Mortality Risk Factors For Multi-drug Resistant Klebsiella Pneumoniae And Resistance Mechanism And Virulence Characteristics Of Carbapenem Resistant Klebsiella Pneumoniae

Enterobacteriaceae（e.g.,Klebsiella and Enterobacter）are common causes of severe and hospital-acquired infections.Enterobacteriaceae bacteria are often carry genes that induce three or more antimicrobial resistance,making them multi-drug resistant.The multi-drug resistant Enterobacteriaceae bacterium attract worldwide attention,especially the carbapenem-resistant Klebsiella pneumoniae.And antimicrobial resistant infections often undermine the effective treatment.Hence,understanding the mortality risk factors of Enterobacteriaceae and carbapenem resistant Klebsiella pneumoniae infections,the antimicrobial resistance and the primary sequence type of carbapenem resistant Klebsiella pneumoniae is critical.We attempted to determine the mortality risk factor for multiple drug resistant Enterobacteriaceae infection,analyze the region’s major epidemic clone and the resistance and virulence characteristics of carbapenem resistant Klebsiella pneumoniae,then provide a favorable drug regimen for clinic and appropriate interventions.一、The mortality risk factors of multi-drug resistant Klebsiella pneumoniae infection in the tertiary hospital of Shanxi from 2015 to 2019Objective:This study was initiated to investigate the mortality risk factors for multi-drug resistant Enterobacteriaceae infection in a tertiary hospital from 2015 to 2019.Methods:1.Strain and clinical data collection:All multi-drug resistant Enterobacteriaceae strains were reviewed and collected in the tertiary hospital of Shanxi from January 2015 to June2019.Electronic medical records of patients who underwent multi-drug resistant Enterobacteriaceae infection were reviewed.The mortality risk factors of multi-drug resistant Enterobacteriaceae and multi-drug Klebsiella pneumoniae infection were analyzed,including demographic,outcome of infection,treatment regimens.2.Statistic analysis:Usingχ²test or Fisher’s exact test to compared categorical variables.Variables,which p value<0.1,were analyzed in the Logistic model analysis,to identify the independent mortality risk factors of multi-drug resistant Enterobacteriaceae infection and multi-drug resistant klebsiella pneumoniae infection.We analyzed the variables with value<0.2 in the Cox regression analysis,to determine the independent predictors of 30-day mortality.Aiming to analyze the effect of usage of aminoglycosides on the 30-day survival,patients were divided into two groups according to with or without the usage of aminoglycosides.Results:1.Strains and Clinical information:A total of 91 cases and 91 multi-drug resistant Enterobacteriaceae strains were collected.2.Distribution of departments,specimen types and types of infection:28.57%of 91patients were in the intensive care unit.The sputum specimen was the main isolated specimen（64.84%）.The majority types of infection were pneumoniae infection（43.96%）.In 2018,multi-drug resistant Enterobacteriaceae had the highest prevalent rate,with 49 strains（53.85%）isolated.In 2018,the proportion of MDR-KP was 36.26 and the proportion of CRKP in MDR-KP was 98.3.3.Mortality risk factors of multi-drug resistant Enterobacteriaceae infection:In univariate analysis,it was revealed that the death group was more likely to have cardiovascular disease（P=0.039）,cerebrovascular disease（P=0.039）and invasive procedures.Among them,mechanical ventilation,central venous catheter,tracheal cannula,tracheotomy,urinary catheter had statistical difference（P<0.05）.In the multivariate Logistic regression analysis,cerebrovascular disease（OR,4.046;95%CI,1.434–11.418;P=0.008）and central venous catheter OR,4.543;95%CI,1.338–15.425;P=0.015)were severed as the independent factor associated with death.Cox regression analysis revealed that the usage of aminoglycosides was associated with 30-day survival of patients who infected with multi-drug resistant Enterobacteriaceae（HR,0.351;95%CI,0.118–1.044;P=0.06）.The survival time of the group without aminoglycoside was lower than that of the group with aminoglycoside antibiotics（P=0.06）.4.Mortality risk factors of MDR-KP infection:The mechanical ventilation,central venous catheter,tracheal cannula,tracheotomy,urinary catheter had statistically significant（P<0.05）.History of immunosuppressant use was associated with death group with borderline statistical significance（p=0.056）.Multivariate Logistic regression analysis identified tracheal cannula（OR,4.654;95%CI,1.5–14.438;P=0.008）as the independent factor associated with death.Cox regression analysis revealed that the usage of aminoglycosides was associated with 30-day survival with borderline statistical significance（HR,0.235;95%CI,0.053–1.044;P=0.057）.二、Resistance and virulence gene analysis of CRKP and hv KP strains isolated from aseptic body fluid in 2018Objective:The resistance mechanism and virulence characteristics of carbapenem-resistant Klebsiella pneumoniae（CRKP）and hypervirulent Klebsiella pneumoniae（hv KP）,which were isolated from aseptic body fluid,were discussed.Methods:1.Strain collection:The KP strains isolated from part 1 were selected for further analysis.2.Antimicrobial sensitive test in clinical common antibiotics:Antimicrobial sensitive test was evaluated by the agar dilution and broth micro-dilution methods.3.Phenotypic characterization of carbapenemases:The phenotypes of carbapenemases were explored with modified Carbapenem Inactivation Method（m CIM）.Virulence phenotype was detected by the string test,when the string length>5mm,the results was positive.4.PCR amplification of resistance,virulence and capsular serotype genes:The presence of genes encoding resistance mechanism（Class A、Class B、Class D、Amp C、ESBLs）,virulence（iro N、rmp A、rmp A2、iut A、iuc A）and capsular serotype（wzi）were detected by PCR.The positive PCR amplicons were visualized by agarose gel electrophoresis and sequenced.Using the NCBI Gen Bank database and Pasteurs database to BLAST the DNA sequences.5.Next generation sequencing:The genomes extracts were sequenced by Ion S5 plus（Thermo fisher）.De novo assembly was performed using SPAdes genome assembler.Antimicrobial resistance and virulence genes were screened and annotated using CARD and VFDB tools.Results:1.Strain collection:12 KP strains were collected.Among them,eleven strains were CRKP.The remaining one strain was hv KP.2.Antimicrobial sensitive tests:11 CRKP strains showed highly resistance rates to third-or fourth-generation cephalosporins and CSL.More than 90.9%of the CRKP strains had high MICs（≥32 mg/L）forβ-lactam combination agents.Twelve strains showed highly sensitive rates to tigecycline（TGC）and amikacin（AMK）.The hv KP strain showed lower resistance to antibiotics.3.Detection of resistance mechanism:A total of 58.3%（7/12）of CRKP strains were detected with carbapenemase genes.NDM（33.3%,4/12）was the main resistance mechanism,whereas 41.7%（5/12）of isolates were not found any resistance genes.The MICs of ATM and quinolones in NDM-1 CRKP and NDM-5 CRKP strains were different.4.PCR amplification of resistance,virulence and capsular serotype genes:Only 2strains were positive for virulence gene amplification.ST11 was the main type.The serotype of the majority strains was KL22KL37（58.3%,7/12）,hv KP stain belonged to K64.5.Next generation sequencing analysis:CRKP strains harbored plasmid-mediated quinolone resistance genes（oqx A,oqx B,qnr S,qnr B）,β-lactams(bla _CTX-M-3),aminoglycosides,type I and type III fimbriae genes,siderophore genes,and transporter and pumps.SIM-producing ST1764 K64 showed typical features of hv KP,showing hypermucoviscosity phenotype.The virulence genes,including rmp A2,alls and aerobactin genes,linked to hv KP,were found in ST1764 hv KP.hv KP was sensitive to quinolone;also,oqx A gene was detected.三、Analysis of therapeutic strategies of clinically isolated strains of CRKP Objective:The tigecycline-based combination regimens were preformed through in vitro combination sensitivity test,in order to provide an effective selection of drug in CRKP and hv KP treatment.Methods:In vitro combinations antimicrobial sensitivity testing:Synergistic activity of tigecycline–imipenem（TGC+IPM）,tigecycline–meropenem（TGC+MEM）,and tigecycline–aztreonam（TGC+ATM）combinations were performed by microdilution checkerboard method.Concentrations of each antimicrobial tested in combination range from 1/32×MIC to 4×MIC.The effects of antibiotics in combination were quantified by the fractional inhibitory concentration（FIC）index.The FIC index≤0.5 was interpreted as synergy,>0.5 to≤4.0 as additive,and>4.0 as antagonism.Results:TGC+IPM,TGC+MEM,and TGC+ATM combinations were high synergistic against100%（11/11）,90.1%（10/11）,and 90%（9/10）of CRKP strains,respectively.Antagonism was not observed in these isolates in either of the combinations.四、Homology analysis of CRKP in four tertiary hospital in Taiyuan Objective:Analyzing the relationship of CRKP in four tertiary hospitals,to identify the homology of CRKP in different hospital.Methods:1.Strain collection:From September 28,2017,to December 31,2018,non-duplicated clinical CRKP isolates were selected from four tertiary hospitals.2.Homology analysis:The housekeeping genes of MLST were detected by PCR.Using the Pasteurs database to identify the DNA sequences.MEGA-X and goe BURST method were selected to analyze the main CRKP clones in Taiyuan.To analyze the relationship between ST4564 and high-risk clones.3.Sensitive test:We used broth microdilution method to assess the antibiotics sensitive of ST4564 CRKP strain.4.Phenotypic characterization and resistance gene amplifications:The method is the same as in Part 2.5.Phenotypic characterization and amplify genes of virulence:The method is the same as in Part 2.6.Next-Generation Sequencing:The method is the same as in Part 2.Results:1.Strain collection:A total of 57 non-duplicated clinical strains of CRKP were obtained from 4 hospitals in Taiyuan.2.Homology analysis:ST11（56.1%,32/57）was the main prevalent clone.One novel sequence type was found,named as ST4564.MEGA-X analysis showed that CRKP strains were divided into 5 clusters.There was some homology among different strains,which were isolated from different hospitals.Compared the allele of ST4564 with high-risk clones we found that ST4564 was not related to any of high-risk clones.3.Antimicrobial susceptibility testing in clinical common antimicrobial agents:ST4564strain exhibited resistance to carbapenems,3rd,4th generation cephalosporins,β-lactam combination agents and ciprofloxacin,levofloxacin but remained susceptible to AMK,TGC and COL.4.Detection of resistance mechanism:ST4564 strain was positive for bla _NDM-1 and bla _CTX-M-14,17 by PCR amplification and sequencing.5.Characterization of virulence:ST4564 strain was positive for the string test,it showed hypermucoviscosity phenotype.ST4564 strain was negative for the iuc A,iut A,rmp A,iro N,rmp A2 gens by PCR and sequencing.Moreover,none of the serotypes were identified.6.Genetic determinants of resistance and virulence:ST4564 strain harbored efflux pump genes（acr A,acr B）,which were associated with multiple drug resistance phenotype.ST4564 harbored multiple genes encoding resistance toβ-lactams(bla_CTX-M-14,bla _CTX-M-17,bla _NDM,bla _TEM,bla _LEN),aminoglycosides[aac（3）-IIb,aph（6）-Id,aph（3’）-Ia,aad A6/aad A10],quinolones（qnr Bs）,macrolides（mph A,mrx）,and sulfonamide（sul1,sul2）.ST4564 also harbored multiple virulence genes,including the genes encoding type-I fimbriae cluster,siderophore genes（iut A,fep D,iro E,ent）,transporter and pumps[ABC transporter,RND efflux system（acr AB,rcs AB）],and the genes encoding pullulanase secretion protein（T6SS）genes.Conclusion:1.Cerebrovascular disease and central venous catheter were the independent mortality factors for death in patients infected with multi-drug resistant Enterobacteriaceae bacteria.The usage of aminoglycosides was associated with 30-day survival of patients.The independent mortality factor for death groups,patients infected with multi-drug resistant Klebsiella pneumoniae,was tracheal cannula.The usage of aminoglycosides was associated with 30-day survival with borderline statistical significance.2.NDM was the major resistance mechanism of CRKP.3.The synergism of the combination of tigecycline with imipenem,meropenem and aztreonam is more than 90%,and the combination of TGC+IPM could serve as an available treatment for CRKP infections.4.ST11 was the main prevalent clone in four tertiary hospitals in Taiyuan.5.One novel sequence type was detected in this study.ST4564 was associated with multiple drug resistance phenotype.It was not related to high-risk clones.However,infection control of the new clone was still be needed,to prevent it from becoming a high-risk clone of CRKP.