Molecular Epidemiology And Resistant Mechanism Of Carbapenem-resistant Klebsiella Pneumoniae

Objective:Due to the widespread use of carbapenems,carbapenem-resistant Klebsiella pneumoniae(CRKP)has been increasingly reported and has posed a serious public health threat.Here,we carried out molecular epidemiological studies on CRKP,and determined the predominant STs of the CRKP in the Second Affiliated Hospital of Kunming Medical University.In addition,our study will establish the detection method of resistance gene in CRKP strains in our hospital and to investigate molecular mechanism of drug resistance.And we can provide theoretical and clinical application for developing the prevention and control strategy of CRKP in our hospitcal and guiding the proper clinical use of antibiotics.Methods:The 147 CRKP strains isolated from the clinical samples of the Second Affiliated Hospital of Kunming Medical University from January 2015 to December 2017.The biochemistric phenotypes and drug sensitivity tests of these strains were identified by VITEK-2 Compact automatic bacterial identification system.At the same time,species identification of the strains was performed by PCR and sequencing of 16S rRNA gene.And the multi locus sequence typing(MLST)was used to classify the detailed Sequence types(ST)of CRKP.Furthermore,PCR method was used to detect the existence of carbapenem,including blaIMP,blaVIM,blaSPM,blaNDM,blaKPC,blaBIC,blaAIM,blaGIM,blaSIM and blaDIM genes and verify by sequencing.Results:The 147 CRKP strains isolated from sputum at most,accounting for 58.50%,followed by urine 15.65%,secretion 10.20%,other 7.48%and blood 6.80%.The 147 CRKP strains originated from the ICU department,accounting for 29.25%;followed by 27.21%for neurosurgery,10.88 for urology,8.84%for burn unit,8.84%for hepatobiliary and pancreatic surgery,and 14.97%for other departments.The resistance rates to ceftazidime,amikacin,aztreonam tazobactam and levofloxacin were 92.31%?89.74%?100%?100%?97.44%in 2015.The resistance rates to ceftazidime,amikacin,aztreonam,tazobactam and levofloxacin were 100%?88.89%?95.56%?97.78%?95.56 in 2016.The resistance rates to ceftazidime,amikacin,aztreonam,tazobactams and levofloxacin were 96.83%?85.71%?96.83%?96.83%?98.41 in 2017,which showed multiple drug resistance or pan drug resistance.The results of MLST showed that 147 CRKP strains were divided into 13 STs,and the resistant strains showed clonal diversity.In 2015,39 strains were subgrouped into ST11(29 strains),ST23(2 strains),ST37(2 strains),ST375(2 strains),ST65(1 strain),ST731(1 strain),ST2193(1 strain)and ST2260(1 strain).In 2016,45 strains were ST11(39 strains),ST37(2 strains),ST231(2 strains),ST86(1 strain)and ST420(1 strain).In 2017,63 strains were ST11(58 strains),ST23(1 strain),ST25(1 strain),ST37(1 strain),ST86(1 strain)and ST792(1 strain).ST11 is the main dominant type of this study.Compared with previous reports in Kunming,there is no change in major epidemic strains.The drug resistance gene test result showed that the positive rate of KPC gene was the highest,100%,followed by NDM2.56%in 2015.The positive rate of KPC gene was the highest,95.56%,followed by NDM4.44%in 2016.The positive rate of KPC gene was the highest,100%,followed by NDM6.35%in 2017,while BIC,VIM,SIM,IMP,SPM,AIM,GIM and DIM genes were not detected.The high positive rate of carbapenem gene is one of the most important reasons for the multiple drug resistance of strains in this study.Conclusion:The 147 CRKP strains isolated from sputum at most,accounting for 58.50%,followed by urine 15.65%.The 147 CRKP strains originated from the ICU department,accounting for 29.25%;followed by 27.21%for neurosurgery.Klebsiella pneumoniae in this study has a one hundred percent resistance rate to carbapenems,it is also highly resistant to many commonly used antibiotics,such as beta lactamase inhibitors,quinolones,cephalosporins and aminoglycosides.The strain has a high resistance rate to the commonly used antibiotics,and has a slight decrease in Amikacin and tazobactam,but a slight increase in levofloxacin from 2015 to 2017.multiple drug resistant and pan drug resistance lead to CRKP infection to be incurable.The 147 strains of CRKP in this study were divided into 13 STs.There were 8 different STs in 2015,5 different STs in 2016,and 6 different STs in 2017,with being predominant ST11.ST11 accounted for 74.36%in 2015,86.67%in 2016,and 92.06%in 2017,respectively.The rate of ST 11 is increasing year by year.Compared with 2015,ST23,ST65,ST375,ST731,ST2193,ST2260 were not found in 2016,but ST86,ST231,ST420 appeared.Compared with 2016,ST86,ST231 and ST420 were not found in 2017,but ST23,ST25 and ST792 appeared.The resistant strains showed the characteristics of clonal diversity,and we should focus on the prevalence of other cloned strains.Detection of 10 carbapenem genes in CRKP isolated from our hospital showed that the positive rate of KPC was 100%in 2015,95.56%in 2016,and 100%in 2017.In three years,the detection rate of KPC did not decrease.There was no downtrend in the detection rate of KPC,and the detection rate of NDM showed a slight upward trend.In addition,BIC,VIM,SIM,IMP,SPM,AIM,GIM and DIM were not detected.The existence of carbapenem is the most important reason for the multiple drug resistance in this study.CRKP,with KPC,can hydrolyze carbapenem antibiotics,and is an important drug resistance mechanism in Kunming.Multiple drug resistance or widespread drug resistance has been severe challenges to clinical treatment.It is necessary to strengthen the further research on the surveillance and epidemiology of CRKP and take effective measures to prevent the epidemic of hospital infection caused by CRKP.