Study On Drug Resistance Genes Of Aminoglycosides Modifying Enzymes And 16S RRNA Methylase Among Klebsiella Pneumoniae

Objective To investigate the drug resistance in Klebsiella pneumoniae from Xiangya Hospital of Central South University.To detect aminoglycosides modifying enzymes and 16s rRNA methylase genes in Klebsiella pneumoniae. Study resistance mechanism of aminoglycoside antibiotics in order to provide laboratory evidence for the clinical use of antibiotics.Methods1.96 non-repetitive clinical isolates of Klebsiella pneumoniae strains were collected from Xiangya hospitals of Central South University in January 2009 to July 2009. All of the clinical isolates were identifi cated and antibacterial susceptibility tested by Biomerieus automate microbiological-assay system VITEK-2,using GN cards and AST GN-13 cards respectively. MICs of gentamycin, amikacin and Tobramycin were determined by agar dilution method.2. The genotype of aminoglycosides modifying enzymes (aac(3)-Ⅰ,aac(6')-Ⅰb,ant(3")-Ⅰ,aph(3')-Ⅵa) and 16srRNA methylase genes (arm A,rmtA,rmtB,rmtC,rmtD,npmA) was detected by PCR.3. the PCR products were purificated and sequenced by automatic sequencer. The sequences acquired were compared with the known sequence in GenBank datebase in order to determine the gene subtype by BLAST searching.4. Investigate the resistance genes and resistance phenotypes compareing 96 strains of Klebsiella pneumoniae resistant genotypes and phenotypes.Result1.Of 96 clinical isolates of Klebsiella pneumoniae strains,the drugs resistence rates to gentamycin,amikacin and tobramycin were 63.5%,21.9%and 41.7% respectively. Strains resistant to amikacin were resistant to gentamicin and tobramycin. MIC50 of three aminoglycoside drugs and MIC90 were≥256μg/ml and> 512μg/ml.2. Among aminoglycosides modifying enzymes isolates of Klebsiella pneumoniae through PCR amplification,aac(3)-Ⅰ,aac(6')-Ib and ant(3")-Ⅰisolates accounted for 18.8%,57.3% and 3.1% respectively.3.aac(6')-Ⅰb is the most common genotype.The prevalence of aac(6')-Ib in ciprofloxacin-resistant and susceptible Isolates were 79.1% (19/24) and50%(36/72). Through Chi-Square Tests, the difference was statistically significant (x2=6.26, P<0.05)4.Among 16s rRNA methylase gene by PCR amplification and sequence analysis, there are 22 strains carried armA.17 strains harboring armA 16S rRNA methylase genes were highly resistant to gentamicin, amikacin and to bramycin(MICs≥512μg/ml). RmtA,rmtB,rmtC,rmtD,npmA and aph(3')-Ⅵa were not identified from 96 isolates. Conclusion1.aac(3)-Ⅰ,aac(6')-Ⅰb,ant(3")-Ⅰaminoglycosides modifying enzymes and armA 16s rRNA methylase gene had emerged for the first time in Human Province, aac(6')-Ⅰb is the most common genotype. armA 16S rRNAMEsthylase resistance gene carried high.2.Klebsiella pneumoniae resistant to the aminoglycoside antibiotics is very serious, the strains resistant to amikacin in Klebsiella pneumoniae were resistant to gentamicin and tobramycin drugs. amikacin can be used as the screening of antibacterial drugs to aminoglycoside resistance gene.3.Producing aac(6')-Ib gene of Klebsiella pneumoniae resistant to ciprofloxacin was significantly higher than that Sensitive to ciprofloxacin.Klebsiella pneumoniae producing armA Type 16s rRNA methylase gene was most highly resistant to aminoglycosides antibiotic.