Characteristic Analysis Of Carbapenem-Resistant Klebsiella Pneumoniae And Study On The Mechanism Of Tigecycline Resistance

Klebsiella pneumoniae is an opportunistic pathogen that inhabits normal human microflora and is the main cause of hospital-acquired infections.The emergence of carbapenem-resistant K.pneumoniae isolates has led to increased hospitalization,mortality,and morbidity.The efficacy of carbapenem antibiotics as the last line of defense against the production of extended-spectrum beta-lactamases(ESBLs)has been lost and carbapenem-resistant bacteria belonging to the family Enterobacteriaceae are constantly emerging.In particular,carbapenem-resistant K.pneumoniae(CRKP),which rapidly spreads mainly through plasmids,has become an important public health concern worldwide.Tigecycline is a new class of glycyltetracycline antibiotics,with broad-spectrum antimicrobial activity.It has been targeted for the treatment of bacterial infections exhibiting multi-drug resistance(MDR)and pan-drug resistance(PDR).It is also the most important antibiotic for the treatment of CRKP in China.However,clinical reports of a number of tigecycline-resistant strains are a cause of concern.The emergence of tigecycline-resistant K.pneumoniae(TRKP)may put CRKP-infected patients at serious risk.In this context,in the present study,which is divided into two parts,we have first discussed the epidemic situation and drug resistance characteristics of CRKP in our hospital,and have subsequently provided a description of our investigations on the new mechanism of drug resistance to tigecycline in CRKP strains,with the aim of providing an important reference for clinical treatment of CRKP and TRKP strains.In addition,the study of the tigecycline resistance mechanism is expected to be helpful in the prevention and treatment of TRKP and should also provide references for discovery of new drug targets.In the first part,the characteristics of CRKP strains,including drug susceptibility,molecular epidemiological characteristics,and drug-resistant genes,were analyzed to obtain data for further guiding the clinical use of the drug.For these studies,we used an existing bacterial library,which contains thousands of strains of K.pneumoniae.Among these,172 strains with carbapenem resistance have been selected and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS).We selected 166 strains of CRKP in the present research.The microbroth dilution method was used to determine the minimum inhibitory concentration(MIC)of tigecycline for the selected strains.We used multilocus sequence typing(MLST)to isolate the sequence type(ST)strains and construct phylogenetic trees.Pulse field gel electrophoresis(PFGE)was used to analyze the homology of CRKP strains in the pediatric intensive care unit(NICU),and finally the antibiotic resistant genes were detected using quantitative PCR.The results were positive for CRKP in 15.74%(166/1055)of the cases in our hospital.All the 166 CRKP strains exhibited MDR.The resistance to amikacin was the lowest(41.60%),followed by the resistance to tobramycin(61.60%),minocycline(64.00%),gentamicin(68.80%),trimethoprim/sulfamethoxazole(69.20%),cefoperazone/sulbactam(69.60%),and other common antibiotics(more than 90.00%);the resistance to imipenem was observed in all the strains.Test for the drug resistance genes was positive for KPC-2 in 75.90%(126/166),CTX-M9 in 68.07%(113/166),and CTX-M1 in 7.83%(13/166)of the selected strains.The deletion rates were 4.82%(8/166)for OmpK35,93.98%(156/166)for OmpK36,and 5.42%(9/166)for OmpK37.Three genes,namely IPM-4,NDM-1,and OXA-48 were not detected.The microbroth dilution assay showed that the MIC of tigecycline was mostly around 0.25 and 0.5 μg/mL for about 73.5% of the selected strains.No strain was found to be resistant to tigecycline(FDA standard: drug resistance,MIC ≥ 8 μg/mL).Twenty-four ST strains were identified by MLST.The main ST type in our hospital was ST11,accounting for 80.12%(133/166)of the strains.Homology analysis of 15 strains suspected to have caused nosocomial infection outbreaks in the pediatric ICU revealed that the outbreak strains were ST37 and ST006.Analysis using PFGE revealed that the outbreak strains corresponded to Class A and Class C,and the homology was more than 90%.The results showed that the epidemic situation of CRKP in our hospital was severe,owing to the drug resistance level being higher than the national average.The mechanism of drug resistance was mainly based on the production of KPC-2 enzyme.Tigecycline was found to be an effective drug for CRKP in our hospital.ST was mainly found to be ST11,consistent with the ST of epidemic strains in China.In the second part of the study,we investigated the new mechanism of resistance to tigecycline in bacterial strains by transcriptome sequencing,bioinformatics analysis,and knockout and complementation of differentially expressed genes.Three CRKP strains,namely KPN222(Group A),KPN114(Group B),and KPN315(Group C),were randomly selected for screening of tigecycline-resistant mutants in vitro,and transcriptome sequencing of mutants and primary strains was performed to identify the differentially expressed genes.Validation of the identified genes and detection of other genes related to tigecycline resistance was performed by qRT-PCR.The cusS–cusR gene deletion strain was constructed using a suicide plasmid pKO3-Km,and the cusR gene complement strain was constructed using pGEM-T-easy plasmid.The changes in the MIC of tigecycline before and after gene editing were determined,and in vitro bacteriostasis of TRKP was assessed using a combination of efflux pump inhibitors and determination of MICs for tigecycline.The following results were obtained: 1)Three groups(9 strains)of K.pneumoniae were screened in vitro—Group A(KPN222),comprising A1,A2,and A3;Group B(KPN114),comprising B1,B2,and B3;Group C(KPN315),comprising C1,C2,and C3.A1,B1,and C1 were the original strains,and A2,A3,B2,B3,C2,C3 were the resistant mutants.Drug susceptibility analysis showed that the MICs of tigecycline were 0.5,16,and 32 μg/mL,respectively.The strains were sensitive only to aminoglycosides and polymyxins and showed resistance to other common antibiotics.2)Transcriptome sequencing and qRT-PCR validation showed that the expression of acrAB–tolC efflux pump system increased in all the three groups but increased significantly in Group B(P < 0.01);the expression of cusS–cusR metal resistance system increased significantly in Group C(P < 0.01).3)Detection of other genes of K.pneumoniae related to tigecycline resistance revealed no mutation in ramR regulatory genes,and texT,Tet(A),oqxA,oqxB,acrE,acrF,and oprD were also not highly expressed.4)The cusR gene knockout(cusR)and its gene complement(C-cusR)mutants were successfully constructed;the MICs of tigecycline were 4 and 32 μg/mL,for cusR and C-cusR,respectively.5)The assessment of bacteriostasis of TRKP using a combination of an efflux pump inhibitor and tigecycline showed that phenylalanine arginine beta-naphthalamide(PAβN)had a significant inhibitory effect,whereas 1-methyl-2-pyrrolidone(NMP)and carbonyl-cyanogen-m-chlorophenylhydrazone(CCCP)caused no significant inhibition.Studies have shown that different strains exhibit different antibiotic resistance mechanisms.Although the phenotypes of antibiotic-resistant strains are similar,the mechanisms of antibiotic resistance differ.This is the first report of the role of the cusS–cusR system,a metal resistance system(copper and silver resistance),is in the mechanism of antibiotic resistance of K.pneumoniae.Moreover,we report the acrAB–tolC efflux pump system as a multi-drug efflux system that participates in most of the mechanisms of tigecycline resistance.