Secondary Metabolites From The Endophytes Of Polygonum Capitatum Active Against Urinary Tract Infection

Objective:To search for the antimicrobial ingredients from the endophytic fungi of Polygonum capitatum.Methods:Endophytic fungi of P.capitatum were isolated and purified by microbial purification method and plate scribing method.Endophytic bacteria were preserved by glycerol preservation method.The activity of metabolites of endophytic fungi against resistant bacteria generally found in urinary tract infection was determined by the Oxford Cup method and the checkboard microdilution method was used to evaluate the synergistic effect between the metabolites and the antibiotics on clinical drug-resistant bacteria.The endophytes with metabolites active against multidrug-resistant bacteria were identified by Internal transcribed spacer of rDNA?ITS?method.The activity-guided isolation method was used to separate and purify the antibacterial substances from the endophytic fungi fermentation and their structures were identified by MS,NMR and other spectroscopic techniques;the antibacterial effect of the obtained compounds on clinical drug-resistant bacteria was tested by a 96-well plate method to screen for the active ingredient and the effect of the active ingredient on the MICs of antibiotic?against multi-drug-resistant strain?was evaluated at a concentration with no antibacterial effect to see if it can reverse the resistance of the bacteria with respect to the antiboitcs;the lipids obtained from the Aspergillus terreus were analyzed by GC-MS.Results:56 endophytic fungi were obtained from P.capitatum,of which 16possssed MICs below 50 mg·mL^-11 against clinical drug-resistant urological bacteria.Twenty one compounds were isolated from three endophytic fungal strains?i.e.,Gibberella intermedia,Aspergillus terreus and Alternaria tenuissima?with lowest MICs.Among the isolated compounds,fusarium acid?FA?,helvolic acid?HA?,emodin,5-methoxyheptoryporphyrin,altenusin and linoleic acid showed varied degrees of inhibition against 12 clinically resistant bacteria,with minimal inhibitory concentrations?MICs?of 4²56?g·mL^-1.Meanwhile,FA and HA possessed the effect of reversing drug resistance of the clinically resistant bacteria.FA at its 1/8 MIC concentration reduced the MICs of levofloxacin and ciprofloxacin?against clinically resistant E.coli?by 4-and 2-fold,respectively;at a concentration of 1/4 MIC,FA reduced the MICs of levofloxacin and ciprofloxacin against the resistant strain Proteus mirabilis by 2 and 4 folds,respectively,and the MIC values of clinically resistant Staphylococcus aureus are reduced.2 times.HA also reversed the multi-drug resistance of Escherichia coli,Proteus mirabilis,and Staphylococcus aureus at its 1/2MIC concentration,resulting in the decreases of the MICs of ceftriaxone by 8,4,and8 folds,respectively,and those of the MICs of ampicillin by 4,4,and 16 folds,respectively.Fourteen compounds were identified from the lipid fraction of A.terreus,with palmitic acid?29.35%?,stearic acid?10.87%?,linoleic acid?21.94%?,and oleic acid?34.85%?being the main components.The 96-well plate antibacterial test showed that the lipid fraction had inhibitory activity against the multi-drug resistant E.coli and Klebsiella pneumoniae,with MICs of 12.5 and 50 mg·mL^-1,respectively.The MICs range from 0.25 to 1.0 mg·mL^-1.Conclusion:Fusarium acid is the main active component of G.intermedia against drug-resistant bacteria with multi-drug resistance reversing activities against Escherichia coli,Proteus mirabilis and Staphylococcus aureus.Wihle for A.terreus,helvolic acid and emodin are the main active components with same activity.5-Methoxytryptorytosine,altenusin and linoleic acid were the main active components of A.tenuissima against clinically resistant bacteria.The study laid the foundation for the treatment of multidrug-resistant urinary tract infections and the improvement of the efficacy of quinolones and?-lactam antibiotics in the treatment of urinary tract infections.