Study On Synergistic Effects Of Eight Plant Antibacterials Combined With Clinical Antibacterial Agents Against Drug-resistant Strains

[Objective]1. To determine the alone and synergistic effects of eight plant antibacterial activity components (emodin, aloe-emodin, rhein, cryptotanshinone, Usnea acid, alcohol, matrine and oxymatrine) in combination with conventional antibacterial agents against clinical MRSA, as well as ESBLs and AmpC enzyme producing drug-resistant strains in order to investigate antibacterial synergy and the potentiality of reversing the resistance.2. To investigate the acute toxicity in mice of four compounds (aloe-emodin, rhein, cryptotanshinone and alcohol).[Methods]1. Screening the potential synergistic antibacterial agents when they were combined used with the8compounds by K-B disk diffusion method firstly. Then the checkerboard microdilution and time-killing curve methods were performed to evaluate the modes of antibacterial interaction.2. The different doses of compound were administered in kunming mice via intragastric. The toxic reactions were observed and recorded, the median lethal doses were calculated.[Results]1. The six compounds (emodin, aloe-emodin rhein, cryptotanshinone, usnic acid and sclareol) showed good activity against MRSA used alone. Their combined results with eighteen antibacterial agents showed that:(1) The disk diffusion tests screened out nine different classes of antibacterial agents, including aminoglycosides (amikacin, streptomycin, etimicin, glycopeptides (vancomycin,teicoplanin), tetracyclines (minocycline) and penicillin (penicillin, ampicillin, piperacillin/tazobactam).(2) The checkerboard microdilution method exhibited that emodin combined with vancomycin, teicoplanin, piperacillin/tazobactam and minocycline showed synergy, additivity or indifference with the FICI ranges as0.313～1.500. The aloe-emodin and rhein combined with vancomycin, teicoplanin, amikacin, streptomycin, minocycline showed synergy, additivity or indifference with the FICI ranges as0.125～1.125and0.094～1.500, respectively. The cryptotanshinone combined with vancomycin, teicoplanin, amikacin streptomycin, penicillin, piperacillin/tazobactam and minocycline with the FICI ranges as0.125～2.250, and most of which were additivity or synergy. The usnic acid combined with vancomycin, teicoplanin, etimicin, amikacin, streptomycin and minocycline showed synergy, additivity or indifference with the FICI ranges as0.516～1.500. The sclareol combined with vancomycin, teicoplanin, etimicin, amikacin, penicillin, ampicillin, piperacillin/tazobactam and minocycline showed synergy, additivity or indifference with the FICI ranges as0.188～1.500.(3) The results of time-killing curves at24h showed that:combination of emodin with antibacterial agents showed synergy or additivity (1.249～2.064log10CFU/mL increase in killing). Combinations of aloe-emodin with antibacterial agents showed synergy or additivity, except its combination with minocycline showing indifference (0.931～3.2441og10CFU/mL increase in killing). Combinations of rhein with antibacterial agents showed synergy, except the additivity with teicoplanin (1.898～3.556log10CFU/mL increase in killing). Most combinations of cryptotanshinone with antibacterial agents showed synergy or additivity, some showed indifference (0.066～2.253log10CFU/mL increase in killing). Combinations of usnic acid with antibacterial agents showed synergy or additivity (1.249～2.064log10CFU/mL increase in killing). Combinations of sclareol with antibacterial agents mostly showed synergy, together with some showing indifference (0.368～5.253log10CFU/mL increase in killing).2. The three compounds (sclareol, matrine and oxymatrine) showed good activity against ESBLs (extended-spectrum β-lactamases) and AmpC (Ampler class C β-lactamase) enzyme producing multi-drug resistant strains. Their combined results with fourteen antibacterial agents showed that:(1) The K-B disk diffusion tests screened out five antibacterial agents amikacin, gentamicin, levofloxacin, minocycline and cefoperazone/sulbactam.(2) The results of checkerboard microdilution method through combined tests between the individually active compounds and the five antibacterial agents showed that:the FICIs of sclareol, matrine and oxymatrine combined with conventional antibacterial agents were ranged0.313-2.031,0.313-1.500,0.266-2.016, respectively. And most of which showed additivity, some showed synergy and a few showed indifference.(3) The results of time-killing curves at24h showed that:combinations of sclareol and matrine with amikacin showed additivity (1.176log10CFU/mL increase in killing). Combinations of matrine with antibacterial agents minocycline, sulbactam/cefoperazone showed synergy (2.347,4.136log10CFU/mL increase in killing, respectively). Combinations of oxymatrine with antibacterial agents amikacin, minocycline, sulbactam and cefoperazone showed synergy or additivity (1.477,2.347,2.020log10CFU/mL increase in killing, respectively).3. The acute toxicity results showed that:the LD50of aloe-emodin, rhein, cryptotanshinone, sclareol was LD5o=128.65mg/kg, LD5o=74.47mg/kg, LD50=75.86mg/kg, LD50:=669.89mg/kg, respectively.[Conclusion]1. The results of the synergistic effects of six plant compounds combined with antibacterial agents against MRS A were reported so far for the first time. In which the cryptotanshinone could reverse the resistance of amikacin against MRS A; the aloe-emodin, rhein and usnic acid could reverse the resistance of minocycline against MRSA. The six compounds combined with the other tested antibacterial agents could also enhance the antibacterial agents’ activities against MRSA.2. The synergistic results of the three compounds against ESBLs and AmpC enzyme producing multi-drug resistant strains demonstrated that sclareol, matrine and oxymatrine combined with antibacterial agents mostly showing additivity among which matrine enhance the antibacterial agents’activities significantly. This is also the first time reported, which provided an experimental basis for their further clinical application investigations.3. The acute toxicity results showed that aloe-emodin, rhein and cryptotanshinone were moderately toxic compounds, while sclareol with low toxicity, and preliminarily evaluated the toxicity of tested components.