Characterize The Molecular Biology Of Carbapenem-Resistant Klebsiella Pneumoniae Isolated From Clinical And Study On Kinetics Of Enzyme-Inhibited Reactions

ObjectivesTo study the drug resistance,molecular typing,dissemination characteristics and clinical distribution of carbapenem-resistant Klebsiella pneumoniae?CRKP?pathogenic bacteria,so as to clarify the molecular biological characteristics of CRKP pathogenic bacteria and their clinical significance.To observe the captopril in vivo and in vitro to reverse the drug resistance of metallo-?-lactamases?MBLs?-producing CRKP bacteria and to sensitize the antimicrobial agents.In-depth study of the inhibitory kinetics of captopril on MBLs IMP-4 to characterize the affinity of captopril for IMP-4,and to open up a new way to explore the drug resistance treatment of MBLs-producing bacteria.Materials and MethodsIsolates:327 CRKP strains were continuously isolated from our hospital from 2013 to 2016?exclude the repeated duplicates and missing data?,and 98 strains of CRKP were collected for this experiment.The control strains were Escherichia coli ATCC 25922,Klebsiella pneumoniae ATCC 700603,ATCC 1705 and ATCC 1706.Methods:?1?Re-identification and drug susceptibility test were monitored by Vitek 2 automatic microbial identification instrument,the sensitivity of the strain to carbapenems was detected by K-B disc diffusion method,and the minimum inhibitory concentration?MIC?of the antimicrobial agents was detected by agar dilution method.The phenotype of carbapenemase was determined by modified Hodge test and carbapenemase inactivation method?mCIM combined with eCIM?;The main drug resistance genes were detected by PCR amplification and sequencing;Multi-site sequence type?MLST?and pulsed-field gel electrophoresis?PFGE?were used for molecular typing and homology analysis;Combined with the clinical distribution characteristics of the strain,the mode of transmission was analyzed.?2?To investigate the clinical data of 87 strains,30 days of death and ST11 CRKP as observation points,establish a logistic regression model to analyze the impact of dominant ST11 CRKP on the prognosis.In addition,the drug resistance genes,molecular typing and clinical distribution characteristics of 63 cases of CRKP pathogens associated with nosocomial pneumonia as specific group were analyzed.Captopril acts on in vitro and in vivo experiments on MBLs-producing resistant bacteria:Screening 5 clinically isolated MBLs-producing strains of CRKP,and conducting combined experiments with captopril and meropenem in vitro to analyze whether captopril can inhibit the resistance of drug-resistant bacteria to antimicrobial agents.The MBL NDM-1 of KP0106 and the MBL IMP-4 of KP27630 were used as the research objects?the two strains had the same MIC for meropenem?,and the infection model of the G.mellonella was established respectively.The captopril combined meropenem or single-drug administration method was used to analyze the survival curve of the larvae and the difference of the bacterial load in the body,and to evaluate whether the captopril in vivo can effectively restore the sensitivity of the drug-resistant bacteria to the antibiotics.The IMP-4 protein was obtained by constructing the expression vector pET28b-IMP4-BL21?DE?,induced by IPTG,purified by affinity chromatography,identified by SDS-PAGE and Western Blot.The half maximal inhibitory concentration IC₅₀0 was determined by the concentration of the inhibitor and the residual activity of the enzyme.The concentration of captopril and substrate was changed,the concentration of the enzyme was kept unchanged,and the data were determined.According to Lineweaver-Burk,the double reciprocal graph was used to determine the enzyme inhibition kinetic parameters,and the inhibition constant Ki was solved to characterize the captopril to MBL IMP-4 affinity.ResultsThe drug susceptibility results showed that 98 strains of CRKP were all resistant to carbapenem antibiotics?two strains were moderately resistant to imipenem?,MICs ranged from 2?g/mL to 64?g/mL,and the remaining 11 antimicrobial agents,except for all sensitive to tigecycline?two mediators?,the isolates are highly resistant to most other antimicrobial agents.Phenotypic analysis showed that 74 strains were positive for carbapenase,among which 10 were MBLs-positive klebsiella pneumoniae,6 were carrying bla_NDM-1and 4 were carrying bla_IMP.The remaining 60 strains all carried bla_KPC-2.Moreover,the detection rate of KPC-2 enzyme in the 3 groups showed an increasing trend year by year?p<0.001?.Among the 98 pathogenic strains of CRKP,a total of 24ST types were found,among which 55 strains were ST11?56.12%,55/98?.The detection rate of serial ST11 in the 3 groups was consistent with that of bla_KPC-2,which also showed an increasing trend year by year,showing significant differences.The 95strains of CRKP bacteria?3 strains were removed for nucleic acid degradation?were divided into 59 different types?PFGE01-59?,the 46 strains had unique PFGE bands,indicating the polymorphism of the bacterial gene.The MLST classification showed that 29 of the three major dominant-type clones were ST11?93.55%,29/31?.The 55strains of ST11 were divided into 23 different cloned bands,suggesting that there are multiple ST-type strains for out-of-hospital input.At the same time,multiple groups of ST11 strains have the same clonal banding pattern,suggesting the presence of intra-hospital spread.?2?Through the investigation of the molecular biology of CRKP pathogens,it was found that clinically reasonable empirical medication and total hospitalization time were independently correlated with the prognosis of CRKP infection?30-day death?,and the ST11 strain was analyzed as an independent risk factor of prognosis for CRKP infection.There were 63 strains of CRKP pathogens associated with nosocomial pneumonia,and 53 strains carrying KPC-2 enzymes were divided into6 ST types,of which ST11 had 47 strains?88.68%?and ST23 accounted for 2 strains?3.77%?.ST15,ST528 and ST661 each accounted for 1 strain,suggesting that there are fewer heterogeneous cloned backgrounds.The proportion of dominant clone ST11 in each ward was 72%in ICU,80.8%in medical ward and 100%in emergency department,indicating the prevalence and severity of in-hospital clonal transmission of nosocomial pneumonia.MBLs-producing Klebsiella pneumoniae,under the action of captopril,can significantly reduce the MIC value of carbapenems,which can be reduced to 1?g/mL?equivalent to1/16¹/32 times of the original MIC value?,and captopril inhibited NDM-1 and IMP-4MBLs in a dose-dependent manner in vitro.The NDM-1-producing KP0106 and IMP-4-producing KP27630 were concentration dependent on the lethality of the G.mellonella.When the IMP group infected the larvae of G.mellonella,the survival rates were significantly higher in the combined meropenem group compared with the other monotherapy groups?p<0.0001 or p=0.0121?,whereas no survival was observed in the NDM-1 group.Within 24 hours of single administration,the bacterial load in the captopril combination group was significantly lower than that in the monotherapy group,and the IMP-4 group was more prominent than the NDM-1 group,indicating that captopril inhibited the MBL IMP-4 more than NDM-1;Single administration after 48h,there was no difference in the inter-group bacterial load of the two strains infected with the G.mellonella,indicating that the effect of the antimicrobial agents was time-sensitive.The expression vector pET28b-IMP4-BL21?DE?was successfully constructed and used as the optimal induction time of pET28b-IMP4-BL21?DE?at 16°C for 8 h.The expression and purification were confirmed to be IMP-4 protein.The concentration of captopril inhibitor increased continuously,and the residual activity of IMP-4 decreased continuously,showing a curve correlation.Based on the LOGIT fit test,the IC₅₀0 was calculated to be 26.34?M,and the 95%confidence was 19.82³7.98?M.The double reciprocal solution in the Michaelis-Menten equation is derived.Under the action of competitive inhibitors,regardless of how the inhibitor concentration[I]changes,the longitudinal intercept 1/Vmax is theoretically unchanged.According to the change of the cross section in different[I],the Ki value was calculated to be 37.14?M.ConclusionThe resistance mechanism of CRKP pathogens is mainly KPC-2 carbapenemase,which is increasing year by year.The genotyping of its strains has genetic polymorphism,but the sequence type ST11 is still its prevalent dominance and is characterized by a significantly upward trend in clonal propagation.Clinical isolation of ST11-type CRKP pathogens not only serves as an independent risk factor for the prognosis of CRKP infection.,but also reveals that non-heterogeneous clonal transmission is the key to dissemination of nosocomial pneumonia.Captopril can effectively reverse the drug resistance of the MBLs-producing CRKP bacteria in vitro and in vivo,restore the sensitivity of the strain to antimicrobial drugs,and develop a new direction for the research of drug resistance of MBLs-produing resistant organism.Enzyme inhibition kinetics quantitatively and accurately evaluated the affinity of captopril for MBL IMP-4 which comfirmed that captopril has a good inhibitory effect on MBL IMP-4 activity,laying the foundation for clinical treatment.