus faecalis and explore its phage therapy for periapical inflammation caused by Enterococcus faecalis.In this study,a lytic phage,named ZXL-01,was isolated from lake water with Enterococcus faecalis as the host bacterium,which belonged to the order of tailed phages and the family of long-tailed phages,with a diameter of about 48 nm and a tail of about 203 nm.The host specificity of phage ZXL-01 is strong.The optimal infection complex of phage ZXL-01 is 0.1.The incubation period is 20 min and the lysis period is Phage ZXL-01 can maintain high activity at 50°C and 60°C.Its activity decreases substantially at 70°C and finally remains low.It will be completely inactivated at 80°C for 10 min..Phage ZXL-01 is active in the environment of p H4～11.Its activity decreases slightly in the environment of p H 9,but its activity decreases dramatically in the environment of p H 5,but a large number of phages still survive.The whole genome sequencing of phage ZXL-01 has been completed and uploaded to Genbank with the sequence number ON113334.The full length of the genome of phage ZXL-01 is 40,804 bp with 35% GC content,and no virulence genes have been found in the genome of phage ZXL-01;the genome contains 63 ORFs,of which 39 ORFs are positive and 26 ORFs are negative;44 ORFs are unknown functions and 19 ORFs are known functions,which can be classified into three modules: cleavage module,DNA regulatory module and structural protein module.Evolutionary analysis showed that phage ZXL-01 belonged to Enterococcus faecalis phage and had the closest evolutionary relationship with Enterococcus faecalis phage LY0322.From morphological observation,it was found that phage ZXL-01 could both inhibit the formation of E.faecalis biofilm and have a lytic effect on the already formed E.faecalis biofilm.On this basis,an in vitro model of Enterococcus faecalis root canal infection was constructed using an isolated tooth as a carrier to investigate the phage treatment of periapical inflammation caused by Enterococcus faecalis.After scanning electron microscope observation,it was found that ZXL-01 could destroy the mature biofilm formed in the root canal of the tooth and effectively kill the planktonic Enterococcus faecalis and the Enterococcus faecalis in the biofilm.In conclusion,a strain of Enterococcus faecalis phage ZXL-01 with high lytic activity,high temperature and acid-base tolerance was obtained in this study.Phage ZXL-01 has great potential in the treatment of periapical inflammation caused by Enterococcus faecalis.