Targeted Stress Modulation in S.aureus to Combat Resistant Infections via Bioactive Hybrid Therapy

Transcript:

1 in 4 patients die within three months of getting detected with resistant bloodstream infection majorly caused by a bacteria called S. aureus.

Current antibiotics often fail because they only target one bacterial stress pathway that cannot effectively break down biofilms.

To solve this, I developed a bioactive hybrid therapy that targets multiple bacterial stress pathways at once.

I combined an antimicrobial peptide, DJK-5, with natural compounds derived from hop plants, and tested them against both free-floating bacteria and biofilms using disk diffusion and MIC assays.

My previous study’s combination treatment was proven to be more effective against free-floating bacteria, while my hybrid therapy showed significant and consistent disruption of biofilms, which is the main cause of resistance.

This shows the benefits of shifting from a single-target antibiotic to multi-target therapies.

Ultimately, this approach has the potential to improve outcomes for patients battling resistant infections.

Why Antibacterial Resistance in Staphylococcus aureus (S. aureus)?

Antibacterial resistance is a growing global health crisis, causing ~2.8 million infections and 35,000 deaths annually in America [2]. Among the most concerning pathogens, S. aureus causes 60%-70% of these challenging infections due to its ability to form biofilms that protect it from antibiotics (Fig.1) [3,5]. In severe cases, 1/4 of patients die within 3 months, highlighting the need for more effective treatment[ 1].

Problem

Conventional antibiotics are monotherapies that target a single mechanism in actively growing bacteria (planktonic), failing to disrupt biofilms, which persist through quorum-sensing and stress-response signalling.

My previous study showed concentrated hop(Humulus lupulus) extract with antibiotics inhibited planktonic S. aureus, but had variable effects against S. aureus biofilms, with some technical limitations (Fig.2) [4]. To address these limitations, I used natural hop with multiple bioactive compounds, as observed from the HPLC in Fig.3, with an improved methodology to better investigate S. aureus biofilms.

Approach: Bioactive Hybrid Therapy

This study also develops a hybrid therapy, combining natural compounds (hop) with DJK-5(AMP) to target multiple bacterial survival pathways simultaneously (Fig.4) [6,7,8]. Therefore, in combination, these mechanisms would shift our current single-target treatments to a multi-target approach, to decrease mortality rates and increase the quality of life for patients.

Objectives:

Investigate the individual and combined effects of hop compounds and DJK-5 against S. aureus.

Compare efficacy against both planktonic and biofilm forms.

Compare hybrid therapy against conventional treatments.

Hypothesis:

The combination of hop and DJK-5 inhibit S. aureus growth (both planktonic and biofilm) more effectively than single treatments, due to the multi-target disruption of bacterial survival pathways within 24 to 48hrs.

For investigating this, two assays were performed:

Disk Diffusion[9]

This assay tests planktonic S. aureus inhibition across 5 treatment groups, with each tested 8 times for reliable data and statistical comparison with the 7 treatment groups (previous study).

Treatment Groups:

Controls: Water, Ethanol

Individual: Hop, DJK-5

Combined: Hop+DJK-5

Treatment Preparation:

DJK-5 (Solvent: Water):

Concentration: 2.5 mg in 2.5 mL = 1 mg/mL stock

50 µL stock + 4950 µL water = 10 µg/mL

Hop Extract (Solvent: Ethanol then Water):

Spent hop extraction in ethanol = 100 mg/mL stock, as seen in Fig.5 [10].

Concentration: 40 µL stock + 3.96 mL water = 1 mg/mL

32 µL + 3.968 mL water = 8 µg/mL

Application:

A 100µL bacterial suspension per TSA plate and 20 µL of treatment/disk, incubated at 37°C for 24 hours. After which, the zone of inhibition was measured for data collection (Fig.6).

Limitation Resolved:

The precipitation of hop was resolved by low working concentrations and the residual ethanol maintaining solubility of hydrophobic hop compounds[11].

Minimum Inhibitory Concentration[12,9]

This assay tests S. aureus biofilms across 3 treatment groups and 11 concentrations, with each concentration tested 12 times for accurate data and comparison with the 5 treatment groups(previous study).

Treatment Groups:

Control: No drug column, only biofilm growth

Individual: Hop, DJK-5

Combined: Hop+DJK-5

Serial Dilution of Treatments:

To achieve the required concentrations, a 2-fold serial dilution method was used(Fig.7).

Hop Extract Concentrations: 2048µg/mL to 2µg/mL

DJK-5 Concentrations:  20µg/mL to 0.019µg/mL

After incubation, biofilms were detected using CV Staining (Fig.8)[13].

Results

Combination Treatment Showed Moderate Inhibition on Planktonic S. aureus

The combination of spent hop and DJK-5 produced measurable zones of inhibition against planktonic S. aureus, but it did not exceed the efficacy of concentrated hop and ciprofloxacin or ofloxacin, as seen in Fig.9. This shows how antibiotic-based combinations may provide a stronger inhibition against planktonic S. aureus. Additionally, spent hop and DJK-5 individually showed low activity, with neither outperforming the combination. However, as observed from the box plot, there was a statistical difference between the individual antibiotics and the combination of spent hop and DJK-5, suggesting comparable efficacy despite different mechanisms. Finally, the negative controls showed negligible inhibition, proving that observed effects were due to the treatments and not the solvents Fig.10. One-way ANOVA and Tukey’s HSD test (indicated by the alphabets) confirmed significant differences(p<0.05) between treatment groups, highlighting the importance of multi-target approaches Fig.9.

Combination Treatment Significantly Reduces Biofilm Formation

The MIC results demonstrated that the combination treatment (spent hop and DJK-5) showed statistically significant reduction in biofilm growth across concentrations as seen in Fig.11. The Heatmap reveals a clear gradient pattern, indicating a controlled and progressive inhibition (lighter colour in majority: higher inhibition) for the combination treatment, compared to the individual treatments (darker colour in majority: lower inhibition). In comparison to the previous study’s combination (concentrated hop with ciprofloxacin or ofloxacin), which appears visually effective, but produces irregular and variable inhibition of biofilm, which proves the treatment to be ineffective under different environmental factors Fig.12.

Limitation Resolved:

In this study, the previous inability to perform statistics on MIC data was resolved. I combined data sets for each study separately and analyzed the mean and standard deviation to assess variability and trends. Then I further ran a one-way ANOVA test followed by Tukey’s HSD test, which confirmed that the spent hop and DJK-5 combination showed statistically significant inhibition(p<0.05), compared to individual treatments(p>0.05), Fig.11.

When the same process was replicated for the previous study, it was evident that the combination (concentrated hop and ciprofloxacin or ofloxacin) was not statistically significant, proving how the current hybrid treatment is the most consistent and robust in disrupting S. aureus biofilms Fig.12.

Key Findings:

The combination of spent hop and DJK-5 showed significant inhibition of S. aureus biofilms, which is the major cause of current antibiotic resistance. This supports the hypothesis that multi-target disruption through membrane destabilization, QSI and ppGpp degradation enhances antimicrobial activity. On the other hand, the hypothesis was true in part, as the same combination (spent hop and DJK-5) produced measurable inhibition, but was still less effective than the combination of concentrated hop with ciprofloxacin or ofloxacin against planktonic S. aureus, highlighting that efficacy can vary with the bacterial state/infection type. Statistical analysis also supported that, despite these differences, the combination treatments(for both current and previous studies) consistently outperformed individual treatments, showing the potential of multi-target therapies to improve treatment consistency and robustness.

Discussion and Implications:

This study shows that antimicrobial effectiveness depends on both the treatment and bacterial state being targeted. While hop with antibiotics strongly inhibit planktonic S. aureus, the hop with DJK-5 treatment showed an effective eradication of S. aureus biofilms, which are the main drivers of persistent resistant infections.

S. aureus infection can be either planktonic or biofilm-based, depending on the disease Fig.13 [16]. Meaning that, treatments can be selected and based on the infection/severity, which allows us to provide more precise and effective treatments(addressing the current gap) Fig.14 [17].

While for evolving infections, a sequential dual-therapy approach can be more effective [16]. Alternating between therapies that target planktonic cells, followed by treatments that disrupt biofilms, could eradicate evolving S. aureus infections. Overall, this study shows the benefits of shifting from single-target antibiotics to multi-target therapies. Compared to last-resort antibiotics that exponentially reduce the quality of life for the patients and are currently less effective due to resistance, the approach outlined in this study offers more targeted and sustainable treatment alternatives.

Conclusion:

This research shows that the combination of hop with DJK-5 or antibiotics has a strong potential as a multi-target strategy against resistant S. aureus infections. While the effectiveness varies by bacterial state, the findings of this study demonstrate that treatments can be strategically selected or combined as a sequential dual-therapy for maximum impact. With further research on the clinical applications, this approach could lead to more effective and less harmful therapies for resistant infections.

Future Directions

Pharmacokinetics & Bioavailability:

Exploring how both the combination of hop with DJK-5 and hop with antibiotics would react and metabolize in the human body, through Cytotoxicity assays. This would provide insights for clinical applications.

Currently in the works:

Contacting drug development professionals and start-ups to confirm the relevance and rationalize the pathway of commercialization, so that my research can contribute towards sustainably eradicating resistance to uplift the quality of life for patients.

Also, I am working towards virtual-docking of these molecules to observe molecular interactions and the potential pharmacokinetics & bioavailability of these treatments for potential delivery.

I am grateful to the STEM project grant for providing me with the necessary financial aid for my research.

Also, a HUGE thank you to the KPU Applied Genomics Centre’s faculty members, especially Dr.Assogba, who supported my research initiative and let me continue my project from last year, providing me with his time, laboratory space, and invaluable guidance.

I also thank Dr. R.E.W. (Bob) Hancock (Director of Microbial Diseases and Immunity Research at UBC) for accepting my cold-email and supporting my research not only through a guidance meeting, but also by providing me with the DJK-5.

I also thank Steel & Oak for providing me with the spent hop that made this study possible. Finally, thank you Micheal, for your feedback and guidance whenever I needed it.

It is because of their and my parents’ support that my project was able to become a reality.

THANK YOU!

All the information was derived from peer-reviewed journals, internationally recognized organizations and from standardized laboratory methodologies as seen from the sources cited below:

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