| The presence of bacterial efflux pumps reduces the content of antibiotics and other drugs in cells,further enhancing the survival ability of bacteria.This is one of the reasons why bacteria such as Staphylococcus aureus develop high and multiple drug resistance.Effluent pump inhibitors are an effective way to reverse bacterial resistance,so finding a low toxicity and efficient efflux pump inhibitor is the key to solving the problem of bacterial resistance.Andrographolide,as a traditional Chinese medicine that can treat various diseases,has shown inhi-bitory effects on various bacteria and has made significant breakthroughs in the formation of biofilms and quorum sensing systems in Staphylococcus aureus.Therefore,Andrographolide is expected to a potential efflux pump inhibitor,providing a new approach to address the resistance of Staphylococcus aureus.1.The micro broth dilution method recommended by CLSI was used to detect the MIC values of 87 strains of Staphylococcus aureus which stored in the laboratory against 8 antibacterial drugs.PCR was used to detect the carrying status of six efflux pump genes.Then analyze the correlation between the drug-resistant phenotype of Staphylococcus aureus and efflux pump genes.The drug sensitivity test showed that the drug resistance of penicillin was the most prominent,with the drug resistance rate of 83.9%,followed by erythromycin,with the drug resistance rate of 52.8%.The lowest is doxycycline and amikacin,were both 11.5%.The PCR detection results showed that the proportion of norA,norB,norC,sepA,mepA,and mdeA genes carried simultaneously was 24.1%.The detection rate of norB gene was the highest,reaching 87.4%,followed by norC and norA,with detection rates of 87.2%and 82.8%.The statistical analysis results showed that there was a correlation between the antibiotic resistance phenotype of Staphylococcus aureus and the carrying of efflux pump genes.Among them,the erythromycin and amikacin resistance phenotype had a very significant correlation with the carrying of mdeA gene(P<0.01),the tetracycline and gentamicin resistance phenotype had a significant correlation with the norB gene,and the penicillin resistance phenotype had a significant correlation with thecarrying of norC gene(P<0.05).2.The synergistic effect of 8 antibiotics and Andrographolide was detected by drug sensitive disk and chessboard method.The effect of Andrographolide on the accumulation of ciprofloxacin in Staphylococcus aureus was detected using fluorescence spectrophotometry.The mechanism of action of the combination of ciprofloxacin and Andrographolide was predicted by network pharmacology.The results showed that Andrographolide had no bacteriostatic effect within the measured concentration range(8-4096 μg/mL).The diameter of the bacteriostatic zone of some strains increased by 4-6 mm compared with ciprofloxacin alone,and the FICI≤0.5 after combined with ciprofloxacin.Indicating that the combination of Andrographolide and ciprofloxacin had synergistic effect,combined with tetracycline,doxycycline,levofloxacin and penicillin showed additive effect The fluorescence spectrophotometer detection results showed that Andrographolide can increase the accumulation of ciprofloxacin in Staphylococcus aureus at 12 minutes significantly,which was 61%higher than the control group(P<0.0001).Network pharmacology prediction shows that STAT3,MAPK1,IL6,PIK3R1,etc.are core targets for the treatment of Staphylococcus aureus with Andrographolide,where MAPK1 and IL6 are enriched in signaling pathways related to drug resistance.3.RT-PCR method was used to determine the effect of Andrographolide on the expression levels of six efflux genes in Staphylococcus aureus.Homologous modeling was performed on the efflux proteins NorA,NorB,and NorC of fluoroquinolone drugs.The interaction between Andrographolide and the three efflux proteins was analyzed using molecular docking technology and validated using kinetic simulations.The results showed that after the action of Andrographolide,the expression levels of efflux genes in Staphylococcus aureus were significantly downregulated,and norB reaching a concentration of 256 μg/mL and 512 μg/mL in Andrographolide,the decrease was the largest,91%lower than the control group(P<0.001).Molecular docking results showed that Andrographolide formed four hydrogen bonds with the amino acid residues PHE166,LYS279,GLY278 and GLY168 of NorB protein,and the binding energy was the lowest Kinetic simulation showed that Andrographolide had the effect of stabilizing the conformation of NorB.In summary,Andrographolide has a certain inhibitory effect on the drug-resistant phenotype and gene expression level of Staphylococcus aureus.That means,Andrographolide can reduce the expression level of efflux genes in Staphylococcus aureus,inhibit the transcription level of efflux genes,and increase the intake of antibiotics by reducing the efflux of ciprofloxacin in the bacteria,enhancing the sensitivity of Staphylococcus aureus to antibiotics.Therefore,Andrographolide may serve as a new type of efflux pump inhibitor,providing an effective solution for the prevention and treatment of Staphylococcus aureus. |
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