Synergistic Effect and Antibiofilm Activity of a Skin and Wound Cleanser

Ann Beal Salamone, MA; Joseph C. Salamone, PhD; Rebecca Erin McMahon, PhD; Suprena Poleon, BS; Nina Bionda, PhD; Peter D'Arpa, PhD


Wounds. 2020;32(8):208-216. 

In This Article

Abstract and Introduction


Introduction: Biofilm in chronic wounds impedes the wound healing process. Each biofilm has differing characteristics requiring a multifaceted approach for removal while maintaining a surrounding environment conducive to wound healing.

Objective: In this study, 3 of the components in a wound cleanser are tested to determine synergy in eradicating biofilms of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa in vitro.

Materials and Methods: The 3 components assessed for synergy were ethylenediamine tetraacetic acid sodium salts (EDTA), vicinal diols (VD; ethylhexylglycerin and octane-1,2-diol), and polyhexamethylene biguanide (PHMB). Each component was assessed individually and in combination while dissolved in a base solution. The Calgary assay method was used for biofilm growth and treatment. Kull Equation analysis for synergy was conducted using viable count results.

Results: Synergy is defined as the interaction of components to produce a combined effect greater than the sum of their separate effects. The base solution containing all 3 components (EDTA, VD, and PHMB) reduced biofilm viability by more than 5 logs, demonstrating statistically significant synergy. The 3 components tested individually in the base solution resulted in the following: EDTA did not reduce bacteria viability; VD reduced viability by about 1 log; and PHMB reduced P aeruginosa viability by about 2.5 logs and MRSA viability by about 4 logs. Of importance, the MRSA biofilm failed to regrow in the recovery plates after combined treatment, indicating complete elimination of the biofilm bacteria.

Conclusions: The experimental and calculated results indicate the 3 components (VD, EDTA, and PHMB) when used together act synergistically to eradicate MRSA and P aeruginosa biofilms in vitro.


In most natural environments, free-floating bacteria exist transiently and only as a minor population, while the predominant form is biofilm,[1] aggregates of microorganisms within a self-created polymeric matrix wherein they are resistant to host defenses and antimicrobial agents. Biofilm has been reported to be involved in 78% to 90% of human chronic wounds[2,3] and is associated with delayed wound healing[4] and other negative wound healing outcomes.[5–7]

Solutions for the elimination of biofilm have demonstrated significant challenges, in part due to the complexity of the biofilm structure. Biofilm is the construct (microbial community) created through the attachment of microorganisms to substrata within an extracellular polymeric substance (EPS).[8,9] This construct is stabilized by electrostatic interactions, hydrogen bonds, and London dispersion forces.[8,9] The EPS comprises about 50% to 90% of the total biofilm organic matter and varies depending on the microorganisms, environment, and biofilm age.[8,10,11] The EPS of biofilm in wounds is comprised of dead host tissues, in addition to the substances the microorganisms secrete, as well as proteins, nucleic acids, lipids, polysaccharides, and humic substances.[8,10,11] These substances and their interactions are targets for biofilm elimination from wounds.

Control of the wound bioburden and biofilm involves multiple treatment modalities and components that impact microbial activity and the integrity and attachment of EPS.[12,13] Optimally, components are synergistic not just additive. In other words, with synergy, 1 plus 1 is greater than 2 (1 + 1 > 2).

Components of the wound cleanser product studied herein were selected based on the authors' experiences in eye care and water treatment as well as taking into consideration the wound milieu to target a breadth of biofilms. These components comprise: (1) polyhexamethylene biguanide (PHMB), a broad-spectrum polycationic wound care antimicrobial[14–16] that also is used in multipurpose contact lens solutions,[17] water treatment, and numerous consumer products; (2) ethylenediamine tetraacetic acid sodium salts (EDTA), a chelator of divalent metal ions used in wound care, contact lens cleaners, and numerous personal care products;[18] and (3) vicinal diols (VD), ethylhexylglycerin and octane-1,2-diol, which are amphiphilic surfactants with moisturizing, antimicrobial, and odor-reducing functions used in underarm deodorants.[19,20] The 3 components (PHMB, EDTA, and VD) were theorized to synergistically disrupt EPS, providing access to the microbes, and then to synergistically permeabilize cell membranes and impair processes needed for viability.

Each component was evaluated individually and compared with the 3 components combined in the cleanser product for synergistic activity in disrupting and killing Pseudomonas aeruginosa and Staphylococcus aureus biofilms. Each test solution was dissolved in a base solution containing a non-ionic surfactant poloxamer 407 (P-407), a mucoadhesive hydroxypropylmethylcellulose (HPMC), and sodium chloride (NaCl). A statistically significant synergy index of less than 1 was determined, which proved synergy.