A Cumulative Spore Killing Approach: Synergistic Sporicidal Activity of Dilute Peracetic Acid and Ethanol at Low pH Against Clostridium difficile and Bacillus subtilis Spores

Effective hand hygiene is essential to prevent transmission of pathogens [1, 2]. Due to their efficacy and convenience, alcohol-based hand sanitizers have become the primary method of hand hygiene in healthcare settings, and they are commonly used in the community [3]. The antimicrobial activity of alcohols is due to their ability to denature proteins, resulting in germicidal activity against vegetative bacteria and many fungi and enveloped viruses [4]. However, alcohols lack activity against bacterial spores, and use of alcohol hand sanitizer does not reduce levels of spores on hands [5, 6]. This gap in the spectrum of activity of alcohols is of critical importance because the spore-forming anaerobe Clostridium difficile is a major pathogen in healthcare facilities and in the community [7, 8]. In addition, the intentional use of Bacillus anthracis spores as an agent of bioterrorism remains a threat, and ethanol is ineffective in reducing levels of a surrogate of B anthracis on hands [9, 10]. Although soap and water hand washing and application of chlorine or hypochlorous acid solutions can reduce levels of C difficile and Bacillus spp spores on hands [5, 6, 10–12], these hand hygiene methods lack the convenience and efficiency of alcohol hand sanitizers. As a novel approach for development of sporicidal hand hygiene solutions, we have investigated the potential to induce sporicidal activity in alcohol and other skin disinfectants through alteration of physical or chemical conditions that might degrade or allow penetration of spore coats [13, 14]. Chlorhexidine, a nonsporicidal cationic bisbiguanide, exhibited activity against C difficile spores in the presence of denaturing chemical and physical conditions such as elevated temperature, elevated pH, or alcohol [13]. Moreover, acidification, alkalinization, and heating of ethanol induced rapid sporicidal activity against C difficile and to a lesser extent Bacillus thuringiensis and Bacillus subtilis [14]. The sporicidal activity of acidified ethanol was enhanced by increasing ionic strength and mild elevations in temperature [14]. On skin, sporicidal acidified ethanol formulations with pH 1.5–2 were as effective as soap and water hand washing in reducing levels of C difficile spores [14]. Although these findings are promising, it is anticipated that sporicidal ethanol formulations with less acidic pH will be required to assure safety and tolerability with repeated use. Peracetic acid (PAA) is a sporicidal oxidizing agent used for disinfection of hard surfaces, waste water, and heat-labile instruments [15–17]. Although irritating to the skin and respiratory tract at high concentrations, PAA is well tolerated on skin at concentrations of ≤2000 ppm (0.2% w/v) [6, 18]. Peracetic acid at 2000 ppm combined with surfactant (90-second application time) was more effective than a tap water wash (30-second application) for removal of C difficile spores from skin [6]. Alcohol enhances the efficacy of PAA [19, 20]. Dilute PAA combined with ethanol is used for disinfection of tissue and bone grafts [21, 22], and 2000 ppm PAA combined with 80% ethanol has been proposed as a hand sanitizer due to its efficacy against enveloped viruses [23]. It has recently been demonstrated that B subtilis spores with defective coats are greatly sensitized to PAA, purportedly because PPA is allowed greater access to its site of sporicidal action on the inner membrane [24]. Based on these data, we hypothesized that the combination of dilute PAA and acidified ethanol would result in synergistic killing of C difficile and Bacillus spp spores, and the addition of dilute PAA would result in an effective sporicidal acidified ethanol hand hygiene solution with pH > 3.