| Objective:In order to provide new ideas for the understanding of cigarette smoke exposure and respiratory infections,a bacteria pneumonia mice model were established.On this basis,further works were carried out to investigate the effects of cigarette smoke extract(CSE)on the virulence and drug resistance of Pseudomonas aeruginosa and its potential mechanism.Methods:Part I:Preparation of CSE:3R4F reference cigarette burned cigarette smoke was injected into tryptone soy broth(TSB)to prepare CSE.The concentration of 10 ml of broth was injected into the smoke of one cigarette as 100%CSE concentration,which was evaluated by measuring OD260 CSE repeatability between batches.Logarithmic seudomonas aeruginosa was exposed to 10%CSE(diluted with TSB)as the CSE xposure group,while blank TSB broth cultured Pseudomonas aeruginosa was used as the control group.The CSE exposure group and the control group were measured by OD620 to evaluate the differences in the growth status of the two groups of bacteria,and then the cells were centrifuged and washed 3 times with sterile phosphate buffered aline(PBS).The turbidity of the bacteria was adjusted to 0.5 McGonstein concentration(OD600=0.1)for dilution plate culture.The above two groups of bacteria were used to construct a mouse acute lung infection model,and the general state,mortality,lung issue pathology,lung and spleen tissue bacterial load,alveolar lavage fluid,and serum TNF-?and IL-6 were observed.The virulence changes of Pseudomonas aeruginosa after CSE treatment were evaluated.The mechanism of virulence of CSE on Pseudomonas eruginosa was explored by transcriptome sequencing combined with bioinformatics analysis.Part 2:According to the CSE exposure method in Part 1,perform the ollowing experiments.The minimum inhibitory concentration(MIC)of Pseudomonas aeruginosa and the minimum bactericidal concentration(MBC)of the coating plate ethod were detected by the drug susceptibility test of the micro broth dilution method.Next,we evaluated the changes in CSE-treated P.aeruginosa resistance to common linical antimicrobials and performed PCR validation of related genes in conjunction with transcriptome sequencing results.Results:1.Effect of CSE on the virulence of Pseudomonas aeruginosaThe CSE-exposed group and the control group were cultured to logarithmic phase,and the bacterial concentration was adjusted to 1×10~8 cfu/ml.A pneumonia model was successfully constructed by tracheal intubation and 1×10~5 cfu of P.aeruginosa in mice.Compared with the control group,mice infected with Pseudomonas aeruginosa in the CSE exposure group showed different characteristics as the disease evolved.1.General status of mice:The mice infected with Pseudomonas aeruginosa in the CSE exposure group had significantly reduced activity,purulent secretions appeared in the corners of the eyes,and some mice had diarrhea from 24 hours after infection.2.Mortality:At the end of the 10-day observation period,the mortality of mice infected with Pseudomonas aeruginosa in the CSE-exposed group was significantly higher than that in the control group(70%to 30%,p<0.05).3.Pulmonary histopathology:The lung tissue of mice nfected with Pseudomonas aeruginosa in the CSE exposure group showed a more evere exudation of red blood cells,and a more severe inflammatory cell infiltration and alveolar unit destruction were observed under a light microscope.4.Bacterial load in ung and spleen tissue:The bacteria load in the lung tissue of mice infected with seudomonas aeruginosa in the CSE exposure group was significantly increased.Unlike the spleen tissue of the control group,the spleen tissue of the CSE-exposed group can lso culture Pseudomonas aeruginosa.5.Detection of serum and alveolar lavage fluid inflammatory factors:Alveolar lavage fluid of mice infected with Pseudomonas eruginosa in the CSE exposure group was red,and red blood cells were visible.The levels of TNF-?and IL-6 in the serum and alveolar lavage fluid of mice infected with Pseudomonas aeruginosa in the CSE exposure group were higher than those in the PBS control group(p<0.05).Compared with the PA infection group,the levels of TNF-?and IL-6 in the alveolar lavage fluid of the CSE-PA infection group were more ignificant(p<0.05).6.Transcriptome sequencing results suggest that CSE induces high flagellar assembly related genes,high expression of the two-component regulatory ystem PhoQ/P,and low expression of genes related to the type III secretion system.2.Effect of CSE on drug resistance of Pseudomonas aeruginosaBoth the paper diffusion method and the micro broth dilution method suggest that CSE can increase the minimum inhibitory concentration of Pseudomonas aeruginosa on levofloxacin,imipenem,tigecycline and minocycline.The bacterial solution obtained from the microbiological dilution of the microbiological dilution method was applied to tryptone soy agar(TSA)and further confirmed that CSE could increase the minimum bactericidal concentration of Pseudomonas aeruginosa to imipenem and levofloxacin.PCR results showed that CSE induced low expression of the transcription factor mvaT in P.aeruginosa,up-regulated mexE/mexF-opr N and down-regulated oprD.Conclusion:Cigarette smoke extract induces the transformation of P.aeruginosa to a ore virulent phenotype,manifested by more severe lung lesions,higher lung and blood circulation bacterial loads,and more severe sepsis in mouse models and higher ortality.Its mechanism is related to the flagellar assembly related genes of P. eruginosa,the high expression of the two-component regulatory system PhoQ/P,and the low expression of T3SS-related genes.Cigarette smoke extract enhances P. eruginosa resistance to levofloxacin,imipenem,tigecycline,and minocycline.The mechanism is to induce low expression of the transcription factor mvaT,up-regulate mexE/mexF-oprN and down-regulate oprD.Transformation of Pseudomonas aeruginosa to an nfxC resistant phenotype. |
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