Investigating the Synergistic Antimicrobial Potential of Natural Extracts

Hi everyone, this is my intro video! I'm Zunaira and natural antibiotics are the future solution to AMR. If you have any questions, feel free to ask!

Why I Did This Project

Antimicrobial resistance (AMR) is one of the most urgent global health threats, with bacteria evolving faster than new antibiotics are being developed. This crisis feels especially important to me, not only because of its global impact, but because of my own personal connection to the field. My grandfather, who passed away two years ago, dedicated his PhD in medicine to researching new natural antimicrobial agents. He ended up discovering a new plant with antimicrobial properties. Learning about his research inspired me to explore this field myself and honor the work he was so passionate about.

Hypothesis

This led me to my central hypothesis: natural antimicrobial compounds, when used in combination, may produce synergistic or additive effects that enhance bacterial inhibition more effectively than when used individually, offering a potential alternative strategy against AMR.

Objective

The objective of this study was to investigate the antimicrobial activity of garlic, honey, mangosteen extract, and berberine, both individually and in combination, using a checkerboard microdilution method. I aimed to determine whether specific combinations could increase efficacy at lower concentrations against both Gram-positive and Gram-negative bacteria.

Impact

This research could benefit healthcare systems and communities facing increasing antibiotic resistance, particularly in resource-limited settings where access to new antibiotics is limited. By identifying accessible, natural alternatives, this work may contribute to reducing reliance on conventional antibiotics and slowing the progression of resistance.

Background Research

I began by reviewing scientific literature on antimicrobial resistance (AMR) and natural antimicrobial agents. The common method to evaluate antimicrobial activity is through broth microdilution, which allows for accurate determination of minimum inhibitory concentrations (MIC).To assess interactions between compounds, fractional inhibitory concentration (FIC) analysis is used. FIC is important because it quantifies whether combinations produce additive, synergistic, or antagonistic effects, making it a reliable way to evaluate how compounds work together. This method was chosen because it is standardized, widely accepted, and allows for meaningful comparison of antimicrobial interactions across different bacterial types.

Experimental Procedure

To test bacterial inhibition and interactions between the compounds, a broth microdilution assay was used. First, solutions of each natural compound were prepared at a concentration of 1x, then serial two-fold dilutions were performed to a concentration of 1/16x. A sterile 96-well microplate was then prepared and filled with the bacterial dilution as well as Lysogney Broth to make sure the conditions were consistent and ideal for growth. For combination testing, the selected combinations were tested for bacterial inhibition in a checkerboard pattern. Each compound was also tested individually across the dilution range for bacterial inhibition. Positive control wells (ampicillin) and negative control wells (bacteria only) were included. The plate was incubated at 37°C for exactly 24 hours, after which bacterial growth was observed.

Data Analysis

The bacterial growth was assessed using visual turbidity comparison. Minimum inhibitory concentration (MIC) values were identified as the lowest concentration with no visible growth. The MIC was then input into a calculation for the fractional inhibition concentration index (FICI) which evaluated the synergistic interactions by comparing MIC values between single-compound and combination treatments, focusing on reductions in MIC when compounds were used together. The data was organized to compare individual versus combined antimicrobial effects.

Main Results and Findings

The microdilution assay demonstrated measurable antimicrobial activity across all four natural compounds with variation in inhibitory strength depending on compound type and concentration. In single-compound testing, garlic and berberine showed the strongest antibacterial effects, achieving the lowest minimum inhibitory concentrations (MICs). This aligns with their known mechanisms, where allicin in garlic disrupts bacterial enzymes and berberine interferes with membrane integrity and cellular function.

Mangosteen showed moderate antibacterial activity, while honey demonstrated the weakest but still consistent inhibition, likely due to osmotic pressure, acidity, hydrogen peroxide production (honey), and membrane-disrupting xanthones (mangosteen). Across both Gram-positive and Gram-negative bacteria (E. coli and B. subtilis), similar trends were observed, although slight differences in inhibition suggest variation in bacterial susceptibility due to structural differences.

A key finding is that combining these natural compounds produced consistent shifts in antibacterial activity. FIC analysis showed mainly additive and indifferent interactions, with berberine-containing combinations maintaining strong inhibitory effects across both bacterial types.

The novelty of this study lies in its systematic evaluation of four natural antimicrobial agents in combination using a standardized checkerboard microdilution method, including combinations that are rarely or not previously reported in literature.

Overall, the results suggest that natural antimicrobials act differently depending on bacterial structure and compound pairing, supporting the idea that they should be studied as interacting systems rather than isolated compounds in the fight against antimicrobial resistance.

Data Analysis Approach

Antibacterial effectiveness was assessed using minimum inhibitory concentration (MIC), defined as the lowest concentration where no visible bacterial growth occurred. Growth was recorded visually across dilution wells.

To evaluate interactions between antimicrobials, the Fractional Inhibitory Concentration (FIC) index was calculated. In this analysis, MIC A and MIC B refer to the MIC values of each antimicrobial when tested individually (alone), while the “MIC in combination” refers to the concentration required when both substances are used together in the same well.

FICa = (MIC of A in combination) / (MIC of A alone)

FICb = (MIC of B in combination) / (MIC of B alone)

FIC Index = FICa + FICb

Interpretation followed standard criteria:

≤ 0.5 = synergy

0.5–2 = additive

2–4 = indifferent

4+ = antagonistic

This allowed direct comparison between individual and combined antimicrobial performance under identical conditions.

Discussion and Key Findings

All four natural compounds showed measurable antibacterial activity, with garlic and berberine demonstrating the strongest effects (lowest MICs). Mangosteen showed moderate inhibition, while honey had weaker but consistent effects. Differences between E. coli and B. subtilis suggest that bacterial structure influences susceptibility.

Combination testing showed mainly additive and indifferent interactions, with berberine-containing combinations maintaining strong inhibitory effects. This indicates that combining compounds can consistently enhance antibacterial activity, even without strong synergy.

Implications

These findings highlight the value of combination-based approaches in antimicrobial research. Natural compounds may become more effective when used together, revealing new properties not seen individually. This suggests that studying interactions between compounds is an important and underexplored area in addressing antimicrobial resistance (AMR).

Conclusion

This study shows that natural antimicrobials can work effectively both individually and in combination. Importantly, consistent additive effects—not just rare synergy—can improve antibacterial outcomes.

In the context of AMR, where new antibiotics are limited, optimizing combinations of natural compounds offers a practical and scalable strategy. Overall, this research supports shifting focus toward reliable combination-based approaches to help combat antimicrobial resistance.

Future work could include increasing bacterial strains tested, including clinically relevant antibiotic-resistant strains to better model AMR. Repeating the microdilution assay in trials would improve statistical reliability and allow stronger synergy analysis using fractional inhibitory concentration (FIC) indices.

Improvements could also include testing a wider range of concentration ratios for each compound combination to better map interaction strength. Additionally, incorporating time-kill assays or biofilm models would provide deeper insight into how these compounds perform in more realistic infection conditions. Future research could also isolate active components within each natural extract to identify which molecules drive synergistic effects.

There are multiple people that I would like to acknowledge and thank.

First, I thank the Peel Region Science Fair for giving me the opportunity to represent Peel and present this project at CWSF. I would like to sincerely thank my parents for their continuous support throughout this experiment, which made it possible for me to stay motivated and focused. I am grateful to UWaterloo Nanoengineering student at Timothy Gavris for his assistance in the lab and guidance during experimental procedures. Special thanks to Dr. Atiyeh Ahmadi from UWaterloo for her valuable suggestions and scientific insight. I also appreciate Professor Brian Ingalls from UWaterloo for his supervision, resources, and overall academic support throughout the project. I acknowledge the University of Waterloo for providing access to laboratory space and materials essential for this study. Most importantly, I thank my uncle, without whom this experiment could not have been done.

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