Mode of Action of a Novel Antibacterial Material CPC-Clay

Objectives: We have assessed the mode of action of CPC-Clay, a novel particulate antibacterial material consisting of Cetylpyridinium chloride (CPC) and Clay, against a common oral cariogenic bacteria S.mutans, by using perturbation of membrane polarity and integrity.
Methods: In this study, we have used fluorescent probes bis-oxonal DiBAC₄ (DiBAC₄) and Propidium Iodide (PI) to monitor the changes in bacterial membrane polarity and membrane integrity. Log phase culture of S.mutans UA159 was mixed with DiBAC₄ and PI, test actives were added and the fluorescence monitored using a TECAN plate reader at 37⁰C. The fluorescence readings over time was plotted to evaluate membrane depolarization and rupture.
Results: CPC-Clay and CPC showed a significant increase in fluorescence for both DiBAC₄ and PI. No change in fluorescence was observed either with clay alone or with buffer, which was added as a negative control.
Conclusions: The polarity and integrity of bacterial membrane are critical for its function and disruptions to these caused by any agent can compromise survival of bacteria. CPC works by binding to the negatively charged surface of the bacterial cell membrane, altering and rupturing it, resulting in loss of cytoplasmic material leading to cell death. In this study, increase in fluorescence of DiBAC₄ indicates depolarization of the bacterial membrane, while that of PI indicates compromised bacterial membrane integrity following addition of the test active. We have shown that CPC-Clay alters the bacterial membrane potential and membrane integrity, similar to that of CPC, but clay alone does not have any effect. This effect on the bacterial membrane could lead to leaching out of cellular components and eventually lead to a bactericidal action. The results indicate that particulation of CPC on clay does not alter the mode of action of CPC.