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Cold-Air Atmospheric Pressure Plasma Against Clostridium difcile Spores: A Potential Alternative for the Decontamination of Hospital Inanimate Surfaces Tânia Claro, Orla J. Cahill, Niall O’Connor, Stephen Daniels and Hilary Humphreys Infection Control & Hospital Epidemiology / FirstView Article / March 2015, pp 1 - 3 DOI: 10.1017/ice.2015.39, Published online: 17 March 2015
Link to this article: http://journals.cambridge.org/abstract_S0899823X15000392 How to cite this article: Tânia Claro, Orla J. Cahill, Niall O’Connor, Stephen Daniels and Hilary Humphreys Cold-Air Atmospheric Pressure Plasma Against Clostridium difcile Spores: A Potential Alternative for the Decontamination of Hospital Inanimate Surfaces. Infection Control & Hospital Epidemiology, Available on CJO 2015 doi:10.1017/ice.2015.39 Request Permissions : Click here
Downloaded from http://journals.cambridge.org/ICE, IP address: 216.255.247.52 on 27 Mar 2015
infection control & hospital epidemiology
concise communication
Clostridium difficile is the main causative agent of healthcareassociated colitis. Although the vegetative form under anaerobic conditions produces toxins associated with pathogenicity, it is in the endospore form that C. difficile is transmitted.1 C. difficile spores can be acquired from the environment near a contaminated patient, through direct contact with a symptomatic patient, or via the hands of healthcare workers. Spores may persist on surfaces for months.2 However, eradication through traditional disinfection techniques or through relatively new decontamination methods such as hydrogen peroxide or ultraviolet radiation remains a challenge. Furthermore, hydrogen peroxide and ultraviolet radiation are associated with substantial limitations such as toxicity, the need to vacate the area of patients and staff, unsuitability with some materials, and relative cost.3,4 Preliminary work in our laboratory has demonstrated the bactericidal activity of a cold-air atmospheric pressure plasma (CAPP) single-jet plume against vegetative bacteria.5 Such CAPP systems are sources of reactive atoms and molecules—for example, atomic oxygen, ozone, superoxide, oxides of nitrogen, and intense electric fields. These properties disrupt and etch the cell wall, interfere with transport within the cell, and/or induce DNA damage. We present an in vitro evaluation of a CAPP single- and multi-jet for the decontamination of C. difficile spores from 2 surfaces found in hospitals in the absence and presence of human serum albumin (HSA), a marker for organic load.
Fulton Spore Stain Kit (Sigma Aldrich). Suspensions were adjusted to a concentration of approximately 1 × 106 to 1 × 108 colony-forming units (CFU) per mL (1 colony equivalent to 1 spore) and 100 µL were added to sections of mattress (Meditec Medical), the surface of which is polyurethane coated, permeable to vapour but water resistant, and sections of stainless steel; the size for both mattress and stainless steel was 25 cm2 (5 × 5 cm). Sections of mattress and stainless-steel were decontaminated beforehand as described previously.5 Three percent HSA (Sigma Aldrich, Ireland) was also added to the inoculum to mimic the organic load characteristically found in the hospital inanimate environment.7 All surfaces were airdried more than 1 h before CAPP treatment in a laminar flow cabinet. The artificially inoculated sections were exposed for 30 s, 60 s, and 90 s to the CAPP single-jet 5 and CAPP multi-jet (9 jets arranged in 10 × 10 cm array) (Figure 1). In the CAPP multi-jet prototype each plasma jet is driven by a separate sinusoidal, high-voltage power supply. The drive frequency is 8 kHz, the amplitude of the voltage is approximately 1.5 kV, and each supply consumes an average of 15 W. The operating gas is air that flows into the device at 13 standard liters per minute. The air was drawn from the ambient into a compressor that contained a water trap to actively remove moisture before the air entered the plasma source cavity. No further treatment of the air took place, the room in which the plasma was operated was well ventilated, and no modification of the immediate environment was observed. The plume temperature did not exceed 45°C, the distance between the plume and the test surface was 1 cm, and the plume was moved around to cover the entire surface, to mimic as far as possible the potential use of such a prototype. All experiments were carried out on 3 independent occasions in duplicate. The entire area of treated and untreated surfaces was sampled using phosphate-buffered saline premoistened nylon flocked-swabs (Copan) and placed in 3 mL phosphate-buffered saline, vortexed, and cultured on to C. difficile–selective plates (Oxoid).6 One-in-ten serial dilutions were performed when needed to determine the total viable count—that is, the number of CFU/ mL. All plates were incubated anaerobically for up to 48 hours at 37°C for spore enumeration. Statistical analysis was performed using the GraphPad Prism, version 5.00 (GraphPad Software). The means of the C. difficile spore log reduction following CAPP single- and multi-jet treatment with and without HSA were compared by 1-way analysis of variance and further Tukey’s multiple comparison tests when significant (ie, P < .05).
m e th o d s
resul ts
C. difficile spores ATCC 700057 (Cruinn Diagnostics) were prepared as previously described.6 Spore numbers and purity were determined by microscopy using the Schaeffer and
Unlike for vegetative cells,5 the CAPP single-jet had very little consistent effect on C. difficile spores over 90 s on either mattress or stainless steel (Figure 2). In contrast, the CAPP
Cold-Air Atmospheric Pressure Plasma Against Clostridium difficile Spores: A Potential Alternative for the Decontamination of Hospital Inanimate Surfaces Tânia Claro, PhD;1 Orla J. Cahill, PhD;2 Niall O’Connor, PhD;2 Stephen Daniels, PhD;2 Hilary Humphreys, MD1,3
Abstract Clostridium difficile spores survive for months on environmental surfaces and are highly resistant to decontamination. We evaluated the effect of cold-air plasma against C. difficile spores. The single-jet had no effect while the multi-jet achieved 2–3 log10 reductions in spore counts and may augment traditional decontamination. Infect Control Hosp Epidemiol 2 01 5 ;0 0( 0 ): 1– 3
2
infection control & hospital epidemiology
figure 1. diagram.
Multi-jet cold-air pressure plasma prototype schematic
figure 2. Single- and multi-jet cold-air pressure plasma sporicidal activity, expressed in log10 (colony-forming units [CFU]/mL) reduction, against Clostridium difficile spores on mattress and stainless steel over 90 s in the presence and absence of 3% human serum albumin (HSA). Each bar represents the mean of at least 3 individual experiments (n ≥ 3), and error bars represent the standard error of the mean.
multi-jet prototype treatment resulted in a time-dependent reduction of the C. difficile spore load on both materials. Maximum log10 reductions expressed in CFU/mL of 2.69 ± 0.50 and 1.81 ± 0.18 were achieved following 90 s treatment on mattress and stainless steel, respectively. The CAPP multi-jet prototype was more effective in reducing the load of C. difficile spores compared with the single-jet (P < .01). The addition of HSA to mimic the clinical environment had minimal impact on the effectiveness of the CAPP multi-jet (P = nonsignificant) (Figure 2). Maximum log reductions varied from 2.69 ± 0.50 to 1.59 ± 0.38 on mattress and 1.81 ± 0.18 to 2.55 ± 0.41 on stainless-steel following the addition of HSA. Although the differences between these results on the 2 surfaces were not statistically significant, they may in part be explained by the porosity of the mattress compared with the stainless steel. Furthermore, the effects of the CAPP multi-jet in the absence or presence of HSA were also independent of the type of material surface used.
d i s c u s s io n Recent studies evaluating decontamination systems against C. difficile spores showed maximum reductions of 4 log10.
The use of hydrogen peroxide, however, involves significant contact and aeration time (>1 h 30 min), and requires the evacuation of the clinical area to be decontaminated.4 In the case of chemical disinfectants7 and ultraviolet radiation3 the presence of an organic load significantly limits efficacy. In the present study, the single-jet CAPP had minimal effect on C. difficile spores whereas the CAPP multi-jet system reduced C. difficile spore counts by 2–3 log10. This is not surprising because in the multi-jet configuration, the effective treatment areas of the jets will overlap and the effect of the plasma in a unit area will be multiplied on the surface being treated. The efficacy of the plasma was not significantly affected by the presence of organic material as represented by HSA, and the log reduction was accomplished in just 90 s. However, its potential future use would be to complement and not replace routine decontamination. The low temperature, nontoxicity, and fast working conditions of this type of technology have been studied by others with encouraging results. Gas discharge plasma inactivated C. difficile spores in less than 3 min,8 dielectric barrier plasma achieved log reductions of Geobacillus spores of greater than 6 log10 in 20 min,9 and recently surface micro-discharge CAPP removed C. difficile spores in less than 6 min.10 The results achieved in this study using CAPP multi-jet against C. difficile spores on both mattress and stainless-steel dry surfaces are promising and may provide a potentially rapid and practical measure to augment traditional decontamination methods in hospitals. Nonetheless, further improvements are needed in order to achieve the best efficacy in the shortest time. Additional studies are required with alternative markers for organic loads, with mixed bacteria including C. difficile spores, with smaller inocula that may better reflect the bioburden in hospitals, and over shorter contact times. Finally, the technology needs to be evaluated in a busy clinical setting.
acknowledgments Financial support. Translational Research Award from Science Foundation Ireland and the Health Research Board (TRA/2010/10). Potential conflicts of interest. H. H. reports that he has research collaborations with Steris Corporation, Inov8 Science, Pfizer, and Cepheid and has also received lecture and other fees from Novartis, AstraZeneca, and Astellas. All other authors report no conflicts of interest relevant to this article.
Affiliations: 1. Department of Clinical Microbiology, Royal College of Surgeons in Ireland, Dublin, Ireland; 2. School of Electronic Engineering and National Centre for Plasma Science Technology, Dublin City University, Dublin, Ireland; 3. Department of Microbiology, Beaumont Hospital, Dublin, Ireland. Address correspondence to Tânia Claro, Department of Clinical Microbiology, Education and Research Centre, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland ([email protected]). Presented in part: 9th Healthcare Infection Society International Congress; Lyon, France; November, 2014. Received November 24, 2014; accepted February 5, 2015 © 2015 by The Society for Healthcare Epidemiology of America. All rights reserved. DOI: 10.1017/ice.2015.39
cold-air pressure plasma against c. difficile
references 1. Lawley TD, Croucher NJ, Yu L, et al. Proteomic and genomic characterization of highly infectious Clostridium difficile 630 spores. J Bacteriol 2009;191:5377–5386. 2. Otter JA, French GL. Survival of nosocomial bacteria and spores on surfaces and inactivation by hydrogen peroxide vapor. J Clin Microbiol 2009;47:205–207. 3. Nerandzic MM, Cadnum JL, Pultz MJ, Donskey CJ. Evaluation of an automated ultraviolet radiation device for decontamination of Clostridium difficile and other healthcare-associated pathogens in hospital rooms. BMC Infect Dis 2010;10:197. 4. Barbut F, Menuet D, Verachten M, Girou E. Comparison of the efficacy of a hydrogen peroxide dry-mist disinfection system and sodium hypochlorite solution for eradication of Clostridium difficile spores. Infect Control Hosp Epidemiol 2009;30:507–514. 5. Cahill OJ, Claro T, O'Connor N, et al. Cold air plasma to decontaminate inanimate surfaces of the hospital environment. Appl Environ Microbiol 2014;80:2004–2010.
3
6. Claro T, Daniels S, Humphreys H. Detecting Clostridium difficile spores from inanimate surfaces of the hospital environment: which method is best? J Clin Microbiol 2014;52: 3426–3428. 7. Speight S, Moy A, Macken S, et al. Evaluation of the sporicidal activity of different chemical disinfectants used in hospitals against Clostridium difficile. J Hosp Infect 2011;79: 18–22. 8. Tseng S, Abramzon N, Jackson JO, Lin WJ. Gas discharge plasmas are effective in inactivating Bacillus and Clostridium spores. Appl Microbiol Biotechnol 2012;93:2563–2570. 9. Mastanaiah N, Johnson JA, Roy S. Effect of dielectric and liquid on plasma sterilization using dielectric barrier discharge plasma. PLOS ONE 2013;8:e70840. 10. Klämpfl TG, Shimizu T, Koch S, et al. Decontamination of nosocomial bacteria including Clostridium difficile spores on dry inanimate surface by cold atmospheric plasma. Plasma Process Polym 2014;11:974–984.
Control & Hospital Epidemiology:
Email alerts: Click here Subscriptions: Click here Commercial reprints: Click here Terms of use : Click here
Cold-Air Atmospheric Pressure Plasma Against Clostridium difcile Spores: A Potential Alternative for the Decontamination of Hospital Inanimate Surfaces Tânia Claro, Orla J. Cahill, Niall O’Connor, Stephen Daniels and Hilary Humphreys Infection Control & Hospital Epidemiology / FirstView Article / March 2015, pp 1 - 3 DOI: 10.1017/ice.2015.39, Published online: 17 March 2015
Link to this article: http://journals.cambridge.org/abstract_S0899823X15000392 How to cite this article: Tânia Claro, Orla J. Cahill, Niall O’Connor, Stephen Daniels and Hilary Humphreys Cold-Air Atmospheric Pressure Plasma Against Clostridium difcile Spores: A Potential Alternative for the Decontamination of Hospital Inanimate Surfaces. Infection Control & Hospital Epidemiology, Available on CJO 2015 doi:10.1017/ice.2015.39 Request Permissions : Click here
Downloaded from http://journals.cambridge.org/ICE, IP address: 216.255.247.52 on 27 Mar 2015
infection control & hospital epidemiology
concise communication
Clostridium difficile is the main causative agent of healthcareassociated colitis. Although the vegetative form under anaerobic conditions produces toxins associated with pathogenicity, it is in the endospore form that C. difficile is transmitted.1 C. difficile spores can be acquired from the environment near a contaminated patient, through direct contact with a symptomatic patient, or via the hands of healthcare workers. Spores may persist on surfaces for months.2 However, eradication through traditional disinfection techniques or through relatively new decontamination methods such as hydrogen peroxide or ultraviolet radiation remains a challenge. Furthermore, hydrogen peroxide and ultraviolet radiation are associated with substantial limitations such as toxicity, the need to vacate the area of patients and staff, unsuitability with some materials, and relative cost.3,4 Preliminary work in our laboratory has demonstrated the bactericidal activity of a cold-air atmospheric pressure plasma (CAPP) single-jet plume against vegetative bacteria.5 Such CAPP systems are sources of reactive atoms and molecules—for example, atomic oxygen, ozone, superoxide, oxides of nitrogen, and intense electric fields. These properties disrupt and etch the cell wall, interfere with transport within the cell, and/or induce DNA damage. We present an in vitro evaluation of a CAPP single- and multi-jet for the decontamination of C. difficile spores from 2 surfaces found in hospitals in the absence and presence of human serum albumin (HSA), a marker for organic load.
Fulton Spore Stain Kit (Sigma Aldrich). Suspensions were adjusted to a concentration of approximately 1 × 106 to 1 × 108 colony-forming units (CFU) per mL (1 colony equivalent to 1 spore) and 100 µL were added to sections of mattress (Meditec Medical), the surface of which is polyurethane coated, permeable to vapour but water resistant, and sections of stainless steel; the size for both mattress and stainless steel was 25 cm2 (5 × 5 cm). Sections of mattress and stainless-steel were decontaminated beforehand as described previously.5 Three percent HSA (Sigma Aldrich, Ireland) was also added to the inoculum to mimic the organic load characteristically found in the hospital inanimate environment.7 All surfaces were airdried more than 1 h before CAPP treatment in a laminar flow cabinet. The artificially inoculated sections were exposed for 30 s, 60 s, and 90 s to the CAPP single-jet 5 and CAPP multi-jet (9 jets arranged in 10 × 10 cm array) (Figure 1). In the CAPP multi-jet prototype each plasma jet is driven by a separate sinusoidal, high-voltage power supply. The drive frequency is 8 kHz, the amplitude of the voltage is approximately 1.5 kV, and each supply consumes an average of 15 W. The operating gas is air that flows into the device at 13 standard liters per minute. The air was drawn from the ambient into a compressor that contained a water trap to actively remove moisture before the air entered the plasma source cavity. No further treatment of the air took place, the room in which the plasma was operated was well ventilated, and no modification of the immediate environment was observed. The plume temperature did not exceed 45°C, the distance between the plume and the test surface was 1 cm, and the plume was moved around to cover the entire surface, to mimic as far as possible the potential use of such a prototype. All experiments were carried out on 3 independent occasions in duplicate. The entire area of treated and untreated surfaces was sampled using phosphate-buffered saline premoistened nylon flocked-swabs (Copan) and placed in 3 mL phosphate-buffered saline, vortexed, and cultured on to C. difficile–selective plates (Oxoid).6 One-in-ten serial dilutions were performed when needed to determine the total viable count—that is, the number of CFU/ mL. All plates were incubated anaerobically for up to 48 hours at 37°C for spore enumeration. Statistical analysis was performed using the GraphPad Prism, version 5.00 (GraphPad Software). The means of the C. difficile spore log reduction following CAPP single- and multi-jet treatment with and without HSA were compared by 1-way analysis of variance and further Tukey’s multiple comparison tests when significant (ie, P < .05).
m e th o d s
resul ts
C. difficile spores ATCC 700057 (Cruinn Diagnostics) were prepared as previously described.6 Spore numbers and purity were determined by microscopy using the Schaeffer and
Unlike for vegetative cells,5 the CAPP single-jet had very little consistent effect on C. difficile spores over 90 s on either mattress or stainless steel (Figure 2). In contrast, the CAPP
Cold-Air Atmospheric Pressure Plasma Against Clostridium difficile Spores: A Potential Alternative for the Decontamination of Hospital Inanimate Surfaces Tânia Claro, PhD;1 Orla J. Cahill, PhD;2 Niall O’Connor, PhD;2 Stephen Daniels, PhD;2 Hilary Humphreys, MD1,3
Abstract Clostridium difficile spores survive for months on environmental surfaces and are highly resistant to decontamination. We evaluated the effect of cold-air plasma against C. difficile spores. The single-jet had no effect while the multi-jet achieved 2–3 log10 reductions in spore counts and may augment traditional decontamination. Infect Control Hosp Epidemiol 2 01 5 ;0 0( 0 ): 1– 3
2
infection control & hospital epidemiology
figure 1. diagram.
Multi-jet cold-air pressure plasma prototype schematic
figure 2. Single- and multi-jet cold-air pressure plasma sporicidal activity, expressed in log10 (colony-forming units [CFU]/mL) reduction, against Clostridium difficile spores on mattress and stainless steel over 90 s in the presence and absence of 3% human serum albumin (HSA). Each bar represents the mean of at least 3 individual experiments (n ≥ 3), and error bars represent the standard error of the mean.
multi-jet prototype treatment resulted in a time-dependent reduction of the C. difficile spore load on both materials. Maximum log10 reductions expressed in CFU/mL of 2.69 ± 0.50 and 1.81 ± 0.18 were achieved following 90 s treatment on mattress and stainless steel, respectively. The CAPP multi-jet prototype was more effective in reducing the load of C. difficile spores compared with the single-jet (P < .01). The addition of HSA to mimic the clinical environment had minimal impact on the effectiveness of the CAPP multi-jet (P = nonsignificant) (Figure 2). Maximum log reductions varied from 2.69 ± 0.50 to 1.59 ± 0.38 on mattress and 1.81 ± 0.18 to 2.55 ± 0.41 on stainless-steel following the addition of HSA. Although the differences between these results on the 2 surfaces were not statistically significant, they may in part be explained by the porosity of the mattress compared with the stainless steel. Furthermore, the effects of the CAPP multi-jet in the absence or presence of HSA were also independent of the type of material surface used.
d i s c u s s io n Recent studies evaluating decontamination systems against C. difficile spores showed maximum reductions of 4 log10.
The use of hydrogen peroxide, however, involves significant contact and aeration time (>1 h 30 min), and requires the evacuation of the clinical area to be decontaminated.4 In the case of chemical disinfectants7 and ultraviolet radiation3 the presence of an organic load significantly limits efficacy. In the present study, the single-jet CAPP had minimal effect on C. difficile spores whereas the CAPP multi-jet system reduced C. difficile spore counts by 2–3 log10. This is not surprising because in the multi-jet configuration, the effective treatment areas of the jets will overlap and the effect of the plasma in a unit area will be multiplied on the surface being treated. The efficacy of the plasma was not significantly affected by the presence of organic material as represented by HSA, and the log reduction was accomplished in just 90 s. However, its potential future use would be to complement and not replace routine decontamination. The low temperature, nontoxicity, and fast working conditions of this type of technology have been studied by others with encouraging results. Gas discharge plasma inactivated C. difficile spores in less than 3 min,8 dielectric barrier plasma achieved log reductions of Geobacillus spores of greater than 6 log10 in 20 min,9 and recently surface micro-discharge CAPP removed C. difficile spores in less than 6 min.10 The results achieved in this study using CAPP multi-jet against C. difficile spores on both mattress and stainless-steel dry surfaces are promising and may provide a potentially rapid and practical measure to augment traditional decontamination methods in hospitals. Nonetheless, further improvements are needed in order to achieve the best efficacy in the shortest time. Additional studies are required with alternative markers for organic loads, with mixed bacteria including C. difficile spores, with smaller inocula that may better reflect the bioburden in hospitals, and over shorter contact times. Finally, the technology needs to be evaluated in a busy clinical setting.
acknowledgments Financial support. Translational Research Award from Science Foundation Ireland and the Health Research Board (TRA/2010/10). Potential conflicts of interest. H. H. reports that he has research collaborations with Steris Corporation, Inov8 Science, Pfizer, and Cepheid and has also received lecture and other fees from Novartis, AstraZeneca, and Astellas. All other authors report no conflicts of interest relevant to this article.
Affiliations: 1. Department of Clinical Microbiology, Royal College of Surgeons in Ireland, Dublin, Ireland; 2. School of Electronic Engineering and National Centre for Plasma Science Technology, Dublin City University, Dublin, Ireland; 3. Department of Microbiology, Beaumont Hospital, Dublin, Ireland. Address correspondence to Tânia Claro, Department of Clinical Microbiology, Education and Research Centre, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland ([email protected]). Presented in part: 9th Healthcare Infection Society International Congress; Lyon, France; November, 2014. Received November 24, 2014; accepted February 5, 2015 © 2015 by The Society for Healthcare Epidemiology of America. All rights reserved. DOI: 10.1017/ice.2015.39
cold-air pressure plasma against c. difficile
references 1. Lawley TD, Croucher NJ, Yu L, et al. Proteomic and genomic characterization of highly infectious Clostridium difficile 630 spores. J Bacteriol 2009;191:5377–5386. 2. Otter JA, French GL. Survival of nosocomial bacteria and spores on surfaces and inactivation by hydrogen peroxide vapor. J Clin Microbiol 2009;47:205–207. 3. Nerandzic MM, Cadnum JL, Pultz MJ, Donskey CJ. Evaluation of an automated ultraviolet radiation device for decontamination of Clostridium difficile and other healthcare-associated pathogens in hospital rooms. BMC Infect Dis 2010;10:197. 4. Barbut F, Menuet D, Verachten M, Girou E. Comparison of the efficacy of a hydrogen peroxide dry-mist disinfection system and sodium hypochlorite solution for eradication of Clostridium difficile spores. Infect Control Hosp Epidemiol 2009;30:507–514. 5. Cahill OJ, Claro T, O'Connor N, et al. Cold air plasma to decontaminate inanimate surfaces of the hospital environment. Appl Environ Microbiol 2014;80:2004–2010.
3
6. Claro T, Daniels S, Humphreys H. Detecting Clostridium difficile spores from inanimate surfaces of the hospital environment: which method is best? J Clin Microbiol 2014;52: 3426–3428. 7. Speight S, Moy A, Macken S, et al. Evaluation of the sporicidal activity of different chemical disinfectants used in hospitals against Clostridium difficile. J Hosp Infect 2011;79: 18–22. 8. Tseng S, Abramzon N, Jackson JO, Lin WJ. Gas discharge plasmas are effective in inactivating Bacillus and Clostridium spores. Appl Microbiol Biotechnol 2012;93:2563–2570. 9. Mastanaiah N, Johnson JA, Roy S. Effect of dielectric and liquid on plasma sterilization using dielectric barrier discharge plasma. PLOS ONE 2013;8:e70840. 10. Klämpfl TG, Shimizu T, Koch S, et al. Decontamination of nosocomial bacteria including Clostridium difficile spores on dry inanimate surface by cold atmospheric plasma. Plasma Process Polym 2014;11:974–984.
