The Mechanism Of Fitness Costs Of Phage Resistance Mutation Triggered By Outer Membrane Porin Deficiency In Klebsiella Pneumoniae K7R~B

The fitness costs of bacteriophage resistance are regarded as a basis for judging the ability of phage-resistant mutant strains to spread and formulating“phage-antibiotic”combination therapy.As an important zoonotic pathogen,Klebsiella pneumoniae poses severe challenges to the human public health and the animal husbandry.Although K.pneumoniae has many cases of phage therapy and is also very prone to phage resistance mutations,its fitness costs of resisting against phage infection are rarely reported.Derived from the interation between K.pneumoniae K7 and phage GH-K3,K7R^Bis an outer membrane porin-deficient phage-resistant mutant strain triggered by omp C₇₁₂deletion,exhibits expression inhibition of Omp C,Omp N,KPN＿02430,and Omp F,but it still maintains“big and smooth”colony.Therefore,in this study,phage-resistant mutant strain K.pneumoniae K7R^Bwith Omp C deficiency was selected to reveal the fitness costs and regulatory mechanisms of K2 serotype K.pneumoniae resisting to the phage infection.The biological characteristics,drug resistance,and virulence of K7R^Bwere revealed firstly:There was no significant difference in growth characteristic and capsular type between K7R^Band K7.The results of animal experiment showed that compared with K7 group,mice infected with K7R^Bexhibited a less rapidly disease progression,but still all died within 10 days.The fitness cost in K7R^Bis limited.However,the competition in vitro and resistance to some cephalosporins of K7R^Bwere increased.To explore the inner regulating mechanisms of K7R^B,transcriptomic and metabonomic analyses were subsequently performed.The transcriptomic analysis showed that the transcriptional level of metabolism-related genes had changed significantly,which major involving the transcriptional suppression of related genes in glyceride metabolism and nitrogen metabolism,and elevated transcriptional level of related genes in permeases,TCA cycle and phenylalanine metabolism.However,virulence factor and drug resistance factor had not been changed significantly.The metabolomic analysis showed that differential metabolites were mainly enriched in TCA cycle and energy metabolism,amino acid metabolism,phospholipid metabolism,and nucleotide and cofactor metabolism.The inner gene regulating and metabolic adjustments of K7R^Bwere thus clarified,which laid a foundation for further exploring the generation of fitness costs in this strain.Then,the roles which four porins—Omp C,Omp N,KPN＿02430,and Omp F played in regulating fitness costs of K7R^Bwere identified.Artificially constructed K7(Δomp C₇₁₂)confirmed that the suppression of Omp N,KPN＿02430,and Omp F was related to the suppression of Omp C.Omp A may indirectly regulate the interaction of Omp C,Omp N,KPN＿02430,and Omp F according to STRING analysis.K7（Δomp A）and K7（Δomp A）C-omp A^K7were constructed separately to verify Omp A has a negative regulatory relationship with the four outer membrane proteins.The overexpression of Omp N,KPN＿02430,and Omp F restored the sensitivity of K7R^Bto antibiotics to varying degrees.However,the overexpression of Omp C increased the resistance to antibiotics（especiallyβ-lactams）.In addition,the virulence of the bacteria is mainly related to the type of outer membrane protein,rather than capsular production.Omp C and Omp F were shown to play an important role in maintaining strain virulence,while overexpression of Omp N and KPN＿02430 reduced bacterial virulence.In conclusion,this study elucidates the fitness cost and its regulatory mechanism of K2serotype K.pneumoniae with phage resistance—K.pneumoniae K7R^B（with outer membrane protein deficiency）.This study enriches the theoretical study of the fitness cost of bacterial resistance to phage infection,and provides a scientific basis for improving the“phage-antibiotic”combination treatment for the prevention and control of bacterial infection.