| ObjectiveCarbapenem-resistant Klebsiella pneumoniae（CRKP）is one of the reasons for inducing nosocomial infection and has the characteristic of extensively drug-resistance and rapid spread,which has become a challenge of clinical treatment.Tigecycline and polymyxin B are the main antibiotics to cure CRKP,but many cases of developing resistance during the treatment have been observed.Heteroresistance,a unique phenotype,is one of the reasons for drug treatment failure because of the lack of measures to detect this phenotype.Our study is to explore the effect of polytherapy and mechanisms of dual-heteroresistant Klebsiella pneumoniae clinical isolates.Methods1.We collected the clinical isolates of carbapenem-resistant Klebsiella pneumoniae and detected the presence of resistance-related genes by PCR.To screen polymyxin B and tigecycline sensitive strains,we performed the micro broth dilution method and used population analysis profile（PAP）to confirm the heteroresistance of polymyxin B and tigecycline.The subpopulations were collected to confirm the MIC by the broth microdilution method,and the stability was observed through continuous subcultures on an antibiotic-free medium.Pulsed-field gel electrophoresis（PFGE）was used to detect the homology of the parent strains and subpopulations.2.The efflux pump inhibitor CCCP was used to perform the broth microdilution method to determine the MIC of tigecycline.Real-time PCR was carried out to detect the expression levels of the two-component system（pmr A,pmr B,pho P and pho Q）and efflux pump genes（acrA、acrB、tol C、oqx A and oqx B）.3.Time-kill curve was performed to detect the therapeutic effect of polymyxin B and tigecycline in combination on heteroresistant Klebsiella pneumoniae.Results1.Among 95 CRKP clinical isolates,we selected 90（90/95,94.7%）polymyxin B heteroresistant strains,50 tigecycline heteroresistant strains（50/95,52.6%）,and 46strains（46/95,48.4%）that were heteroresistant to both polymyxin B and tigecycline.The frequency of heteroresistance to polymyxin B ranged from 4.41×10-7 to 3.66×10-6,and the frequency of heterogeneous resistance to tigecycline ranged from 2.14×10-6 to5.12×10-6.PCR founded that all strains（95/95,100%）had bla KPC,17strains（17/95,17.9%）carried bla CTX-M-1 and 1 strain（1/95,1.1%）carried both bla KPC and bla NDM.PFGE analysis confirmed the homology between the parent strain subpopulation,respectively.The findings indicated that the mechanism of polymyxin B and tigecycline resistance of the double heteroresistant strains was independent,based on the MIC values of dual-heteroresistant subpopulations to polymyxin B and tigecycline.2.In the efflux pump inhibition assay,there were two subpopulation strains（KP230and KP250）with heteroresistance to tigecycline,and the MIC emerged 8-fold and 16-fold reduction after adding CCCP,respectively.Real-time PCR showed that the efflux pump gene acrA,acrB,and tol C of tigecycline heteroresistant strains were up-regulated to varying degrees compared with parent strains,which were 2.4-3.2 folds（P<0.05）and3.0-5.7 folds（P<0.05）,respectively.The expression levels of pmr A and pmr B in the polymyxin B heteroresistant strains were higher than their parent strains,which were 3.2-214.6 folds（P<0.05）and 13.2-407.3 folds（P<0.05）,respectively.3.By analyzing time-kill curves,the effect of using polymyxin B or tigecycline alone to treat heteroresistant strains was not effective.Although at a higher concentration（2×MIC）,the monotherapy groups had a short-term bactericidal effect,the bacteria may emerge re-growth after 10h.In the combination of polymyxin B and tigecycline,even if a lower concentration combination（PMB 0.125mg/L+TGC 1mg/L）was robust.This combination could achieve the effect of completely removing bacteria and maintain the bactericidal effect until 48h.ConclusionsWe obtained nearly half of all strains that were heteroresistant to both polymyxin B and tigecycline.In the dual heteroresistant strains,the expression levels of the efflux pump genes acrA,acrB,tol C,and the two-component system pmr A,pmr B,pho P,and pho Q were higher than those of the parental strains,suggesting that the heteroresistance may be related to the two-component system（Pmr AB and Pho PQ）and efflux pump system（Acr AB-Tol C）.Time-kill curve indicated that the effect of monotherapy（PMB or TGC）on heteroresistant strains was not ideal,but the combination treatment could eliminate bacteria in the early stage.This study explored the CRKP mechanisms of dual-heteroresistance and the effect of polytherapy for providing a strategy for the clinical treatment to cure heteroresistant strains. |
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