The Mechanisms Of Polymyxin Heteroresistance In Carbapenem Resistant Klebsiella Pneumoniae

Objective:Carbapenem-resistant Klebsiella pneumoniae(CRKP)infection has emerged as a significant clinical challenge worldwide.Polymyxin is often the “last-line” choice of chemotherapy.Researches have demonstrated the emergence of polymyxin heteroresistance(HR)in CRKP,which can lead to treatment failure.In this study,we investigated 1)the prevalence of HR,proportion and resistance level of subpopulations in CRKP to polymyxin E,2)in vitro and in vivo bactericidal effect of polymyxin against CRKP;3)the mechanisms of polymyxin heteroresistance in CRKP.So as to provide primary evidences for the detection of heteroresistance of polymyxin and the rational treatment choice for multi-drug resistant bacterial infections.Materials and Methods:1.A total of 36 CRKP clinical strains including 31 sequence types(ST)were selected from all CRKP strains with whole genome sequence(WGS)data preserved in Infectious Diseases Laboratory,Center of Infectious Disease,West China Hospital of Sichuan University from September,2014 to May,2018.The MIC values of polymyxin were measured by broth microdilution(BMD)method.Modified population analysis profiles(PAPs)were conducted to determine wether heteroresistance(HR)existed or not for these strains.2.CRKP 085072 strain,with dominant sequence type ST11 was selected as primary strain for variability analysis including the colony morphology,MIC values,HR and stability of resistance for 9subpopulations.3.The time-kill kinetics of polymyxin against 4 heteroresistant and susceptible strains were investigated with 0,0.5×MIC,1×MIC,2×MIC concentrations to compare the in vitro bactericidal effect of polymyxin E against CRKP.4.The Galleria mellonella infection model was established to compare the in vivo bactericidal effect of polymyxin E to HR and susceptible CRKP.5.One subpopulation was respectively chosen from HR CRKP020018,020034 and 020052 strains and were grown daily in polymyxin-free broth to gain passages.The reference strains,subpopulations and passages of these three strains were sequenced to gain WGS data.The reference strain,9 subpopulations and 9 passages of 085072 strain were sequenced to gain WGS data.All reference and derivative strains were compared to analyze heteroresistance mechanisms.Results:1.The MIC ranges of polymyxin E against all 36 isolates by BMD were within 0.5 μg to 2 μg/ml.PAPs revealed heteroresistance in 28/36(77.8%)isolates.The MIC values of polymyxin E against resistant subpopulations were 8μg to >64 μg/ml,much higher than that of HR primary strains.2.The 9 resistant subpopulations of CRKP 085072 strain showed variability in colony morphology,MIC values,HR and resistance stability to polymyxin E.3.Polymyxin E killed >99% of bacteria(except CRKP 115029 and CRKP83 strains)in 2h,even at 0.5×MIC of polymyxin.While there was regrowth in almost all strains(mostly after 4-6h persistent incubation),even at 2×MIC of polymyxin.Regrowth of heteroresistant strains was earlier than susceptible strains.4.The survival rate of Galleria mellonella infection model infected with 3 CRKP strains had no significant increase after polymyxin E treatment in vivo.5.Single nucleotide polymorphism(SNPs)were found in crr B,pho P and pmr B genes in derivative strains of 020018,020034,020052.Different SNPs were found in different resistant subpopulations of CRKP 085072 strain.A 1,196bp-long IS element was found at nucleotide position 75 of mgr B gene in085072R2X4/085072R2X4G200 which led to Mgr B disruption and inactivation.No amplification/duplication causing heteroresistance of CRKP to polymyxin were found in structural variation analysis.Resistant subpopulations lost some genes involved in resistance to other antibacterial agents while being resistant to polymyxin.The derivative strains of CRKP 020034 even lost the entire p IMP4 plasmid and lost a large segment about 25 kb long in p Qnr B91 plasmid;The derivative strains of CRKP 020052 also lost the entire p IMP4 plasmid.Conclusion:The phenomenon that a somewhat wide range of CRKP were heteroresistant to polymyxin and the high level of resistance of resistant subpopulations to polymyxin deserve clinical highly attention.The low proportion of resistant subpopulations are likely missed by clinically commonly used susceptibility test.Single colony isolation commonly used in clinical practice may not fully reflect the characteristics of all bacteria in a clinical specimen.Resistant subpopulations may cause regrowth and lead to treatment failure after susceptible subpopulations are killed by polymyxin.Galleria mellonella infection model may be or not be suitable for the study of low-proportion heteroresistance need further study.Mutation of crr B gene rather than replication/amplification is a common mechanism that mediates heteroresistance of CRKP to polymyxin.Resistant subpopulations lost other resistant genes while being resistant to polymyxin,that may be helpful for antibacterials selection in chemotherapy and prevention of multi-drug resistent organisms(MDRO)infections.