Efficacy of concurrent application of chlorhexidine gluconate and povidone iodine against six nosocomial pathogens Michele J. Anderson, PhD,a Mary E. Horn, BS,a Ying-Chi Lin, PhD,a PatrickJ.Parks,MD,PhD,a,b and Marnie L. Peterson, PharmD, PhDa Minneapolis and St. Paul, Minnesota Background: Chlorhexidine gluconate (CHG) and povidone iodine (PI) are rarely used concurrently despite a lack of evidence regarding functional incompatibility of these agents. Methods: CHG and PI, alone and combined, were evaluated against Staphylococcus aureus (methicillin-suseptible S aureus [MSSA] and methicillin-resistant S aureus [MRSA]), Staphylococcus epidermidis (MRSE), Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli using checkerboard microbroth dilution techniques. Minimum bactericidal concentration (MBC) was the con- $ centration (percent wt/vol) that reduced bacterial burden 5-log10 colony-forming units/mL at 2 hours when compared with bac- terial densities in growth controls. Fractional bactericidal concentration indexes (FBCIs) were calculated to determine CHG and PI compatibility. Additionally, tissue plugs from freshly excised porcine vaginal mucosa were infected with S aureus (MSSA), treated for 2 hours with CHG 3%, PI 5%, or CHG 3% and PI 5% combined and then viable bacteria on the tissue plugs enumerated. Results: In broth, CHG demonstrated dose-dependent bactericidal activity, whereas PI activity was all-or-none. All isolates studied were similarly susceptible to CHG (MBCs: 0.0078% 6 0.0019%, 0.0069% 6 0.0026%, 0.0024% 6 0.0005%, 0.0024% 6 0.0005%, 0.0059% 6 0.0%, and 0.0029% 6 0.0%, respectively). The MBCs of PI were identical (0.625%) for all isolates. Overall, FBCI calculations showed indifference. Treatment of MSSA-infected porcine tissue for 2 hours demonstrated that the CHG-PI com- bination was superior to either antiseptic alone. Conclusion: FBCIs, determined in broth culture, indicate that combining CHG and PI had no negative impact on antisepsis. More- over, data from an ex vivo porcine mucosal infection model suggest a potential benefit when combining the 2 antiseptic agents. Key Words: Chlorhexidine gluconate; povidone iodine; antiseptic; bacteria; fractional bactericidal concentration index. Copyright ª 2010 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved. (Am J Infect Control 2010;38:826-31.) Nosocomial infection prevention involves a multi- bacteria will develop resistance, whereas antibiotics pronged approach: infection control, antibiotic pro- tend to have specific intracellular targets. Also, resis- phylaxis, and antisepsis.1 The emergence of tance to antiseptics may be overcome by increasing multidrug-resistant organisms underscores the impor- the concentration or time of exposure.3 Chlorhexidine tance of using antiseptic agents as a strategy for infec- gluconate (CHG) and polyvinylpyrrolidone iodine (po- tion prevention.2 In general, antiseptics have a broader vidone iodine; PI) are 2 of the most commonly used spectrum of activity than antibiotics. Antiseptics often antiseptic agents for antimicrobial skin preparation have multiple targets, reducing the likelihood that prior to surgery and central venous catheter (CVC) in- sertion.4 Many studies have compared the individual a From the University of Minnesota, Minneapolis, MN ; and 3M Company, antiseptic activity of these 2 compounds, but few stud- St. Paul, MN.b ies have evaluated the activity of the compounds in Address correspondence to Marnie L. Peterson, PharmD, PhD, Depart- combination. In addition, studies investigating the ment of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Room 4-212 McGuire Translational Research combined effects of CHG and PI have applied the anti- Facility, Minneapolis, MN 55455. E-mail: [email protected]. septics sequentially. Langgartner et al concluded that Supported by a grant from the 3M Company, St. Paul, MN (to M.L.P.). skin disinfection with a propanol/CHG solution fol- Previously presented as an abstract at The Society for Healthcare lowed by PI was superior to either regimen alone in Epidemiology of America (SHEA) meeting, 2009, No. 449. preventing CVC colonization, whereas Guzel et al Conflicts of interest: P.J.P. is an employee of 3M Company and provided found that cleaning the skin with 15% CHG followed editorial assistance during manuscript preparation. The remaining by 10% PI was safe and effective for skin antisepsis authors report no conflicts of interest. prior to neurosurgical intervention.5,6 Our study exam- 0196-6553/$36.00 ined the antimicrobial effects of an aqueous solution Copyright ª 2010 by the Association for Professionals in Infection containing both CHG and PI. Therefore, the bacteria Control and Epidemiology, Inc. Published by Elsevier Inc. All rights were exposed to the 2 compounds simultaneously. reserved. CHG is a polycationic bisbiguanide with broad- doi:10.1016/j.ajic.2010.06.022 spectrum antimicrobial activity. CHG damages the 826 www.ajicjournal.org Anderson et al. 827 Vol. 38 No. 10 outer bacterial surface layers, thereby promoting its forming units (CFU)/mL in cation-adjusted Mueller Hinton own uptake and subsequently attacking the cytoplas- broth. The number of bacteria in the culture was verified mic or inner membrane of the organism.3,7 At low using standard saline dilution and plating techniques. concentrations, CHG reduces membrane fluidity, os- The adjusted bacterial suspensions were used to inoculate moregulation, and metabolic capacity.8 At higher con- the 96-well microtiter plates resulting in inoculums of centrations, such as those in commercially available ;106 CFU/mL. preparations, the membrane becomes liquid crystal- MSSA (MN8) was chosen as the model organism in line and loses its integrity.8 CHG has broad-spectrum the studies utilizing the ex vivo porcine model of infec- activity against bacteria and yeast. It has low antiviral tion. A 1-mL aliquot of an overnight culture grown in activity and is not sporicidal but can prevent spore Todd Hewitt broth at 378C was centrifuged to pellet development.3 In addition to its direct effect on mi- the cells and washed in RPMI 1640 media (no supple- crobes, CHG can bind to the outermost layer of the ments). The washed cell pellet was resuspended in ; epidermis and to mucous membranes, which provides RPMI 1640 media to an OD595 of 0.5, which resulted a lingering or persistent antimicrobial effect.9,10 in ;1 3 106 CFU in the 2-mL volume used to infect the PI is a complex of iodide and a solubilizing carrier, tissue plugs. polyvinylpyrrolidone, which acts as a reservoir of ‘‘free’’ iodine (the active component). The iodine is Antiseptics slowly released and delivered to the bacterial cell surface Aqueous CHG 3% wt/vol was prepared from a where it penetrates the cell membrane and inactivates 20% wt/vol stock (Sigma-Aldrich Corporation, St. Louis, key cytosolic proteins, fatty acids, and nucleotides.11 MO) in water and further diluted before addition to mi- Slow release of iodine from the PI complex in solution crotiter plates. CHG was tested over a concentration minimizes iodine toxicity towards mammalian cells. range of 0.0018% to 0.375%. The concentration (per- Iodine has broad-spectrum antibacterial activity, as cent wt/vol) of CHG in commercial products ranges well as activity against fungi, protozoa, viruses, and from 0.12% in oral rinses to 4% in topical skin some bacterial spores.12 cleansers. CHG and PI have different cellular targets and differ- Polyvinylpyrrolidone iodine (PI) 10% wt/vol (Sigma- ent mechanisms of action. These differences may Aldrich Corporation) was prepared from powder, in wa- prove beneficial when using these 2 antiseptics in com- ter. PI was tested over a concentration range of 0.078% bination. CHG damages the outer membrane, which, in to 2.5%. The concentration of PI in commercial prepa- turn, would augment access to the intracellular targets rations is usually 10%, but ophthalmic preparation so- necessary for the bactericidal action of PI. Moreover, lutions contain only 5%. PI’s activity is more immediate, whereas CHG’s activity Higher concentrations were required for antiseptic occurs later and lingers,9,13 implying that these 2 com- activity in the porcine ex vivo mucosal experiments pounds may work cooperatively. In clinical practice, due to the interaction of CHG and PI with mucosal pro- the use of CHG and PI in combination is commonly teins. The concentrations of CHG and PI alone were 3% avoided despite a lack of evidence regarding functional and 5%, respectively. After combining a 6% solution of incompatibility of these agents. The aim of this study CHG with an equal volume of 10% PI, the final concen- was to determine whether the combined activity of trations of CHG and PI in the mixture were the CHG and PI against clinically relevant pathogens is same as the individual concentrations: 3% and 5%, inferior to the activity of either agent alone. respectively. Microdilution assay MATERIALS AND METHODS Bacteria The checkerboard method was used to determine the minimum bactericidal concentration (MBC) of Clinical isolates of methicillin-sensitive and methicillin- each antiseptic agent, both alone and in combination. resistant Staphylococcus aureus (MSSA and MRSA, respec- We defined the MBC as the concentration that reduced tively), methicillin-resistant Staphylococcus epidermidis the bacterial burden by $5-log10 CFU/mL when com- (MRSE), multidrug-resistant Acinetobacter baumannii