Study On Resistance Mechanisms And Molecular Epidemiology Of Carbapenem Resistant Klebsiella Pneumoniae

Objective:The aim of our study is to investigate the antibiotic resistance of Carbapenem-resistant Klebsiella pneumoniae?CRKP?strains and to detect related antibiotic resistance genes in resistant strains in the First Affiliated Hospital of Chongqing Medical University?CQMU?from2012 to 2015,and to explore the resistance mechanism and molecular epidemiological characteristics of CRKP so as to contribute to the development of a better approach reducing CRKP infection.Methods:CRKP strains were identified using the VITEK2 compact or VITEK MS systems?bioMerieux,Hazelwood,MO,USA?at the department of laboratory medicine of the First Affiliated Hospital of Chongqing Medical University from January 2012 to December 2015.The minimal inhibitory concentrations?MICs?of carbapenem antibiotics were tested by broth microdilution method.The polymerase chain reaction?PCR?and sequencing were adopted to amplify the antibiotic resistant genes of bacterial strains,including carbapenem genes,extended spectrum?-lactamases?ESBLs?,aminoglycosides resistance determinants?ARDs?,fluoroquinolones resistance determinants?QRDs?,Intl-1 intergons,and the loss of porin genes including Ompk35 and Ompk36.Multilocus sequence typing?MLST?and pulsed-field gel electrophoresis?PFGE?were used to explore the epidemiology of strains at the molecular level.Results:A total of 78 Carbapenem-resistant Klebsiella pneumoniae were isolated in the First Affiliated Hospital of CQMU from 2012 to 2015,mainly from the Department of Hepatobiliary Surgery?18/78,23.1%?and the Respiratory Medicine?13/78,16.7%?.The most common specimen types were urine and sputum,accounting for 29.5%and 25.6%,respectively.Of the 78 CRKP strains,24?30.8%?strains were resistant to meropenem,and 29 strains were resistant to imipenem.All strains were resistant to ertapenem.In addition,28.2%of the CRKP were resistant to all of the three carbapenem antibiotics.There were 78.2%and 75.6%of the isolated strains were resistant to ceftriaxone and ceftazidime respectively,and 41 strains were resistant to cefepime.62.8%and 61.5%of bacterial strains were resistant to aminoglycosides and fluoroquinolones respectively.Among the CRKP strains collected,30?38.5%?strains produced carbapenemases,including 22 isolates possessing bla_NDM-1,eight isolates containing bla_KPC-2,and three isolates haboring bla_IMP-4.There were 63?80.8%?strains producing ESBLs,including 53 strains of bla_SHVHV and 46strains of bla_CTX-M.There were also 47.4%strains of Amp C positive,of which bla_DHA-1,bla_CMY-2,and bla_MOX-3accounted for 42.3%,1.3%,and 3.8%,respectively.There were 56?71.8%?bacterial strains detected with quinolone-resistant genes,mainly including qnrB,qnrS,and aac?6'?-Ib-cr,and 51?65.4%?bacterial strains detected with aminoglycoside resistance genes.The absence of membrane pore protein were found in 8 strains,3strains with lack of omp K35 and 5 strains with loss of ompK36.66.7%of bacterial strains were found to have Int I.PFGE results showed that the 78CRKPs collected in our hospital could be categorized into 67 types,and there was no epidemiological evidence of outbreaks among the strains.MLST was performed on the strains producing carbapenemase.The results showed that the most common type was ST11,followed by ST147,ST290 and ST2133.Conclusions:1.The positive rate of carbapenemase gene in the CRKP strain in the First Affiliated Hospital of CQMU was relatively high,with38.5%,of which NDM-1 accounted for 73.3%?22/30?.2.The high expression of ESBLs combined with AmpC may play an important role in carbapenem resistance Klebsiella pneumoniae in our hospital.The co-expression of multiple drug resistance genes and the high detection rates of IntI partly explained the multidrug resistance mechanism of Carbapenem-resistant Klebsiella pneumoniae.3.The carbapenem-resistant K.pneumoniae isolated from our hospital were sporadic and not from the same colony.Molecular epidemiology has not found the outbreak among the strains.