| Objective:Klebsiella pneumoniae is an important conditional pathogen and one of the iatrogenic infection bacteria.When the body’s immunity declines,it will cause infection in various systems of the body.As more and more Klebsiella pneumoniae combined with high drug resistance and high virulence was discovered,and the serious infection and treatment difficulties pose great challenges to clinical treatment.Therefore,this study explored the molecular mechanism of Klebsiella pneumoniae with high virulence and high drug resistance(carbampenem-resistance).Methods:1.The carotenoid-resistant Klebsiella pneumoniae isolated from clinical specimens from the Second Affiliated Hospital of Kunming Medical University from January 2015 to December 2017 was collected.Previous studies have tested the MLST typing and drug resistance phenotype of Klebsiella pneumoniae.In this experiment,Klebsiella pneumoniae was divided into hypervirulent Klebsiella pneumoniae and high-resistance Klebsiella pneumoniae,carbapenem-resistant genes blaIMP,blaVIM,blaSPM,blaNDM blaKPC,blaBIC,The blaAIM,blaGIM,blaSIM and blaDIM genes and four related virulence genes of rmpA,rmpA2,iucA and iroN were detected by PCR and verified by sequencing.The virulence genes rmpA and rmpA2 were used to determine whether the strain contained virulence plasmid..Analyze and compare the pLVPK virulence plasmid carrying between Klebsiella pneumoniae high-resistance strains and highly virulent strains,and preliminarily judge the drug resistance and virulence,and provide a basis for further research and screening of high-resistance and high-virulence strains of Klebsiella pneumoniae.2.Through the screening results of high virulence and high drug resistance K.pneumoniae,one strain of ST65 high virulence high drug resistance strain No.63 and one strain ST11 high drug resistance and high virulence can be selected strain No.26 was used as the experimental group,NTUH-k2044 was used as the positive control group,and normal saline was used as the normal control group.The relevant virulence experiments were performed on the mice,and Klebsiella pneumoniae in the logarithmic growth phase was formulated with physiological saline.0.5 McDonald’s(108 cfu/mL),normal saline was diluted to 108,107,106,105,104,103 and 102cfu/mL in turn,and intraperitoneal injection of 0.5 mL per group of 8 BARB/C mice The LD50 value of the mice was observed for 7 days,and the liver,lung,spleen and kidney of the mice were stained with HE,To compare the virulence between high-drug resistant plasmids and highly virulent strains to obtain highly resistant plasmid strains 3.According to the previous part of the study,it is known that the highly virulent strains obtain high virulence plasmid strains and the highly virulent strains have high virulence plasmids,and the highly virulent strains obtain high drug resistance plasmid strains.The virulence of strains with high virulence plasmids was higher than that of strains with high virulence.Therefore,two strains of ST65,two strains of ST23 and two strains of ST11 were selected from the strains,including the former part of the mice.The strains of the virulence experiment were numbered 63,66,24,77,26,and 110,respectively.Six genome-wide Klebsiella pneumoniae genome-wide assays were performed to analyze the factors affecting drug resistance and virulence by genome-wide sequencing to explore the virulence and high drug resistance of strains that caused high virulence to obtain high drug resistance.Molecular mechanisms of virulence differences in virulence strains.Results:1.This study classified K.pneumoniae into highly virulent strains and highly resistant strains based on 147 strains of K.pneumoniae resistant to K.pneumoniae.Among them,9 strains of highly virulent strains accounted for 6.12%,a total of 138 high-resistance strains,accounting for 93.88%.In the detection of carbapenem-resistant genes of Klebsiella pneumoniae,136 strains containing drug resistance genes accounted for 98.55%,of which two strains did not contain drug resistance genes,accounting for 1.46%.Among the high-resistance strains,one strain containing one drug resistance gene was the most,with 90 strains(65.69%);the least containing three drug resistance genes,two strains(1.46%).Among the 9 strains of high-virulence Klebsiella pneumoniae resistant to carbapenem resistance,9 strains(100%)contained drug-resistant genes,including one drug-resistant gene,6(66.67%).%),no strain contains three drug resistance genes.The detection rate of rmpA2 in 138 strains of Klebsiella pneumoniae virulence gene was the highest,49 strains(35.50%)were detected,the detection rate of iucA was the lowest,and 5 strains(3.62%)were detected,carrying virulence plasmid.The number of strains was 27,accounting for 19.57%of the highly resistant strains.The four virulence genes of rmpA,rmpA2,iroN and iucA in 9 strains of highly virulent Klebsiella pneumoniae were detected in 9 strains,the detection rate was 100%,and the number of strains carrying virulence plasmids was 9 Strain,accounting for 100%of highly virulent strains.There were 36 strains with high virulence combined with high drug resistance,accounting for 24.49%of all strains.The analysis of the relationship between virulence gene distribution and drug resistance gene distribution of 147 strains of Klebsiella pneumoniae showed significant differences,and there was a negative correlation between the number of virulence genes and the number of drug resistance genes.2.After intraperitoneal injection of BARB/C mice in each group,the survival rate of 102cfu/mL mice in ST65 type Klebsiella pneumoniae strain group was 12.5%within 7 days.It is speculated that the strain LD50<0.5 × 102cfu,positive control group NTUH-k2044 The LD50 of the strain in this experiment was 4.8 × 10 4 cfu,and the ST11 strain could cause half of the death of the mice under the measurement of 5×107 cfu,and the LD50 value could not be calculated,but it could be proved that it had certain virulence to the mice,from the mouse.In the virulence experiment,the virulence of the three strains to mice ST65 strain>NTUH-k2044 strain>ST11 strain can be seen.HE staining of ST65 strain group and NTUH-k2044 strain group showed that the lung and liver of mice were infected and damaged to different extents.No obvious abnormalities were found in HE staining of ST 11 strain group and normal control group.3.Six strains of Klebsiella pneumoniae carried a variety of drug resistance genes and virulence genes,and six strains were analyzed by genome sequencing.All of the six strains carried the carbapenem-resistant gene blaKPC-2 gene,among which ST65(No.66)and ST11 The(No.110)strain carries the blaKPC-2 gene.Although it is resistant to imipenem and ertapenem,it is not resistant to meropenem.The ST23(No.77)strain does not carry aminoglycosides.Related genes,but resistant to aminoglycoside drugs.ST23(No.77)strain and ST65(No.66)strain carry sulfa resistance genes and quinolone resistance genes,respectively,but they are not resistant to sulfonamides and quinolones.A virulence gene correlation analysis of six strains of Klebsiella pneumoniae revealed that the fimE/A gene associated with adhesion function was carried,and the virulence genes associated with the iron uptake system were the most,including the iroN/D/C/B of the Salmochelin,entA/B/E/S and fepA/B/C/D/G of Enterobactin,fyuA,ybtA/E/T/U/S/P/Q/and irp1/2 of Yersiniabactin,iutA,iucA/B/C/D of Aerobactin.Except for ST23(24)strains,they carried the fimE/A gene associated with adhesion function,and ST 1(26,110)type strains compared to ST65(63,66)and ST23(77,24).The four strains lacking the virulence-related genes of gas-producing bacteria and salmonella.Conclusion:1.147 strains of carbapenem-resistant Klebsiella pneumoniae ST classification of Klebsiella pneumoniae into high-virulence strains and high-resistance strains,of which 9 strains(6.12%)of highly virulent strains,high drug resistance There were 138 strains(93.88%).The carbapenem-resistant genes of 10 Klebsiella pneumoniae were tested.Both high-resistance and high-virulence strains carried only three genes,blaKPC,blaBIC and blaNDM,and carried multiple drugs at the same time.The situation of genes is rare.The four virulence genes of the highly virulent Klebsiella pneumoniae rmpA,rmpA2,iroN and iucA carry higher proportions than the highly resistant Klebsiella pneumoniae,and all of the highly virulent Klebsiella pneumoniae carry virulence Plasmid..2.Highly resistant strains obtained high virulence plasmids and highly virulent strains obtained virulence differences between high-resistance plasmid strains and high virulence strains obtained high-drug resistant plasmid strains with higher virulence than high-resistant strains A strain of a virulence plasmid.3.The difference in virulence between high-drug resistant plasmids and highly virulent strains obtained by highly resistant plasmids is mainly due to the high virulence strains obtained by highly resistant plasmid strains carrying Aerobactin and Salmochelin... |
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