Bactericidal Quantitative Suspension Assay of Chlorhexidine-Based Disinfectant according to EN Standard 13727


  • Çinel Köksal Karayildirim Ege University, Science Faculty, Biology Department, 35100 Bornova-Izmir, Turkey
  • Ebru Sanci Ege University Center for Drug Research & Development and Pharmacokinetic Applications, 35100 Bornova-Izmir, Turkey



 EN 13737, Bactericidal activity, Chlorhexidine, Biocides, Escherichia coli K12, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus hirae


Purpose: Hand hygiene with biocidal products is an effective strategy for preventing healthcare-associated infection in medical areas. It is a first-line defense in the prevention and control of microbial growth. In comparison to biocide efficacy, methods for biocide susceptibility testing of microorganisms have to be standardized for routine use. One of the aims of this study was to formulate a chlorhexidine-based hand disinfectant as a biocidal product with a low and effective concentration.

Methods: This biocidal product was tested against E. coli K12, P. aeruginosa, S. aureus, and E. hirae to evaluate their susceptibility at different concentrations of chlorhexidine (0.1 %, 0.04 %) according to European (EN) Standard.

Results: The results of the dilution-neutralization method indicated that 0.1 % chlorhexidine-based disinfectant represented strong antibacterial activity with a 5 decimal logarithmic reduction under clean conditions against four reference strains. All experimental analyses and conditions were supported by pre-validation and verification studies

Conclusion: This was the first study on chlorhexidine-based biocidal product analysis which was conducted according to the EN 13727 Standard.


Le Y, Hou P, Wang J, Chen FJ, Controlled release active antimicrobial corrosion coatings with Ag/SiO2 core–shell nanoparticles. Materials Chemistry and Physics 2010; 120: 351-355.

Urbanczyk MM, Bester K, Borho N, Schoknecht U, Bollmann EU. Influence of pigments on phototransformation of biocides in paints. Journal of Hazardous Materials 2019; 364: 125-133.

ECHA Guidance on the Biocidal Products Regulation Volume II Efficacy - Assessment and Evaluation (Parts B+C) Version 3.0 April 2018.

Low A. Regulation of sporicides under the European Biocidal Products Directive. Journal of Hospital Infection 2011; 77: 189- 192.

Fraser SN, Johnson KA, Wilborn RR, Dujovne AG, Leland N. Evaluation of chlorhexidine hydrochloride treatment on endometrial health of normal mares. Theriogenology 2017; 88: 61-66.

World Health Organization, Model Lists of Essential Medicines (EML) essentialmedicines/en/ 2017.

Zhang Y, Zhao Y, Xu C, Zhang X, Li J, Dong G, Cao J, Zhou T. Chlorhexidine exposure of clinical strains of Klebsiella pneumoniae leads to acquired resistance to this disinfectant and colistin. International Journal of Antimicrobial Agents 2019; 53(6): 864-867.

Bock LJ, Wand ME, Sutton JM. Varying activity of chlorhexidinebased disinfectants against Klebsiella pneumoniae clinical isolates and adapted strains. Journal of Hospital Infection 2016; 93: 42-4.

Liu Q, Zhao H, Han L, Shu W, Wu Q, Ni Y. Frequency of biocideresistant genes and susceptibility to chlorhexidine in high-level mupirocin-resistant, methicillin-resistant Staphylococcus aureus (MuH MRSA). Diagnostic Microbiology and Infectious Disease 2015; 82: 278-283.

Sipahi RO, Uysal S, Aydemir ?S, Pullukçu H, Ta?bakan M, Tu?nger A, Çilli FF, Yazmazhan T, Arda B, Sipahi H, Ulusoy S. Antibacterial resistance patterns and incidence of hospitalacquired Staphylococcus aureus bacteremia in a tertiary care educational hospital in Turkey:a perspective from 2001 to 2013. Turk J Med Sci 2017; 47: 1210-1215.

Sipahi RO. Economics of antibiotic resistance. Expert Rev Anti Infect Ther 2008; 6: 523-539.

Bourafa N, Loucif L, Boutefnouchet N, Rolain MJ. Enterococcus hirae, an unusual pathogen in humans causing urinary tract infection in a patient with benign prostatic hyperplasia: first case report in Algeria. New Microbes New Infect 2015; 8: 7-9.

Sankalp Malhotra S, Limoli HD, English EA, Parsek RM, Wozniak JD. Mixed Communities of Mucoid and Nonmucoid Pseudomonas aeruginosa Exhibit Enhanced Resistance to Host Antimicrobials. American Society for Microbiology 2018; 9(2): e00275-18.

Karay?ld?r?m KÇ, Çelenk UV. Antibacterial Efficiency of Benzalkonium Chloride Base Disinfectant According To European Standard 13727 Chemical Analysis and Validation Studies. CBÜ Fen Bilimleri Dergisi 2016; 12: 39-47.

EN 13727, Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity in the medical area - Test method and requirements (phase 2, step 1) 2014.

Hsu SC, Chen MI, Liang CK, Shih HC. Evaluation of disinfection efficiency in pet's hospital by using chlorine dioxide. Sustainable Environment Research 2016; 26: 191-195.

Chow A, Arah AO, Chan PS, Poh FB, Krishnan P, Kian W, Choudhury S, Chan J, Ang B. Alcohol handrubbing and chlorhexidine handwashing protocols for routine hospital practice: A randomized clinical trial of protocol efficacy and time effectiveness. American Journal of Infection Control 2012; 40: 800-5.

Ferraz CC, Gomes BP, Zaia AA, Teixeira FB, Souza-Filho FJ. In vitro assessment of the antimicrobial action and the mechanical ability of chlorhexidine gel as an endodontic irrigant. J Endod 2001; 27: 452-455.

Gomes BP, Vianna ME, Zaia AA, Almeida JF, Souza-Filho FJ, Ferraz CC. Chlorhexidine in endodontics. Braz Dent J 2013; 24: 89-102.

Panpaliya PN, Dahake TP, Kale JY, Dadpe VM, Kendre BS, Siddiqi GA, Maggavi RU. In vitro evaluation of antimicrobial property of silver nanoparticles and chlorhexidine against five different oral pathogenic bacteria. Saudi Dental Journal 2019; 31: 76-83.

Baraldi MM, Gnatta RJ, Padoveze CM. Risks and benefits of using chlorhexidine gluconate in handwashing: A systematic literature review. American Journal of Infection Control 2018; 1- 11.

Leikin JB, Paloucek FP. Chlorhexidine Gluconate Poisoning and Toxicology Handbook (4th ed.), Informa 2008; 183-84.

Dumville JC, McFarlane E, Edwards P, Lipp A, Holmes A, Liu Z. Preoperative skin antiseptics for preventing surgical wound infections after clean surgery. The Cochrane Database of Systematic Reviews 2015; 4: 39-49.

Fiorentino MAF, Corrêa AM, Salgado NRH. Development and validation of a microbiological assay for determination of chlorhexidine digluconate in aqueous solution. Brazilian Journal of Pharmaceutical Sciences 2013; 49: 351-358.

Barnes S, Stuart R, Redley RNB. Health care worker sensitivity to chlorhexidine-based hand hygiene solutions: A cross-sectional survey. American Journal of Infection Control 2019; 00: 1-5.

Morrissey I, Oggioni MR, Knight D, Curiao T, Coque T, Kalkanci A, Martinez JL. BIOHYPO Consortium Evaluation of epidemiological cut-off values indicates that biocide resistant subpopulations are uncommon in natural isolates of clinically relevant microorganisms. PLoS One 2014; 9(1): e86669.