UNIVERSIDAD DE COSTA RICA SISTEMA DE ESTUDIOS DE POSGRADO DESINFECCIÓN DE SUPERFICIES CONTAMINADAS CON ESPORAS DE Clostridioides difficile CON DISOLUCIONES ACTIVADAS ELEC- TROQUÍMICAMENTE Tesis sometida a la consideración de la Comisión del Programa de Estudios de Posgrado en Biología para optar al grado y título de Maestría Académica en Biología. ALEJANDRO MEDAGLIA MATA Ciudad Universitaria Rodrigo Facio 2019 DEDICATORIA A mis padres, a mis hermanos, a mi novia Marian y a mi familia. ii AGRADECIMIENTOS El autor desea dejar constancia de su agradecimiento a las siguientes personas e institucio- nes, que de alguna u otra forma colaboraron para la culminación de este proyecto: A mis padres Alejandro Medaglia Chaverri y Chris Mata Quirós, quienes siempre me han apoyado e impulsado al éxito en mi vida. A mis hermanos Mariella y Fabrizzio, quienes han sido mis acompañantes en cada una de las actividades que he emprendido a lo largo de estos años. Al Dr. César Rodríguez Sánchez por sus provechosos consejos y por todo el apoyo, aten- ción y compromiso demostrado hacia mi persona durante el desarrollo de este trabajo. A la M.Sc. Ethel Sánchez Chacón por acceder a fungir como mi asesor en este proyecto, así como por todas las sugerencias y consejos que me brindó en términos de procesamiento de muestras. Al Dr. rer. nat. Ricardo Starbird Pérez por acceder a fungir como mi asesor en este proyecto, así como por todo el tiempo y atención que dedicó para atender cualquier duda que tuve. Al Centro de Investigación en Enfermedades Tropicales y el Laboratorio de Investigación en Bacteriología Anaerobia de la Universidad de Costa Rica, por permitirme el uso de sus instalaciones, equipos y reactivos para el desarrollo de este trabajo. Así como a todos sus funcionarios quienes me ayudaron de manera atenta en cada consulta que tuve. Al Laboratorio Institucional de Microscopía del Instituto Tecnológico de Costa Rica por el apoyo recibido, así como por permitirme hacer uso de sus instalaciones y equipos para culminar el proyecto. iii HOJA DE APROBACIÓN “Esta tesis fue aceptada por la Comisión del Programa de Estudios de Posgrado en Biolo- gía de la Universidad de Costa Rica como requisito parcial para optar al grado y título de Maestría Académica en Biología” iv ÍNDICE GENERAL DEDICATORIA ............................................................................................................................ II AGRADECIMIENTOS .............................................................................................................. III HOJA DE APROBACIÓN .......................................................................................................... IV RESUMEN ................................................................................................................................. VII ABSTRACT .............................................................................................................................. VIII ÍNDICE DE CUADROS .............................................................................................................. IX ÍNDICE DE FIGURAS................................................................................................................. X LISTA DE ABREVIACIONES .................................................................................................. XI CAPÍTULO I. TRADITIONAL AND NEW DISINFECTION METHODS OF C. DIFFICILE VEGETATIVE CELLS AND SPORES ON HOSPITAL SURFACES .............. 1 INTRODUCTION ............................................................................................................................... 1 METHODOLOGY .............................................................................................................................. 2 RESULTS AND DISCUSSION ............................................................................................................. 2 TRADITIONAL METHODS ................................................................................................................ 2 ALTERNATIVE METHODS ............................................................................................................... 8 CONCLUSION ................................................................................................................................. 12 CAPÍTULO II. DESINFECCIÓN DE SUPERFICIES CONTAMINADAS CON ESPORAS DE CLOSTRIDIOIDES DIFFICILE UTILIZANDO DISOLUCIONES ACTIVADAS ELECTROQUÍMICAMENTE ................................................................................................... 13 INTRODUCTION ............................................................................................................................. 13 MATERIALS AND METHODS ......................................................................................................... 14 CHEMICALS .................................................................................................................................... 14 BACTERIAL STRAINS AND CULTURE CONDITIONS .......................................................................... 15 SPORE ISOLATION ........................................................................................................................... 15 ECAS SYNTHESIS ........................................................................................................................... 15 CHLORINE QUANTIFICATION .......................................................................................................... 16 v VOLTAMMETRY ANALYSES ............................................................................................................ 16 SPORICIDAL ACTIVITY TESTS ......................................................................................................... 16 TRANSMISSION ELECTRON MICROSCOPY (TEM) .......................................................................... 17 STATISTICAL ANALYSES ................................................................................................................ 17 RESULTS AND DISCUSSION ........................................................................................................... 18 ECAS CHARACTERIZATION ........................................................................................................... 18 SPORICIDAL ACTIVITY TESTS AND STATISTICAL ANALYSIS ........................................................... 21 ELECTRON MICROSCOPY ................................................................................................................ 24 CONCLUSIONS ............................................................................................................................... 25 REFERENCIAS BIBLIOGRÁFICAS ....................................................................................... 26 APÉNDICES ................................................................................................................................ 42 vi RESUMEN Clostridioides difficile es el principal agente causal de diarrea nosocomial. Esta bacteria se diferencia bajo ciertas condiciones adversas en endosporas, las cuales son difíciles de con- trolar debido a su alta resistencia a múltiples agentes desinfectantes. Agentes oxidantes fuertes y compuestos derivados del cloro (p.ej. hipoclorito de sodio (NaOCl), aldehídos y ácido peracético) han demostrado ser efectivos en la eliminación de esporas de C. difficile en suspensión y en superficies. Sin embargo, su uso se asocia a numerosas desventajas, como corrosión de superficies, mal olor, irritación de ojos, piel y mucosas, entre otras. Estas características indeseables están ausentes en las llamadas disoluciones electroquími- camente activadas (ECAS), las cuales muestran actividad bactericida y esporicida, bajos costos de producción y almacenamiento, capacidad de ser preparadas in situ, y alta com- patibilidad ambiental. En esta investigación se comparó la eficacia de ECAS derivadas de NaCl (0.19% w/v NaOCl, pH=9.6-10.3), cloro comercial (2.83% w/v NaOCl, pH=5.6) e isocianurato de sodio (NaDCC, pH=6.8) para inactivar esporas de diversas cepas de C. difficile sobre superficies inanimadas de acuerdo con la prueba estándar cuantitativa apli- cada US EPA MB-21-03. Los valores de reducción logarítmica (VRL) obtenidos fueron estadísticamente diferentes entre los tratamientos (F3,40=76.09, p<0.0001), las cepas (F9,40=16.42, p<0.0001) y la su interacción (F27,40=4.43, p<0.0001). Para efectos de una potencial futura aplicación en campo, las ECAS mostraron VRLs similares o mejores (0.40-5.56) que los obtenidos con NaOCl (0.12-5.50) o NaDCC (0.10-5.12). Análisis por TEM revelaron que la morfología de esporas expuestas a ECAS, NaOCl y NaDCC es si- milar, pero distinta a la de esporas no tratadas. En este contexto, no se observó una disrup- ción evidente de la ultraestructura de las esporas, por lo que es probable que el daño cau- sado por los desinfectantes probados ocurra a nivel funcional. Sin importar la cepa ni el tiempo de activación de las ECAS, un análisis factorial confirmó que 15 minutos de expo- sición son suficientes para reducir al menos en 5 log10 los recuentos en plato de esporas de la cepa más tolerante ensayada. Estos resultados posicionan a las ECAS derivadas de NaCl como una alternativa prometedora para la desinfección de esporas de C. difficile en super- ficies ambientales duras no porosas y dispositivos médicos vii ABSTRACT Clostridioides difficile is the causal agent of nosocomial diarrhea and arises as an important cause of community-acquired diarrhea. Under adverse conditions, this bacterium differenti- ates into endospores, which are resistance structures with high tolerance to multiple disin- fectant agents and therefore troublesome to control. Strong oxidizing agents and chlorine- derived compounds, such as hypochlorite (ClO-1), sodium dichloroisocyanurate (NaDCC), glutaraldehyde, o-phthalaldehyde, hydrogen peroxide and peracetic acid, have proven effec- tive in the inactivation of spores of C. difficile. However, their use is associated with numer- ous drawbacks, including corrosion of surfaces, bad odor, and irritation of eyes, skin and mucous membranes, among others. These undesired characteristics are not present in the so- called electrochemically activated solutions (ECAS), which show bactericidal and sporicidal activity, low production and storage costs, possibility to be prepared in situ, and high envi- ronmental compatibility. In this study, the efficacy of NaCl-derived ECAS (0.18% w/v NaOCl, pH=9.6-10.3), commercial chlorine (2.83% w/v NaOCl, pH=5.6) and NaDCC (pH=6.8) to inactivate spores of various strains of C. difficile on inanimate surfaces was com- pared using the US EPA MO-21-03 standard quantitative test. The logarithmic reduction values (LR) recorded were significantly different between treatments (F3,40 = 76.09, p<0.0001), strains (F9,40=16.42, p<0.0001) and the interaction of both factors (F27,40=4.43, p<0.0001). For the purposes of a potential future field application, ECAS showed similar or better LR values (0.40-5.56) than NaOCl (0.12-5.50) or NaDCC (0.10- 5.12). Transmission Electron Microscopy (TEM) analyses revealed that the morphology of spores exposed to ECAS, NaOCl and NaDCC was similar but distinct to that of untreated spores. No evident ultrastructural disruptions were seen, so the damage likely occurs at a functional level. Regardless of the strain or the ECAS activation time, a factorial design con- firmed that 15 minutes of exposure time are sufficient to reduce spore plate counts from the most tolerant strain assayed in at least 5 log10 units. These results highlight NaCl-derived ECAS as a promising alternative for the disinfection of C. difficile spores on hard non-porous environmental surfaces and medical devices. viii ÍNDICE DE CUADROS Table 1. Commonly used agents for the disinfection of C. difficile in healthcare facilities. .3 Table 2. Summary of the principal characteristics of each disinfection study on the reduction of C. difficile. ........................................................................................................11 Table 3. Susceptibility of C. difficile strains to sodium hypochlorite (NaOCl), sodium dichloroisocyanurate (NaDCC) and electrochemically activated solutions (ECAS) as determined by a carrier test disinfection method. .................................................................22 ix ÍNDICE DE FIGURAS Figure 1. Cyclic voltammograms obtained for (A) sodium chloride (NaCl), (B) sodium hypochlorite (NaOCl), and (C) electrochemically activated solutions (ECAS). Measurements obtained with platinum electrodes and no reference electrode. ...................20 Figure 2. Logarithmic reduction of C. difficile spores caused by sodium hypochlorite (NaOCl, blue points), sodium dichloroisocyanurate (NaDCC, red boxes), and electrochemically activated solutions (ECAS, brown triangles). Black lines represent the mean of the treatment. ..........................................................................................................23 Figure 3. Logarithmic spore reduction as function of ECAS exposure and activation times. (A) LIBA-5757 (Low ECAS susceptibility) (B) LIBA-5758 (High ECAS susceptibility). 24 Figure 4. TEM micrographs of C. difficile LIBA-5758 exposed to ECAS (B), NaOCl (C), and NaDCC (D). An unexposed control is shown in (A). ....................................................25 Supplementary Figure 1. Mean logarithmic reduction of C. difficile spores from different strains caused by sodium hypochlorite (NaOCl), sodium dichloroisocyanurate (NaDCC), and electrochemically activated solutions (ECAS). Bars represent means with standard deviation. (Green dotted line: mean for each strain treatment, red dotted line: 5 log10 reduction). .............................................................................................................................42 x LISTA DE ABREVIACIONES ECAS: Electrochemically activated solutions CDI: Clostridioides difficile infection ORP: Oxidation-reduction potential NaCl: Sodium chloride NaOCl: Sodium hypochlorite NaDCC: Sodium dichloroisocyanurate MLST: Multi-locus sequence typing TSA: Trypticase soy agar TEM: Transmission electron microscopy PBS: Phosphate buffer saline ANOVA: Analysis of variance LR: Logarithmic reduction CFU: Colony forming unit xi 1 Capítulo I. Traditional and new disinfection methods of C. difficile vegetative cells and spores on hospital surfaces Introduction Clostridioides difficile is an anaerobic, endospore-forming, Gram-positive bacterium that has been isolated from human and animal feces, soils and sediments, and surfaces or per- sonnel in healthcare facilities, among other sources (Barbut & Petit, 2001; Levinson, 2004). Under certain conditions, including the use of antibiotics, gut colonization with C. difficile may progress into C. difficile infections (CDI), which are nowadays a leading cause of nosocomial infectious diarrhea (Dworkin & Falkow, 2006; Blossom & McDonald, 2007). In 2011 about half a million CDI cases and 30,000 deaths attributed to CDI were docu- mented only in the United States (Lessa et al., 2015), where CDI expenditures have been estimated to reach $433-793 million every year (Ghantoji, Sail, Lairson, DuPont, & Garey, 2010). Bacterial endospores are resistance structures. They are composed of a core with DNA, RNA, and enzymes, an inner membrane, a peptidoglycan-based cortex region, a coat de- marcated by an outer membrane, and, in some strains, an exosporium with hair-like pro- jections (Lawley et al., 2009; Paredes-Sabja, Shen, & Sorg, 2014). These structures are characterized by a low water content, high levels of dipicolinic acid, and their DNA is bound to small acid soluble proteins that prevent the formation of thymine dimers induced by UV radiation (Driks, 2002; Paredes-Sabja et al., 2014). Therefore, they are highly re- sistant to heat, chemical and physical agents (Weber, Rutala, Miller, Huslage, & Sickbert- Bennett, 2010), and can persist in the environment (e.g. floor, walls, and tables) for as far as five months (Lautenbach, Woeltje, & Malani, 2010; Kavaler & Alexander, 2014; Lessa F et al., 2015). Spores are critical for the acquisition and spread of C. difficile in hospitals (Wyllie, Hyams, & Kay, 2011). Traditional disinfection methods for the control of nosocomial pathogens in healthcare facilities include manual cleaning and the use of chemical agents (Zhang & Gamage, 2010; Schneider, 2013). However, these practices are often ineffective because: 2 i) they are restricted to accessible surfaces, ii) many disinfectants are incompatible with certain materials and/or toxic for humans, and iii) many disinfectants do not inactivate spores (Maclean et al., 2015). In this review we compare the efficiency of traditional methods for the inactivation of C. difficile spores in the hospital environment and present modern alternative agents to limit the spread and thereby control this emerging pathogen. Methodology A search of peer-reviewed journal articles or book chapters in English or Spanish published between 1993 and 2016 was performed using the Web of Science database. This time span was selected because most advances in C. difficile disinfection methods were published during this time window. The following search were used: ‘Clostridium difficile infection’, ‘Clostridium difficile disinfection’, ‘Clostridium difficile spores’, ‘Clostridioides difficile spores’, ‘Clostridioides difficile disinfection’, ‘chlorhexidine’, ‘chlorine-based com- pounds’, ‘aldehydes’, ‘peroxygens’, ‘hydrogen peroxide vapor’, ‘electrochemically acti- vated solutions’, ‘atmospheric pressure plasma’, and ‘photocatalytic disinfection’. The analysis was restricted to publications that report results of C. difficile disinfection tests. Moreover, due to the heterogeneity of experimental conditions applied across the studies, only logarithmic reductions and exposure times were compared. Results and Discussion Traditional methods Guanidines, chlorine-releasing compounds, aldehydes, and peroxides have been used to sanitize hands, sterilize instruments, or disinfect surfaces contaminated with C. difficile spores (Table 1). On the other hand, alcohol-based hand-rub solutions are not effective against C. difficile spores and it has been hypothesized that they may have contributed to 3 increasing the incidence of CDI (Siqueira et al., 2007; Weinstein, Milstone, Passaretti, & Perl, 2008). Table 1. Commonly used agents for the disinfection of C. difficile in healthcare facilities. Recommended Agent Recommended use Sporicidal Reference dose (Bettin, Clabots, Mathie, Chlorhexidine Hand cleaning 4% (m/v) No Willard, & Gerding, 2007) (Orenstein, Aronhalt, Sodium hypo- Surface disinfection 10% (v/v) Yes McManus, & Fedraw, chlorite 2011) Sodium dichloroi- Surface disinfection 1000 ppm Yes (Ascenzi, 1995) socyanurate Acidified sodium Surface disinfection 200 ppm Yes (Goda et al., 2017) chlorite Instrument steriliza- (Wullt, Odenholt, & Glutaraldehyde 2% (v/v) Yes tion Walder, 2003) Ortho-phthalalde- Instrument steriliza- (William Rutala & Weber, 0.55% (v/v) Yes hyde tion 2001) Instrument steriliza- Hydrogen perox- tion and surface dis- 3-6% (v/v) Yes (Alfa et al., 2010) ide infection Peracetic acid Surface disinfection 0.26-0.35% (v/v) Yes (Block, 2004) 4 Chlorhexidine Chlorhexidine (CHX) is a cationic bis guanidine with broad activity against Gram-positive and Gram-negative bacteria, yeasts, and some enveloped viruses. It shows low toxicity to humans; hence it is one of the most common active ingredients of antiseptics, particularly in hand washing and oral products (Mcdonnell, Russell, & Block, 1999; Siqueira et al., 2007; Weinstein et al., 2008; Shen, Stojicic, & Haapasalo, 2011). CHX is a strong alkali, practically insoluble in water. In disinfectant formulations, CHX is present as water-solu- ble salt forms such as chlorhexidine diacetate, chlorhexidine digluconate, or chlorhexidine dihydrochloride (Karpiński & Szkaradkiewicz, 2015). At low concentrations, CHX exerts a bacteriostatic effect due to protein cross-linking (Gomes et al., 2001). At high concentra- tions, instead, CHX exhibits a bactericidal effect by adsorbing onto the negative groups of the cell wall, causing leakage of cellular components. Though CHX lacks sporicidal activ- ity, Nerandzic & Donskey (Nerandzic & Donskey, 2015) showed that C. difficile spores immersed in a CHX solution become susceptible to heat killing at 80 ºC. Other studies have suggested that the use of CHX in hospital bathing can decrease CDI by killing C. difficile vegetative cells and through inhibition of spore germination (Rupp et al., 2012). Chlorine-based compounds Chlorine-releasing formulations for disinfection purposes often contain sodium hypo- chlorite (NaOCl) or sodium dichloroisocyanurate (NaDDC). These compounds possess an- timicrobial and sporicidal activity and their disinfection efficiency depends on the release of free available chlorine in the form of hypochlorous acid (HOCl) in aqueous solutions (Fraise, 1999; Clasen & Edmondson, 2006; Pfafflin & Ziegler, 2006). Hypochlorous acid (HOCl) is a strong oxidizing agent that reacts with proteins (Pattison & Davies, 2001, 2005; Pattison, Hawkins, & Davies, 2007) and free amino acids (Stadtman & Levine, 2003) by the formation of monochloramides and dichloramides with exposed amide functional groups of amino acids (Pullar, Vissers, & Winterbourn, 2000). Hypochlorous acid (HOCl) penetrates membranes by passive diffusion; hence most of its antimicrobial activity is in function of its concentration (Fukuzaki, 2006). 5 Sodium hypochlorite (NaOCl), also known as household bleach, is often commercialized as a 5.25% w/v solution with pH 12-13 (Greenberg, 2003). It shows activity against micro- bial spores (de Almeida et al., 2005) but is easily inactivated by organic matter (Kearns, Freeman, & Lightfoot, 1995). Orenstein et al. (2011) demonstrated that daily cleaning of hospital surfaces with chlorine-releasing agents reduced significantly the incidence rates of CDI and increased the time between hospital-acquired cases of CDI from 8 to 80 days. (Barbut et al. (2009) obtained up to 4.33 log10 CFU reduction of C. difficile spores with 20 minutes of exposure to a 0.5% NaOCl solution. Sodium dichloroisocyanurate (NaDCC) is used for surface decontamination or in industrial sanitizing products. It is often used as an alternative to sodium hypochlorite (NaOCl), be- cause it is more stable and less susceptible to inactivation by organic material (Ascenzi, 1995). Although, it is not as effective against C. difficile spores as NaOCl, it shows activity against spores from this pathogen (Bloomfield & Arthur, 1992). This activity is more strongly influenced by concentration rather than by contact time (Ungurs et al., 2011). Sodium chlorite is a strong oxidizing agent linked to antimicrobial effects (Lu, Luo, Turner, & Feng, 2007). It is approved by the Food and Drug Administration (FDA) for disinfection in poultry and red meat processing plants, and of raw agricultural commodities (Federal Register, 1998, 1999). Studies by Kobayashi, Iwashita, & Suzuki (1989) showed that acid- ification of a sodium chlorite solution increases its antimicrobial properties. Goda et al. (2017) proved the sporicidal effect of a weakly acidified chlorite acid water (WACAW) against C. difficile, with >3 log reductions in presence of organic matter (0.5% polypeptone) after 1 minute of contact time at a concentration of 200 ppm. Aldehydes The activity of the aldehydes is defined by the presence of a carbonyl group. One of the simplest aldehydes is acrolein, which is very active but also toxic, thus alternatives such as formaldehyde, glutaraldehyde, and ortho-phthalaldehyde have been used in disinfection (Ascenzi, 1995). 6 Glutaraldehyde (GTA) is a dialdehyde used for cold sterilization of medical equipment. It is usually commercialized as a 50% acidic aqueous solution and exhibits several ad- vantages, including a broad spectrum of antimicrobial activity, rapid inactivation of micro- organisms, and activity in the presence of organic matter (Leung, 2001; W Rutala, Gergen, & Weber, 1993; Simons et al., 2000; Ünal et al., 2006). It is a surface-acting disinfectant that crosslinks external proteins with a concomitant loss of function (Baba et al., 2002; McDonnell & Burke, 2011). GTA solutions are very effective against C. difficile vegetative cells and spores (Russell, 1999). For instance, GTA-based sterilization of C. difficile spores with a 2% v/v solution for 30 minutes has led to a 4-log reduction (Wullt et al., 2003). However, GTA must be activated prior to use because their sporicidal activity is only achieved at alkaline pH (Fraud, Maillard, & Russell, 2001). Unfortunately, it is linked to adverse effects such as irritation and sensitization of the eyes, skin, and respiratory tract (Takigawa & Endo, 2006; Vonberg et al., 2008). Ortho-phthalaldehyde (OPA) is an aromatic dialdehyde with both antimicrobial and spor- icidal activity that has been reported as an alternative to GTA in high-level disinfection (Fraud et al., 2001; Cabrera-Martinez, Setlow, & Setlow, 2002). OPA, which is usually found as a pale-blue solution containing 0.55% of the active compound, does not require activation prior to its use and has excellent stability over a wide range of pH (Pala & Moscato, 2013). It has shown activity against both Gram-positive and Gram-negative bac- teria and is not inactivated by organic matter (Babb & Bradley, 1999; Bridier et al., 2011). Its antimicrobial activity is due to strong cross-linking with primary amines of proteins (Simões et al., 2003, 2006). Simons et al. (2000) reported that OPA is less reactive for nucleophilic addition reactions than aliphatic aldehydes such as GTA, leading to a less efficient reaction with proteins. However, the lipophilic nature of OPA promotes its transport through lipids and compensates its lower cross-linking efficiency. The activity of OPA against C. difficile has not been widely studied (Barah, 2013). Nevertheless, a study conducted by Gonçalves et al. (2013) evidenced OPA efficacy of 100% for the inactivation of C. difficile spores within 3 minutes of exposure time, yet, no information regarding the logarithmic density of the initial inoculum was reported in this research. 7 Peroxygens Peroxygens include relatively simple biocides such as hydrogen peroxide (H2O2) and peracetic acid (PAA). These compounds have been used for antiseptic and disinfecting purposes on account of their capacity to oxidize proteins, lipids, and nucleic acids (McDonnell, 2007). H2O2 is commonly used as a high-level disinfectant for the disinfection and sanitization of medical equipment in healthcare facilities (William Rutala & Weber, 2004; Brudzynski et al., 2011). It is marketed as a liquid containing 3-6% v/v of the active compound (Andersen et al., 2006). H2O2 may also promote the oxidation and removal of organic matter present on surfaces (Ksibi, 2006). Its antimicrobial activity is due to the formation of hydroxyl radicals (•OH) (Labas et al., 2008) that disrupt membrane integrity and damage membrane proteins by the oxidation of amino acids such as histidine, methionine, cysteine, and phe- nylalanine, leading to protein malfunction (Coyle & Puttfarcken, 1993; Labas et al., 2008). Hydroxyl radicals may also cause lipid peroxidation, leading to structural alteration of cel- lular membranes (Dix & Aikens, 1993) as well as oxidation of nucleic acids, particularly at guanine residues (Hofer et al., 2005). Some microorganisms are more difficult to inacti- vate with H2O2 due to intrinsic catalase and peroxidase activities, which convert H2O2 to water (Finkel & Holbrook, 2000; Otter & French, 2009). Logarithmic reduction rates as high as 6 log10 can be achieved through a 10 min exposure of C. difficile spores to H2O2 (Pérez, Springthorpe, & Sattar, 2005). Congruently, Alfa et al. (2010) achieved 3 log10 reductions within one minute of exposure to a 7% v/v H2O2 formulation, establishing H2O2 as an alternative for killing C. difficile spores on bathroom surfaces. Although H2O2 is an effective disinfectant agent, its application is affected by the fact that the rooms requiring disinfection must be empty of patients and personnel (Rutala & Weber, 2011; Rutala, Gergen, & Weber, 2012). PAA consists of an equilibrium solution of peracetic acid, acetic acid, H2O2 and water usu- ally found in concentrations ranging from 12 to 15% v/v (Watada et al., 2005; Lindler, Lebeda, & Korch, 2007; Boyce et al., 2008). It is a strong oxidizing agent used as a high- level disinfectant due to its broad-spectrum antimicrobial effect, even in the presence of organic matter. Its biocidal activity is associated with its undissociated acid, which acts by 8 oxidation of sulfhydryl (-SH) and sulfur (S-S) bonds of proteins, such as those present in microbial spores.(Liberti, López, & Notarnicola, 1999; W Rutala & Weber, 1999; Santoro et al., 2007). PAA inactivates catalase; an enzyme that detoxifies free hydroxyl radicals (Mcdonnell et al., 1999). Wullt et al. (2003) achieved a 4 log10 reduction of C. difficile spores within 5 minutes of exposure time to a 0.26% PAA solution and it showed to be more toxic to C. difficile spores on stainless steel surfaces than NaDCC (Block, 2004). PAA formulations are slightly corrosive (Bielanski, 2005), hence they are not suitable for high-frequency application in healthcare facilities. Moreover, their sporicidal concentra- tion causes skin and eye damage (Boyce & Pittet, 2002; Wheeldon et al., 2008; Edmonds et al., 2013). Alternative methods Hydrogen peroxide vapor Hydrogen peroxide vapor (HPV) is an emerging technology for surface sterilization and disinfection which according to the Center for Chemical Process Safety (2010) is safer and easier to contain than chlorine dioxide and ethylene oxide. It degrades into water and oxy- gen after its application, is effective in low-temperature sterilization, and maintains its properties at high temperatures (Lindler et al., 2007). Boyce et al. (2008) achieved a con- siderable reduction of C. difficile in hospital rooms that were decontaminated with HPV at a concentration of hydrogen peroxide of 30% and between 3-4 hours of exposure time per patient room, as well as a reduction in the incidence of CDI during the intervention period. More recently, it has been shown that C. difficile spores with logarithmic densities as high as 6.4 log CFU can be inactivated with HPV in 90 minutes exposure time (Otter & French, 2009). HPV has been shown to be highly effective at lower concentrations than liquid H2O2, with the benefit of breaking down into harmless components, However, it holds certain disadvantages such as that rooms need to be sealed and that its levels must be closely mon- itored to allow workers and patients re-entry. Finally, HPV disinfection costs are consid- erably high (Boyce et al., 2008). 9 Electrochemically activated solutions (ECAS) ECAS are produced through electrolysis of a diluted salt solution (electrolyte) by applica- tion of direct current. This treatment leads to the conversion of the electrolyte into an acti- vated ‘metastable' state in which its chemical reactivity is increased (Oxidation-reduction potential (ORP) from +800 mV to +1200 mV) (Thorn, Lee, & Robinson, 2011; Garg & Garg, 2013). The electrochemical chamber usually consists of two submerged electrodes separated by a central compartment (O’Donnell et al., 2009; Liato et al., 2015). The elec- trolysis process involves two different phenomena, namely, reduction reactions on the neg- ative electrode (“cathode”) and oxidation reactions on the positive electrode (“anode”) (Aider et al., 2012). When the mineral component is NaCl, the solution obtained at the negative electrode exhibits detergent properties, with NaOH as principal component, and the solution obtained at the positive electrode consists of a mixture of oxidants (e.g hypo- chlorous acid). According to Thorn et al. (2011), outer and inner cell membranes are likely primary targets for ECAS, where they cause permeabilization, alteration of protein struc- ture, and ultimately cell lysis. Since NaCl-derived ECAS are mainly composed of hypo- chlorite, hydrochloric acid, and hypochlorous acid, their high effectivity is associated with their ORP, which generates osmotic unbalance, damaging membranes (Helme et al., 2010; Thorn et al., 2011). Robinson et al. (2010) reached a 5 log10 reduction in the recovery of C. difficile spores after a 20 seconds exposure to an ECAS that was derived from NaCl and had an ORP of approximately +1170 mV. ECAS can be generated on-site in the required quantities at low expenses, reducing the operating costs associated with transport, storage of oxidizing agents, as well as environmental impacts. Nevertheless, they cannot be stored for long periods of time and its effectiveness relies on the electrochemical cell capabilities (Thorn et al., 2011). Photocatalytic disinfection with TiO2 nanomaterials Photocatalysis is a process in which a chemical reaction is accelerated by the action of light and a catalyst such as TiO2, ZnO, CdS, and Fe2O3 (Taicheng An, Zhao, & Wong, 2016). According to Foster et al. (2011) TiO2 nanocomposites can act as a disinfectant because 10 they absorb photons to form thereby reactive oxygen species that disturb cell membrane phospholipids, lipoproteins, and nucleic acids, leading to function loss and eventually cell death. TiO2 nanocomposites can be conjugated with other compounds to increase their an- tibacterial and sporicidal properties, for example, Krishna et al. (2005) showed that multi- wall carbon nanotubes coated with titanium dioxide were more effective and required lower disinfection times for disinfection of Bacillus cereus spores than titanium dioxide nanopowders. With regard to C. difficile, Dunlop et al. (2010) obtained 2 log10 reductions of C. difficile spores in 5 hours exposure time using TiO2 thin films. TiO2 nanocomposites can be synthesized at low cost. Moreover, their products have low toxicity and high pho- tostability (Taicheng An et al., 2016). However, no standard methods for disinfectant test- ing have been designed so far, making it difficult to compare results across studies (Foster et al., 2011). Atmospheric-pressure plasma (APP) discharge Plasma, considered as the fourth state of matter, is an ionized gas with ions, electrons, and uncharged particles such as atoms, molecules, and radicals (Dunlop et al., 2010). There is thermal and cold plasma (CP), and in the latter, the electrons are at a hotter temperature than the heavy particles, which remain at room temperature. So-called CP jets have been used for disinfection purposes (Mai-Prochnow, Murphy, Mclean, Kong, & Ken, 2014). According to Vatansever (Vatansever et al., 2013), microorganism inactivation by CP oc- curs through direct permeabilization of membranes, oxidative damage to proteins, and chemical damage of nucleic acids. APP application holds promising advantages, such as a targeted disinfection in short periods of time (Mai-Prochnow et al., 2014; Vatansever et al., 2013). Nonetheless, not many studies have confirmed the disinfection potential of APP or have characterized plasma, its active species, and phototoxic side reactions (Mai-Prochnow et al., 2014). A study by Claro et al. (Claro, 2015) demonstrated the effectiveness of CP against C. difficile spores, with a time-dependent activity and a 2.69 log10 reduction with a 90-seconds exposure time. Table 2 summarizes features of selected disinfection agents for C. difficile. 11 Table 2. Summary of the principal characteristics of each disinfection study on the reduc- tion of C. difficile. Concentra- Test Contamination Log reduction Disinfectant Exposure time Reference tion method conditions achieved Spore- Sodium hypo- (Barbut et 0.5% 20 min carrier Clean 4.33 chlorite al., 2009) test Spore- Sodium dichloroiso- (Ungurs et 6000 ppm 2 min carrier Dirty 2.39 cyanurate al., 2011) test (Goda et Sodium chlorite 200 ppm 1 min Solution Dirty >3 al., 2017) (Wullt et Glutaraldehyde 2% 30 min Solution Clean 4.1 al., 2003) Spore- (Gonçalves Ortho-phthalalde- 0.55% 3 min carrier Clean n.r. et al., hyde test 2013) Spore- (Pérez et Hydrogen peroxide 7% 10 min carrier Dirty 6 al., 2005) test (Wullt et Peracetic acid 0.26% 5 min Solution Clean 4 al., 2003) Spore- (Otter & Hydrogen peroxide n.r. 30 min carrier Dirty 6.4 French, vapor test 2009) 12 (G. Robinson ECAS n.r. 20 sec Solution Clean 5 et al., 2010) (Dunlop et Titanium dioxide - 5 hours Surface Clean 2 al., 2010) Atmospheric-pres- Spore- (Claro et sure plasma dis- - 90 sec carrier Dirty 2.69 al., 2015) charge test n.r: not reported. Conclusion We compared the sporicidal activity of chemical agents of diverse nature against C. difficile spores. The traditional methods and alternative disinfection methods revised can achieve similar reduction levels (2.39 -6 log units vs. 2-6.4 log units) and require comparable ex- posure times (5-30 min vs. 1.5-90 min) (Pérez et al., 2005; Barbut et al., 2009; Otter & French, 2009; Claro et al., 2015). Therefore, it is not possible to discriminate them on the basis of efficacy. Unlike the modern alternative disinfectants, the traditional methods re- quire specific activation conditions, are easily inactivated by organic matter, show high toxicity, and in some cases, are hard to implement in hospitals (e.g. rooms must be emptied for very long periods). Moreover, the modern alternative disinfectants can be cheaper and/or more practical and may overcome the resistance developed by some strains to, for instance, chlorine-based compounds (Cherchi & Gu, 2011). However, some of them are linked to poorly understood properties and therefore require further investigation. 13 Capítulo II. Desinfección de superficies contaminadas con esporas de Clostridioides difficile utilizando disoluciones activadas electroquímicamente Introduction Clostridioides difficile is an important causal agent of diarrhea in hospitals and the com- munity (Lydyard et al., 2009; Harold & Dupont, 2019). This illness is triggered by a desta- bilization of the gut microbiota and its functioning, often due to antibiotic consumption (Ramirez, Liggins, & Abel-Santos, 2010; Feuerstadt, 2015). According to Garey et al. (2008), the number of cases and the severity of C. difficile infections (CDI) is increasing worldwide. Moreover, CDI are associated with high morbidity (Kwon, Olsen, & Dubberke, 2015) and high recurrence rates (McFarland, Elmer, & Surawicz, 2002), hence they are linked to a significant economic burden for healthcare systems and negatively impact the productivity of a country. A crucial step in the pathogenesis of CDI is the oral ingestion of endospores, which show structural and physiological properties that render them high resistance to physical agents, such as radiation or high temperatures, and to most chemical agents commonly used in hospital disinfection (Pankey, 2000). Furthermore, spores are able to survive and maintain their germinative potential on inanimate surfaces for long periods of time, acting as reser- voirs for the infection of personnel and patients in the hospital environment (Lautenbach et al., 2010). Chlorhexidine (Bettin et al., 2007), glutaraldehyde (Dyas & Das, 1985), peracetic acid (Bridier et al., 2011), hydrogen peroxide (Labas et al., 2008), and chlorine-derived com- pounds (A. Fraise, 1999) are the most common chemical agents used in hospitals and healthcare centers for disinfection of inanimate surfaces contaminated with C. difficile. However, their application is linked to certain disadvantages, including long exposure times, erratic efficiency, high toxicity, and damage to hospital infrastructure (Rutala et al., 1993; Thorn et al., 2011; William Rutala et al., 2012). Based on their accessibility, low cost, broad spectrum of antimicrobial activity, and the possibility of on-site preparation, different electrochemically activated solutions (ECAS) have emerged as an alternative for traditional disinfection methods (Helme et al., 2010; 14 Thorn et al., 2011). They are produced by electrolysis of a dissolved salt, with the conse- quent generation of strong oxidizing and reducing agents and free radicals in a metastable state that retains its biocidal activity for days or even months (Thorn et al., 2011). ECAS are usually classified according to its pH into acidic, neutral or alkaline ECAS. Ro- binson et al. (2010) successfully killed vegetative cells of Staphylococcus aureus and Pseu- domonas aeruginosa as well as spores of C. difficile and Bacillus atrophaeus using acidic ECAS. Likewise, Helme et al. (2010) reported a high killing activity of acidic, neutral, and alkaline ECAS with a NaOCl concentration of as low as 0.006% against various strains of B. cereus, Candida albicans, Escherichia coli, Enterococcus faecalis, Klebsiella oxytoca, P. aeruginosa, and S. aureus. In the case of NaCl-derived ECAS, the oxidation reactions that occur on the anode yield the formation of chlorine species, which react with water to form HOCl and HCl in an acidic solution (Barry-Ryan, 2012). On the cathode, by contrast, hydrogen and antioxidant compounds in an alkaline solution are produced (Thorn et al., 2011). The physicochemical properties of the ECAS vary depending on the voltage and amperage used in their synthesis; hence it is relevant to characterize the process and the end product of the electrolysis (Robinson, Thorn, & Reynolds, 2012). Robinson et al. (2010) showed that acidic ECAS inactivate C. difficile vegetative cells and spores in exposure periods of 30 seconds. However, their ECAS characterization was lim- ited to pH and oxidation-reduction potential (ORP) and the disinfection method was ap- plied directly to spore suspensions. Aiming to test the potential application of ECAS for C. difficile disinfection on healthcare facilities, we compared the logarithmic reduction of C. difficile spores on contaminated inanimate surfaces caused by a NaCl-derived ECAS and two chlorine-derived compounds widely used in hospitals (5000 ppm sodium hypochlorite, NaOCl and 1000 ppm) sodium dichloroisocyanurate, NaDCC). Materials and Methods Chemicals All chemicals were analytical grade and used without further purification. Platinum foil, sodium chloride (NaCl), sodium hypochlorite (NaOCl), sodium dichloroisocyanurate 15 (NaDCC), and sodium taurocholate were purchased from Sigma Aldrich (St. Louis, Mis- souri, USA). Brucella Agar with hemin and vitamin K1 was purchased from Beckton, Dickinson & Company (Franklin Lakes, New Jersey, USA). Bacterial strains and culture conditions All experiments were performed with reference C. difficile strains or WGS-typed field iso- lates (LIBA) from MLST Clades 1 to 5: 630 (Clade 1), LIBA-6276 (Clade 1), LIBA-5758 (Clade 2), LIBA-5757 (Clade 2), LIBA-6507 (Clade 3), LIBA-7110 (Clade 3), M68 (Clade 4), LIBA-7719 (Clade 4), M120 (Clade 5), and LIBA-7854 (Clade 5). When required, bac- teria were subcultivated in TYT broth (3% w/v Bacto tryptone, 2% w/v yeast extract, and 0.1% w/v sodium thioglycolate, pH 6.8). All incubations were done at 37ºC into an anaer- obic chamber (Bactron, Shel Lab, Cornelius, Oregon, USA) with a controlled atmosphere composed of 90% N2, 5% CO2 and 5% H2. Spore isolation The spore isolation procedure followed was based on protocols published by Paredes-Sabja et al. (2008) and Fraise et al. (2015). In detail, liquid cultures containing vegetative cells were inoculated onto TSA plates (1.5% pancreatic digest of casein, 0.5% peptic digest of soybean meal, and 0.5% NaCl) and incubated for 120 h at 37ºC. The resulting biomass was thereafter resuspended in PBS (0.01 M, pH 7.4) and spores were separated from vegetative cells by centrifugation in a Histodenz™ gradient. To remove Histodenz™ traces and pro- mote spore maduration, spore pellets were washed with PBS, resuspended into PBS + BSA 1% w/v, and stored at 4ºC for 15 days. Prior to their use, the purity and number of viable spores in the suspensions were determined through cultivation on Brucella Agar plates supplemented with 0.5% sodium taurocholate for 120 h at 37ºC. In addition, all spore sus- pensions were observed under a light microscope to confirm the absence of vegetative cells. ECAS synthesis Fifty ml of a 10% NaCl solution was placed into an electrochemical chamber equipped with platinum electrodes (Sigma Aldrich, St. Louis, Missouri, USA) and lacking a mem- brane between the anode and the cathode. A current of 3 A and a voltage of 20 V was 16 applied for 30 minutes using an Analog DC Power Supply (Goldstar, GP-303, Yeouido- dong, Seoul, South Korea). Chlorine quantification Total and available chlorine in ECAS, household bleach (3.15% w/v in label), and NaDCC (Sigma Aldrich, St. Louis, Missouri, USA) were quantified by titration with the standard procedure ASTM D 2022 (ASTM International, 2003) and sodium thiosulphate 0.1 M (Na2S2O3, Sigma Aldrich, St. Louis, Missouri, USA). This procedure was done by triplicate. A 10% NaCl solution (Sigma Aldrich, St. Louis, Missouri, USA) was used as a standard for quality control purposes. Titration results were reported as geometric means with 95% confidence intervals. Voltammetry analyses The electrochemical behavior of the synthesized ECAS, the 10% NaCl solution used for ECAS synthesis, and a 10% NaOCl solution (Sigma Aldrich, St. Louis, Missouri, USA), was measured by triplicate with an electrochemical workstation (AUTOLAB, model: PGSTAT-302, Utrecht, Netherlands) set to measure cyclic voltammetry from -5 to 5 V at a 100 mV/s scan rate. This workstation was equipped with a platinum electrode as the working electrode (1.875 cm2) and platinum foil as a counter electrode. (Hubler, Baygents, Chaplin, & Farrell, 2014; M. Spasojević, Krstajić, Spasojević, & Ribić-Zelenović, 2015). This series of electrochemical experiments was carried out in a 50 mL voltammetry cell, and the open circuit potential (OCP) of each solution was obtained before and after the analysis. Sporicidal activity tests The sporicidal activity of the synthesized ECAS, household bleach, and NaDCC was meas- ured using the Standard Operating Procedure (SOP) MB-21-03 of the United States Envi- ronmental Protection Agency (EPA) (US EPA, 2017). Prior to disinfection, all spore sus- pensions were placed in a water bath at 65C for 10 min to eliminate remaining vegetative cells (Kenters et al., 2017). Stainless-steel pieces (5x5x1 mm) were inoculated with 10 L of spore suspensions adjusted to contain 1x105 CFU/mL. These test surfaces were carefully 17 placed into 1.5 mL plastic microcentrifuge tubes containing 400 L of synthesized ECAS, 5000 ppm NaOCl, or 1000 ppm NaDCC, and after 5 minutes 600 L of ice-cold Luria Bertani (LB) broth was added as neutralizing agent. After this exposure, spores were har- vested by centrifugation at 13,000 rpm for 6 min, washed with ice-cold LB broth, resus- pended, and diluted for plating on Brucella Agar supplemented with 0.5% sodium tau- rocholate. These plates were incubated for 5 days under anaerobic conditions. Log reduc- tion values were obtained by subtracting the CFU/mL of each disinfection treatment to the control CFU/mL count. As a control, inoculated stainless-steel pieces were exposed to phosphate buffer saline (PBS) with 0.1 w/v Tween 80 instead of disinfectant agents. These tests were done by duplicate. Transmission Electron Microscopy (TEM) Spores from all treatments and the control assay were processed for TEM. To this end, spores were pelleted by centrifugation and fixed using an aldehyde solution composed of 2.5% glutaraldehyde and 2% paraformaldehyde in phosphates buffer 0.1 M, pH 7.4. Pellets were first washed with phosphates buffer (0.1 M, pH 7.2) and post-fixed with 1% osmium tetroxide (OsO4) in phosphates buffer (0.1 M, pH 7.4). Thereafter, they were washed with distilled water and dehydrated with acetone. Fixed pellets were embedded into Spurr’s resin (Spurr, 1969), cured at 70 ℃ for 48 hours, cut into ultrathin sections (80 nm), and observed under a transmission electron microscope (JEM-2100, JEOL, Tokyo, Japan). Statistical analyses A Two-way ANOVA test followed by Tukey multiple comparison tests were performed to determine whether the treatments and strain responses differed. P <0.05 was considered to be significant. All tests were done with the GraphPad Prism 6 software. A full-factorial design was performed by duplicate on isolates LIBA 5758 (highly susceptible to disinfect- ants) and LIBA 5757 (more tolerant to disinfectants) for it can provide information on factor significance and be exploited for response optimization. In this regard, three levels of the factor exposure time (5, 10, and 15 min) and ECAS activation time (10, 20, and 30 min) were tested. 18 Results and discussion ECAS characterization As determined by titration, household bleach (2.97% w/v, 95% CI 2.41-3.58) contained more NaOCl than the synthesized ECAS (0.19% w/v, 95% CI 0.171-0.21). This anticipated result is caused in non-divided cells by competing oxidation and reduction reactions that lead to cell inefficiencies by oxygen generation (Abdel-Aal, Sultan, & Hussein, 1993; M. Spasojević et al., 2015). However, as the method only quantifies total chlorine and NaOCl, other unidentified oxidant agents that may have been generated during the electrolysis pro- cess could have contributed to boost the disinfectant potential of ECAS. The disinfection tests were performed with a NaOCl concentration of 5000 ppm, which is the recommended concentration for surface disinfection to be deployed during CDI out- breaks (Barra-Carrasco & Paredes-Sabja, 2014). However, such high concentration carries several risks, including skin and mucosa irritation, corrosion of metallic surfaces, and dam- age of sensitive equipment. Our ECAS contained a lower NaOCl concentration than bleach, turning them into an attractive alternative for control of C. difficile spores on surfaces. In agreement with a previous report (Helme et al., 2010), the pH of the ECAS ranged be- tween 9.6 and 10.3. This alkaline pH can be attributed to the configuration of the open electrochemical cell used for the synthesis, as it does not prevent the flux of ions between the anolyte and the catholyte. In such non-balanced cells, pH increases because water elec- trolysis is a non-balanced process and also due to the release of chlorine during electrolysis, as shown in Eq. 1 (Bergmann & Koparal, 2005; Baniasadi, Dincer, & Naterer, 2013). 2 Cl- + 2 H2O → H2 + Cl2 + 2 OH - (1) In this regard, as the NaCl concentration is increased, the current spent on chlorine and hypochlorite generation also increases, at oxygen evolution expenses (El-Ashtoukhy, Amin, & Abdelwahab, 2009). This process may be tuned by using ruthenium oxide- (RuO2) or titanium oxide- (TiO2) as catalysts during the electrolysis process (Ibl & Landolt, 1967; M. D. Spasojević, Trišović, Ribić-Zelenović, & Spasojević, 2013). 19 Figure 1 shows the electrochemical behavior measured for each of the solutions. Most of the oxidation and reduction processes in the NaCl reaction (Fig. 1A) were observed be- tween -2 and 2 V. Theoretically, this behavior is associated with the production of chlorine (Cl2), hypochlorite (ClO -), chlorite (ClO2), chlorate (ClO - 3 ), and perchlorate (ClO - 4 ) [Eq. 8–10] in both the anode and the cathode as shown below (Ibl & Landolt, 1967; Krstajić, Nakić, & Spasojević, 1991; Wang & Margerum, 1994) : On the anode: 2 Cl- → Cl2 + 2 e (1.36 V) (2) On the cathode: 2 H2O + 2 e → 2 OH - + 2 H+ (-0.83 V) (3) In the bulk solution: Cl2 + H2O → HClO + Cl - + H+ (4) HClO → H+ + ClO- (5) The chlorine generated at the anode (Eq. 2) diffuses throughout the bulk solution and sub- sequently undergoes hydrolysis reactions that lead to the production of hypochlorous acid (HOCl), chloride ions, and protons (Eq. 4) (Krstajić et al., 1991; M. Spasojević et al., 2015). If the bulk solution pH is neutral or alkaline, HOCl dissociates into its ions (Eq. 5), which hold disinfectant properties (Szpyrkowicz, Cherbanski, & Kelsall, 2005; Cheng & Kelsall, 2007;). On the other hand, the electrochemical behavior of NaOCl (Fig. 1B) and the syn- thesized ECAS (Fig. 3C) was rather similar between -2 and 2 V, as expected for solutions enriched in HOCl (Robinson et al., 2012). Despite this similarity, the ECAS cyclic volt- ammogram showed some unique oxidation/reduction processes that can be attributed to the generation of products other than HOCl during the electrolysis (Cai, 2005; Chinello, Hashemi, Psaltis, & Moser, 2019). 20 Figure 1. Cyclic voltammograms obtained for (A) sodium chloride (NaCl), (B) sodium hypochlorite (NaOCl), and (C) electrochemically activated solutions (ECAS). Measure- ments obtained with platinum electrodes and no reference electrode. The open circuit potential (OCP) or zero-current potential is defined as the potential at which no appreciable current flows through the electrochemical cell because an equilib- rium has been established (Bard, Faulkner, Leddy, & Zoski, 1980). The OCP of the three solutions were measured to obtain further information regarding their electrochemical be- havior. The NaOCl (540 mV) and ECAS (500 mV) solutions showed a positive potential, similar to OCP previously reported for a hypochlorite/hypochlorous solution (Ordeig et al., 2005). Instead, the OCP of the NaCl system (-190 mV) had a tendency towards negative 21 values, but as the cyclic voltammetry was performed, it varied from -190 mV to 440/540 mV. No OCP change was recorded despite extensive cleaning of the electrodes and agita- tion of the solution to avoid any residual charge in the electrode surface. This result indi- cates that ECAS activation likely occurs at the beginning of the process. Sporicidal activity tests and statistical analysis We noted significant differences among the logarithmic reductions of the ten strains (F9,40=16.42, p<0.0001), the four treatments (F3,40=76.09, p<0.0001), and the interaction of strain and treatment (F27,40=4.43, p<0.0001). The highest overall logarithmic reduction was achieved with ECAS (3.22 log10 reduction, 95% CI, 2.00-4.43), followed by NaOCl (2.74 log10 reduction, 95% CI, 1.62-3.86) and NaDCC (2.02 log10 reduction, 95% CI, 0.91-3.12) (Figure 2). ECAS and NaOCl performed statistically equally (q40=3.52); therefore ECAS could be used as an alternative to NaOCl on the disinfection of surfaces contaminated with C. difficile spores in healthcare facilities. It was possible to distribute the strains in three different groups depending on their suscep- tibility levels to the disinfectants tested (Table 3 and Supplementary Figure 1). The first group included strains LIBA-7854 (4.23 log10 reduction, 95% CI=2.73-5.70) and LIBA- 6507 (4.23 log10 reduction, 95% CI=2.76-5.69), which exhibited the higher overall suscep- tibility. The second group was characterized by moderately susceptible strains, such as 630 (2.91 log10 reduction, 95% CI=1.17-4.68), M68 (2.62 log10 reduction, 95% CI 1.14-4.10), M120 (2.80 log10 reduction, 95% CI 2.31-3.29), LIBA-7110 (2.30 log10 reduction, 95% CI 1.54-3.06), LIBA-6276 (3.020 log10 reduction, 95% CI 1.71-4.33), and LIBA-5758 (3.89 log10 reduction, 95% CI 1.83-5.96). Finally, the least susceptible strains were LIBA-5757 (0.33 log10 reduction, 95% CI 0.18-0.50) and LIBA-7719 (0.36 log10 reduction, 95% CI 0.17-0.55). 22 Table 3. Susceptibility of C. difficile strains to sodium hypochlorite (NaOCl), sodium di- chloroisocyanurate (NaDCC) and electrochemically activated solutions (ECAS) as deter- mined by a carrier test disinfection method. MLST Log10 CFU reduction (95% confidence intervals) Strain Clade NaOCl NaDCC ECAS 630a 3.50 (2.23-4.77) 1.15 (0.88-3.18) 4.08 (3.95-4.21) 1 LIBA 6276a 4.360 (4.11-4.61) 2.03 (1.90-2.16) 2.67 (1.53-3.81) LIBA 5757b 0.41 (0.03-0.79) 0.16 (0.05-0.85) 0.42 (0.28-0.54) 2 LIBA 5758ab 4.56 (4.31-4.81) 1.56 (1.43-1.69) 5.56 (4.72-6.49) LIBA 7110a 2.33 (0.97-5.63) 1.49 (1.11-1.87) 3.07 (2.94-3.20) 3 LIBA 6507c 4.12 (3.87-4.37) 5.12 (4.86-5.36) 3.11 (2.87-3.37) M68a 1.85 (0.77-2.93) 1.78 (0.13-3.67) 4.24 (3.99-4.49) 4 LIBA 7719b 0.18 (0.06-0.87) 0.34 (0.27-0.40) 0.57 (0.32-0.82) M120a 2.49 (1.01-5.18) 2.56 (1.29-3.83) 3.36 (3.11-3.61) 5 LIBA 7854c 3.60 (2.33-4.87) 3.97 (3.84-4.10) 5.12 (4.87-5.38) *Different letters indicate statistical differences between strains Patients with CDI may release up to 1x107 spores per gram of stool (Smits, Lyras, Lacy, Wilcox, & Kuijper, 2016). Consequently, the level of contamination of surfaces in healthcare centers may reach up to 1 to 3 log10 CFU depending on the quantification method (Barbut, 2015). It is widely accepted that an effective disinfection method for C. difficile spores must achieve a > 5 log10 reduction in a minimum of 5 minutes (Fraise et al., 2015). Therefore, none of the disinfectants tested here can be considered appropriate for all strains. However, this interpretation might be inaccurate, as most results in the literature were obtained with spore suspensions rather than with artificially contaminated surfaces. 23 Figure 2. Logarithmic reduction of C. difficile spores caused by sodium hypochlorite (NaOCl, blue points), sodium dichloroisocyanurate (NaDCC, red boxes), and electro- chemically activated solutions (ECAS, brown triangles). Black lines represent the mean of the treatment. Surface response plots were used to identify ECAS activation and exposure times that op- timize the logarithmic reduction of spores from strains LIBA-5757 (highly tolerant) and LIBA-5758 (highly susceptible). Regardless of the selected activation time, 15 minutes of exposure time were sufficient to maximize the logarithmic reduction observed for LIBA- 5757 (5.170 log10 reduction, Fig 3A). This time period does not deviate much from the minimum contact time recommended for disinfection of hospital surfaces with NaOCl, which is 10 minutes (Dubberke et al., 2008). The logarithmic reduction obtained for the susceptible strain LIBA-5758 with an exposure time of 5 minutes was maximal only when the activation time was 30 minutes (Fig. 3B). However, when the ECAS exposure time 24 exceeded 10 minutes, activation time seemed not to be a determining factor because all factor combinations reached the maximum disinfection value (5.490 log10 reduction). Figure 3. Logarithmic spore reduction as function of ECAS exposure and activation times. (A) LIBA-5757 (Low ECAS susceptibility) (B) LIBA-5758 (High ECAS suscepti- bility). Electron microscopy Our TEM analyses revealed only minor differences in the structure of spores exposed to the treatments. As opposed to the control sample (Fig. 5A), in which the core of the spore, the membranes, the cell wall, and the outer layers were conserved, spores exposed to ECAS (Fig. 5B) NaOCl (Fig. 5C) and NaDCC (Fig. 5D) showed a greater electron density in the core, as well as a ripple in their outermost layers. None of the treated samples showed physical ruptures in the inner and the outer layers. We therefore conclude that the ECAS likely cause functional damages previous to spore germination. According to Loshon et al. 25 (2001), this damage could be related to protein and fatty acid oxidation leading to perme- abilization of the spore membrane and therefore loss of germinative capacity. Figure 4. TEM micrographs of C. difficile LIBA-5758 exposed to ECAS (B), NaOCl (C), and NaDCC (D). An unexposed control is shown in (A). Conclusions Our NaCl-derived ECAS showed sporicidal activity against C. difficile spores, however most of the strains did not reached the required 5 log10 reduction required by EPA. Our ECAS performed equally than NaOCl in disinfection tests, yet the former is accessible, more environmentally friendly, and safer for its application in healthcare facilities. Con- sidering the TEM qualitative results we suggest that the action mechanism of ECAS may be associated to functional damage rather than physical rupture of the spores. Our results 26 also provide novel insights in the electrochemical characterization of ECAS. In this regard, the electrochemical behavior of the ECAS solutions seems to be responsible for its in- creased disinfection potential. 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