| ObjectiveThe drug resistance of K.pneumoniae（Kpn）has been increasing year by year.It has been widely used in clinic with various antibacterials with high curative effect and wide antibacterial spectrum.According to the theory of mutant selection window（MSW）combined with the literature,we selected Amikacin（AK）,BIApenem（BIA）,Levofloxacin（LEV）,piperacillin/ZBP（Piperacillin/Tazobactam,TZP）four commonly used anti-Kpn anti-bacterial drugs in five combinations were studied in order to achieve the following objectives:1.To determine the minimal inhibitory concentration（MIC）and the mutant prevention concentration（MPC）of the tested strains corresponding to AK,BIA,LEV and TZP4 alone or in combination with different combinations of two.To explore the change of MSW in the single and combined use of drugs,and to evaluate the in vitro antibacterial activity of different combinations of antibacterials against Kpn in order to facilitate clinical reference and help Kpn infection develop a rational antibacterial drug combination program.2.To study the mechanism of resistance of Kpn mutants to the best combination of bactericidal drugs in vitro and to analyze the mechanism of molecular resistance and gene mutation of drug-resistant mutants at the gene level to provide a basis for clinical treatment and prevention of Kpn infection.Methods1.The diameter of the zone of inhibition was measured by disk diffusion method,and the susceptibility of the tested strains to 15 commonly used antibiotics was analyzed.The diameter of the zone of inhibition was judged according to the recommended method of the American Society for Clinical Laboratory Standards（CLSI）:S（sensitivity）,I（mediation）,and R（resistance）.The results of the combination of drugs based on the determination of the shape of the inhibition zone of two drug sensitive paper.This completes the review and screening of the preserved CRKP strains and decides the combination of the drugs.2.The broth microdilution method makes the concentration of drug in each well of the 96-well bacterial culture plate be gradient,and the MIC of the test strain corresponding to the combination of AK,BIA,LEV and TZP alone or in different combinations is calculated,and the corresponding Fractional inhibitory concentration index（FIC）.3.The concentration of Kpn was more than 3*1010 CFU ml-1 by broth method,in turn inoculated to the drug concentration from low to high order sterile agar plate.The corresponding selection index（SI）was calculated by measuring the MPC of the tested bacteria corresponding to AK,BIA,LEV and TZP alone or in combination with different combinations.4.The mechanism of levofloxacin,piperacillin/tazobactam and combination of levofloxacin,piperacillin/tazobactam was analyzed by PCR amplification and DNA sequencing.Results1.The minimum inhibitory concentrations of Kpn standard strain ATCC700603 for AK,BIA,LEV and TZP were 0.5μg·mL-1,0.125μg·mL-1,0.5μg·mL-1,8μg·mL-1;Compared with the single drug,the combined use of the MIC,MPC values decreased significantly,showing a synergistic effect.The MIC range of 36 strains of clinical ESBLs-positive CRK strains with AK combined with BIA and TZP were 0.125-4μg·mL-1,0.0625-8μg·mL-1,respectively.When combined with AK,the MIC range of BIA The range of MIC of TZP was 16512μg·mL-1 for 18μg·mL-1 and the range of MIC for LEV combined with AK,BIA and TZP for 36 strains of clinical ESBLs-positive CRKP was 0.25-32μg·mL-1,0.2564μg·mL-1,0.25-32μg·mL-1;MIC of AK was 0.0625-8μg·mL-1 in combination with LEV,MIC was in the range of 0.5-16μg·mL-1.The MIC range of 8256μg·mL-1.AK combined with BIA and TZP,respectively,showed synergistic effects and additive effects on 36 clinical ESBLs positive CRKPs,of which 47.2%（17/36）and 36.1%（13/36）had synergistic antibacterial effects respectively.The additive effects accounted for 33.5%（12/36）and 27.8%（10/36）respectively.No effect was observed in 19.5%（7/36）and36.1%（13/36）.The synergistic effects of synergistic antimicroBIAl effect on 36 CRBLs positive for ESBLs in 36 clinical isolates were 36.1%（13/36）,38.9%（14/36）and 44.4%（16/36）,respectively.The additive effects accounted for 36.1%33.6%（11/36）,27.8%（10/36）and 33.4%（12/36）respectively.The unrelated effects were 33.3%（12/36）,33.3%（12/36）and 22.2%),No antagonism.3.ATCC700603 standard strains and mutants produced by PCR amplification after sequencing,only the TZP mutant drug-resistant strains alone in the 800bp gel near the area has a bright band carrying bla CTX-M gene;ESBLs-positive CRKP resistance The rates of detection of two or more genes in drug-mutated strains were 47.2%（for LEV alone）,25%（for TZP alone）and 22.3%（LEV+TZP）.4.The results of sequencing showed that the sequences of the strains carrying blaKPC-2,bla CTX-M-1 and bla CTX-M-2 were detected with the corresponding strains（ie JQ838154.1,MG948566.1,KJ803939.1）As a result,the percentage of 100%homology was 89.7%（44/49）,82.6%（19/23）and 81.8%（18/22）,respectively,and 4 of the remaining nucleotide sequences Different point mutations:two types of blaKPC-2 gene mutations were detected;bla CTX-M-1,bla CTX-M-2 gene mutations were detectedin the type of each one.ConclusionThe antibacterial activities of Kpn against AKP and BIA,LEV and TZP in combination with two different antibacterials were mainly synergistic in drug sensitivity test in vitro,followed by additive effect.Compared with the single use of antimicroBIAl agents,Klebsiella pneumoniae MIC,MPC values were significantly reduced,the mutant strains of drug-resistant gene mutation rate was significantly reduced,at the same time,mutation selection window in the combination of antimicroBIAl agents significantly Narrowed to achieve the desired effect of effectively avoiding the emergence of drug-resistant mutant strains.Although some strains appeared new subtypes with many kinds of amino acid mutations in this project,the combination of antimicroBIAl agents can reduce the incidence of mutant strains compared with the single drug.In short,the combination of antimicroBIAl drugs should attract the attention of clinical pharmacy staff and clinicians. |
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