(T1030-05-28) Characterizing the Effects of a Novel Surfactant on Growth Inhibition and Preservation of Different Bacterial Species

Purpose: Amphiphilic surfactants (amphipols) are known to stabilize membrane proteins in aqueous solutions. A novel amphipol was found to stabilize live viruses, antibodies, and enzymes in a solid film matrix at room temperature for extended periods of time [1]. Throughout the formulation development process, no microbial growth was visually detected on the films despite high content of sugars and water (~10%) that could support growth. Amphipols are unique in that they can stabilize cellular membranes at certain concentrations, and can cause mild permeabilization effects at other concentrations [2]. This led to the hypothesis that the amphipol used in these studies is responsible for preventing external microbial growth on solid thin films stored at varying temperatures. The purpose of this study was to determine the impact of this excipient within a solid film matrix on the growth kinetics, inhibition, membrane permeability, and viability of different bacterial strains.

Methods: Three bacterial strains utilized in the USP < 51 > Antimicrobial Effectiveness Test (S. aureus, E. coli, and P. aeruginosa) were used in this study. The minimum inhibitory concentration (MIC₅₀) of the amphipol with respect to pH was determined by broth microdilution and spectrophotometric analysis. Treatment time effects were assessed by calculating the optical density (OD) growth reduction percentages for bacterial suspensions treated with 10% w/v and 35% w/v amphipol over a 24-hour period. Bacterial cell viability before and after film reconstitution was determined by colony count method over an 8-week period. Additionally, scanning electron microscopy (SEM) analysis was performed to visualize the cell morphology of bacteria within the film matrix.

Results: The MIC₉₀ of amphipol against gram positive S. aureus was achieved at 35% w/v, while for gram negative E. coli and P. aeruginosa, the amphipol achieved an MIC₉₀ at 30% w/v. Solution pH did not significantly impact the ability of the amphipol to inhibit microbial growth (p < 0.05). At 24 hours post-treatment, 10% w/v amphipol resulted in 7.2% ± 2.2,  12.9% ± 1.3, and 46.6% ± 5.5 growth reduction forS. aureus, E. coli, and P. aeruginosa, respectively. The 35% concentration further reduced growth rate the most in all three strains (Figure 1). Upon reconstitution of films with bacteria, a decline in cell viability of all three bacterial strains was observed with the control films (0% w/v), likely from loss due to drying (Figure 2). As the amphipol concentration increased in the film, there was a more statistically significant reduction in cell viability for gram negative bacteria (em>E. coli, P. aeruginosa) after film reconstitution.

Conclusion: Since amphipols are relatively new compounds, there are limited characterization studies currently published regarding their effects on the membranes of live microorganisms. Use of other standardized antimicrobial assays, like the disk diffusion test, were inconclusive due to solubility limitations of the amphipol and assay incompatibility. Study results suggest that amphipol concentration may influence bacterial growth and viability, with lower MIC₉₀ needed for gram negative bacteria compared to gram positive bacteria.

References: 1. Bajrovic, I., Schafer, S. C., Romanovicz, D. K., & Croyle, M. A. (2020). Novel technology for storage and distribution of live vaccines and other biological medicines at ambient temperature. Science Advances, 6(10), eaau4819.
2. Marie, E., Sagan, S., Cribier, S., & Tribet, C. (2014). Amphiphilic macromolecules on cell membranes: from protective layers to controlled permeabilization. The Journal of membrane biology, 247, 861-881.

Acknowledgements: This project was funded by the American Foundation for Pharmaceutical Education (AFPE) Gateway to Research Award. Jurata Thin Films provided the amphipol and supported these studies through a sponsored research agreement (MAC).