Study On The Mechanism Of Polymyxin Resistance Related To Lipopolysaccharide Modification In Carbapenem-resistanct Klebsiella Pneumoniae

Objective:Polymyxin is currently one of the few antibacterial drugs for the clinical treatment of carbapenem-resistant Klebsiella pneumoniae(CRKP)infections.In recent years,polymyxin-resistant CRKP has increased,which undoubtedly brings unprecedented challenges to clinical anti-infective treatment.The purpose of this study was to investigate the specific mechanism of polymyxin resistance in CRKP by detecting LPS modifications and the changes in LPS of clinical polymyxin-resistant CRKP isolates,in order to provide a theoretical basis for the anti-infection treatment of polymyxin-resistant CRKP.Methods:1.Bacterial identification and drug sensitivity: Matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS)was used for bacterial identification.VITEK-2 Compact automatic bacterial drug sensitivity identification system,Kirby-Bauer(K-B),E-test method,broth microdilution method and 96-well plate method were used for antimicrobial susceptibility testing(AST).2.Enzyme type detection and homology analysis: the carbapenemase type of the clinical isolates was detected by polymerase chain reaction(PCR),and their ST type of multilocus sequence typing(MLST)was detected for homology analysis.3.Study on the mechanism of polymyxin resistance related to lipopolysaccharide modification in CRKP:(1)Detection of plasmid-mediated mobile drug-resistance genes by conjugation assay and mcr genes detection.(2)Expression of two-component systems(TCS)genes was measured using quantitative real-time PCR(q RT-PCR).(3)The whole genome was sequenced(WGS),compared with the reference sequence of polymyxin sensitive CRKP HS11286 in the NCBI database.The mutations related to LPS modifications were analyzed and annotated using Snp Eff software,ISEScan software,KEGG database,CARD database,Pfam database,etc.Partial mutation sites were verified by PCR and Sanger sequencing.(4)Detection of LPS: SDS-PAGE electrophoresis silver staining and MALDI-TOF-MS were performed to analyze changes in LPS.4.Detection of efflux pump and outer membrane porin proteins related drug-resistance genes: The drug-resistance genes related to efflux pump and outer membrane porin proteins were detected by WGS.Results:1.Bacterial identification and drug sensitivity: CRKP with polymyxin resistance showed multidrug resistance(MDR)high level of resistance to polymyxin(MIC value ≥ 32 μg/ml).2.Enzyme type detection and homology analysis: CRKP-1～CRKP-7 strains were KPC-2-type carbapenemase-producing CRKP,STs of those isolates belonged to ST-11 clonal complex.3.The resistance mechanism of polymyxin related to LPS modifications in CRKP:In this study,polymyxin-resistant CRKP mainly occurred mutations of two-component regulatory system mediated by chromosomes.(1)Plasmid-mediated mobile drug-resistance genes were not detected in polymyxin-resistant CRKP: the result of conjugation assay was negative and no mcr gene(mcr-1 to mcr-8)was detected.(2)Changes in relative expression of genes of two-component regulatory system:Compared with the reference strain,expression of pmr A,pmr B and pmr C genes at the transcriptional level was increased in all investigated isolates,and expression of pho P/pho Q genes in 6 strains increased,expression of crr A/crr B gene of the three strains decreased,expression of the four strains to the mgr B gene decreased.(3)Results of WGS analysis: 3677 common mutation sites were detected,of which 2272 were synonymous mutations.The sense mutation sites related to the regulation pathway of the two-component system of the drug-resistant strains were mainly located in KPHS＿09430,KPHS＿35900,KPHS＿39520,KPHS＿52420,etc.pmr A,pmr B,pmr C,pho P,pho Q,crr A and crr B genes were all wild types without mutation.ISkpn14 insertion sequence was detected in the mgr B gene of CRKP-6strain,and the specific insertion site was obtained through PCR and Sanger sequencing,which was between the+123 and+124 nucleotides of the mgr B gene coding region of CRKP-6 strain.(4)Changes in the structure of LPS were observed: The results of SDS-PAGE electrophoresis and silver staining showed that there was no significant difference in the LPS bands of each strain.In MALDI-TOF-MS analysis,compared with the sensitive strain,the polymyxin-resistant CRKP strains showed unique ions at m/z1955 and 1971,which meant the L-Ara4 N modification of natural lipid A in LPS observed at m/z 1824 and 1840(?m/z131).4.The drug-resistance genes related to efflux pump and outer membrane porin proteins: The same mutation sites in the efflux pump Mdt ABC gene and the outer membrane porin Omp K36 gene were detected in CRKP-1～CRKP-7 strains,and even multiple mutation sites were found in the same outer membrane porin gene Omp K36.Conclusion:1.In this study,the main mechanism of CRKP resistance to polymyxin was LPS modification caused by mutations of two-component regulatory system mediated by chromosome.2.Mutations in the two-component regulatory system of different strains had different effects on polymyxin-resistance.