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Journal of Global Antimicrobial Resistance
Volume 24, 2021, Pages 148-157

Synergistic activity of tetrasodium EDTA, ethanol and chlorhexidine hydrochloride against planktonic and biofilm cells of clinically relevant pathogens

Murugesan Sivaranjania,b, Fangning Liua, Aaron P. Whitea,b

Vaccine and Infectious Disease Organization, Saskatoon, Saskatchewan, Canada.



Biofilms associated with implantable medical devices and wounds are clinically relevant, often requiring repeated use of antibiotics without success. A search for non-antibiotic antimicrobial and antibiofilm solutions is warranted, in line with antimicrobial stewardship. Our study aimed to evaluate the broad-spectrum antimicrobial efficacy of tetrasodium EDTA, ethanol and chlorhexidine hydrochloride (HCl) alone and in combination against clinically relevant planktonic and biofilm cells of bacterial and fungal pathogens.


MICs and MBCs were determined for tetrasodium EDTA, ethanol and chlorhexidine HCl against planktonic cells of test pathogens. The MBEC Assay® biofilm inoculator device was used to evaluate the biofilm eradication ability of test antimicrobials alone and in combination against clinically relevant pathogens. The checkerboard microbroth dilution assay was performed to analyze the synergism between test antimicrobials.


Against planktonic cells, the combination of tetrasodium EDTA with ethanol or chlorhexidine HCl resulted in synergistic to indifferent activity, with no antagonism observed. Against mature biofilms, all combinations were synergistic. The MBEC of each test antimicrobial was decreased from 4- to -64-fold when used in combination as compared to when agents were used alone. We optimised the concentration of antimicrobials to achieve rapid eradication of pre-formed biofilms. A triple combination of 3% tetrasodium EDTA, 20% ethanol and 2.5 μg/mL chlorhexidine HCl completely eradicated 48-h-old biofilms of all test strains within 2 h.


All three antimicrobial agents can be used together for prevention and treatment of biofilms and biofilm-related infections. The observed in vitro efficacy should be tested further through in vivo and clinical studies.

Keywords: EDTA, Ethanol, Chlorhexidine, Biofilm, Bactericidal, Fungicidal.

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