| Objective:Construction of CR-hvKP strains induced by imipenem at gradient concentrations,and determination of virulence phenotype and pathogenicity changes before and after induction;Detection of differences in sRNA expression profiles before and after CR-hvKP induced drug resistance;To explore the mechanism of sRNA-051 in drug resistance and virulence in CR-hvKP bacterial infection mouse.It provides a new way for clinically controlling the production and epidemic of CR-hvKP strains.Methods:Construction of CR-hvKP strain with gradient concentration of imipenem.Detection of strain homology before and after induction by pulsed field gel electrophoresis(PFGE)and multi-site sequence typing(MLST).The MIC value of the strain before and after induction was detected by the micro broth dilution method to confirm that the CR-hvKP strain was successfully constructed.Detection of differences in sRNAs expression profiles between wild-type hvKP strains and CR-hvKP strains induced by imipenem induction by sRNA high-throughput sequencing.Screening of target sRNAs that affect drug resistance,virulence and pathogenicity of CR-hvKP strains by genetic recombination.Construction of target sRNAs gene-deficient strains using gene knockout technology.Detection of imipenem MIC value of all sRNAs gene-deficient strains by micro broth dilution method.High-viscosity phenotype,serum resistance,anti-neutrophil phagocytosis,mouse LD50 and other tests to detect the virulence of target sRNA-deficient strains.Construction of CR-hvKP?Hfq deletion strain by gene knockout technology,q RT-PCR to detect the relative expression of sRNA-051 before and after?Hfq deletion,to explore its dependence on Hfq chaperone protein.Animal experiments verify whether sRNA-051 gene knockout can reduce the virulence and pathogenicity of CR-hvKP strain.About 30 SPF BALB/c mice were selected and randomly divided into negative control group,CR-hvKP strain group,CR-hvKP strain group with sRNA-051 gene deletion(?sRNA-051 deletion strain group),10 mice in each group.An animal model of infection was established by injecting the bacterial solution into the tail vein.The infection amount was 3×10~9cfu/ml.Observe the incidence of mice every 2 hours and record changes in body weight of the mice.Mice were sacrificed by eyeball blood extraction after infection.Detection of serum C-reactive protein(CRP),procalcitonin(PCT),interleukin-1(IL-1),IL-6 by enzyme-linked immunosorbent assay.Hematoxylin-eosin staining(HE)staining was used to observe the pathological and morphological changes of lung tissue.Results:After the HvKP strain was induced into CR-hvKP by imipenem,its MIC value changed from(MIC<0.5?g/ml)to(MIC=16?g/ml);The hypermucoviscosity phenotype test,MLST and PFGE sequencing confirmed that the capsular genotyping was ST23 type K1 capsular serotype Klebsiella pneumoniae,and simultaneously expressed the virulence genes rmp A,iro N,aerobactin,mrk D.It is a strain of Klebsiella pneumoniae with hypermucoviscosity phenotype and high toxicity;Through serum resistance,anti-neutrophil phagocytosis ability and LD50 test in mice,we further found that HvKP still maintains its high pathogenicity characteristics after induction of CR-hvKP strain formation.High-throughput sRNA sequencing revealed that 184 sRNAs were up-regulated and 75 sRNAs were down-regulated.RT-q PCR verification revealed that 9 of these sRNAs were up-regulated in CR-hvKP strains produced after imipenem induction.By knocking out 9 sRNAs by genetic recombination technology,it was found that sRNA-051 can significantly reduce the MIC value of CR-hvKP to imipenem(from 16?g/ml to 4?g/ml),and can also significantly reduce the Virulence and pathogenicity.In this study,25 strains of CR-hvKP and 40 strains of hvKP were collected.CR-hvKP?Hfq deletion strains were constructed by gene knock-out technology.The results showed that the expression level of sRNA-051 in the CR-hvKP?Hfq deletion strain was significantly reduced,about 30%of the wild strain,indicating that the sRNA-051 in the CR-hvKP strain was Hfq chaperone-dependent.The expression level of sRNA-051 in CR-hvKP?Hfq deletion strains was significantly reduced,about30%of that in wild strains,indicating that sRNA-051 in CR-hvKP strains was Hfq chaperone-dependent.Animal experiments showed that the mice in the control group were active and had normal diet.The mice in the CR-hvKP strain group started to develop disease after 4 hours,swollen their faces,and died in 20 hours.?sRNA-051-deleted group of mice began to develop symptoms after 20 hours,and the symptoms of mice became worse after 30 hours.The body weight of mice in the control group at 20h and 40h was(31.57±1.03)g and(32.23±1.22)g,which were higher than those before infection(29.81±1.12)g(p<0.05).The body weights of mice in the CR-hvKP strain group at 20h and?sRNA-051 deletion strain group at 20h and 40h were(25.56±0.93)g and(28.22±0.97)g,and(25.98±0.79)g were lower than the corresponding period of the control group(p<0.001)(p<0.001).The body weight of mice in the CR-hvKP strain group at 20 h was lower than that of the pre-infection and?sRNA-051 deletion strain group at 20 h(p<0.001).With time,the body weight of the?sRNA-051-deleted group decreased gradually(p<0.001).The CRP,PCT,IL-1,and IL-6 in the CR-hvKP strain group after infection were(56.78±1.48)ug/ml?(28.71±7.93)ng/ml?(178.62±47.93)pg/ml?(30.25±6.43)pg/ml and?sRNA-051 deletion strains were(21.41±5.28)ug/ml?(8.20±2.37)ng/ml?(115.18±30.19)pg/ml?(14.61±4.08)pg/ml are both higher than the control group(p<0.001).The CRP,PCT,IL-1,and IL-6 in the CR-hvKP strain group were higher than those in the?sRNA-051 deletion group(p<0.001).In the CR-hvKP strain group,inflammatory exudate appeared in the lung tissue of the mice,a large number of inflammatory cells were infiltrated in the alveolar cavity,and the alveolar structure was blurred.A small amount of inflammatory cells infiltrated in the alveoli of the?sRNA-051 deleted strain group,and the alveolar structure was slightly blurred.Conclusion:HvKP bacteria can still maintain its high virulence characteristics after imipenem induced resistance.sRNA-051 gene knockout can significantly reduce the MIC value of CR-hvKP to imipenem(from 16?g/ml to 4?g/ml),and can also significantly reduce the virulence and pathogenicity of CR-hvKP.Inhibiting the expression of sRNA-051 of CR-hvKP strains can reduce the resistance,virulence and pathogenicity of CR-hvKP bacteria.The expression level of sRNA-051 in the CR-hvKP?Hfq deletion strain was significantly reduced,about 30%of that in the wild strain,indicating that the sRNA-051 in the CR-hvKP strain was Hfq chaperone-dependent.Animal experiments show that tail vein injection of CR-hvKP strain and?sRNA-051-deleted CR-hvKP strain can cause mice to lose weight,increase expression of inflammatory factors such as CRP,PCT,IL-1,IL-6,and lung inflammatory cell infiltration.However,the CR-hvKP strain(?sRNA-051 deletion strain group)with gene knockout sRNA-051 caused this series of reactions to a lesser degree than the non-knockout CR-hvKP strain.It indicated that inhibiting the expression of sRNA-051 of CR-hvKP strain can reduce the drug resistance,virulence and pathogenicity of CR-hvKP bacteria.It means that knocking out the gene expression of CR-hvKP strain sRNA-051 can reduce the drug resistance,virulence and pathogenicity of CR-hvKP bacteria,Its mechanism of action may be by inhibiting the interaction of sRNA-051 with Hfq protein,affecting its formation of RNA-protein complexes,thereby changing the stability of m RNA and ultimately affecting protein translation;This mechanism can provide a new way for clinically effective control of the production and epidemic of CR-hvKP strains and point out new directions for effective treatment. |
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