downloaded from on april 19, 2020 by guest · 145 (sparks lab supplies, dublin, ireland) for 30min,...
TRANSCRIPT
1
Cold Air Plasma to Decontaminate Inanimate Surfaces of the Hospital Environment 1
2
3
Orla J. Cahilla#, Tânia Clarob, Niall O’Connora, Anthony A. Cafollac, Niall T. Stevensb, 4
Stephen Danielsa, Hilary Humphreysb,d 5
6
School of Electronic Engineering and National Centre for Plasma Science Technology, 7
Dublin City University, Dublin, Irelanda. Department of Clinical Microbiology, Royal 8
College of Surgeons in Ireland, Dublin, Irelandb. School of Physical Sciences, Dublin 9
City University, Dublin, Irelandc. Department of Microbiology, Beaumont Hospital, 10
Dublin, Irelandd. 11
12
Running title: Air plasma for decontamination of hospital surfaces 13
14
#Corresponding Author: Dr. Orla J. Cahill ([email protected]) 15
O.J.C. and T.C. contributed equally to this work. 16
17
18
19
20
21
22
AEM Accepts, published online ahead of print on 17 January 2014Appl. Environ. Microbiol. doi:10.1128/AEM.03480-13Copyright © 2014, American Society for Microbiology. All Rights Reserved.
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
2
ABSTRACT 23
The hospital environment harbours bacteria that may cause healthcare-associated 24
infections. Microorganisms, such as multi-resistant bacteria, can spread around the 25
patient’s inanimate environment. Some recently introduced bio-decontamination 26
approaches in hospitals have significant limitations due to the toxic nature of the 27
gases and the length of time required for aeration. This study evaluated the in vitro 28
use of cold-air plasma as an efficient alternative to traditional bio-decontamination 29
methods of hospital surfaces. Cultures of methicillin-resistant Staphylococcus aureus 30
(MRSA), vancomycin-resistant enterococci (VRE), extended spectrum β-lactamase 31
(ESBL)-producing Escherichia coli and Acinetobacter baumannii were applied to 32
different materials, similar to those found in the hospital environment. Artificially 33
contaminated sections of marmoleum, mattress, polypropylene, powder-coated 34
mild steel and stainless steel were then exposed to a cold-air pressure plasma single 35
jet for 30s, 60s and 90s, operating at approximately 25W and 12L/min flow rate. 36
Direct plasma exposure successfully reduced the bacterial load by log 3 for MRSA, 37
log 2.7 for VRE, log 2 for ESBL-producing E. coli and log 1.7 for A. baumannii. The 38
present study confirms the efficient anti-bacterial activity of a cold-air plasma single 39
jet plume on nosocomial bacteria contaminated surfaces over a short period of time 40
and highlights its potential for routine bio-decontamination in the clinical 41
environment. 42
43
Keywords: decontamination, surfaces, cold-air pressure plasma, MRSA, VRE, E. coli, 44
A. baumannii 45
46
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
3
INTRODUCTION 47
48
In 2011 the World Health Organization (WHO) stated that in Europe alone, 49
approximately 4.5 million patients are affected by healthcare-associated infections 50
(HCAIs) each year, resulting in 16 million extra-days of hospital stay, at an estimated 51
cost of €7 billion, with a mortality rate of 37,000 deaths (1). The inanimate 52
environment and “high-touch” surfaces have been verified as common reservoirs of 53
bacteria causing HCAIs (2, 3). The onset of a HCAI usually occurs approximately 48 to 54
72h or more after hospital admission but the risk increases significantly by 50 to 75% 55
if the prior occupants of the ward had a HCAI (4). 56
57
Within the hospital environment, contaminated surfaces have been demonstrated to 58
play an important role in the transmission of microorganisms causing healthcare- 59
associated infections (5). The bacterial infections associated with primary surface 60
colonisation include methicillin-resistant Staphylococcus aureus (MRSA), 61
vancomycin-resistant enterococci (VRE) and extended-spectrum beta-lactamase 62
(ESBL)-producing Gram-negative organisms, such as E. coli and A. baumannii, which 63
prevail in the hospital environment for extended periods i.e. months in viable form. 64
Contaminated objects include, hospital bed rails and bed linen, mattresses, patient’s 65
gowns and clothing, curtains, over-bed tables and stethoscopes (6-13). These 66
pathogens may survive on dry surfaces for extended periods and thus facilitate 67
transmission between patients and healthcare workers (14). Primary transmission 68
onto surfaces originates from hands, patients, hospital water systems and airborne 69
sources (15-19). Infection prevention and control practices to prevent HCAIs include 70
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
4
the use of bio-decontamination. However, current sterilisation and disinfection 71
methods have critical limitations in terms of efficacy, environmental impact, clinical 72
downtime and economic cost. In addition, more aggressive decontamination 73
approaches, such as the use of hydrogen peroxide gas and ultra violet (UV) radiation, 74
pose logistical difficulties as both require, the evacuation of patients and healthcare 75
staff for a number of hours (20, 21). Therefore, new approaches that would 76
combine safety and efficiency in terms of minimal disruption in clinical areas are 77
needed. One such method being evaluated involves cold atmospheric pressure 78
plasma (CAPP). CAPP has numerous chemical and physical properties which can 79
affect microbicidal outcomes. Depending on the plasma generating mechanism (e.g. 80
plasma jet, dielectric barrier discharge etc.), CAPP systems are sources of positive 81
and negative ions, reactive atoms and molecules (e.g. atomic oxygen, ozone, 82
superoxide and oxides of nitrogen), intense electric fields and UV radiation. In many 83
cases CAPP sources produce a 'cocktail' of all of the above listed physicochemical 84
properties at the same time, in varying proportions and densities. Positive and 85
negative ions can lead to electrostatic disruption of bacterial cell walls. Oxidative 86
atoms and compounds (e.g. atomic oxygen and ozone) can physically etch the cell 87
wall and interfere with transport within the cell. Furthermore such reactive 88
compounds can induce DNA double and single breakage. Sufficiently intense electric 89
fields can result in electroporation, whereas UV radiation (particularly sub 260nm 90
UV) is well known to induce damage to DNA and intracellular proteins (22). 91
92
The biomedical and clinical applications of CAPP have been evaluated in various 93
areas, such as dermatology and wound treatment (23-25), bone regeneration, 94
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
5
implant treatments (26, 27) and dental procedures including bleaching and root 95
canal disinfection (28-30). However, CAPP has an innate antibacterial activity making 96
it an interesting decontamination technique and a possible solution for 97
environmental decontamination, particularly in the clinical environment. In this 98
study, we describe an in vitro evaluation of a CAPP single jet system for the 99
decontamination of materials commonly found in the clinical environment. 100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
6
METHODS 119
120
Bacterial strains and growth conditions 121
Two Gram-positive organisms (MRSA and VRE) and two Gram-negative organisms (E. 122
coli and A. baumannii) were chosen for this study. The MRSA strain 43300 and ESBL-123
positive E. coli strain CL2 are clinical strains from our collection, the VRE clinical 124
strain was provided by the Beaumont Hospital Microbiology Department and the A. 125
baumannii reference strain 19606 was sourced from the American Type Culture 126
Collection (ATCC). 127
128
Bacteria were stored at -20⁰C on cryovial preservation beads (Microbank, Pro-Lab 129
Diagnostics, Merseyside, UK). MRSA and A. baumannii strains were revived on 130
Columbia blood agar (CBA) (Oxoid Ltd, Basingstoke, UK) plates, the E. coli strain on 131
Mueller-Hinton (MH) (Fluka, Sigma-Aldrich, Ireland Ltd) agar plates and the VRE 132
strain on Trypticase soy broth (TSB) (Oxoid Ltd, Basingstoke, UK) agar plates before 133
each experiment. Overnight (16-18 h) bacterial cultures were grown aerobically at 134
37⁰C, with rotation, in TSB supplemented with 5% NaCl, for MRSA and VRE only, or 135
brain heart infusion (BHI) broth for A. baumannii or MH broth for E. coli strains. 136
137
Test surfaces preparation 138
The test surfaces used in this study were 5cm2 sections of marmoleum flooring 139
(Forbo flooring, Dublin 18, Ireland) and polyurethane mattress (Meditec Medical, 140
Dublin 24, Ireland) commonly used in hospitals and provided by Beaumont Hospital, 141
Dublin, polypropylene (GoodFellow Cambridge Ltd., UK), powder-coated mild steel 142
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
7
(Watermark Engineering, Ireland) and stainless steel. To decontaminate before use, 143
the soft surfaces, i.e. marmoleum and mattress were placed in a 1% virkon solution 144
(Sparks Lab Supplies, Dublin, Ireland) for 30min, rinsed three times in distilled water 145
and dried in the laminar flow cabinet for 1h. The solid surfaces, i.e. polypropylene, 146
powder-coated mild steel, and stainless steel were soaked and wiped with 70% 147
ethanol and left to dry in a laminar flow cabinet. All surfaces were then placed into 148
Petri dishes and placed under UV light for 30min. 149
150
Preparation of the bacterial inoculums 151
A volume of 25ml of the appropriate broth was inoculated with one isolated colony 152
from an overnight culture plate. Fresh overnight cultures were used for each 153
assessment. Overnight cultures were centrifuged for 10min at 15,500g (11,000rpm) 154
(eppendorf centrifuge 5804R) and washed three times with sterile PBS. The bacterial 155
concentration was adjusted to a 3 to 4 McFarland standard (approximately 8 to 9 156
log10 colony forming units (CFU) per ml) into 3ml of sterile PBS, from which 50µl 157
were taken to inoculate each of the test surfaces. 158
159
CAPP Single jet system Experimental design 160
The CAPP single jet system, shown in Figure 1, consists of a hollow, cylindrical 161
polyether ether ketone (PEEK) body with a grounded stainless steel conical nozzle. A 162
high voltage (HV) stainless steel pin electrode runs through the axis of the PEEK 163
cylinder, which is sealed at the end opposite to the nozzle. A sinusoidal high voltage 164
is applied to the centre pin at a frequency of 8kHz and amplitude of approximately 165
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
8
2.5kV. Compressed air is forced through an orifice perpendicular to the jet axis at a 166
flow rate of 12 standard litres per minute (slm). 167
168
CAPP single jet treatment 169
The artificially inoculated test surfaces were exposed to the plasma jet plume for 170
30s, 60s and 90s, operating at approximately 25W and 12L/min flow rate. The plume 171
temperature did not exceed 45⁰C. The distance between the plume and the test 172
surface was 1cm (31). All experiments were carried out at least three times in 173
duplicate. The plasma system was maintained within a fume hood installed with an 174
ozone detector. 175
176
Bacterial recovery and enumeration 177
The entire area of both test and control (non-treated) surfaces were swabbed using 178
flocked eSwabs (Copan, Italy). Swabs were placed into falcon round bottom tubes 179
(BD Bioscience, UK) with 3 ml of PBS, briefly vortexed and cultured on to CBA plates 180
for MRSA and A. baumannii, ESBL brilliance agar plates (Oxoid Ltd, Basingstoke, UK) 181
for ESBL-positive E. coli and VRE brilliance agar plates (Oxoid Ltd, Basingstoke, UK ) 182
for VRE for bacterial enumeration. One in ten serial dilutions were performed when 183
needed to determine a total viable count (TVC), i.e. the number of CFU/ml of one 184
sample (30 to 300 countable colonies on the plate). 185
186
Atomic force microscopy (AFM) 187
Atomic force microscopy images were completed in ambient air with a Dimension 188
3100 AFM microscope controlled by a Nanoscope IIIa controller, (Digital Instruments, 189
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
9
Santa Barbara, CA, USA), operated in Tapping-Mode, using standard silicon 190
cantilevers (Budget Sensors, Bulgaria) with a 7nm radius of curvature and a 42N/m 191
spring constant (nominal values) to assess the physical effects of the plasma on the 192
bacterial cells. Samples were prepared as above, plasma-treated and AFM 193
performed. Multiple areas (approximately 10 areas per surface) were imaged to 194
assure good representation of the total surface inoculated. Images were then 195
examined and edited using WxSM software (Nanotec Electronica S.L, Madrid, Spain) 196
to generate phase and profile data (32). Gwyddion software was also used to 197
perform data analysis on the AFM scans [www.gwyddion.net]. The original 2D scans 198
obtained from the AFM were corrected by removing the polynomial background; 199
this obtains an accurate zero value on the surface therefore verifying the exact 200
height distribution of the cells on the surface. Rt analysis was also carried out. Rt is 201
defined as the maximum peak-to-peak-valley height. This statistically analyses the 202
absolute value between the highest and lowest peaks indicative of the roughness 203
and height of the cells as they are distributed across the surface. To further evaluate 204
the AFM images, height distribution data analysis was also performed. This provides 205
an overall comparison of the root mean square analysis of the cell on the surface, 206
which is the quadratic mean, a statistical measure of the magnitude of a varying 207
quantity of points. 208
209
Statistical analysis 210
Statistical data analysis was carried out using GraphPad Prism 5.00 software. The 211
means of the log (CFU/mL) between recovered control and plasma treated over 30s, 212
60s and 90s were compared by one-way analysis of variance (ANOVA). 213
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
10
RESULTS 214
215
Bactericidal effect of CAPP single jet on A. baumannii, ESBL-producing E. coli, 216
MRSA and VRE inoculated on various surfaces 217
218
The bactericidal effect of the plasma on A. baumannii, ESBL-producing E. coli, MRSA 219
and VRE inoculated on to marmoleum, mattress, polypropylene, powder-coated 220
mild steel and stainless steel is summarised in Figure 2. For all the microorganisms 221
and surfaces tested the effect of CAPP single jet was dependent upon length of 222
exposure to the plasma, with the maximum log reduction achieved at 90s. For each 223
set of data a clear trend was observed over time correlating with the duration of 224
exposure time and effect. There were, however, different effects noted depending 225
upon the types of surface material. 226
227
Following exposure to the CAPP single-jet the highest log (CFU/ml) reductions 228
compared to the recovered inoculum for A. baumannii were observed on the soft 229
surfaces, mattress and marmoleum, of 3.18±1.26 and 3.12±0.57, respectively. On 230
stainless steel and polypropylene there were log reductions of 2.97±0.27 and 231
2.73±0.27, respectively, followed by 1.66±0.50 on powder coated mild steel. 232
233
For ESBL-producing E. coli the effect of CAPP single-jet was more effective after 234
shorter exposure times with a complete killing after 90s for all surfaces except on 235
powder-coated mild steel. Following a 60s exposure time, high log reductions of 236
3.40±0.20 on stainless steel and 2.78±0.93 on the marmoleum were observed. 237
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
11
Similarly, a 60s exposure reduced the log (CFU/ml) numbers by 3.40±0.20 on the 238
polypropylene and by 2.44±0.43 on the mattress. Ninety second treatments of the 239
powder-coated mild steel reduced the numbers of ESBL-producing E. coli by log 240
2.71±0.24. 241
242
For MRSA the best results were achieved on polypropylene with a log reduction of 243
approximately 5.87±0.6 and log reductions of 4.08±0.32, 3.95±0.89, 3.82±0.15 and 244
3.42±0.90 achieved on mattress, stainless steel, marmoleum flooring and powder 245
coated mild steel, respectively, after 90s. 246
247
The effects of the plasma on VRE following 90s treatments resulted in the best log 248
reduction on marmoleum flooring of approximately 5.19±0.86, followed by log 249
reductions of 5.01±0.35, 4.02±0.45, 2.80±0.56 and 2.21±0.08 on polypropylene, 250
mattress, stainless steel and powder-coated mild steel, respectively. 251
252
The bacterial log reduction as an outcome of the effect of the CAPP was confirmed 253
to be statistically significant for all microorganisms inoculated on all surfaces, P<0.05 254
following one-way ANOVA analysis. 255
256
Atomic force microscopy imaging of the bactericidal effect of CAPP single jet 257
Atomic force microscopy imaging of all microorganisms inoculated on powder-258
coated mild steel before and after 90s exposure to CAPP is shown on Figure 3. 259
Powder coated mild steel was chosen as the model surface to image as some of the 260
other surfaces cannot be imaged using AFM due to forces exerted between the 261
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
12
surface and the cantilever. Micrographs A and B illustrate the 2D topography of the 262
applied cells, while C and D illustrate the 3D topography of the applied cells before 263
and after CAPP treatment respectively. Each micrograph represents an area of 5μm, 264
edge to edge, and is representative of multiple experiments (n=10). Plots E and F 265
represent the surface topography in Rt measurements and height distributions, 266
respectively, of untreated and CAPP treated cells on powder-coated mild steel. 267
268
A. baumannii cells before treatment (A and C) were observed as cellular aggregates, 269
indicative of pellicle formation, a morphological characteristic of biofilm forming A. 270
baumannii 19606 (33-36) whereby the secretion of exopolysaccharide causes the 271
cells to clump together. This characteristic is considered to extend the survival of 272
the organism in the environment. Following 90s exposure of A. baumannii to CAPP 273
(micrographs B and D), a significant disruption of the cell aggregates can be observed 274
with single cells showing disruption of the cell wall and leakage of cellular content. In 275
panel E and F the changes in the surface topography, registered in Rt measurements 276
and height distribution, respectively, can be seen. The noise in the measurement of 277
the treated cells is indicative of the severe etching effect by the plasma 278
corresponding to surface damage of the cells. Cell disruption is verified by a 279
reduction in the Rt value and the median height distributions from the untreated 280
(151.8nm and 153.2nm) to the treated cells (118.7nm and 118.9nm). 281
282
ESBL-producing E. coli AFM micrographs show smooth and individual cells for the 283
untreated control in A and C. However, severe cellular disruption can be seen with 284
only cell debris left on the surface, and no residual intact cells, following 90s 285
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
13
exposures to CAPP (B and D). In panels E and F, the surface topography in Rt and 286
height distributions measurements show a considerable reduction in Rt value and 287
cell height of 302nm and 312nm compared to the untreated cells 139.6nm and 288
131.2nm, consistent with the physical disruption of the bacterial cells. 289
290
AFM imaging of the MRSA cells inoculated on powder-coated mild steel show 291
smooth and morphologically intact cells with no disruption visible both on the 2D 292
and 3D micrographs, respectively (A and C). Following 90s of CAPP treatment, 293
cellular distortions can be seen in B and D with obvious cellular debris present and 294
very few intact cells remaining. The surface topography and roughness assessed in Rt 295
measurements (E) and height distributions (F) show an increase in the Rt and height 296
measurements from 262.0nm and 260.0nm to 414.0nm and 415.4nm, possibly due 297
to a build up of cell debris on the surface following CAPP treatment, indicative of the 298
physical disruption of the cells by the air plasma jet. 299
300
Finally, untreated VRE cells appear intact and slightly oval shape cells, which is 301
characteristic of Enterococcus spp., on the corresponding 2D and 3D micrographs of 302
A and C respectively. Cellular malformations arose in VRE treated cells after 90s 303
exposure to CAPP, with cells appearing distorted and what could possibly be 304
intracellular material leaching out of damaged cells (micrographs B and D). In panels 305
E and F, the surface roughness expressed in Rt values and median of height for the 306
untreated and treated cells were 290.5nm and 291.0nm and 294.3nm and 193.9nm, 307
respectively. 308
309
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
14
DISCUSSION 310
311
The present study aimed to evaluate the antimicrobial effect of a CAPP single jet 312
prototype on bacteria of clinical significance including MRSA, VRE, ESBL-producing E. 313
coli and A. baumannii on inanimate surfaces commonly found in the clinical setting. 314
A recent review on environmental contamination has highlighted the importance of 315
this source as a primary mode of transmission of HCAI. Current effective 316
decontamination methods pose logistical difficulties and limitations, but the results 317
presented here suggest that the use of CAPP is a promising tool for environmental 318
bio-decontamination achieving a >log 5 reduction for some bacteria on certain 319
materials after 90s. Although other studies have been performed on biomedical 320
device materials, skin models, pagers and in solution, this is the first study 321
performed on materials of surfaces of clinical relevance (37-40). 322
323
Previous studies on the antimicrobial effects of plasma involved different treatment 324
exposure times mainly due to the physical state of the bacteria and demonstrated 325
shorter times for planktonic cells in solution (41) and longer times for cells dried on 326
test surfaces and in biofilms. In this study, the optimum antimicrobial activity of the 327
air plasma was observed after 90s, producing log reductions of 3 to 5 for MRSA, log 2 328
to 5 for VRE, log 2 to 3 for E. coli and log 1.7 to 3 for A. baumannii, all of which were 329
air-dried on each test surface. Maisch et al., (42) evaluated the efficacy of a CAPP 330
device on MRSA and E. coli contaminated porcine skin and showed that longer 331
exposure times were required to achieve similar log reductions to our study, i.e. 332
6min for log 3 reduction and 8min for a log 5 reduction of both strains but these are 333
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
15
relatively prolonged periods in the busy clinical environment for surface 334
decontamination. Similarly, the efficacy of a plasma micro jet in killing S. aureus and 335
Enterococcus faecalis inoculated on agar found that treatment exposure times of 4 336
to 5min were required to achieve a log 4 reduction. For biofilms, the treatment 337
times increase significantly, in some cases taking as long as 30min to achieve a log 3 338
reduction (43). Recently, remote plasma exposure of MRSA strains, in biofilm 339
form, has proven to be effective also. However, the treatment in this case, 340
required up to 1.5h to inactivate the biofilm completely (44). Few studies 341
have assessed the effects of CAPP on A. baumannii, but one found that this 342
bacterium was more resistant to plasma than S. aureus and other Gram-343
negative organisms (45). 344
345
The isolation of Gram-negative bacteria from the environment poses challenges as 346
they may enter a viable but non-culturable state, and this may partly explain why 347
they are isolated less frequently than Gram-positive bacteria. Although still capable 348
of causing infection recovering them from the environment is difficult in this state 349
(46). This was also reflected in our results as there was an evident decline in log 350
numbers between the applied inoculum and the recovered control after air-drying. 351
Morphological cellular effects following plasma exposure were observed for Gram-352
negative bacteria as seen in Figure 3 for treated A. baumannii and ESBL-producing E. 353
coli AFM images 2D and 3D (panels B and D) compared to the un-treated controls 354
(panels A and C). CAPPs produce numerous reactive ions including reactive oxygen 355
species (ROCs), reactive nitrogen species (RONs) and UV, which, as originally suggested 356
by Laroussi et al. 2003 (47), chemically and physically alter various bacterial, fungal 357
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
16
cells, tissues and surfaces. These species not only affect bacterial cells on a surface 358
level but also intracellularly causing a cascade of effects leading to cell wall 359
disruption, cytoplasm leakage, lipid peroxidation and DNA damage (48-50). For both 360
MRSA and VRE, cellular disruption and physiological changes were observed 361
whereby following 90s of treatment few intact cells remained, with visible cellular 362
debris observed (Figure 3, MRSA and VRE panels B and D). 363
364
Montie et al, (50) suggested that leakage of the cytoplasm occurs due to initial 365
“etching” or physical damage of the bacterial cell wall, then once compromised the 366
reactive oxygen species filter through into the cell causing oxidative damage, 367
eventually leading to cell death. The rates at which this occurs differ between Gram-368
positive and Gram-negative bacteria chiefly due to metabolic and biochemical 369
pathway differences, in addition to the differences in the amount of peptidoglycan 370
present in the cell walls. Another publication by Yusupov et al., (48) verified the 371
disruption of important C-N, C-O and C-C bonds in peptidoglycan by O3, O2 molecules 372
and O atoms following plasma treatment. As the thickness of the peptidoglycan layer 373
differs between Gram-positive (20 to 30nm) and Gram-negative bacteria (6 to 7nm) 374
it can be speculated that the effects of the plasma on the cell wall may be more 375
pronounced in Gram-negative bacteria. In the present study both ESBL-producing E. 376
coli and A. baumannii showed more severe physical damage and in some cases total 377
cell disruption as seen in AFM micrographs of ESBL-producing E. coli (Figure 3, panel 378
B and D) where only cell debris can be seen following treatment of E. coli cells for 379
90s. Similar effects were seen for A. baumannii, (Figure 3, A. baumannii B and D). 380
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
17
Height distribution measurements produced a “noisy” graph that may be indicative 381
of significant etching of the cell walls (Figure 3, A. baumannii E). 382
383
The data presented in this study has verified the efficacy of CAPPs for use as a bio-384
decontaminating agent in clinical environments. The air plasma source used shows 385
significant bactericidal effects on both Gram-positive and Gram-negative organisms 386
with a maximum log reduction of approximately >log 5 after 90s. In addition, the 387
design and configuration of the plasma jet used here produces and delivers reactive 388
species in a controlled manner. This suggests that the use of such a system could 389
greatly enhance infection control procedures currently existing in the clinical setting. 390
391
In conclusion we have shown that CAPP significantly reduces bacterial numbers on a 392
range of surfaces commonly found in the clinical environment within 90s. Further 393
work is required to develop a prototype that could be used in the clinical 394
environment and to evaluate this against spore-forming bacteria such as Clostridium 395
difficile, and mixtures of bacteria with protein and other substances that mimic 396
contamination in a clinical setting. If efficacy is confirmed, CAPP would represent an 397
important and valuable alternative to surface decontamination in healthcare 398
facilities. 399
400
ACKNOWLEDGEMENTS 401
The Health Research Board Ireland and Science Foundation Ireland funded this 402
research through grant TRA/2010/10. We are also grateful to advisors and 403
collaborators for their participation in his project. 404
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
18
REFERENCES 405
406
1. Allegranzi B, Bagheri Nejad S, Combescure C, Graafmans W, Attar H, 407
Donaldson L, Pittet D. 2011. Burden of endemic health-care-associated 408
infection in developing countries: systematic review and meta-analysis. 409
Lancet 377:228-241. 410
2. O'Brien D, Richards J, Walton KE, Phillips MG, Humphreys H. 2009. Survey of 411
teaching/learning of healthcare-associated infections in UK and Irish medical 412
schools. J. Hosp. Infect. 73:171-175. 413
3. Otter JA, Yezli S, Perl TM, Barbut F, French GL. 2013. The role of 'no-touch' 414
automated room disinfection systems in infection prevention and control. J. 415
Hosp. Infect. 83:1-13. 416
4. Drees M, Snydman DR, Schmid CH, Barefoot L, Hansjosten K, Vue PM, 417
Cronin M, Nasraway SA, Golan Y. 2008. Prior environmental contamination 418
increases the risk of acquisition of vancomycin-resistant enterococci. Clin. 419
Infect. Dis. 46:678-685. 420
5. Otter JA, Yezli S, French GL. 2011. The role played by contaminated surfaces 421
in the transmission of nosocomial pathogens. Infect. Control Hosp. Epidemiol. 422
32:687-699. 423
6. Ali S, Moore G, Wilson AP. 2012. Effect of surface coating and finish upon 424
the cleanability of bed rails and the spread of Staphylococcus aureus. J. Hosp. 425
Infect. 80:192-198. 426
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
19
7. Hooker EA, Allen S, Gray L, Kaufman C. 2012. A randomized trial to evaluate 427
a launderable bed protection system for hospital beds. Antimicrob Resist 428
Infect Control 1:27. 429
8. Ferreira AM, de Andrade D, de Almeida MT, Cunha KC, Rigotti MA. 2011. 430
Egg crater mattresses: a deposit of methicillin-resistant Staphylococcus 431
aureus? Rev. Esc. Enferm. USP 45:161-166. 432
9. Creamer E, Humphreys H. 2008. The contribution of beds to healthcare-433
associated infection: the importance of adequate decontamination. J. Hosp. 434
Infect. 69:8-23. 435
10. Bache SE, Maclean M, Gettinby G, Anderson JG, MacGregor SJ, Taggart I. 436
2013. Quantifying bacterial transfer from patients to staff during burns 437
dressing and bed changes: implications for infection control. Burns 39:220-438
228. 439
11. Ohl M, Schweizer M, Graham M, Heilmann K, Boyken L, Diekema D. 2012. 440
Hospital privacy curtains are frequently and rapidly contaminated with 441
potentially pathogenic bacteria. Am. J. Infect. Control 40:904-906. 442
12. Falagas ME, Thomaidis PC, Kotsantis IK, Sgouros K, Samonis G, 443
Karageorgopoulos DE. 2011. Airborne hydrogen peroxide for disinfection of 444
the hospital environment and infection control: a systematic review. J. Hosp. 445
Infect. 78:171-177. 446
13. Russell A, Secrest J, Schreeder C. 2012. Stethoscopes as a source of hospital-447
acquired methicillin-resistant Staphylococcus aureus. J. Perianesth. Nurs. 448
27:82-87. 449
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
20
14. Morgan DJ, Liang SY, Smith CL, Johnson JK, Harris AD, Furuno JP, Thom KA, 450
Snyder GM, Day HR, Perencevich EN. 2010. Frequent multidrug-resistant 451
Acinetobacter baumannii contamination of gloves, gowns, and hands of 452
healthcare workers. Infect. Control Hosp. Epidemiol. 31:716-721. 453
15. Salama MF, Jamal WY, Mousa HA, Al-Abdulghani KA, Rotimi VO. 2013. The 454
effect of hand hygiene compliance on hospital-acquired infections in an ICU 455
setting in a Kuwaiti teaching hospital. J Infect Public Health 6:27-34. 456
16. FitzGerald G, Moore G, Wilson AP. 2013. Hand hygiene after touching a 457
patient's surroundings: the opportunities most commonly missed. J. Hosp. 458
Infect. 84:27-31. 459
17. Breathnach AS, Cubbon MD, Karunaharan RN, Pope CF, Planche TD. 2012. 460
Multidrug-resistant Pseudomonas aeruginosa outbreaks in two hospitals: 461
association with contaminated hospital waste-water systems. J. Hosp. Infect. 462
82:19-24. 463
18. Yaslianifard S, Mobarez AM, Fatolahzadeh B, Feizabadi MM. 2012. 464
Colonization of hospital water systems by Legionella pneumophila, 465
Pseudomonas aeroginosa, and Acinetobacter in ICU wards of Tehran 466
hospitals. Indian J. Pathol. Microbiol. 55:352-356. 467
19. Emmerich SJ, Heinzerling D, Choi J-i, Persily AK. 2013. Multizone modeling of 468
strategies to reduce the spread of airborne infectious agents in healthcare 469
facilities. Build Environ 60:105-115. 470
20. Chmielarczyk A, Higgins PG, Wojkowska-Mach J, Synowiec E, Zander E, 471
Romaniszyn D, Gosiewski T, Seifert H, Heczko P, Bulanda M. 2012. Control 472
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
21
of an outbreak of Acinetobacter baumannii infections using vaporized 473
hydrogen peroxide. J. Hosp. Infect. 81:239-245. 474
21. Liao CM, Lin YJ, Cheng YH. 2013. Modeling the impact of control measures 475
on tuberculosis infection in senior care facilities. Build Environ 59:66-75. 476
22. Kong MG, Kroesen G, Morfill G, Nosenko T, Shimizu T, Dijk Jv, Zimmermann 477
JL. 2009. Plasma medicine: an introductory review. New J Phys 11:115012. 478
23. Metelmann H-R, Vu TT, Do HT, Le TNB, Hoang THA, Phi TTT, Luong TML, 479
Doan VT, Nguyen TTH, Nguyen THM, Nguyen TL, Le DQ, Le TKX, von 480
Woedtke T, Bussiahn R, Weltmann K-D, Khalili R, Podmelle F. 2013. Scar 481
formation of laser skin lesions after cold atmospheric pressure plasma (CAP) 482
treatment: A clinical long term observation. Clin Plasma Med 1:30-35. 483
24. Emmert S, Brehmer F, Hänßle H, Helmke A, Mertens N, Ahmed R, Simon D, 484
Wandke D, Maus-Friedrichs W, Däschlein G, Schön MP, Viöl W. 2013. 485
Atmospheric pressure plasma in dermatology: Ulcus treatment and much 486
more. Clin Plasma Med 1:24-29. 487
25. Daeschlein G, Scholz S, Ahmed R, von Woedtke T, Haase H, Niggemeier M, 488
Kindel E, Brandenburg R, Weltmann KD, Juenger M. 2012. Skin 489
decontamination by low-temperature atmospheric pressure plasma jet and 490
dielectric barrier discharge plasma. J. Hosp. Infect. 81:177-183. 491
26. Choi Y-R, Kwon J-S, Song D-H, Choi EH, Lee Y-K, Kim K-N, Kim K-M. 2013. 492
Surface modification of biphasic calcium phosphate scaffolds by non-thermal 493
atmospheric pressure nitrogen and air plasma treatment for improving 494
osteoblast attachment and proliferation. Thin Solid Films 547:235-240. 495
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
22
27. Shon WJ, Chung SH, Kim HK, Han GJ, Cho BH, Park YS. 2013. Peri-implant 496
bone formation of non-thermal atmospheric pressure plasma-treated 497
zirconia implants with different surface roughness in rabbit tibiae. Clin. Oral 498
Implants Res. 499
28. Park JK, Nam SH, Kwon HC, Mohamed AA, Lee JK, Kim GC. 2011. Feasibility 500
of nonthermal atmospheric pressure plasma for intracoronal bleaching. Int. 501
Endod. J. 44:170-175. 502
29. Schaudinn C, Jaramillo D, Freire MO, Sedghizadeh PP, Nguyen A, Webster P, 503
Costerton JW, Jiang C. 2013. Evaluation of a nonthermal plasma needle to 504
eliminate ex vivo biofilms in root canals of extracted human teeth. Int. Endod. 505
J. 46:930-937. 506
30. Pan J, Sun K, Liang Y, Sun P, Yang X, Wang J, Zhang J, Zhu W, Fang J, Becker 507
KH. 2013. Cold plasma therapy of a tooth root canal infected with 508
Enterococcus faecalis biofilms in vitro. J. Endod. 39:105-110. 509
31. O'Connor N, Cahill OJ, Galvin S, McDonnell C, Stevens N, Hare NO, 510
Humphreys H, Daniels S. 2012, p 2P-155-152P-155. Plasma Science (ICOPS), 511
2012 Abstracts IEEE International Conference on. 512
32. Horcas I, Fernandez R, Gomez-Rodriguez JM, Colchero J, Gomez-Herrero J, 513
Baro AM. 2007. WSXM: a software for scanning probe microscopy and a tool 514
for nanotechnology. Rev. Sci. Instrum. 78:013705. 515
33. Marti S, Nait Chabane Y, Alexandre S, Coquet L, Vila J, Jouenne T, De E. 516
2011. Growth of Acinetobacter baumannii in pellicle enhanced the expression 517
of potential virulence factors. PLoS One 6:e26030. 518
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
23
34. Dubrovin EV, Popova AV, Kraevskiy SV, Ignatov SG, Ignatyuk TE, Yaminsky 519
IV, Volozhantsev NV. 2012. Atomic force microscopy analysis of the 520
Acinetobacter baumannii bacteriophage AP22 lytic cycle. PLoS One 7:e47348. 521
35. Mussi MA, Gaddy JA, Cabruja M, Arivett BA, Viale AM, Rasia R, Actis LA. 522
2010. The opportunistic human pathogen Acinetobacter baumannii senses 523
and responds to light. J. Bacteriol. 192:6336-6345. 524
36. Otter JA, Yezli S, Salkeld JA, French GL. 2013. Evidence that contaminated 525
surfaces contribute to the transmission of hospital pathogens and an 526
overview of strategies to address contaminated surfaces in hospital settings. 527
Am. J. Infect. Control 41:S6-11. 528
37. Burts ML, Alexeff I, Meek ET, McCullers JA. 2009. Use of atmospheric non-529
thermal plasma as a disinfectant for objects contaminated with methicillin-530
resistant Staphylococcus aureus. Am. J. Infect. Control 37:729-733. 531
38. Boxhammer V, Morfill GE, Jokipii JR, Shimizu T, Klampfl T, Li YF, Koritzer J, 532
Schlegel J, Zimmermann JL. 2012. Bactericidal action of cold atmospheric 533
plasma in solution. New J Phys 14:113042. 534
39. Isbary G, Köritzer J, Mitra A, Li YF, Shimizu T, Schroeder J, Schlegel J, Morfill 535
GE, Stolz W, Zimmermann JL. 2013. Ex vivo human skin experiments for the 536
evaluation of safety of new cold atmospheric plasma devices. Clin Plasma 537
Med 1:36-44. 538
40. Banerjee KK, Kumar S, Bremmell KE, Griesser HJ. 2010. Molecular-level 539
removal of proteinaceous contamination from model surfaces and 540
biomedical device materials by air plasma treatment. J. Hosp. Infect. 76:234-541
242. 542
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
24
41. Thiyagarajan M, Sarani A, Gonzales X. 2013. Atmospheric pressure resistive 543
barrier air plasma jet induced bacterial inactivation in aqueous environment. 544
J Appl Phys 113:093302-093302-093312. 545
42. Maisch T, Shimizu T, Li YF, Heinlin J, Karrer S, Morfill G, Zimmermann JL. 546
2012. Decolonisation of MRSA, S. aureus and E. coli by cold-atmospheric 547
plasma using a porcine skin model in vitro. PLoS One 7:e34610. 548
43. Marchal F, Robert H, Merbahi N, Fontagne-Faucher C, Yousfi M, Romain CE, 549
Eichwald O, Rondel C, Gabriel B. 2012. Inactivation of Gram-positive biofilms 550
by low-temperature plasma jet at atmospheric pressure. J Phys D Appl Phys 551
45:345202. 552
44. Yu QS, Huang C, Hsieh FH, Huff H, Duan Y. 2007. Bacterial inactivation using 553
a low-temperature atmospheric plasma brush sustained with argon gas. J 554
Biomed Mater Res B Appl Biomater 80:211-219. 555
45. Heller LC, Edelblute CM, Mattson AM, Hao X, Kolb JF. 2012. Inactivation of 556
bacterial opportunistic skin pathogens by nonthermal DC-operated afterglow 557
atmospheric plasma. Lett. Appl. Microbiol. 54:126-132. 558
46. Galvin S, Dolan A, Cahill O, Daniels S, Humphreys H. 2012. Microbial 559
monitoring of the hospital environment: why and how? J. Hosp. Infect. 560
82:143-151. 561
47. Laroussi M, Mendis DA, Rosenberg M. 2003. Plasma interaction with 562
microbes. New J Phys 5:41. 563
48. Yusupov M, Neyts EC, Khalilov U, Snoeckx R, van Duin ACT, Bogaerts A. 564
2012. Atomic-scale simulations of reactive oxygen plasma species interacting 565
with bacterial cell walls. New J Phys 14:093043. 566
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
25
49. Korachi M, Aslan N. 2011. The Effect of Atmospheric Pressure Plasma Corona 567
Discharge on pH, Lipid Content and DNA of Bacterial Cells. Plasma Sci Technol 568
13:99-105. 569
50. Montie TC, Kelly-Wintenberg K, Roth JR. 2000. An overview of research 570
using the one atmosphere uniform glow discharge plasma (OAUGDP) for 571
sterilization of surfaces and materials. Ieee T Plasma Sci 28:41-50. 572
573
574
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
26
FIGURE LEGENDS 575
576
Figure 1 - Atmospheric pressure air plasma jet. The nozzle is 1mm in diameter. The 577
luminous plasma jet extends approximately 25mm along the axis of the jet 578
body, when allowed to expand into air. When a substrate is placed in the expansion 579
field of the jet, it spreads to a diameter of circa 20mm over the substrate. 580
581
Figure 2- Bactericidal effects of the CAPP single jet on A. baumannii, ESBL E. coli, 582
MRSA and VRE inoculated on various surfaces over 30, 60 and 90 seconds (n≥3). 583
Appl: initial inoculums applied to the surface, Rec: number of bacteria recovered 584
from surface before use of CAPP. 585
586
Figure 3- AFM images of A. baumannnii, ESBL E. coli, MRSA and VRE inoculated on 587
powder-coated mild steel before and after 90s CAPP treatment: A and B correspond 588
to the 2D images and C and D to the 3D images. Micrograph plots (E and F) are 589
representative of the surface topography Rt measurements and height distribution 590
of untreated and treated cells on the powder-coated mild steel surface. 591
592
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
2D AFM images 3D AFM images Surface topography
Untreated 90s treated Untreated 90s treated Rt measurements Height distributionUntreated 90s treated Untreated 90s treated Rt measurements Height distribution
A. baumannii
E. coli
MRSA
VRE
on April 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from