Agar Microdilution Procedure: A Promising Technique for Antimicrobial Susceptibility Test of Colloidally Unstable Nanostructures

Abstract

Background: Broth microdilution is the only available microscale endpoint technique used to evaluate the antimicrobial potency of nanostructures. In the case of unstable nanostructures or at high concentrations of nanostructures, this technique is not applicable due to aggregation and sedimentation issues. Most nanostructures can absorb visible light, and this optical feature can interfere with the OD600 measurement that is commonly employed for the evaluation of microbial cells growth. The demand for high-tech plate readers is another limitation of the broth microdilution procedure. Agar microdilution can be a promising novel procedure to overcome all these technical difficulties. Objective: In the current experiment, the agar microdilution procedure was developed and introduced to be employed for the evaluation of antimicrobial potency of colloidally unstable nanostructures even at extremely high concentrations. Methods: Thymus daenensis herbal nanoparticles (HrbNPs) were fabricated through a top-down approach and were tested against methicillin-resistant Staphylococcus aureus (MRSA). Also, the particles were fortified with povidone-iodine and peracetic acid as potent antimicrobial compounds to achieve the enhanced antimicrobial activity. Results: Viscose molten agar media prevented the particles from sedimentation during 96-well plate preparation. By agar solidification, the nanoparticles were immobilized in the media, and no aggregation or sedimentation could occur. After incubation, the bacterial growth was recognizable in the well as a thin creamy layer. The MIC of HrbNPs against MRSA was found to be 20 mg/ml. Fortification with povidone- iodine had no impact on the antimicrobial potency of HrbNPs. However, fortification of HrbNPs with peracetic acid resulted in a four-fold increase in the antimicrobial potency of nanoparticles, and MIC was reduced to 5 mg/mL. Conclusions: Results indicated that agar microdilution can be a promising procedure for the antimicrobial susceptibility test of nanostructures at extremely high concentrations. Also, colloidally unstable nanostructures can be tested via this procedure without any concern for possible aggregation and sedimentation. On the other hand, it was found that fortification with antimicrobial compounds can be an effective approach to increasing the antimicrobial potency of HrbNPs against superbugs.