Mechanism Of Antibiotics Resistance And Molecular Epidemiology Of Carbapenem Non-Susceptible Klebsiella Pneumoniae

Objective:To explore the characteristics of resistance phenotype and the mechanism of antibiotics resistance in carbapenem-non-susceptible (CNS) Klebsiella pneumoniae (K. pneumoniae) isolates collected during 2012-2014 in a teaching hospital of Chongqing. Meanwhile, molecular epidemiology of these isolates was also investigated.Methods:Carbapenem-non-susceptible Klebsiella pneumoniae isolates, indentified by VITEK2 Compact automated bacterial identification and susceptibility testing system, were collected from January 2012 to December 2014, and the antibiotic suscepbilities to carbapenem were validated by broth dilution susceptibility test. Carbapenemase genes including blaKPC blaSME,blaVIM,blaIMP, blaNDM-1 and blaOXA-48, extended spectrum β-lactamases (ESBLs) genes including blaTEM,blaSHV,blaCTX-M-1’ and blaCTX-M-9’,QRD genes including qnrA, qnrB, qnrC, qnrD, qnrS and aac(6’)-Ib-cr, and ARD genes including aac(6’)-Ib, armA and rmtB of these isolates were determined by PCR and sequencing, and Class I integron gene Intl-1 as well as porin genes ompK35 and ompK36 were also identified. Pulsed Field Gel Electrophoresis (PFGE) and Multiple Locus Sequence Typing (MLST) were employed for the molecular epidemiology.Results:1. Among the 2,131 K. pneumoniae isolates identified in our laboratory from 2012 to 2014,56 (2.63%) non-duplicated CNS K. pneumoniae isolates were collected. Most of the strains were isolated from urine (32.14%) and sputum (25.00%) samples, most of which colledted from surgery Ward (51.79%) and Intensive Care Unit (ICU) (25.00%).2. Of all these isolates,56 (100%) were ETP non-susceptible,27 (48.21%) were IPM non-susceptible, and 21 (37.50%) were MEM non-susceptible.82.14%(46/56) of these strains were resistance to at least one kind of cephalosporins, and the rates of resistance to aminoglycosides and quinolones were 69.64%(39/56) and 64.29% (36/56), respectively.3. Of all 56 CNS K. pneumoniae isolates,10 (17.86%) harbored carbapenemase genes:blaNDM-1(6/56,10.71%),blaIMP-4(1/56,1.79%), blaKPC-2(3/56,5.36%); 52 (92.86%) carried ESBLs genes:blaTEM(20/56, 35.71%), blaSHW(49/56,87.5%), blaCTX-M(30/56,53.57%). Moreover,39 (69.64%) harbored quinolone genes:qnrB(27/56,48.21%), qnrS(10/56, 17.86%),aac(6’)-Ib-cr(16/56,28.57%), and 34(60.71%) carried aminoglycoside genes:aac(6’)-Ib(25/56,44.64%), armA(10/56,17.85%), rmtB(3/56,5.36%). Intl-1 gene was detected in 37 (66.07%) stains, and deletions in ompK35 and ompK36 were found in three (5.36%) and four (7.14%), respectively. No strain was detected to possess deletions in the two porin genes simultaneously. Among the seven strains with porin gene deletion, four strains showed obviously high MICs of ETP (128μg/ml or 256μg/ml).4. Of the 10 carbapenemase positive isolates, the co-expression rates of ESBLs, QRD, ARD and Intl-1 were 90%,100%,80% and 80%, respectively, and the loss rate of porin genes was 20%. Of the 52 isolates haboring ESBLs genes, the postive rates of fluoroquinolone, aminoglycoside resistant genes and integron genes were 71.15%,63.46% and 67.31%, respectively. Moreover, the rate of porin deletions was 11.54%. This is the first discovery of a new resistance genes combination indentified in one strain:co-expression of blaKPC-2,blaSHV,blaCTX-M-15’, aac(6’)-Ib-cr, aac(6’)-Ib and Intl-1 combined with ompK35 deletion.5. PFGE results showed that the 56 CNS K. pneumoniae isolates were divided into 52 different types, named 1-52. Notably, type 45 had 3 subtypes incluing 45a,45b and 45c. MLST results suggested that the ten carbapenemase positive isolates could be classified into seven STs, including ST11, ST15, ST29, ST290, ST323, ST412 and ST1641.Conclusion:1. Compared with imipenem and meropenem, the non-susceptible rate to ertapenem of the CNS K. pneumoniae was more high, and the minimum inhibitory concentration (MIC) to ertapenem increased gruadually from 2012 to 2014. Most of the 56 isolates were multiple drug resistant.2. The positive rate of carbapenemase genes was not very high, and expression of ESBLs genes combined with deletions in porin genes might be the primary mechanism of carbapenem non-susceptible in our hospital. Co-expression of multiple resistant genes and extensive expression of Intl might closely related to multiple drug resistance.3. Most of the strains were isolated from urines and sputums, most of which were isolated from surgery Ward and ICU. Molecular epidemiology analysis revealed clonal diversities in these 56 strains.