Polymyxin Resistance Mechanism Of Carbapenem-Resistant Klebsiella Pneumoniae In Zhejiang Province

Purpose:To determine the distribution of polymyxin MICs and the drug resistance rate in carbapenem-resistant Klebsiella pneumonia（CRKP）in Zhejiang Province during the period from March 1,2017 to July 31,2017.To clarify the polymyxin resistance mechanism in CRKP and help to reduce the isolation rate of polymyxin resistant strains.Methods:The clinical isolates of CRKP were continuously collected from patients at 36tertiary hospitals in Zhejiang Province from March 1,2017 to July 31,2017.The CRKP MICs of polymyxin were estimated using microbroth dilution method.The PFGE method was used to analyze the homology of polymyxin-resistant isolates.The two-component system regulate genes（mgr B,pho P,pho Q,pmr A,pmr B,crr AB）and colistin resistant gene mcr-1,were analyzed by PCR and sequencing.The relative expression of regulate genes（mgr B,pho P,pho Q,pmr B,pmr C,pmr K）were compared between polymyxin susceptible group and resistant group using q RT-PCR.Results:A total of 872 non-repetitive isolates of CRKP were collected.The MIC₅₀were<0.125mg/L,the MIC₉₀were 0.25mg/L,and polymyxin resistant rate was 1.49%（13/872）.The 13 polymyxin resistant isolates can be divided into 11 clonotypes,and there was no obvious clone propagation.The PCR results revealed that among the polymyxin resistant isolates,1 strain（KP13223）had mcr-1 gene,and its MIC was4mg/L.In other 12 isolates,the MICs of 75%（9/12）isolates were≥16mg/L.The PCR result shows that 2 isolates had mutation in mgr B gene,which were IS1 family insertion（KP45856）and IS5 family insertion（KP13090）;5 isolates had pmr B gene mutations,including pmr B（D150Y,KP02105）,pmr B（A246T,V287M,KP24030）,pmr B（S85R,KP01101 and KP01224）,pmr B（K220Q,A246T,KP01214）,meanwhile,2 of them had pho P gene mutation at the same time.And in strain KP24030 that is phop（I162V）mutation and in strain KP01214 that is phop（P74L）mutation.The results of q RT-PCR showed that among 12 mcr-1 negative polymyxin-resistant isolates,75%（9/12）of them had higher expression of pmr C gene,and 58.33%（7/12）had higher pmr K gene expression when compared with the sensitive group.Six isolates had increased expression in both pmr C and pmr K genes,meanwhile,5 of them had the MICs of≥16mg/L,showing high level of polymyxin resistance.Compared with the sensitive group,the 5 resistant strains with prm B gene mutations had significantly higher expression of prm C gene（p=0.02）.In the 2 strains with mgr B gene insertions,the expression of mgr B gene was decreased compared with the sensitive group,leading to higher expression of their downstream genes pho P and pmr K.Among the other 5 strains that we didn’t find any mutation,3 of them had higher expression in pmr C genes compared to the sensitive group.Conclusions:The polymyxin resistant rate of CRKP was generally at a low level in Zhejiang Province.In this study,we found that in Zhejiang Province,the polymyxin resitance is more often caused by the gene mutations in two-component system which lead to the higher expression of the pmr C and pmr K.The resistance caused by the mcr-1 gene was rare.