Inhibitory Effect Of Gallium Nitrate On The Growth And Biofilm Of Carbapenem-resistant Klebsiella Pneumoniae

ObjectiveTo use the VITEK 2 Compact automatic microbial analysis system for bacterial identification,and the agar dilution method was used for drug susceptibility test.The clinically isolated carbapenem-resistant Klebsiella pneumonia was screened to understand its resistance to antibiotics.PCR technology was used to detect strain drug resistance genes,and multi-locus sequence typing(MLST)technology was used to classify CRKP.To determine the minimum inhibitory concentration(MIC)of Gallium nitrate(GaN)on CRKP by microbroth dilution method;The BF strong positive strain was screened by 96-well plate crystal violet staining;The inhibitory effect of GaN at subconcentration on BF formation of BF strong positive strains was examined.It provides an effective method for effectively treating infections caused by drug-resistant bacteria.Materials and Methods Isolates he experimental strains were isolated from various specimens of inpatients and outpatients in the 34 hospitals affiliated to the Monitoring Network of Bacterial Resistance Surveillance Center of Anhui Province from 2015 to 2016.Methods1.Collecting KPN isolated from 34 hospitals in Anhui Province from 2015 to2016.VITEK 2 Compact automatic microbial analysis system was used for bacterial identification,and agar dilution method was used for drug sensitivity test to screen out clinically isolated CRKP.2.The ST classification of CRKP from 2015 to 2016 was detected by MLST genotyping technology.Partial resistance genes carried by CRKP in 2016 were detected by PCR.3.The growth curve of the KPN biofilm was drawn by crystal violet staining by constructing a biofilm model in vitro.Based on this,the growth of the biofilm in 28 hours was determined.The biofilm strong positive strains were screened from the experimental strains by crystal violet staining to detect the minimum inhibitory concentration(MIC)of GaN on strong positive strains.4.Using gentamicin(GEN)as a control drug,crystal violet staining,MTT assay,and confocal laser scanning microscope(CLSM)were used to detect the formation of 1/2MIC GaN on biofilm matrix.Changes in the number of viable bacteria and three-dimensional structure of biofilm,and study the phenotypic effects of GaN on the formation of CRKP biofilm.Results1.The proportion of CRKP in Anhui Province in 2016 is higher than that in 2015.82 clinically isolated CRKPs were screened and multidrug-resistant bacteria.2.The ST type of CRKP in Anhui Province is mainly ST11.The production of KPC-2 nzyme CRKP is still the main trend,and all are ST11 type.3.By establishing a biofilm growth curve of the biofilm,it was determined that the iofilm reached maturity in 24 hours.The MIC range of GaN to 82 strains of CRKP as 0.0625~2μmol/mL,of which MIC50 and MIC90 were 0.25 and 0.5μmol/mL,respectively.There were 31 strains of BF strong positive strains in 82 clinical isolates of CRKP,accounting for 37.8%(31/82).4.Sub-inhibitory concentration of GaN can significantly inhibit the formation of biofilms and the number of viable cells in experimental strains,the difference is statistically significant(P<0.05),and can significantly change the morphology of BF,and its inhibition is better than GEN.Statistical significance(P <0.05).Conclusions1.BF strong positive strains accounted for 37.8%(31/82)of clinical isolates of CRKP,indicating that once the human body is infected by drug-resistant bacteria,it will bring huge challenges to clinical treatment.2.The sub-inhibitory concentration of gallium nitrate(1/2MIC)is superior to the sub-inhibitory concentration of gentamicin in inhibiting the formation of biofilm matrix,and can also reduce the number of living cells in the biofilm and its normal morphological structure.It is suggested that gallium nitrate has a certain role in the prevention and treatment of clinical infection caused by CRKP biofilm.