Detection Of ESBLs And Analysis Of KPC In Klebsiella Pneumoniae

Background and Objective:With the popular utilization of extended-spectrum antibiotics, especially the extensive use of the third-generation cephalosporins, the isolation rate of extended spectrum beta-lactamases(ESBLs)-producing Klebsiella Pneumoniae was increased year by year. Most of the ESBLs were produced by gram-negative bacilli, which was a kind of enzyme that can hydrolyzeβ-lactam antibiotics. Drug resistant gene can be spread among bacteria through plasmid by conjugation, transformation and conduction, thus resulted in serious hospital infections and the wide spread of drug resistant strains. K.Pneumoniae was very sensitive to carbapenem antibiotics, but there also existed the drug-resistant strain with the increasing use of carbapenem antibiotics(Imipenem and Meropenem). K.pneumoniae carbapenemases(KPC)is a kind of carbapenemase that was found recently, it can hydrolyze many kinds ofβ-lactam antibiotics, including carbapenems; KPC gene was located in transferable plasmid throuch which KPC is spread between strains. The detection of ESBLs and KPC-producing strains and epidemiological surveillance should be taken seriously in clinic laboratory, and the prevalence and outbreak of related hospital infection should also be prevented and controlled. This study was aimed to investigate the drug resistance of K.pneumoniae and occurrence of ESBLs-producing strains and the optimal screening substrate, to detect and analyze the genotype of KPC from the Imipenem-resistant K.pneumoniae, to understand the mechanism of drug resistance, and to provide the basis for the rational use of antibiotic in clinic. Methods:To make clear the status quo of infection and the situation of drug resistance, 124 strains of K.pneumoniae isolated from Jan 2009 to Oct 2010 were analyzed retrospectively with WHONET 5.4. The initial screening of phenotype of ESBLs was performed with Ceftazidime, Cefotaxime, Aztreonam and Ceftriaxone, double-paper tests was used to detect ESBLs. ESBLs-producing K.pneumoniae was screened with the Modified Hodge Test. KPC gene in K.pneumoniae was identified by polymerase chain reaction (PCR) followed by sequencing, and the genotype of KPC was confirmed by comparison with the internet GenBank database. Homology of KPC was analyzed with pulsed-field gel electrophoresis (PFGE). Plasmid conjugation method was used to determine the transfer of KPC gene by plasmid. Minimal inhibitory concentration (MIC) of carbapenems in the zygote and the initial stains was detected by agar dilution method.Results:The detectable rates of K.pneumoniae in Intensive Care Unit was the highest(17.74%). Infection location was focused on respiratory tract (37.90%). Our work demonstrated that the optimal screen substrate for ESBLs was Cefotaxime, the sensitivity was 95.5%, the missing rate was the highest when Ceftazidime was used as screen substrate only. 42 ESBLs positive strains were detected, accounting for 33.87%. Drug resistance rates of ESBLs-producing strains were higher than those of the non- ESBLs-producing ones. Among the 124 strains, 13(10.48%)showed resistant to Imipenem, 10 KPC-positive strains(76.92%)were screened initially with Modified Hodge Test. KPC positive was found in 2 strains by PCR and sequencing. homologous analysis indicated that they were belonged to KPC-2 type carbapenemase gene. The 2 strains came from the same clone verified by the results of PFGE. Plasmid conjugation method showed that zygomycete acquired the carbapenem-resistant gene, but the MIC value of zygote to carbapenems was decreased compared with the original strain. Conclusion:Respiratory tract was the main pathway of the infection of K.pneumoniae in the hospital, the monitor of bacteria in the air is clinically necessary, and ESBLs resulted in the K.pneumoniae's resistance to penicillin, the third and the fourth generation of Cefquinome; KPC enzyme contributed to the K.pneumoniae's resistance to Carbapenem. K.pneumoniae carrying KPC-2 type carbapenemase gene was firstly isolated in Bengbu district of Anhui, this gene was located in plasmid, the gene can be rapidly spread through plasmid. The result of PFGE indicated that the cross-infection existed in the ward. The resistance of zygote was weaker than the initial strain, suggesting additional mechanisms of resistance to carbapenemase.