Revisiting the Synergistic In Vitro Antimicrobial and Antibiofilm Potential of Chlorhexidine Gluconate and Cetrimide in Combination as an Antiseptic and Disinfectant Agent

Chlorhexidine and cetrimide are often used as antiseptics and disinfectants. While their individual activities are well-documented, their synergism has rarely been evaluated. Here, we attempted to evaluate the antimicrobial and antibiofilm effects of the combination of these two antimicrobial agents against two environment isolates, viz., P. aeruginosa and S. aureus. The synergism was assayed by determining the fractional inhibitory concentrations, while the antibiofilm effects were determined using crystal violet staining and the resazurin assay. Further, the effects on the biofilms were visualized using brightfield and confocal laser scanning microscopy. Our results show that the combination of these antimicrobials resulted in synergistic inhibition of P. aeruginosa growth. When tested at concentrations below the individual MICs (one-quarter of the MICs), the combination was able to significantly reduce the adherence of S. aureus biofilms to a polystyrene surface, while no effect was observed for P. aeruginosa. The combination was also able to significantly reduce the viability of pre-formed biofilms of both bacteria, thereby showing its antibiofilm potential. Next, we evaluated the performance of this combination against a wide array of micro-organisms. This fixed-dose combination formulation exhibited a significant reduction in the viability of an array of clinically relevant micro-organisms, including ESKAPE pathogens, Mycobacterium sp., MRSA, Leptospira, Candida sp., norovirus and adenovirus. Overall, it can be inferred that the combination of chlorhexidine and cetrimide is a potential biocide that continues to be relevant for use in antisepsis and disinfection against infection-causing pathogens.