Study On Resistance And KPC Enzyme Gene In Klebsiella Pneumoniae Isolates

BackgroundIn about 20 years, the rates of ESBLs producers were increased most because of using the third cephalosporins wide, such as cefotaxime, ceftriaxone and ceftazidime. Nosocomial infections caused by ESBLs producing strains increased year by year and have been reported to cause epidemic outbreak in some hospital. ESBLs were produced by gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae etc and could inactivate oxyimino-β-lactams, due to their wider substrates for hydrolysis than penicillinase.gram-negativeThus, understanding the best drug resistance profile in Klebsiella pneumoniae isolates can help us prevent regional spreading of ESBLs and guide the proper clinical therapy, in case of delaying instant therapy too much waste.ObjectiveTo investigate the resistance and prevalence of ESBLs producers in isolates of Klebsiella pneumoniae.MethodsEighty seven clinical isolates of Klebsiella pneumoniae were collected from the first affiliated hospital of Anhui medical university (including 38 in 2005; 49 in 2008). Their resistance to antimicrobial agents was also tested by agar dilution method and ESBLs producers were screened by CLSI phenotype confirmatory test. ResultsThe resistance rates of Klebsiella pneumoniae to piperacillin, cefazolin, cefuroxime and chloramphenicol were more than 70%; to fluoroquinolones except levofloxacin and gatifloxacin were more than 50%; to meropenen and imipenem were o;to piperacillin/tazobactam, ceftazidime and amikacin were no less than 50% in 2005. The resistance rates of Klebsiella pneumoniae to piperacillin, cefazolin, cefuroxime and chloramphenicol were more than or nearly 80%; to fluoroquinolones except gatifloxacin were more than 60%; to meropenen and imipenem were 2%; to piperacillin/tazobactam, ceftazidime and amikacin were no less than 50% in 2008. The resistance rates to cefazolin, cefepime, ofloxacin and levofloxacin in 2008 were significantly higher that in 2005 (P﹤0.05). The totally incidence of ESBLs producers were 48.2% in two years. They were resistant to most antimicrobial agents. ESBLs producers were multi-resistance.Compared with the resistance rates of non-ESBLs producing strains, ESBLs producers were more resistant to piperacillin, cephazolin, cefuroxime, cefotaxime, levofloxacin and ciprofloxacin (P﹤0.01).Conclusions1. The resistance rates of Klebsiella pneumoniae to penicillins, the first and second cephalosporins, cefotaxime, chloramphenicol and most fluoroquinolones were more than 50%; to amikacin, ceftazidime and piperacillin/tazobactam were no less than 50%; to carbapenems was not resistant in 2005 and 2008.2.The incidence of ESBLs producers was high in Klebsiella pneumoniae isolates. The incidence of ESBLs producers in 2008 was no more significant than that in 2008. ESBLs producers were multi-resistance.Compared with the resistance rates of non-ESBLs producing strains, ESBLs producers were more resistant (P﹤0.01).3. Due to ESBLs producers which were resistant toβ-lactamases including the third and fourth cephalosporins, and most fluoroquinolones and ESBLs producers were multi-resistance, we should choose carbapenems such as meropenen and imipenem to therapy. BackgroundIn recent years carbapenem antibiotics, such as meropenen, imipenem and ertapenem, were wider used in clinical practice to therampy gram-negative bacteria, which included Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae etc.Specially it can control serious infection by gram-negative bacteria or multi-resistance efficiently. As a result, carbapenems enzyme which can extensive hydrolyseβ-lactams including meropenen and imipenem were produced and spreaded. Producing KPC enzyme is one of the most resistant mechanism to carbapenems for Klebsiella pneumoniae. KPC enzyme belongs to A class carbapenems enzyme.It can hydrolyse almost could hydrolyze almost allβ-lactamases. It hydrolyzed efficiently ampicillin and piperacillin. It hydrolyzed penicillins, cefazolin, cefuroxime more efficiently than imipenem, meropenem, cefotaxime and aztreanam. It hydrolyzed inefficiently ceftazidime and cefepime.β-lactamases inhibitor can inhibit hydrolyzation.ObjectiveDetecting, Cloning and Expression of KPC enzyme gene of Klebsiella pneumoniae.MethodsAccording to CLSI 2008, Klebsiella pneumoniae have carbapenem(meropenem, imipenem) MICs of 2 mg/L may produce KPC-type. The isolates were choosed which meropenen and imipenem MICs were≥2 mg/L. The KPC encoding gene was amplified by polymerase chain reaction(PCR ) and sequenced by Sanger's dideoxy chain termination composition method .Then blast program was used to identify the genotype at GenBank.The PCR product was linked into the vector E. coli PHSG398 by T4 DNA Ligase, then the recombinant plasmid was introduced into the component cell E. coli JM109 and the transformant was selected on M-H agar containing ampicillin and chloromycetin.Attempt to transfer the plasmid of clinic isolate to the E. coli J53.M-H agar containing ampicillin and Sodium azide was used selected the transconjugant. Agar dilution method was used to determine MICs against clinical strain,transconjugant and transformant.ResultsAmong the 87 isolates, the MIC of 12 isolates( 4 in 2005 and 8 in 2008) to meropenen and imipenem were MIC≥2mg/L. By PCR and sequenced,we identified that was KPC-2 enzyme gene.Clinical isolate was resistant to all of antimicrobial agents. Transconjugant was resistant to piperacillin,ampicillin,cefazolin,cefuroxime and chloramphenicol and it exhibited low lever resistant to the otherβ-lactamase including meropenen and imipenem (more than eightfold concentration increase to E.coli J53 in the MIC).Transformant was resistant to ampicillin,cefazolin and chloramphenicol and it also exhibited low lever resistant to the otherβ-lactamase including meropenen and imipenem(more than eightfold concentration increase to E.coli JM109 in the MIC). andβ-lactamase inhibitor combinations can significantly decrease the resistance.Conclusion1. KPC-2 enzyme gene was firstly found in klebsiella pneumoniae in Anhui province.2.The clinical isolate carrying KPC-2 enzyme gene was multi-resistance. Transformant exhibited low lever resistant toβ-lactams including meropenen and imipenem.β-lactamases inhibitor can inhibit hydrolyzation..3. The success of transconjugant showed KPC-2 gene located in plasmid, which may spread the gene horizontally.