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Objective To investigate the antimicrobial resistant mechanisms of high level carbapenem resistant Klebsiella pneumoniae infection of burn patients .Methods A retrospective study was conducted on totally 18 non-repetitive high level CR-KP which were isolated from burn patients hospitalized between July 2014 and June 2015.MIC of antibiotics were determined by using the GN 13 cards and agar dilution method.The specific PCR and DNA sequence analysis were performed to confirm the β-lactamase type.Plasmid conjugation transfer experiments and southem hybridization were applied to study the mode of carbapenem resistance transmission .Outer membrane proteins ( Omps) were isolated and examined by PCR and ( sodium dodecyl sulfate polyacrylamide gel electrophores ) SDS-PAGE.Pulsed-field gel electrophoresis( PFGE ) and Multilocus sequence typing ( MLST ) was used to determine the genotypes . Results Susceptibility of antimicrobial agents indicated that all these strains with multiple drug resistance . The resistance rate to piperacillin/tazobactam, ceftriaxone, levofloxacin, ciprofloxacin, gentamicin, tobramycin, cefotetan, ceftazidime, cefepime, aztreonam, imipenem, and meropenem was 100％ (18/18).Moreover, the resistance rate of CR-KP isolates to amikacin was 72.2％ ( 13/18 ) , compound sulfamethoxazole was 61.1％(11/18), tigecycline was 0％(0/18).Conjugation study with Escherictda coli J53 resulted in the transfer of significant reduced carbapenem susceptibility from donors (MICs increased at least 8-fold).By PCR, eighteen strains of Klebsiella pneumoniae carried NDM-1 gene, 5 strains carried KPC-2 gene.The blaNDM-1 was transferable by plasmids.Southern blot hybridization indicated that the blaNDM-1 gene was located on plasmid in size of 46 kb.The plasmid belonged to incompatibility group IncX 3.Seven types of CR-KP were detected by PFGE.In addition, MLST assigned them to sequence type ( ST)11, ST395, ST17, ST37, ST263, ST14 and ST76 types.SDS-PAGE and ompK35/36 genes sequence analysis of Omp indicated that there was absence of outer membrane proteins OmpK 36 in ST11, ST395, ST37 strains.However, the other STs strains expressed lower quantities of OmpK 36.Conclusions High level carbapenem resistance in K.pneumoniae causing infection in burn patients is attributable to production of plasmid-mediated metallo· β-laetamase NDM-1 combined with porin OmpK36 deficiency or low expression .The K.pneumoniae with NDM-1 and KPC-2 carbapenemase were detected .
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ObjectiveTo investigate the prevalence and distribution of 16S rRNA methylase gene and research the relationship with drug resistant spectrum.And preliminary explore its role in molecular epidemiology analysis.MethodsCollected 69 clinical isolates of non repetitive ESBL-producing Klebsiella pneumoniae in our hospital from Mar to Sep 2010.Detection 16S rRNA methylation enzyme gene by PCR,and analyze ESBL genetype and integron gene of the positive strains.All PCR products were sequenced for determination.Plasmid conjugation test and plasmid elimination method to determine dissemination of 16S rRNA methylase gene.Then we used ERIC-PCR genotyping technology for the establishment of DNA fingerprinting.ResultsIn sixty-nine strains,twenty isolates were rmtB positive (28.9％),two isolates were armA positive,and two strains coproduce rmtB and armA.All positive isolates carried the CTX-M gene,detemined by sequencing,14 strains of CTX-M-14 gene,6 strains of CTX-M-15 gene,14 strains carried TEM1 gene,8 strains carried SHY gene,sequencing showed that 5 strains of SHV-12 gene,3 strains of SHV-11 gene,3 strains carried OXA-10 gene,3 strains carried VBE-1 gene.In addition,the intl gene was found in 12 isolates of 20 rmtB positive strains.All the intl gene positive strains were divided into five kinds gene cassettes,which contained drfA25,drfA1,drfA12,aadA1,aadA2,sat and blaVEB-1 genes.Respectivily,16S rRNA methylase gene positive strains were divided into five genetypes using ERIC-PCR technology.A genetype was the advantage popular clones.Conjugative plasmid and elimination test found that rmtB gene was located in a plasmid in KP5 and KP16 isolates with A genetype,and can disseminate by conjugation.ConclusionA high prevalence of 16S rRNA methylase gene-rmtB was found among clinical ESBL-producing K.pneumoniae isolates in our hospital,which could lead to resistant to almost all aminoglycoside at a high level.Both horizontal gene transfer and clonal spread were responsible for the dissemination of the rmtB gene.In addition,K.pneumoniae co-producing ESBLs,16S rRNA methylation enzymes and class Ⅰ integron existed and were spreading.
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Objective To investigate mechanisms of carbapenem resistance in Klebsiella pneumoniae. Methods Two carbapenem-non-susceptible Klebsiella pneumoniae Z4 and Z5 isolated from Beijing Hospital in 2008 were investigated. MICs of antibiotics were determined by agar dilution method.Conjugation experiment was carried out in mixed broth cultures. Plasmid DNA preparations were obtained by using an alkalinelysis technique. Elimination of plasmids was performed by repeated SDS treatment. The crude β-lactamase extracts were subjected to IEF. The genotype of β-lactamases were confirmed by PCRs and DNA sequence analysis. Outer membrane proteins (Omps) were isolated and examined by SDS-PAGE.The ompK35 and ompK36 genes were amplified by using PCR and were sequenced. Results MICs of imipenem, meropenem and ertapenem for Z4 and Z5 were 32, 32 and 256 μg/mi, and 1, 1 and 2 μg/ml.Conjugation study with Escherichia coli EC600 resulted in the transfer of significant reduced carbapenem susceptibility from Z4 and Z5 ( MICs increased at least 8-fold). Klebsiella pneumoniae Z4 produced IMP-4 metallo-β-lactamase, TEM-1 and SHV-1 spectrum β-lactamase and Z5 produced IMP-4, TEM-1 and SHV-12 extended-spectrum β-lactamase. E. coli transconjugants of both Z4 and Z5 produced a single IMP-4.Elimination of IMP-4-encoding plasmid from Z5 resulted in carbapenem susceptibility in the isolate,however, Z5 whose IMP-4-encoding plasmid was eliminated exhibited reduced susceptibility to carbapenems ( MICs of imipenem, meropenem and ertapenem were 0. 25 μg/ml,0. 5 μg/ml and 4 μg/ml). Amplification of integron revealed that blaIMP-4 gene of both Z4 and Z5 located within two different class I integrons which were carried on two plasmids with a similar size of approximately 55 000 bp. SDS-PAGE and ompK35/36 genes sequence analysis of Omp indicated that Z4 failed to express OmpK36, because of a nonsense mutation (CAG into TAG) in the ompK36 gene. Conclusion Production of plasmid-mediated metallo-β-lactamase IMP-4 or production of β-lactamase combined with porin OmpK36 deficiency can lead to reduced susceptibility to carbapenems. High-level carbapenem resistance in Z4 is mainly due to production of IMP-4 and the loss of OmpK36.
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0.05).Conclusion Imipenem and meropenem is effective and safe for the treatment of bacterial infection in spontaneous bacterical peritonitis patients.The average time of abatement of fever and treatment time in imipenem groups was shorter than than of meropenem groups.
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2.0 g/L,the ratio of CSF and blood glucose ratio