Molecular Epidemiology Of Carbapenem-resistant Klebsiella Pneumoniae And Antibacterial Mechanism Of Chlorogenic Acid

Objective: Klebsiella pneumoniae is an important pathogen of nosocomial and community-acquired infections.With the increasing trend of clinical isolation of carbapenem-resistant Klebsiella pneumoniae,the systemic multi-focal infection caused by carbapenem-resistant Klebsiella pneumoniae and the multi-drug resistance it carries aggravation the complexity of clinical diagnosis and treatment,posing a great threat to public health.To provide theoretical basis for clinical infection prevention and control through epidemiological investigation and analysis.To explore the molecular mechanism of chlorogenic acid,a natural component,against Klebsiella pneumoniae infection,and to provide appropriate reference for clinical treatment of infection and new drug development.Methods: 1.Fifty-seven non-repetitive strains of carbapenem-resistant Klebsiella pneumoniae isolated from clinical specimens in the Second Affiliated Hospital of Kunming Medical University from October 2020 to September 2021 were collected,and the species and drug sensitivity were identified by VITEK-2 Compact automatic microbial analysis system.Carbapenem-resistant Klebsiella pneumoniae strains were genotyped by multilocus sequence typing(MLST).Ten carbapenem-resistant genes(blaIMP,blaVIM,blaSPM,blaNDM,blaKPC,blaBIC,blaAIM,blaGIM,blaSIM and blaDIM)were amplified by PCR.The results were verified by sequencing.2.KEGG pathway analysis was used to predict the possible signaling pathways related to the activation of immune responses against KPN infection regulated by chlorogenic acid in lung tissues,and verified by mouse alveolar macrophages(MH-S).The expression of PI3 K,AKt,TLR2,TLR4,mTOR,p38 MAPK,MyD88,NF-κBp65,IκBα,AMPK,TRIF,JNK,ERK in the infection group and chlorogenic acid treatment group were detected by real-time quantitative PCR(Q-PCR),and the specific molecular mechanism was explored.Results: 1.Fifty-seven strains of carbapenem-resistant Klebsiella pneumoniae(CRKP)were isolated from 33.33% of sputum,26.32% of urine,10.53% of secretion,10.53% of drainage fluid,5.26% of blood,5.26% of venous catheter,5.26% of bile,and 3.51% of pus.The proportion of departments from which 57 strains of carbapenem-resistant Klebsiella pneumoniae were sent for examination was as follows: ICU 42.11%,neurosurgery 12.28%,neurological rehabilitation 10.53%,burns 8.77%,urology 8.77%,hepatobiliary and pancreatic surgery 7.02%,respiratory medicine 3.51%,geriatrics 3.51%,endocrinology 1.75%,pediatrics 1.75%.The resistance rate of CRKP to at least one drug of carbapenem antibiotics meropenem,imipenem,and ertapenem was 100%,and the resistance rate to three drugs was85.96%.The resistance rates of CRKP to cephalosporin ceftazidime,aminoglycoside amikacin and aztreonam,β-lactamase inhibitor tazobactam,and quinolone levofloxacin were 96.49%,84.21%,96.49%,98.25% and 98.25,respectively.Among the 57 CRKP strains,57(100%)were blaKPC positive,4(7.02%)were blaNDM positive,and 1(1.75%)was blaIMP positive.The blaBIC,blaVIM,blaSIM,blaSPM,blaAIM,blaGIM and blaDIM genes were not detected.Among them,52 strains(91.23%)contained only one resistance gene,5 strains(8.77%)contained two resistance genes,and no strain carried more than two resistance genes.A total of 57 CRKP strains were divided into 5 ST types.The most common ST type was ST11(52strains,91.23%),followed by ST23(1 strain,1.75%),ST37(2 strains,3.5%),ST65(1strain,1.75%),and ST375(1 strain,1.75%).2.After KPN infection,the mRNA expression of PI3 K,AKt,mTOR and p38 MAPK in mouse alveolar macrophages(MH-S)decreased,and the expression of TLR2,TLR4,MyD88,NF-κBp65,IκBα,AMPK,TRIF,JNK and ERK increased.After treatment with chlorogenic acid,the mRNA expression of PI3 K,AKt and mTOR in KPN infected MH-S cells decreased,the expression of TLR4,p38 MAPK,IκBα,TRIF,JNK and ERK decreased first and then increased,and the expression of TLR2,MyD88,NF-κBp65 and AMPK increased.Conclusion: 1.The high isolation rate of 57 carbapenem-resistant Klebsiella pneumoniae specimens was sputum,urine,secretion and drainage fluid.The distribution rate of 57 CRKP isolates was high in ICU,neurosurgery,and neurorehabilitation departments.Therefore,bacterial monitoring and prevention should be strengthened in key departments.Carbapenem-resistant Klebsiella pneumoniae showed high drug resistance rate to a variety of commonly used clinical antibiotics cephalosporins,aminoglycosides,β-lactamase inhibitors and quinolones,with an average drug resistance rate of more than 90%,and presented multiple drug resistance or even total drug resistance.2.57 strains of CRKP were divided into 5ST types,the main ST type was ST11,and the rest were ST23,ST37,ST65 and ST375.The prevalence of CRKP was mainly ST11,accompanied by low prevalence of other ST types.3.The detection rates of carbapenase genes in 57 CRKP strains were 100% in blaKPC,7.02% in blaNDM and 1.75% in blaIMP.No blaBIC,blaVIM,blaSIM,blaSPM,blaAIM,blaGIM and blaDIM genes were detected.The prevalence and multi-drug resistance of CRKP increase the complexity and difficulty of clinical diagnosis and treatment.In addition to strengthening the monitoring and control of bacteria,new drug combination schemes and development of new drugs are discussed to effectively treat Klebsiella pneumoniae infection.4.Chlorogenic acid may activate lung tissue immune response against Klebsiella pneumoniae infection through TLR2/MyD88/NF-κBp65 signaling pathway.