The Network Mechanism Of Staphylococcus Aureus Resistance Against Cefquinome Quantified By PK/PD Parameters

Staphylococcus aureus is a gram-positive pathogenic bacterium,which can cause a variety of life-threatening diseases in wildlife.Cefquinome have potent antimicrobial activity to S.aureus,and to explore its relationship with the resistant S.aureus selective enrichment and network mechanism of resistance,this experiment adopts the PK/PD modeling study in vitro cefquinome resistance under different dosage regimen and enrichment of the relationship.At the same time,drug-resistant bacteria differentially expressed proteins in quantitative determination,screening and analysis to preliminary elucidate the main resistance mechanism of cefquinome.In this experiment,cefquinome extract and S.aureus ATCC6538 were used as materials.The specific steps were as follows:1.In vitro PK/PD model was constructed to administe with different concentrations of 3 μg/mL,5 μg/mL,and 10 μg/mL at 2.5 or 5 h elimination half-life and 12 or 24 h interval.The operation duration of each cycle was 72 h,during which the broth samples in the model were collected at different time points for the determination of drug concentration and bacterial content.2.The low-concentration resistant strains with 2 MIC resistance,the high concentration resistant strains with 8 MIC resistance and the standard sensitive strains were screened with the drug-containing tablet under the same drug regimen for quantitative proteomics of Labelfree(n=3).Through GO analysis,KEGG pathway enrichment analysis and PPI network analysis,differentially expressed proteins were screened and their relationships were demonstrated,so as to better understand the evolutionary process of resistant bacteria and the network mechanism of resistant related proteins.The research results and conclusions were as follows:1.In vitro,the minimum inhibitory concentration(MIC)of cefquinome against S.aureus ATCC6538 was 0.50 μg/mL,the inhibitory concentration(MIC99)was 0.40 p-g/mL,the mutant prevention concentration(MPC)was 1.60 μg/mL and the mutation selection window(MS W)ranged from 0,40～1.60 μg/mL.2.Under different dosage regimens in wildlife,drug resistance mutations were as follows:when the drug delivery interval and elimination half-life were the same,the drug-resistant bacteria appeared earlier with the drug concentration increased.When the drug concentration same with elimination half-life,the longer the drug interval,the earlier the drug-resistant bacteria appeared.The shorter the elimination half-life,the earlier the emergence of resistant bacteria in the same dose interval and concentration.The degree of drug resistance increased gradually with the prolongation of administration time.Bacteria can be killed with high concentration.At the high dose,the bacteriostatic ability did not dependent on concentration.When cefquinome was administered clinically in a variety of animals,the concentration of cefquinoxime was higher than that of MPC,the percentage of the interval time(%T>MPC)was greater than 50%,it had good antibacterial activity and could inhibit the occurrence of resistance.3.A total of 2,065 proteins were identified in S.aureus by Labelfree quantitative proteomics technology.According to the GO functional annotation,it was found that the differentially expressed proteins in the three groups of strains mainly existed in cells and cell membranes,participated in the metabolic process and intracellular process,and had catalytic activity and binding activity.KEGG pathway analysis further confirmed that there were 102 related proteins in the pathway with significantly enriched differentially expressed proteins.Specifically,10 KEGG signaling pathways were involved:ribosomal pathway,vancomycin resistance pathway,lysine biosynthesis pathway,methyl butyrate metabolism pathway,citric acid cycle pathway,riboflavin pathway,oxidative phosphorylation pathway,glyoxalate-dicarboxylic acid metabolism pathway,glutathione metabolism pathway,thiamine metabolism pathway.The metabolic pathways of significantly enriched differentially expressed proteins among strains with different drug resistance were not completely consistent,and there were network correlations among the pathways.PPI analysis revealed 11 pivot proteins that were significantly up-regulated in all pathways,one pivot proteins that were significantly down-regulated,which provided important reference candidate proteins for the study of drug resistance targets of S.aureus against cefquinome.To sum up,cefquinome had an effect on the generation time and growth degree of S.aureus resistance in different dosage regimens in wild animals.When the percentage of time to maintain drug concentration higher than MPC is greater than or equal to 50%of the time between administration,it has well antibacterial activity and the occurrence of drug resistance can be inhibited.When S.aureus evolved resistance,multiple pathways in the bacteria would work together and interrelate to fight the survival pressure brought by drugs.In conclusion,this experiment provided a certain reference basis for rational drug administration after clinical infection of S.aureus in wild animals and preliminarily clarifies the network mechanism of resistance.