| Background: Klebsiella pneumoniae(KP)belongs to Klebsiella of the family Enterobacteriaceae,which widely distributed in the digestive tract of humans and various animals,and is an important opportunistic pathogen.About 30%of hospital-acquired Gram-negative bacterial infections worldwide are caused by Klebsiella pneumoniae and manifest as urinary tract infections,cystitis,pneumonia,and surgical infections.In recent years,the detection rate of drug-resistant Klebsiella pneumoniae has been increasing globally.Resistance mechanisms include limiting and reducing the intake of antibiotics,efflux of antibiotics through active efflux pumps,changing the target of antibiotics,and producing enzymes that can inactivate and break down antibiotics.Exploring the effects of bacterial drug resistance on bacterial metabolism and host cell immune effects from the perspective of bacterial secreted metabolites is a new perspective to explore the mechanisms of drug resistance based on genomics and proteomics.Purpose: This study aimed to explore the bacterial physiology,biochemical and genomic characteristics of drug-resistant Klebsiella pneumoniae and drug-susceptible Klebsiella pneumoniae,and to analyze the genetic and epigenetic differences between drug-resistant and sensitive strains.The differences in the secretory metabolomics may provide ideas for clinical treatment of drug-resistant Klebsiella pneumoniae from the metabolic level.In addition,the immune effects and cell physiology changes of drug-resistant and-sensitive strains in lung adenocarcinoma cell line A549,human-derived lung organoids,and mouse infection models are supposed to studied respectively,to clarify immune interactions between drug-resistant Klebsiella pneumoniae and pulmonary epithelial cells.Methods:(1)Biochemical and drug susceptibility characteristics of a drug-resistant Klebsiella pneumoniae and a drug-sensitive strain were identified.(2)The in vitro growth curves of the two Klebsiella pneumoniae strains were drawn by turbidimetric method,and then the two were mixed with the same initial bacterial amounts for 12 h and 16 h to observe the growth of the two strains under the mixed cultures.(3)Scanning electron microscope was used to observe the surface morphologies,and Image J was used to analyze the relative surface area of ? ? the two.(4)The production of capsular polysaccharide and biofilm formation of drug-resistant and-sensitive strains were quantified and compared by phenol-concentrated sulfuric acid method and crystal violet staining method respectively.(5)Perform whole genome sequencing on the two strains of Klebsiella pneumoniae and compare the reference genomes to determine whether they have new drug resistance gene mutations or drug resistance plasmids.(6)Using non-target metabolomics to detect the secreted metabolites of two Klebsiella pneumoniae strains,combined with univariate statistical analysis and multidimensional statistical analysis,to find the differential secreted metabolites.(7)Establish an infection model of Klebsiella pneumoniae infection on A549 cell line,human adult stem cell-derived lung organoids,and mice.The morphological changes of lung epithelial cells were observed from the ability of bacteria to adhere to lung epithelial cells,the ability of bacteria to proliferate in cells,and immunofluorescence staining of the tight junction proteins and cytoskeletal protein.(8)The transcriptome sequencing was tested on A549 cells infected with Klebsiella pneumoniae,through GO and KEGG pathway analysis,the differential genes were detected between the drug-resistant and-sensitive strain group,and quantitative real-time PCR was performed to validate results from the RNA-seq.Results:(1)The experimental strains were 1 strain of extended-spectrum β-lactamase-producing resistant Klebsiella pneumoniae and 1 strain of drug-resistant Klebsiella pneumoniae.(2)The in vitro growth characteristics of the two strains were relatively consistent,however,in the mixed cultures,the drug-resistant strain showed a competitive growth advantage.(3)Under the scanning electron microscope,the two showed obvious differences.Compared with the sensitive strain,the resistant strain lacked three-dimensional spatial structure,and the bacteria were surrounded by thicker extracellular substances.The comparison of the relative surface area of ? ? the two and the subsequent quantitative experiments of capsular polysaccharide and biofilm formation were consistent with the morphological observation of scanning electron microscope.The relative surface area of resistant strains was smaller than that of sensitive strains(p<0.0001),and the levels of capsular polysaccharide secretion and biofilm formation were higher than those of sensitive strains(p<0.05).(4)Whole genome sequencing of drug-resistant and susceptible strains did not reveal any new drug-resistant gene mutations or the formation of drug-resistant plasmids.(5)Non-target metabolomics detected a total of 306 secreted metabolites from the two Klebsiella pneumoniae strains,including 16 differentially secreted metabolites,including inosine,D-mannose,adenine,adenosine,L-group acid,etc.(6)In the A549 cell and lung organoid infection model,the entrapment rate of drug-resistant Klebsiella pneumoniae was higher than that of the-sensitive strain(p<0.05).(7)The influence of sensitive Klebsiella pneumoniae on the viability of A549 cells was worse than that of drug-resistant strain(p<0.05),and the supplement of supernatant secreted by drug-resistant strain had no significant effect on cell viability.(8)In a certain concentration range,the differential metabolite inosine increased the viability of A549 cells;D-mannose inhibited the viability of A549 cells.(8)The degree of damage to the tight junction protein and cytoskeleton of A549 cells and lung organoids in the susceptible strain infection group was more obvious.The lung pathology of mice in the susceptible strain-infected group also showed more severe lesions.(9)The results of RNA sequencing showed that there were differences between the A549 cells infected with the drug-resistant Klebsiella pneumoniae and the-sensitive strain group.It was verified by quantitative real-time PCR that the mitochondria-encoded c-cytochrome oxidase-1 located in the oxidative phosphorylation pathway and guanylate-binding protein-4 located in NOD receptor-mediated signaling pathway were consistent with RNA sequencing results.(10)The detection results of cytokines and chemokines at 24 hours post infection showed that the secretion of inflammatory factors interleukin-2,interleukin-6,interleukin-9,and macrophage inflammatory protein-1 β in the two Klebsiella pneumoniae infection groups was significantly increase.The secretion level of monocyte chemoattractant protein-1 in the infection group was lower than that in the control.Conclusions: The drug-resistant Klebsiella pneumoniae has the advantage of growth in vitro,and its surface morphology and secretion of extracellular substances increase its adhesion to epithelial cells.The drug-sensitive Klebsiella pneumoniae strain are relatively more virulent to epithelial cells and have stronger intracellular proliferation ability.The difference between the two secreted metabolites in a certain concentration range on the viability of epithelial cells.There are also differences in the immune effects of epithelial cells infected with-resistant and-sensitive Klebsiella pneumoniae strains,such as the secretion of cytokines and chemokines.At the transcriptional level,oxidative phosphorylation pathway and NOD receptor-mediated signaling pathway may also be altered. |
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