| Klebsiella pneumoniae(KP)is a kind of Gram-negative bacteria,which can cause necrotizing pneumonia,liver abscess,respiratory tract,urinary tract,wound and blood infection.Klebsiella pneumoniae is a conditional pathogen.It is a normal flora in the intestinal and urinary system.When the body has low immunity and dysbacteriosis,it will cause various kinds of inflammation.At present,with the extensive use of antibiotics,the isolation rate of multidrug-resistant KP is increasing year by year.Bacteriophage is a virus that infects and kills bacteria.As an antibacterial substance,bacteriophage can specifically recognize and lyse host bacteria.This study included the isolation and purification of Klebsiella pneumoniae,the isolation and identification of phage and the analysis of phage genome.Results six strains of Klebsiella pneumoniae were isolated and purified.The results showed that the six KP strains had the ability to form biofilms,and carried virulence genes and drug resistance genes such as fim、wab G genes in their genomes.The six KP strains were resistant to a variety of antibiotics,and they were all multidrug-resistant Klebsiella pneumoniae.Two lytic phages named ABTNL-1 and ABTNL-2 were isolated from untreated Hospital Wastewater by sewage enrichment method.The results of host spectrum showed that two or more strains of bacteria could be lysed by phages ABTNL-1 and ABTNL-2;Transmission electron microscopy showed that the two phages belonged to Siphoviriade;The optimal multiplicity of infection of phages ABTNL-1 and ABTNL-2 were 0.1 and 1;The one-step growth of ABTNL-1 and ABTNL-2 was 30 min and 40 min respectively,and the amount of cleavage was 200 PFU/cell and 150 PFU/cell,respectively;The two phages were not sensitive to chloroform;When the temperature was between 0 ℃ and 40 ℃,the two phages were in a relatively stable state and could maintain a high titer.When the temperature reached 50 ℃,the phage ABTNL-1 lost its activity and the phage ABTNL-2 decreased its activity.When the temperature reached 60 ℃,the two phages lost their activity;It is stable at neutral pH and loses its activity under the condition of over acid or over alkali;Single phage could significantly inhibit the growth of host bacteria under different infection complex numbers,but the number of bacteria increased after 5 h,suggesting that phage resistant strains might be produced;The phage cocktail was more effective in inhibiting the growth of host cells,and the phage resistant strains appeared for 10 h.The whole genome of phage was obtained by proteinase K/SDS method.The high-throughput second generation sequencing was carried out to obtain the genome sequence and analyze its gene.ABTNL-1 and ABTNL-2 were double stranded linear DNA: the total genome length of ABTNL-1 and ABTNL-2 were 112841 bp and 49159 bp,respectively,with GC content of 45.54% and 51.03% and t RNA number of 25 and 0,respectively.The results of genome function annotation showed that the phages ABTNL-1 and ABTNL-2 contained 144 and 77 ORFs respectively,and the number of ORFs with known functions was 55 and 36 respectively.After comparison,the genomes of the two phages did not contain lysogen gene,antibiotic resistance gene and virulence gene,which proved the safety of the two phages at the gene level.The phylogenetic tree was established by the large subunit of terminal enzyme and major capsid protein genes.The results showed that ABTNL-1 was closely related to Demerecviridae and Sugarcandvirus;The relationship between ABTNL-2 and Drexlerviridae,Webervirus is relatively close.The purpose of this study is to screen lytic phages to kill multidrug-resistant Klebsiella pneumoniae,study their characteristics,and analyze their genomes,so as to provide a basis for the application of phages in Klebsiella pneumoniae infection. |
