Cold Air Plasma To Decontaminate Inanimate Surfaces of theHospital Environment
Orla J. Cahill,a Tânia Claro,b Niall O’Connor,a Anthony A. Cafolla,c Niall T. Stevens,b Stephen Daniels,a Hilary Humphreysb,d
School of Electronic Engineering and National Centre for Plasma Science Technology, Dublin City University, Dublin, Irelanda; Department of Clinical Microbiology, RoyalCollege of Surgeons in Ireland, Dublin, Irelandb; School of Physical Sciences, Dublin City University, Dublin, Irelandc; Department of Microbiology, Beaumont Hospital,Dublin, Irelandd
The hospital environment harbors bacteria that may cause health care-associated infections. Microorganisms, such as multire-sistant bacteria, can spread around the patient’s inanimate environment. Some recently introduced biodecontamination ap-proaches in hospitals have significant limitations due to the toxic nature of the gases and the length of time required for aeration.This study evaluated the in vitro use of cold air plasma as an efficient alternative to traditional methods of biodecontaminationof hospital surfaces. Cultures of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE),extended-spectrum-�-lactamase (ESBL)-producing Escherichia coli, and Acinetobacter baumannii were applied to different ma-terials similar to those found in the hospital environment. Artificially contaminated sections of marmoleum, mattress, polypro-pylene, powder-coated mild steel, and stainless steel were then exposed to a cold air pressure plasma single jet for 30 s, 60 s, and90 s, operating at approximately 25 W and 12 liters/min flow rate. Direct plasma exposure successfully reduced the bacterial loadby log 3 for MRSA, log 2.7 for VRE, log 2 for ESBL-producing E. coli, and log 1.7 for A. baumannii. The present report confirmsthe efficient antibacterial activity of a cold air plasma single-jet plume on nosocomial bacterially contaminated surfaces over ashort period of time and highlights its potential for routine biodecontamination in the clinical environment.
In 2011, the World Health Organization (WHO) stated that inEurope alone, approximately 4.5 million patients are affected by
health care-associated infections (HCAIs) each year, resulting in16 million extra days of hospital stay, at an estimated cost of €7billion, with a mortality rate of 37,000 deaths (1). The inanimateenvironment and “high-touch” surfaces have been verified ascommon reservoirs of bacteria causing HCAIs (2, 3). The onset ofa HCAI usually occurs approximately 48 to 72 h or more afterhospital admission, but the risk increases significantly by 50% to75% if the prior occupants of the ward had a HCAI (4).
Within the hospital environment, contaminated surfaces havebeen demonstrated to play an important role in the transmissionof microorganisms causing health care-associated infections (5).The bacterial infections associated with primary surface coloniza-tion include methicillin-resistant Staphylococcus aureus (MRSA),vancomycin-resistant enterococci (VRE), and extended-spec-trum-beta-lactamase (ESBL)-producing Gram-negative organ-isms, such as Escherichia coli and Acinetobacter baumannii, whichprevail in the hospital environment for extended periods, i.e.,months, in viable form. Contaminated objects include hospitalbed rails and bed linen, mattresses, patients’ gowns and clothing,curtains, overbed tables, and stethoscopes (6–13). These patho-gens may survive on dry surfaces for extended periods and thusfacilitate transmission between patients and health care workers(14). Primary transmission onto surfaces originates from hands,patients, hospital water systems, and airborne sources (15–19).Infection prevention and control practices to prevent HCAIs in-clude the use of biodecontamination. However, current steriliza-tion and disinfection methods have critical limitations in terms ofefficacy, environmental impact, clinical downtime, and economiccost. In addition, more-aggressive decontamination approaches,such as the use of hydrogen peroxide gas and ultraviolet (UV)radiation, pose logistical difficulties, as both require the evacua-tion of patients and health care staff for a number of hours (20,
21). Therefore, new approaches that would combine safety andefficiency in terms of minimal disruption in clinical areas areneeded. One such method being evaluated involves cold atmo-spheric pressure plasma (CAPP). CAPP has numerous chemicaland physical properties which can affect microbicidal outcomes.Depending on the plasma-generating mechanism (e.g., plasma jet,dielectric barrier discharge, etc.), CAPP systems are sources ofpositive and negative ions, reactive atoms and molecules (e.g.,atomic oxygen, ozone, superoxide, and oxides of nitrogen), in-tense electric fields, and UV radiation. In many cases, CAPPsources produce a “cocktail” of all of the physicochemical prop-erties listed above at the same time, in various proportions anddensities. Positive and negative ions can lead to electrostatic dis-ruption of bacterial cell walls. Oxidative atoms and compounds(e.g., atomic oxygen and ozone) can physically etch the cell walland interfere with transport within the cell. Furthermore, suchreactive compounds can induce DNA double and single breakage.Sufficiently intense electric fields can result in electroporation,whereas UV radiation (particularly sub-260-nm-wavelength UV)is well known to induce damage to DNA and intracellular proteins(22).
The biomedical and clinical applications of CAPP have beenevaluated in various areas, such as dermatology and wound treat-ment (23–25), bone regeneration, implant treatments (26, 27),
and dental procedures, including bleaching and root canal disin-fection (28–30). However, CAPP has an innate antibacterial activ-ity, making it an interesting decontamination technique and apossible solution for environmental decontamination, particu-larly in the clinical environment. In this study, we describe an invitro evaluation of a CAPP single-jet system for the decontamina-tion of materials commonly found in the clinical environment.
MATERIALS AND METHODSBacterial strains and growth conditions. Two Gram-positive organisms(MRSA and VRE) and two Gram-negative organisms (E. coli and A. bau-mannii) were chosen for this study. MRSA strain 43300 and ESBL-positiveE. coli strain CL2 are clinical strains from our collection, the VRE clinicalstrain was provided by the Beaumont Hospital Microbiology Depart-ment, and the A. baumannii 19606 reference strain was sourced from theAmerican Type Culture Collection (ATCC).
Bacteria were stored at �20°C on cryovial preservation beads (Micro-bank; Pro-Lab Diagnostics, Merseyside, United Kingdom). MRSA and A.baumannii strains were revived on Columbia blood agar (CBA) (OxoidLtd., Basingstoke, United Kingdom) plates, the E. coli strain was revivedon Mueller-Hinton (MH) (Fluka, Sigma-Aldrich, Ireland Ltd.) agarplates, and the VRE strain was revived on Trypticase soy broth (TSB)(Oxoid Ltd., Basingstoke, United Kingdom) agar plates before each ex-periment. Overnight (16 to 18 h) bacterial cultures were grown aerobicallyat 37°C, with rotation, in TSB supplemented with 5% NaCl, for MRSAand VRE only or brain heart infusion (BHI) broth for A. baumannii orMH broth for E. coli strains.
Test surface preparation. The test surfaces used in this study were5-cm2 sections of marmoleum flooring (Forbo Flooring, Dublin, Ireland)and polyurethane mattress (Meditec Medical, Dublin, Ireland) com-monly used in hospitals and provided by Beaumont Hospital, Dublin,polypropylene (GoodFellow Cambridge Ltd., United Kingdom), powder-coated mild steel (Watermark Engineering, Ireland), and stainless steel.To decontaminate before use, the soft surfaces, i.e., marmoleum and mat-tress, were placed in a 1% Virkon solution (Sparks Lab Supplies, Dublin,Ireland) for 30 min, rinsed three times in distilled water, and dried in thelaminar flow cabinet for 1 h. The solid surfaces, i.e., polypropylene, pow-der-coated mild steel, and stainless steel, were soaked and wiped with 70%ethanol and left to dry in a laminar flow cabinet. All surfaces were thenplaced into petri dishes and placed under UV light for 30 min.
Preparation of the bacterial inoculums. A volume of 25 ml of theappropriate broth was inoculated with one isolated colony from an over-
night culture plate. Fresh overnight cultures were used for each assess-ment. Overnight cultures were centrifuged for 10 min at 15,500 � g(11,000 rpm) (Eppendorf centrifuge 5804R) and washed three times withsterile phosphate-buffered saline (PBS). The bacterial concentration wasadjusted to a 3 to 4 McFarland standard (approximately 8 to 9 log10 CFUper ml) in 3 ml of sterile PBS, from which 50 �l was taken to inoculate eachof the test surfaces.
CAPP single-jet system experimental design. The CAPP single-jetsystem, shown in Fig. 1, consists of a hollow, cylindrical polyether etherketone (PEEK) body with a grounded stainless steel conical nozzle. Ahigh-voltage (HV) stainless steel pin electrode runs through the axis of thePEEK cylinder, which is sealed at the end opposite to the nozzle. A sinu-soidal high voltage is applied to the center pin at a frequency of 8 kHz andan amplitude of approximately 2.5 kV. Compressed air is forced throughan orifice perpendicular to the jet axis at a flow rate of 12 standard litersper min (slm).
CAPP single-jet treatment. The artificially inoculated test surfaceswere exposed to the plasma jet plume for 30 s, 60 s, and 90 s, operating atapproximately 25 W and 12 liters/min flow rate. The plume temperaturedid not exceed 45°C. The distance between the plume and the test surfacewas 1 cm (31). All experiments were carried out at least three times induplicate. The plasma system was maintained within a fume hood in-stalled with an ozone detector.
Bacterial recovery and enumeration. The entire areas of both test andcontrol (nontreated) surfaces were swabbed using flocked eSwabs (Co-pan, Italy). Swabs were placed into Falcon round-bottom tubes (BD Bio-science, United Kingdom) with 3 ml of PBS, briefly subjected to a vortexprocedure, and cultured onto CBA plates for MRSA and A. baumannii,ESBL Brilliance agar plates (Oxoid Ltd., Basingstoke, United Kingdom)for ESBL-positive E. coli, and VRE Brilliance agar plates (Oxoid Ltd.,Basingstoke, United Kingdom) for VRE for bacterial enumeration. One-in-10 serial dilutions were performed when needed to determine a totalviable count (TVC), i.e., the number of CFU/ml of one sample (30 to 300countable colonies on the plate).
AFM. Atomic force microscopy (AFM) images were completed inambient air with a Dimension 3100 AFM microscope controlled by aNanoscope IIIa controller (Digital Instruments, Santa Barbara, CA),operated in tapping mode, using standard silicon cantilevers (BudgetSensors, Bulgaria) with a 7-nm radius of curvature and a spring con-stant of 42 N/m (nominal values) to assess the physical effects of theplasma on the bacterial cells. Samples were prepared as describedabove and plasma treated, and AFM was performed. Multiple areas
FIG 1 Atmospheric pressure air plasma jet. The nozzle is 1 mm in diameter. The luminous plasma jet extends approximately 25 mm along the axis of the jet bodywhen allowed to expand into air. When a substrate is placed in the expansion field of the jet, it spreads to a diameter of circa 20 mm over the substrate.
Air Plasma for Decontamination of Hospital Surfaces
(approximately 10 areas per surface) were imaged to ensure good rep-resentation of the total surface inoculated. Images were then examinedand edited using WxSM software (Nanotec Electronica S.L., Madrid,Spain) to generate phase and profile data (32). Gwyddion software wasalso used to perform data analysis on the AFM scans (www.gwyddion.net). The original two-dimensional (2D) scans obtained from theAFM were corrected by removing the polynomial background; thisresults in an accurate zero value on the surface, therefore verifying theexact height distribution of the cells on the surface. Rt analysis was alsocarried out. Rt is defined as the maximum peak-to-peak-valley height.This statistically analyzes the absolute value of the difference betweenthe highest and lowest peaks indicative of the roughness and height ofthe cells as they are distributed across the surface. To further evaluatethe AFM images, height distribution data analysis was also performed.This provides an overall comparison of the root mean square analysisof the cell on the surface, which is the quadratic mean, a statisticalmeasure of the magnitude of a quantity of various points.
Statistical analysis. Statistical data analysis was carried out usingGraphPad Prism 5.00 software. The means of the log (CFU/ml) valuesfrom comparisons between recovered control and plasma-treated sam-ples over 30 s, 60 s, and 90 s were determined by one-way analysis ofvariance (ANOVA).
RESULTSBactericidal effect of CAPP single jet on A. baumannii, ESBL-producing E. coli, MRSA, and VRE inoculated on various sur-faces. The bactericidal effect of the plasma on A. baumannii,ESBL-producing E. coli, MRSA, and VRE inoculated onto mar-moleum, mattress, polypropylene, powder-coated mild steel, andstainless steel is summarized in Fig. 2. For all the microorganismsand surfaces tested, the effect of the CAPP single jet was dependentupon the length of exposure to the plasma, with the maximum logreduction achieved at 90 s. For each set of data, a clear trend wasobserved over time correlating with the duration of exposure timeand effect. There were, however, different effects noted dependingupon the types of surface material.
Following exposure to the CAPP single jet, the highest log(CFU/ml) reductions compared to the recovered inoculum for A.baumannii were observed on the soft surfaces of mattress andmarmoleum: 3.18 � 1.26 and 3.12 � 0.57, respectively. On stain-less steel and polypropylene, there were log reductions of 2.97 �
0.27 and 2.73 � 0.27, respectively, followed by 1.66 � 0.50 onpowder-coated mild steel.
For ESBL-producing E. coli, the CAPP single jet was more ef-fective after shorter exposure times, with a complete killing after90 s for all surfaces except on powder-coated mild steel. Followinga 60-s exposure time, high log reductions of 3.40 � 0.20 on stain-less steel and 2.78 � 0.93 on the marmoleum were observed. Sim-ilarly, a 60-s exposure reduced the log (CFU/ml) numbers by3.40 � 0.20 on the polypropylene and by 2.44�0.43 on the mattress.Ninety-second treatments of the powder-coated mild steel reducedthe numbers of ESBL-producing E. coli by log 2.71 � 0.24.
For MRSA, the best results were achieved on polypropylene,with a log reduction of approximately 5.87 � 0.6, and log reduc-tions of 4.08 � 0.32, 3.95 � 0.89, 3.82 � 0.15, and 3.42 � 0.90were achieved on mattress, stainless steel, marmoleum flooring,and powder-coated mild steel, respectively, after 90 s.
The effects of the plasma on VRE following 90-s treatmentsresulted in the best log reduction on marmoleum flooring of ap-proximately 5.19 � 0.86, followed by log reductions of 5.01 �0.35, 4.02 � 0.45, 2.80 � 0.56, and 2.21 � 0.08 on polypropylene,mattress, stainless steel, and powder-coated mild steel, respec-tively.
The bacterial log reduction as an outcome of the effect of theCAPP was confirmed to be statistically significant for all microor-ganisms inoculated on all surfaces (P � 0.05 following one-wayANOVA).
Atomic force microscopy imaging of the bactericidal effect ofthe CAPP single jet. Atomic force microscopy imaging of all mi-croorganisms inoculated on powder-coated mild steel before andafter 90-s exposure to CAPP is shown on Fig. 3. Powder-coatedmild steel was chosen as the model surface to image as some of theother surfaces cannot be imaged using AFM due to forces exertedbetween the surface and the cantilever. Micrographs A and B il-lustrate the 2D topography of the applied cells, while micrographsC and D illustrate the 3D topography of the applied cells beforeand after CAPP treatment, respectively. Each micrograph repre-sents an area of 5 �m, edge to edge, and is representative of mul-tiple experiments (n � 10). Plots E and F represent the surface
FIG 2 Bactericidal effects of the CAPP single jet on A. baumannii, ESBL E. coli, MRSA, and VRE inoculated on various surfaces over 30, 60, and 90 s (n � 3). Appl,initial inoculums applied to the surface; Rec, number of bacteria recovered from surface before use of CAPP.
Cahill et al.
2006 aem.asm.org Applied and Environmental Microbiology
topography in Rt measurements and height distributions, respec-tively, of untreated and CAPP-treated cells on powder-coatedmild steel.
A. baumannii cells before treatment (A and C) were observedas cellular aggregates indicative of pellicle formation, a morpho-logical characteristic of biofilm-forming A. baumannii 19606 (33–36) whereby the secretion of exopolysaccharide causes the cells toclump together. This characteristic is considered to extend thesurvival of the organism in the environment. Following 90 s ofexposure of A. baumannii to CAPP (micrographs B and D), asignificant disruption of the cell aggregates can be observed, withsingle cells showing disruption of the cell wall and leakage of cel-lular content. In panels E and F, the changes in the surface topog-raphy, registered in Rt measurements and height distribution, re-spectively, can be seen. The noise in the measurement of thetreated cells is indicative of the severe etching effect caused by theplasma corresponding to surface damage of the cells. Cell disrup-tion is verified by a reduction in the Rt value and the median heightdistributions from the untreated cells (151.8 nm and 153.2 nm) tothe treated cells (118.7 nm and 118.9 nm).
ESBL-producing E. coli AFM micrographs show smooth andindividual cells for the untreated control in panels A and C. How-ever, severe cellular disruption can be seen, with only cell debrisleft on the surface, and no residual intact cells, following 90-sexposures to CAPP (B and D). In panels E and F, the surfacetopography in Rt and height distribution measurements show a
considerable reduction in the Rt value and cell height of 302 nmand 312 nm compared to the untreated-cell values of 139.6 nmand 131.2 nm, consistent with the physical disruption of the bac-terial cells.
AFM imaging of the MRSA cells inoculated on powder-coatedmild steel shows smooth and morphologically intact cells, with nodisruption visible on the 2D and 3D micrographs, respectively(panels A and C). Following 90 s of CAPP treatment, cellulardistortions can be seen in panels B and D, with obvious cellulardebris present and very few intact cells remaining. The surfacetopography and roughness assessed in Rt measurements (E) andheight distributions (F) show increases in the Rt and height mea-surements from 262.0 nm and 260.0 nm to 414.0 nm and 415.4nm, possibly due to a buildup of cell debris on the surface follow-ing CAPP treatment, indicative of the physical disruption of thecells by the air plasma jet.
Finally, untreated VRE cells are seen to be intact and with aslightly oval shape, which is characteristic of Enterococcus spp., onthe corresponding 2D and 3D micrographs of panels A and C,respectively. Cellular malformations arose in treated VRE cellsafter 90 s of exposure to CAPP, with cells appearing distorted andwhat could possibly be intracellular material leaching out of dam-aged cells (micrographs B and D). In panels E and F, the surfaceroughness expressed in Rt values and median of height for theuntreated and treated cells were 290.5 nm and 291.0 nm and 294.3nm and 193.9 nm, respectively.
FIG 3 AFM images of A. baumannnii, ESBL E. coli, MRSA, and VRE inoculated on powder-coated mild steel before and after 90 s of CAPP treatment. Panels Aand B correspond to the 2D images and panels C and D to the 3D images. (E and F) Micrograph plots are representative of the surface topography Rt
measurements and height distributions of untreated and treated cells on the powder-coated mild steel surface.
Air Plasma for Decontamination of Hospital Surfaces
The present study aimed to evaluate the antimicrobial effect of aCAPP single-jet prototype on bacteria of clinical significance, in-cluding MRSA, VRE, ESBL-producing E. coli, and A. baumannii,on inanimate surfaces commonly found in the clinical setting.
A recent review on environmental contamination has high-lighted the importance of this source as a primary mode of trans-mission of HCAI. Current effective decontamination methodspose logistical difficulties and limitations, but the results pre-sented here suggest that the use of CAPP is a promising tool forenvironmental biodecontamination, achieving a �log 5 reduc-tion for some bacteria on certain materials after 90 s. Althoughother studies have been performed on biomedical device materi-als, skin models, and pagers and in solution, this is the first studyperformed on materials of surfaces of clinical relevance (37–40).
Previous studies on the antimicrobial effects of plasma in-volved different treatment exposure times mainly due to the phys-ical state of the bacteria and demonstrated shorter times for plank-tonic cells in solution (41) and longer times for cells dried on testsurfaces and in biofilms. In this study, the optimum antimicrobialactivity of the air plasma was observed after 90 s, producing re-ductions of log 3 to 5 for MRSA, log 2 to 5 for VRE, log 2 to 3 forE. coli, and log 1.7 to 3 for A. baumannii, all of which were air-dried on each test surface. Maisch et al. (42) evaluated the efficacyof a CAPP device on MRSA- and E. coli-contaminated porcineskin and showed that longer exposure times were required toachieve log reductions similar to those seen in our study, i.e., 6min for a log 3 reduction and 8 min for a log 5 reduction of bothstrains, but those are relatively prolonged periods in the busy clin-ical environment for surface decontamination. Similarly, the effi-cacy of a plasma microjet in killing S. aureus and Enterococcusfaecalis inoculated on agar found that treatment exposure times of4 to 5 min were required to achieve a log 4 reduction. For biofilms,the treatment times increase significantly, in some cases taking aslong as 30 min to achieve a log 3 reduction (43). Recently, remoteplasma exposure of MRSA strains, in biofilm form, has proven tobe effective also. However, the treatment in that case required upto 1.5 h to inactivate the biofilm completely (44). Few studies haveassessed the effects of CAPP on A. baumannii, but one found thatthis bacterium was more resistant to plasma than S. aureus andother Gram-negative organisms (45).
The isolation of Gram-negative bacteria from the environmentposes challenges as they may enter a viable but nonculturablestate, and this may partly explain why they are isolated less fre-quently than Gram-positive bacteria. Although they are still capa-ble of causing infection, recovering them from the environment isdifficult in this state (46). This was also reflected in our results asthere was an evident decline in log numbers between the appliedinoculum and the recovered control after air-drying. Morpholog-ical cellular effects following plasma exposure were observed forGram-negative bacteria as seen in Fig. 3 for treated A. baumanniiand ESBL-producing E. coli in the 2D and 3D AFM images (Fig. 3Band D) compared to the untreated controls (Fig. 3A and C).CAPPs produce numerous reactive ions, including reactive oxy-gen species (ROCs), reactive nitrogen species (RONs), and UV,which, as originally suggested by Laroussi et al. (47), chemicallyand physically alter various bacterial, fungal cells, tissues, and sur-faces. These species not only affect bacterial cells on a surface levelbut also intracellularly cause a cascade of effects leading to cell wall
disruption, cytoplasm leakage, lipid peroxidation, and DNA dam-age (48–50). For both MRSA and VRE, cellular disruption andphysiological changes were observed whereby, following 90 s oftreatment, few intact cells remained, with visible cellular debrisobserved (Fig. 3, MRSA and VRE panels B and D).
Montie et al. (50) suggested that leakage of the cytoplasm oc-curs due to initial “etching” or physical damage of the bacterial cellwall and that, once the cell wall has been compromised, the reac-tive oxygen species then filter through into the cell, causing oxi-dative damage and eventually leading to cell death. The rates atwhich this occurs differ between Gram-positive and Gram-nega-tive bacteria chiefly due to metabolic and biochemical pathwaydifferences, in addition to the differences in the amounts of pep-tidoglycan present in the cell walls. Another publication by Yu-supov et al. (48) verified the disruption of important C-N, C-O,and C-C bonds in peptidoglycan by O3 and O2 molecules and Oatoms following plasma treatment. As the thicknesses of the pep-tidoglycan layer differ between Gram-positive bacteria (20 to 30nm) and Gram-negative bacteria (6 to 7 nm), it can be speculatedthat the effects of the plasma on the cell wall may be more pro-nounced in Gram-negative bacteria. In the present study, bothESBL-producing E. coli and A. baumannii showed more-severephysical damage and in some cases total cell disruption as seen inAFM micrographs of ESBL-producing E. coli (Fig. 3B and D),where only cell debris can be seen following treatment of E. colicells for 90 s. Similar effects were seen for A. baumannii (Fig. 3, A.baumannii panels B and D). Height distribution measurementsproduced a “noisy” graph that may be indicative of significantetching of the cell walls (Fig. 3, A. baumannii panel E).
The data presented in this study have verified the efficacy ofCAPPs for use as a biodecontaminating agent in clinical environ-ments. The air plasma source used shows significant bactericidaleffects on both Gram-positive and Gram-negative organisms,with a maximum log reduction of approximately �log 5 after 90 s.In addition, the design and configuration of the plasma jet usedhere produce and deliver reactive species in a controlled manner.This suggests that the use of such a system could greatly enhanceinfection control procedures currently existing in the clinical set-ting.
In conclusion, we have shown that CAPP significantly reducesbacterial numbers on a range of surfaces commonly found in theclinical environment within 90 s. Further work is required to de-velop a prototype that could be used in the clinical environmentand to evaluate this against spore-forming bacteria such as Clos-tridium difficile and mixtures of bacteria with protein and othersubstances that mimic contamination in a clinical setting. If effi-cacy is confirmed, CAPP would represent an important and valu-able alternative to surface decontamination in health care facili-ties.
ACKNOWLEDGMENTS
The Health Research Board Ireland and Science Foundation Irelandfunded this research through grant TRA/2010/10.
We are also grateful to advisors and collaborators for their participa-tion in his project.
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