Nosocomial Infection Of Carbapenem-Resistant Klebsiella Pneumoniae And Its Tigecycline Resistant Mechanism

Klebsiella pneumoniae is a common pathogen in clinical infectious diseases.Because of the abuse of antibiotics and the wide spread of resistance genes,multidrug resistant Klebsiella pneu,rioniae has been a tough problem among clinicians.As it is highly resistant to most common antiobitics,for instance,p-lactam antibiotics,aminoglycosides,macrolides etc.,carbapenems are almost the last line of defense.The battles between new antibiotics and the evolution of resistance mechanisms never stop.The urge of new antibiotic treatments arises along with the increasing emergence of carbapenem-resistant Klebsiella pneumoniae(CRKP)whole worldwide caused by the spread of carbapenemases.Tigecychne,a derivative of minocycline,can overcome the Tet protein related tetracycline resistant mechanisms,and was found effective to several multidrug resistant bacteria.But soon after tigecycline marketing,tigecycline resistance appears among patients.Tigecycline resistance in CRXP is now a great concern and its clinical distribution and resistant mechanisms need further exploration.This study includes three parts,to analyze the the risk factors and clinical distribution of CRKP,the clinical characteristics of tigecyeline-resistant carbapenem-resistant Klebsiella pneumoniae(TR-CRKP),and the tigecycline resistant mechanism in clinical CRKP isolates,respectively.In the first part,we studied the resistance rates of clinical CRKP isolates to several antibiotics and analyzed the possible risk factors of infection of TR-CRKP.We obtained 286 CRKP isolates from inpatients,which only include the first isolate in each patient.Their minimum inhibitory concentration(MIC)of several antibiotics were tested by microdilution broth method and agar dilution method.The correlated clinical informations were collected,including age,gender,height,weight,basic diseases,the length of hospitalization,the length of intensive care unit(ICU)stay,prognosis,invasive operations performed three months previous to isolate collection,antiobiotics received three months previous to isolate collection.Comparisons between non-tigecycline-resistant group(TS-CRKP)versus tigecycline-resistant group(TR-CRKP)(tigecycline MIC>8 mg/L,according to FDA)were performed.Result:1)The resistant rates of 286 CRKP isolates to tigecycline,colistin,ceftazidime/avibactam,fosfomycin,amikacin,aztreonam were 16.4%,4.9%,3.8%,25.5%,41.6%,99.7%,respectively.2)The characteristics of CRKP patients include male,elders and surgical patients and they are mainly nosocomial infection.3)The main CRKP transmission routes include respiratory tract,bile duct,blood and urinary tract.4)Patients infected with TR-CRKP had higher rates of ICU stay,and the risk factors include endotracheal intubation,the use of catheter or gastric tube and the use of tigecycline,while the patients’ basic status is not related.Conclusion:The resistance of CRKP to several drugs tends to be more severe year after year.Tigecycline,colistin,ceftazidime/avibactam and fosfomycin maintained its high efficacy,while Aztreonam was almost useless.CRKP infections are mainly hospital-acquired,and characteristics of CRKP patients include male,elders and surgical patients.The higher rates of ICU stay,and the use of catheter,gastric tube,endotracheal intubation and tigecycline in TR-CRKP infection,gave us the hint about how to control nosocomial infection.In the second part,we studied the connection between TR-CRKP clinical isolates We obtained 47 TR-CRKP isolates from inpatients as mentioned previously.Whole Genome Sequence(WGS),Pulsed Field Gel Electrophorcsis(PFGE)and Multilocus Sequence Typing(MLST)were performed.Result:Up to 47 TR-CRKP isolates were selected from 286 CRKP isolates.43 out of 47 isolates belonged with ST11 Klebsiella pneumoinae,the other four isolates belonged with ST268,ST15,ST1886 and ST485 PFGE showed that 42 out of 47 isolates can be clustered into 5 clone types,while the other five isolates left were clones with low identity.Conclusion:The infection of TR-CRKP is mainly related to nosocomial transmission,while individual cases happen occasionally.In the last part,we studied the tigecycline resistant mechanism in TR-CRKP isolates,which emerged its tigecyeline resistance during the patient’s antimicrobial treatment.We obtained a series of isolates from an inpatient during tigecycline treatment.Antimicrobial sensitivity test,PFGE,MLST,WGS and analysis of resistance genes were performed.The putative gene cause was identidied by the comparing single nucleotide polymorphism(SNP),and plasmid complementation was carried out to ensure the gene is related wtih tigecycline resistance.Result:A series of isolates named A814,A112,A113 were obtained successively from a 60-year-old woman,with tigecyline MIC of 4mg/L,4mg/L,16mg/L,respectively.They belonged with ST11 Klebsiella pneu;oinae and the same PFGE type,had no difference in resistance genes.However,a loss-of-function insertion ISkpn18 was identified in A113,which could be the possible cause of tigecycline resistance.Plasmid pCR2.1-hyg-ramR,carrying wild type ramR and antibiotic marker Hygromycin coding gene,was tranfered into A113.The tigecycline MICs of complemented isolate A113-pCR2.1-hyg-ramR and reference isolate A113-pCR2.1-hyg were 8mg/L and 16mg/L.Conclusion:Tigecycline resistance related with ramR loss-of-function insertion by ISkpn18 was first reported in our research.However,there could be other mechanisms unrevealed considering that A113-pCR2.1-hyg-ramR is still tigecycline resistant.