| With the rapid increase of antimicrobial resistance(AMR)in recent years,bacterial disease prevention and control has become increasingly difficult.Bacteriophages have natural bactericidal ability,can specifically infect and break down bacteria.Phage can be used as a new type of antibiotic supplement,is being reattention.However,in the process of phage therapy,bacteria may develop resistance to the phage,which affects the therapeutic effect.Considering that addressing the issue of bacteriophage resistance in antcbiotic-resistant pathogens can improve treatment outcomes.This study aims to isolate,identify and characterize bacteriophages and comprehensively screen and analyze genetic determinants related to bacteriophage resistance and sensitivity using transposon sequencing(Tn5-Seq)and phenotype verification methods.This will help in selecting appropriate bacteriophage preparations and small molecule inhibitor combinations to treat infections caused by specific pathogens in clinical settings.In this study,a high lytic bacteriophage AH67C600_Q5 was isolated from pig farm wastewater using the model host Escherichia coli C600.The biological characteristics of this bacteriophage were investigated.It was confirmed as a member of Myoviridae by transmission electron microscope.Its optimal infection multiplicity was 0.1,and the one-step growth curve revealed a latency period of approximately 30 minutes and an eruption period of about 50 min.This bacteriophage also exhibits good stability.In the temperature stability test,it maintains good activity between 4-50℃.In the pH stability test,it can tolerate weak alkaline environment between pH6-8.Subsequent sequencing and analysis of the bacteriophage genome revealed that the nucleic acid type of the bacteriophage was double-stranded DNA.The full-length genome of the bacteriophage was discovered by sequencing to be 38,824bp,with a GC content of 48.38%.It encodes 50 CDS regions,and no lysogenic or virulence-related genes were found in the bacteriophage.An evolutionary tree was constructed based on the large subunit of the terminal enzyme,and it was found that this bacteriophage has a close relationship with Escherichia coli bacteriophage CICC 80001 and belongs to the T7-like bacteriophages.Research has shown that T4-like and T7-like bacteriophages are the two most prevalent double-stranded DNA lytic bacteriophage in the environment.To comprehensively screen and analyze genetic determinants related to bacteriophage tolerance,we used the T7-like bacteriophage AH67C600_Q5 characterized in this study,as well as the previously characterized T4-like bacteriophage AH67C600_Q9,to construct a Tn5 transposon mutant library in Escherichia coli C600.The results showed that six genes(qseE,rstB,wbbL,mltC,uxaA and entS)were commonly involved in susceptibility under the pressure of both bacteriophages.Among them,two genes qseE and rstB encoding the histidine kinase of two-component sensor systems,i.e.QseE/QseF system,showed a significant increase in the number of insertions.Meanwhile,nine genes(ydhJ,ompW,glnP,mutM,yeaO,menC,pepB,yraQ and sulA)were commonly involved in bacteriophage tolerance-related genes,among which the gene ydhJ encoding the secretion protein and the gene ompW encoding the outer membrane protein showed a significant decrease in the number of insertions.Go enrichment analysis of these susceptible and resistant genes showed that they were inclined to be enriched in cellular and metabolic process in biological processes.To validate the accuracy of the Tn-Seq results,we selected the susceptible gene qseE for phenotype verification.This gene encodes the histidine kinase of the two-component sensor system QseE/QseF,and we knocked it out and constructed a deletion strain.Phenotype verification was conducted using the phage spot assay.The results showed that compared with the wild type strain,the lytic ability of the bacteriophage to the mutant strain was decreased.In summary,this study isolated a highly lytic T7-like bacteriophage and characterized its biological properties.Genome analysis showed its potential as a potent therapeutic agent for clinical use.Considering that the genetic determinants involved in the resistance and susceptibility of bacterial hosts to phage infections can improve treatment outcomes,this study selected the lytic T4-like phage AH67C600_Q9 and the T7-like phage AH67C600_Q5 to infect a mutant library of Escherichia coli C600.Using Tn-seq,genes associated with phage susceptibility and resistance were identified,and the effectiveness of the method was validated by constructing knockout strains of the target genes and testing them in phage spot assay.The results indicated that Tn5 transposon mutant libraries can serve as effective screening methods for identifying genetic determinants involved phage resistance and susceptibility.Moreover,by understanding these genes and further deciphering their underlying functions,potential adjuvants that can assist phage therapy could be screened,greatly improving the efficacy of phage therapy. |
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