| Objective : To investigate the The correlation between the biofilm formation ability and drug resistance of klebsiella pneumoniae.Distribution of efflux pump genes of multi-drug resistant klebsiella pneumoniae and the expression of acr A in biofilm.Efflux pump inhibitors CCCP effect on the biofilm of klebsiella pneumoniae.Further study the klebsiella pneumoniae biofilm resistance mechanism and To provide scientific basis for effective control of klebsiella pneumoniae infection.Methods:1.61 strains isolated from the clinical infection samples of The Affiliated Hospital of Southwest Medical University from January2017 to June 2017 were collected.The Micro Scan Walk Away 96 Plus automatic microbiological identification/drug sensitivity test system was used for identification.Analysis of the source and distribution of the specimens and analysis of their drug resistance spectrum.2.Crystal violet staining and confocal laser scanning microscopy(CLSM)were used to evaluate the biofilm ability of klebsiella pneumoniae.To analyze the correlation between the strength and resistance of klebsiella pneumoniae biofilm formation.3.The biofilm growth curve of klebsiella pneumoniae was prepared by randomly selecting 16 strains of biofilm forming ability.Use the microbroth dilution method to detect the MIC and MBEC of 16 strains of Cefepime,Amikacin,Ciprofloxacin,Imipenem and Polymyxin B.Analyed the correlation between MIC,MBEC and biofilm formation ability.4.Use PCR(Polymerase Chain Reaction,PCR)technology to detect 28 strains of Multiple drug-resistant bacteria pneumonia klebsiella acr A efflux pump gene,emr B,oqx A,qac E?1 carrying case.and RT-PCR was used to analyze the expression of acr A positive strains in the state of biofilm and floating.5.Analyzed the effect of CCCP on the biofilm formation of klebsiella pneumoniae by crystal violet staining.Results:1.Clinical isolation of 61 strains of klebsiella pneumoniae,It was mainly from 34 strains of blood,accounting for55.74%,followed by sputum 16,accounting for 26.22%,secretion 8,13.11%,and urine 3 strains,accounting for 4.92%.The clinical department distribution was the highest in ICU ward,21.31%,followed by the respiratory ward,and the detection rate was 13.11%.Detection of multiple drug-resistant klebsiella pneumoniae(MDR-Kpn),the detection rate was 45.9%(28/61),33 strains of non-multiple-drug-resistant bacteria,with a detection rate of 54.1%(33/61).MDR-Kpn including Ampicillin and Cefazolin,13 kinds of antibiotic resistant rate over50%,only to the Meropenem,Imipenem,Eritrea,Amikacin and Cefoperazone/sulbactam sensitive.MDR-Kpn(n = 28)and none MDR-Kpn(n =33)comparing two groups of 23 kinds of antibiotic resistance,MDR-Kpn to except Ampicillin,Meropenem,Imipenem,Eritrea.,Amikacin Cefoperazone/sulbactam and so on 17 kinds of antibiotic resistance were significantly higher than the none MDR-Kpn strains(P< 0.05).2.Among 61 strains of Kpn,only Kpn216 and Kpn225 could not form biofilm,and the positive rate of biofilm was 96.72%.Among them,14 strains of biofilm were strongly positive,27 were positive,and 18 were weakly positive.There was no significant difference between multidrug-resistant bacteria and non-multidrug-resistant biofilm formation(P> 0.05).3.Random selection of biofilm formation ability of 8strains of bacteria and biofilm formation ability of 8 strains of bacteria,detection of growth curve,found no obvious difference in growth rates between bacteria,explain the differences between the ability of biofilm formation is not due to different of the bacterial growth rate.The MIC and MBEC of 16 strains were detected quantitatively in the Cefepime,Amikacin,Ciprofloxacin,Imipenem,and polymyxin B,As a result,Kpn formed biofilm and increased resistance to different antibiotics.For Cefepime,MIC was 4-64ug/ml,MBEC was 320-2560ug/ml,and the increase was 20-640 times.For amikacin,MIC is 1-16ug/ml,MBEC is 80-5120ug/ml,and the increase is 40-5120 times.For Ciproxacin,MIC is0.25-8ug/ml,MBEC is 40-5120ug/ml,and the increase is 80-5120 times.For Imipenem,MIC is 0.25-16ug/ml,and MBEC is 160-2560ug/ml,with an increase of 80-5120 times.For polymyxin B,MIC was 0.5-2ug/ml,and MBEC was2-512ug/ml,with an increase of 4-512 times.Through the analysis,different strains MIC value and MBEC with Cefepime(r = 0.628,p = 0.628)and Ciprofloxacin(r = 0.81,p = 0.0001)is positive correlation,namely strain MIC value,the greater the MBEC value after the formation of biofilm.Polymyxin B(r=0.319,p=0.229),Amikacin(r=0.441,p=0.088)and Imipenan(r=0.079,p=0.769)were not correlated.4.28 strains of MDR-Kpn efflux pump gene detection,emr B gene positive 25 strains,the highest positive rate,at 89.29%,followed by positive 22 strains oqx A gene,positive rate was 78.57%,qac E?1strains gene is positive,positive rate of 39.29%,10 strains acr A gene positive,positive rate was 35.71%.28 isolates Kpn215 not only efflux pump gene detection to carry only one type of efflux pump gene have 4 strains,accounting for 14.29%,carry two efflux pump gene 8 strains,accounted for 28.57%,a carry three efflux pump gene 12 strains,accounted for 42.86%,and at the same time carry four efflux pump gene have 5 strains,accounting for 17.86%.After statistical analysis,there was no significant correlation.After sequencing,the homology of the corresponding gene sequences of 4 kinds of efflux pump gene positive amplification products was more than 98%.5.Randomly selected from 6 acr A gene positive strains of klebsiella pneumoniae,contrast state of biofilm and planktonic condition acr A gene expression,found a biofilm condition acr A rise in gene expression exist to varying degrees,the highest increase multiples of 78 times.It is proved that the formation of biofilm can promote the expression of acr A.6.When the CCCP concentration of the efflux pump inhibitor was1.25-10?g/ml,it could promote the formation of klebsiella pneumoniae biofilm.Conclusions : 1.Multiple drug-resistant pneumonia klebsiella bacteriadetection rate is higher,MDR-Kpn only to Meropenem ? Imipenem ?Eritrea.Amikacin Cefoperazone/sulbactam low percentages,as the treatment of klebsiella pneumoniae infection drug use.2.There was no significant difference in the biofilm formation ability of klebsiella pneumoniae biofilm with positive detection rate,and multidrug-resistant bacteria and non-multidrug-resistant Kpn.,The resistance of klebsiella pneumoniae to biofilm increased significantly,and the MIC value of cefepime and ciprofloxacin was positively correlated with MBEC value.3.The detection rate of the efflux pump gene of MDR-Kpn strain was relative increase,and most carried two or more efflux pump genes,and The formation of biofilm can promote acr A expression..4.The efflux pump inhibitor CCCP has a inhibitory effect on the biofilm of klebsiella pneumoniae lower than the working concentration can promote the formation of biofilm at different degrees. |
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