Study On The Mechanism Of Tigecycline Resistance In Klebsiella Pneumoniae

Bacterial resistance often leads to poor or even ineffective clinical treatment of bacterial infection,which has been widely concerned around the world.In order to reduce the influence of antibiotic-resistant bacterial infection on human life expectancy,people have been trying to explain the generation and transmission of resistance mechanism.In this study,four tigecycline-resistant Klebsiella pneumoniae were screened through antibiotic sensitive tests of 32 multi-drug resistant clinical isolates,and the antibiotic resistance mechanisms were preliminarily investigated.The data in this paper not only provide enlightenment for finding or explaining new mechanisms of drug resistance,but also provide theoretical support for subsequent studies on controlling the formation and transmission of antibiotic resistance.In this study,23 strains of K.pneumoniae and 9 strains of Escherichia coli were isolated and identified.Firstly,the antibiotic resistance of 32 strains to 24 common antibiotics was determined by disc diffusion test(K-B method).The results showed that 23 strains of K.had a wider antibiotic resistance spectrum than Escherichia coli:91%(21/23)of K.pneumoniae had a resistance range of 62.5%(15/24)or above to 24 antibiotics,and 7 strains of K.pneumoniae were resistant to 86.9%(20/24)or above of antibiotics.In addition,four strains of K.pneumoniae were insensitively to tigecycline,and the mechanism of drug resistance was further explored.Four strains of tigecycline resistant K.pneumoniae were named KP-291,KP-299,KP-382 and KP-386,respectively.The minimum inhibitory concentrations of tigecycline resistance were determined to be 8,8,4 and 4 mg/L by broth dilution method,respectively.The PCR method was used to amplify the integron and the tigecycline related resistance genes,and the result shows 100%type Ⅰ integron detection rate.In addition,complex type Ⅰ integron was detected in KP-299.The role of resistance-nodulation-cell division efflux pump(RND)of tigecycline resistance was evaluated by real-time fluorescence quantitative PCR and efflux pump inhibitor experiments.The results showed that efflux pump system was not the sole source of tigecycline resistance.In view of the important role of tigecycline in clinical application,the whole genome of the four antibiotic resistant strains was sequenced using the second-generation Illumina sequencing technology.Sequencing results were assembled by SPAdes,then Gene Mark S-2,RAST,CARD,NCB1 and other databases or tools were used for prediction,annotation and analysis of genomic sequence.The annotation results were compared with the reference strain K.pneumoniae HS11286,and the CARD annotation result showed that the four strains had 3-12 more antibiotic resistance genes than K.pneumoniae HS11286.An important RND type efflux pump,AcrAB-TolC,was found in all four strains.KP-291 and KP-299 also contained OqxAB efflux pump,and the background expression levels of the efflux pump were distinctly different,some genes were significantly up-regulated in the presence of IC50 tigecycline.By comparing the specific resistance genes of the four strains relative to K.pneumoniae HS11286,it was determined that the resistance of KP-291 tigecycline was mainly caused by the efflux pump and the resistance gene tet(X4),that the resistance of KP-299 was mainly caused by the combined action of efflux pump and tetA and ramR mutation.By further comparison of proteins,two types of proteins most likely caused resistance to tigecycline in KP-382 and KP-386:copper/silver efflux RND transporter components and ABC superfamily transporters.In conclusion,in this thesis,the important role of efflux pump in tigecycline resistance was further revealed by analyzing the antibiotic resistance mechanism of four tigecycline resistant K.pneumoniae strains.The results of whole genome sequencing and bioinformatics analysis suggest that in addition to the reported mechanisms of tigecycline resistance,there may be other resistance mechanisms that can mediate tigecycline resistance.This work provides a theoretical basis for the formation and spread of tigecycline resistance and the development of new antibiotics in the future.