| Methicillin-resistant Staphylococcus aureus(MRSA)is a pathogenic bacterium that has multiple drug resistance and seriously endangers human health.MRSA is enriched and attached to form the biofilm structure leading to increased pathogen resistance which is the main reason for the failure of antibiotic treatment.Bacterial biofilm is a major problem in anti-infection clinical treatment.Therefore,it is of great significance to find a method capable of inhibiting MRSA biofilm formation and eradicating its mature biofilms.In this study,two anthraquinone secondary metabolites,3,8-dihydroxy-1-methylanthraquinone-2-carboxylic acid(1)and 3,6,8-trihydroxy-1-methylanthraquinone-2-carboxylic acid(2),were isolated from a rare actinomycete strain,Kitasatospora albolonga R62,from the rhizosphere soil of the mangrove plant Kandelia candel(L.)Druce.Among them,compound 1 showed no antibacterial activity against MRSA at the concentration of 200 μg/m L,but its inhibitive activity against biofilm formed by MRSA was higher than 50%,and this anti-biofilm activity has not been reported yet.So,it is speculated that compound 1 has the potential for being a new anti-biofilm agent.Compound 2(200 μg/m L)had no obvious anti-MRSA activity and anti-biofilm activity compared with compound 1.Due to the low yield and difficult to obtain enough amount of compound 1,this research have bought 10 other commercial anthraquinone analogues of compound 1 for further explored antibacterial and anti-biofilm activity,and structure-activity relationship of anthraquinones,including anthraquinone(3),1-hydroxyanthraquinone(4),2-hydroxyanthraquinone(5),anthraquinone-2-carboxlic acid(6),2,6-dihydroxyanthra-quinone(7),alizarin(8),purpurin(9),1,8-dihydroxy-anthraquinone(10),emodin(11)and rhein(12).The antibacterial assay results showed that both compound 6(MIC value of 100 μg/m L)and compound 12(MIC value of 12.5 μg/m L)had antibacterial activity,and the remaining compounds did not have antibacterial activity at the concentration of 200 μg/m L.Anti-biofilm assay results showed that compounds 5,6,8,9,11 and 12 had strong inhibition activity against biofilm formed by MRSA(> 50%)at concentrations of 200 μg/m L and 50 μg/m L,and compounds 6,10 and 12 had moderate eradication activity against mature biofilm of MRSA(> 20%)at concentrations of 200 μg/m L and 50 μg/m L.Based on the above results,structure-activity relationship analysis of the above compounds showed that compounds 6 and 12 containing a carboxyl substituent at the C-2 position of the anthraquinone skeleton had stronger antibacterial activity and biofilm eradication activity than other anthraquinones;the hydroxyl group at the C-2 position was more important than that at the C-1 position during MRSA biofilm formation inhibition test at high concentrations.Combining the above results,compounds 6 and 12 with best eradication activity were selected as target compounds for further research.Crystal violet staining and MTT staining method were employed to explore mature biofilm eradication activity of compounds 6 and 12 at different gradient concentrations(12.5-200 μg/m L),and the results showed that the removal percentage of bacterial cells in the biofilm was higher than that of the total biofilm.At the same time,the scanning electron microscopy imaging and confocal scanning laser microscopy imaging results were verified with the staining results.RNA-seq sequencing was used to investigate the effects of compounds 6 and 12 on the transcription level of bacterial cells inside biofilm:(1)According to GO enrichment analysis,it was found that compound 6 mainly affected the biosynthesis metabolic process and ion transport process-related genes in biological process,membrane related genes in cellular component,and oxidoreductase activity related genes in molecular function.And compound 12 also affected genes related to nitrate reductase complex in cellular component.Moreover,GO enrichment analysis of the overlap significantly differentially expressed genes of both groups found that they all significantly upregulated the genes related to phosphate ion transport and downregulated cell death related genes.(2)KEGG pathway enrichment analysis results showed that the compound 6 treatment group had three mainly influenced metabolic pathways: microbial metabolism in diverse environments,biosynthesis of amino acids and two-component system.Genes mainly affected in compound 12 treatment group were involved in pyrimidine metabolism pathway,two-component system pathway and glycolysis/gluconeogenesis pathway.This study provides data support and analysis basis for investigating anthraquinones’ mechanism of eradicating mature MRSA biofilm. |
PDF Full Text Request