| Klebsiella pneumoniae is a common zoonotic Gram-negative pathogen,which can survive in a variety of ecological environments and mainly cause respiratory diseases in humans,as well as urinary tract infections,bacteremia,meningitis and nosocomial infections.It causes bovine pneumonia,bovine mastitis,mink pneumonia,swine pneumonia in animals.At the same time,the massive abuse of antibiotics has accelerated the production of multi-drug-resistant Klebsiella pneumoniae,known as the superbug NDM-1,and even led to the infection of the bacteria to the point where no drugs are available,which makes clinical treatment difficult.Bacteriophage is a virus that can specifically infect and decompose bacteria.It is a "precise" and "proliferative" living antibacterial substance with a unique antibacterial mechanism.It is a natural "ecological drug" against drug-resistant bacteria.Infections,especially drug-resistant bacterial infections,have broad application prospects and potential.However,in the process of phage therapy,with the game between bacteria and phage,the host bacteria develop resistance to phage,which seriously affects the efficacy.Elucidating the reasons and mechanisms of bacterial resistance to phages is a scientific and practical problem facing phage therapy.In this study,a new phage was isolated from sewage used the host bacteria Klebsiella pneumoniae K7 R and named v B?Kpn S?ZH01(P-K7 R for short)according to the standard nomology,and then a series of studies on P-K7 R were carried out.First,the general biological characteristics of P-K7 R were studied,P-K7 R was determined to be a long-tailed phage by scanning electron microscopy,and the optimal MOI of P-K7 R was determined to be 0.01 by double-layer plate method.One-step growth curve results showed that the incubation period of P-K7 R was 10 min,the burst volume of the cell was about 169 pfu/cell.The phage also showed good stability and maintained good activity between 4 and 50? in temperature stability tests and it kept good activity between p H 3 and 10 in the p H stability test.The lysis profile of this phage showed a relatively narrow lysis profile.In the study,the nucleic acid type and phage group of P-K7 R were sequenced and analyzed,and it was found that the nucleic acid type of P-K7 R was double-stranded DNA.Through sequencing,it was found that the full genome length of P-K7 R was 52111 bp,and the G+C content was 49.13%.The highest homology between the genome sequence and the sequences of other phages in the database is 78.33%.86 open reading frames are predicted,and the functions of the open reading frames are annotated.It is found that the phage does not contain lysogen and virulence-related genes.Through genetic analysis,it was found that enterobacter phage was closely related to P-K7 R.Subsequently,a phage-resistant Klebsiella pneumoniae was induced and named K7R-5P in this study,and a series of identifications were carried out for it.Through general biological characteristics analysis,it was found that there was no difference between it and K7 R in morphology,colony morphology and growth curve under the microscope no difference between K7 R and K7 R.And it was found that this strain was not different from K7 R at the whole genome level.Next,the transcriptomic level of K7 R and K7R-5P was compared in this study,and significant differences were detected in the expression levels of 5 genes between K7 R and K7R-5P.They are omp C,omp F,N5,N102 and N103,respectively.Since the differential genes have adsorption receptors related to bacteria and phage adsorption,the reason for k7R-5P's resistance to P-K7 R may be related to adsorption.Therefore,the adsorption rate of the two strains to phage was further measured,and the test results showed that the adsorption efficiency of K7 R can reach more than 80%,while the adsorption efficiency of K7R-5P is less than 20%.There was a significant adsorption difference between the two strains.Finally,in order to further verify that changes in transcription level would directly cause bacterial resistance to phages,this study verified transcriptome results by q RTPCR,and then constructed overexpressed strains by using overexpressed plasmid PUC18 K to verify whether the recovery of transcription level would restore the sensitivity of resistant bacteria.The results showed that the resistance of K7R-5P to phage P-K7 R disappeared after the omp C gene expression of K7R-5P was restored.In addition,the results of plaque test,plaque test and adsorption efficiency test showed that K7R-5P would also recover to the same level as K7 R.It shows that omp C can cause the resistance of bacteria to phage only by changing the expression level,which proves that omp C plays a key role in the adsorption of Klebsiella pneumoniae and phage.In conclusion,a new Klebsiella pneumoniae phage P-K7 R strain was isolated in this study,which enriched the phage library and induced a phage-resistant Klebsiella pneumoniae K7R-5P in vitro.The mechanism of resistance to this bacteriophage was identified,which laid the foundation for the further application of bacteriophage therapy. |
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