Study On Resistant Mechanisms And Molecular Epidemiology In Klebsiella Pneumoniae

Objectives: To investigate the resistance of Klebsiella pneumoniae (K.pneumoniae)isolated in clinical specimens,strain clusters as well as resistant genes associated withβ-lactamases,aminoglycoside-modifying enzymes and qacE△1-sul1.Meanwhile, the molecular mechanism of K.pneumoniae resistant to carbapenems were further studied.Methods: Bacteria identification and susceptibility tests were detected by phoenix-100TM system. Minimum inhibitory concentrations(MICs) of antimicrobials were determined by E-test.The resistant genes ofβ-lactamases, aminoglycoside-modifying enzymes,qacE△1-sul1 and 2 outer membrane protein(porin)genes were detected by PCR.The amplified products were purified and then sequences were analyzed by DNA sequencer.The porins were extracted by sonication and ultracentrifuge,and analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE).Results: (1)53 strains of K.pneumoniae isolated from clinical specimens were all sensitive to imipenem, and the resistant rate of remaining 9 antimicrobials was higher than 60%.The detecting rates of 8β- lactamase genes including TEM, SHV, CTX-M-1 group, CTX-M-9 group, OXA-1 group, LEN, OKP and DHA in 53 K.pneumoniae were 75.5%, 13.2%, 10%, 7.5%, 11.3%, 5.7%, 1.9% and 41.5%,respectively.Of 53 K.pneumoniae, at least 1β-lactamase gene in 49 strains, more than 2β-lactamase genes in 29 strains,and 6β-lactamase genes in 2 strains.However,only 4 strains didn't carry anyβ-lactamase gene.In addition,3 resistant genes encoding aminoglycoside- modifying enzymes were found in 53 isolates,such as aac(3)-Ⅱ,aac(6′)-Ⅰand ant(3″)-Ⅰb ,and they accounted for 90.6%,49.1% and 24.5%,respectively.The positive rate of qacE△1- sul1 genes was 90.6% in 53 isolates. Cluster analysis indicated that there were two clones in 18 strains and their resistant phenotypes were similar.(2)MICs of piperacillin, piperacillin/ sulbactam, amoxicillin/clavulanic acid, cefoperazone/sulbactam, cefotaxime,cefepime and aztreonam in 5 strains of Kp01,Kp02,Kp03,Kp04 and Kp05 were all higher than 128μg/ml,and those of imipenem or meropenem were higher than 32μg/ml.Five isolates of K.pneumoniae carried blaTEM-1 and blaDHA-1 genes.The gene loss of ompK35 and ompK36 in Kp01 and Kp03 occurred simultaneous.Similarly, the base deletion of ompK35 existed in Kp02 and Kp05.In addition, there were still base insertion of ompK36 in Kp02, Kp04 and Kp05. Compared with GenBank(GU945384), ompK 35 gene mutations of G→C at base 465 and T→C at base 466 in Kp04 lead to Gln to His Substitution at Position 155 and Tyr To His Substitution at Position 156,and it might be a new subtype. Conclusions: (1) K.pneumoniae have carried many resistant genes,such asβ-lactamases and aminoglycoside modifying enzymes,and associated withβ-lactams, aminoglycoside antimicrobials resistance.The cluster analysis indicates that the spread of clonies occurred in our hospital.(2) The production of DHA-1β-lactamase combined with the loss of OmpK36 or/and OmpK35 porin genes may contribute to high-level carbapenem resistance in K.pneumoniae.