Atmospheric pressure, nonthermal plasma ... - Wiley Online Library

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Atmospheric pressure, nonthermal plasma inactivation of MS2 bacteriophage: effect of oxygen concentration on virucidal activity N.H. Alshraiedeh1, M.Y. Alkawareek1, S.P. Gorman1, W.G. Graham2 and B.F. Gilmore1 1 School of Pharmacy, Queen’s University Belfast, Belfast, UK 2 Centre for Plasma Physics, Queen’s University Belfast, Belfast, UK

Keywords atmospheric pressure plasma, disinfection, MS2 bacteriophage, nonthermal plasma, norovirus, plasma sterilization. Correspondence Brendan F. Gilmore, School of Pharmacy, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK. E-mail: [email protected] 2013/0995: received 20 May 2013, revised 26 July 2013 and accepted 13 August 2013 doi:10.1111/jam.12331

Abstract Aims: The main aim of this study was to determine the virucidal inactivation efficacy of an in-house-designed atmospheric pressure, nonthermal plasma jet operated at varying helium/oxygen feed gas concentrations against MS2 bacteriophage, widely employed as a convenient surrogate for human norovirus. Methods and Results: The effect of variation of percentage oxygen concentration in the helium (He) carrier gas was studied and found to positively correlate with MS2 inactivation rate, indicating a role for reactive oxygen species (ROS) in viral inactivation. The inactivation rate constant increased with increasing oxygen concentrations up to 075% O2. 3 log10 (999%) reductions in MS2 viability were achieved after 3 min of exposure to the plasma source operated in a helium/oxygen (9925% : 075%) gas mixture, with >7 log10 reduction after 9 min exposure. Conclusions: Atmospheric pressure, nonthermal plasmas may have utility in the rapid disinfection of virally contaminated surfaces for infection control applications. Significance and Impact of Study: The atmospheric pressure, nonthermal plasma jet employed in this study exhibits rapid virucidal activity against a norovirus surrogate virus, the MS2 bacteriophage, which is superior to previously published inactivation rates for chemical disinfectants.

Introduction Norovirus, a single-stranded RNA, nonenveloped virus of the family Caliciviridae (Green et al. 2001), is the most common etiological agent of acute nonbacterial gastroenteritis outbreaks globally (Frankhauser et al. 2002; Bitler et al. 2013). Norovirus gastroenteritis outbreaks have been occurring with increasing annual frequency in healthcare and community settings in recent years (MacCannell et al. 2011), prompting healthcare providers to develop guidelines to prevent and control such outbreaks, by preventing norovirus transmission. In the USA, it has been estimated that norovirus may be the causative agent of >23 million gastroenteritis cases each year, representing ca. 60% of all acute gastroenteritis cases (Mead et al. 1420

1995). In the UK, according to 2002–2003 figures, norovirus is estimated to cost the National Health Service (NHS) in excess of £100 million per annum, in spike years (Lopman et al. 2004). In addition to hospitals, norovirus infection is associated with community outbreaks of gastroenteritis in schools, restaurants, hotels and cruise ships. Because the main route of transmission of this highly contagious virus is the faecal–oral route via ingestion of contaminated food and water, via environmental transmission by hand-to-mouth contact from contaminated fomites and by aerosolization (Xavier 2011), much of these guidelines focus on patient isolation, hand hygiene and environmental cleaning and disinfection (MacCannell et al. 2011; Health Protection Agency 2012). However,

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N.H. Alshraiedeh et al.

even after cleaning and chemical disinfection in the hospital environment, norovirus can still be detected on surfaces (Morter et al. 2011). Thus, effective approaches to viral inactivation and disinfection of both viable and fomite surfaces are central to the prevention and control of norovirus outbreaks. One promising approach to sterilization and disinfection of both viable and nonviable surfaces is the use of atmospheric pressure, nonthermal plasmas (APNTP). A number of recent studies have examined the use of such plasmas in a range of biomedical applications including microbial inactivation, sterilization and disinfection (Laroussi 2002; Fridman et al. 2008; Kong et al. 2009; Yardimci and Setlow 2010). In addition, the activity of nonthermal plasma against microbial biofilms (Alkawareek et al. 2012a,b), spores (Venezia et al. 2008) and DNA viruses (Venezia et al. 2008; Yasuda et al. 2010) has also been demonstrated. The application of APNTP has potential advantages over standard chemical disinfectants and sterilants. These include simplicity of design and operation (Yardimci and Setlow 2010); utilization of nontoxic gases and an absence of toxic residues (Rossi et al. 2009; Yardimci and Setlow 2010); and production of a large quantity of diverse microbicidal active species including chemically reactive species (for example, reactive oxygen species and reactive nitrogen species), UV and electromagnetic fields (Moisan et al. 2002). To date, there is no suitable cell culture system available to support the replication of norovirus (Duizer et al. 2004; Dawson et al. 2005), and a number of surrogate virus models for evaluation of the virucidal efficacy of chemical disinfectants have been proposed. One such surrogate, the MS2 bacteriophage, is of particular interest because it requires no mammalian tissue culture facilities, and virucidal efficacy assessments can be made using facile protocols in the microbiology laboratory. The Escherichia coli MS2 bacteriophage has been evaluated and validated as an effective surrogate for norovirus on fresh produce (Dawson et al. 2005), in viral persistence models of groundwater and surface water (Bae and Schwab 2008) and drinking water (Allwood et al. 2005) and in chemical disinfectant virucidal efficacy tests (Maillard et al. 1994; Brion and Silverstein 1999; Pinto et al. 2010; Su and D’Souza 2012). The objective of this study was to evaluate the virucidal activity of an in-house-built kilohertz-driven atmospheric pressure, nonthermal plasma jet, using the MS2 coliphage as a surrogate for norovirus. In particular, the effect of variation of oxygen concentration in the helium carrier gas (up to 1% O2) on MS2 virucidal activity was assessed to evaluate the potential of atmospheric pressure, nonthermal plasmas in norovirus disinfection in clinical environments.

Plasma eradication of MS2 bacteriophage

Materials and methods Plasma source The basic design of the plasma source used in this study was previously described (Teschke et al. 2005; Algwari and O’Connell 2011; Alkawareek et al. 2012b). Specifically, the plasma source consists of a capillary dielectric quartz tube (inner and outer diameters of 4 and 6 mm, respectively). Two copper electrodes (2 mm wide) encircle the tube with an interelectrode distance of 25 mm. The output of a high-voltage-pulse source (Haiden PHK-2k), operating at repetition frequency of 20 kHz and voltage amplitude of 6 kV, was applied to the downstream electrode, which is 10 mm from the end of the plasma tube. The upstream electrode was grounded. The plasma jet was operated with in a flowing gas mixture of 00–10% oxygen (0–20 standard cubic centimetres per minute) and 1000–990% helium, at a total flow rate of two standard litres per minute (SLM). Under these conditions, an intense discharge is created between the electrodes and a luminous plume [with a rotational gas temperature of around 40°C (Alkawareek et al. 2012b)], extends from end of the plasma jet tube, reaching the sample to be treated, which was placed 10 mm away from the end of the tube. Strains and growth conditions The commercially available MS2 bacteriophage ATTC 15597-B1 was used in this study and propagated using host strain E. coli ATCC 15597, and both phage and host strain were obtained from LGC Standards (Middlesex, UK) and maintained/propagated according to supplier protocols. Host strain E. coli ATCC 15597 was grown in ATCC 271 medium (broth) consisting of 10 g l 1 of tryptone, 8 g l 1 NaCl and 1 g l 1 of yeast extract dissolved in distilled water. Top and bottom agar layers contained 5 g l 1 (05%) and 15 g l 1 (15%) agar, respectively. In addition, a filter-sterilized supplement (13 ml) containing 10 ml of 10% glucose, 2 ml of 1 mol l 1 CaCl2 and 1 ml of thiamine (10 mg ml 1) was added aseptically to one litre of sterilized ATCC 271 medium. Sample preparation and plasma exposure MS2 phage stock was prepared by extracting bacteriophages from plates with confluent lyses. The soft agar overlay was removed using a sterile scalpel blade and resuspended in quarter strength Ringer’s solution (QSRS). This suspension was then centrifuged at 3000 g for 10 min, and the supernatant was filter-sterilized using 020-lm filters (Minisart syringe filters; VWR international, Leicestershire, UK). The resultant phage stock was stored, protected from


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light, at 4°C for short-term usage. The titre of bacteriophage was enumerated using the standard agar overlay technique (Adams 1959), through serial dilution of stocks and determination of plaque-forming units per millilitre (PFU ml 1). Bacteriophage titre was adjusted to a density of 109 PFU ml 1 in QSRS; then, 50-ll aliquots (sample thickness 1–3 mm) of this phage suspension were exposed to the plasma jet for exposure times of up to 9 min. After plasma exposure for each allocated time point, samples were serially diluted, and the double-layer method (plaque assay) was employed for the enumeration of surviving phages. Essentially, 100 ll of the diluted phage suspension was mixed with 20 ll of a mid-log E. coli culture in 3 ml of molten 271 top agar medium prepared as described above. The molten top agar was vortex-mixed gently and then poured on plates containing a set bottom agar layer. When set, the plates were incubated at 37°C overnight. After incubation, plaques, zones of lysis in the bacterial lawn, were enumerated, and the surviving phage density was calculated. All experiments were performed in triplicate. A gas-only control for each helium/oxygen mixture at each time point was included. The temperature of the plasma effluent was previously determined experimentally (Algwari and O’Connell 2011; Alkawareek et al. 2012b) and found to be 39°C. Statistical analysis Statistical analysis, using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA), was performed using a one-way analysis of variance (ANOVA), where P < 005 was taken to represent a statistically significant difference, and post hoc Tukey’s HSD multiple comparison test was then performed. Results Table 1 shows the log10 reduction in MS2 infectivity/viability over time, when exposed to the plasma source as described, with varying concentrations of oxygen in the carrier gas mixture and for exposure times of 1, 3, 5, 7 and

9 min respectively. Negligible evaporation of the sample was observed at each time point. These data were converted to percentage survival curves and are graphically represented in Fig. 1. In the absence of oxygen (Fig. 1a), there is a significant inactivation of MS2 bacteriophage at each time point, with a maximal reduction of 498 log10 reductions in PFU ml 1 following 9 min of helium-only plasma exposure. The significant virucidal activity of helium-only plasma generated in ambient air is perhaps unsurprising, because a number of studies indicate the production of multiple reactive species in the effluent plume of helium plasmas including reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as OH˙, N2+* and NO˙ (Ries et al. 2011), which can be produced by interactions between the energetic metastable helium species and the surrounding air (predominantly O2, N2 and water vapour). Figure 1b–d shows the effect of increasing oxygen concentration in the carrier gas mixture; from 025 to 1% O2 on MS2 phage inactivation. At 1 min exposures, 025, 05 and 10% oxygen admixtures provide a comparable reduction in MS2 viability, when compared to helium-only, whilst at 075% oxygen, the log10 reduction in MS2 viability is almost double that of helium-only (099 vs 053 log10 reduction in PFU ml 1). At each time point, the addition of 075% O2 to the helium carrier gas resulted in the greatest reductions in phage viability. From Table 1, it can be appreciated that plasma discharge at 075% oxygen/9925% He mixture resulted in a 3 log10 reduction in MS2 bacteriophage viability after only 3 min of exposure, compared with 5 min of exposure required to achieve the same log reduction in the case of plasma operated in He-only discharge and at oxygen concentrations of 025, 05 and 1%, as shown in Table 1. After 3 min of exposure to the plasma source, the log reduction in phage viability increased significantly from 138 005 (helium-only plasma, 0% oxygen) to 306 002 on the addition of 075% oxygen (P < 00001). Increasing the percentage of oxygen in the helium carrier gas to 1% resulted in a significant decrease (compared with 075% oxygen) in the log reduction in phage viability to 173 001 (P < 0016). Interestingly, whilst a concentration of 05% oxygen in the

Table 1 Log10 reduction of MS2 viability following exposure to nonthermal plasma operated at varying oxygen admixtures. Residual infectivity was observed after all exposure times, for each set of plasma operating conditions tested log10 reduction in MS2 bacteriophage viability Exposure time (min)

He/0% O2

1 3 5 7 9

053 138 316 411 498

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001 005 003 004 003

He/025% O2 059 164 322 446 569

002 002 004 003 003

He/05% O2 061 208 376 491 593

005 007 001 002 002

He/075% O2 099 306 390 531 706

004 002 003 003 002

He/1% O2 060 173 330 463 573

001 001 007 002 001




(b)

0 % Oxygen

100 80

% Survival

% Survival

(a)

60 40 20

0·25 % Oxygen

100 80 60 40 20 0

0 0

1

2

3

4

5

0

1

2

0·5 % Oxygen

(d)

100 80

% Survival

% Survival

(c)

60 40 20 0

3

4

5

4

5

Time (min)

Time (min)

0·75 % Oxygen

100 80 60 40 20 0

0

1

2

3

4

0

5

1

2

Time (min) 1 % Oxygen

gas mixture gave inactivation (up to 5 min exposure) of MS2 phage comparable to 075% oxygen, increasing the concentration to 1% oxygen actually reduced the degree of inactivation, giving rates equivalent to the lowest oxygen concentration, 025%, at all time points. This is most likely due to the increasing concentration of the molecular oxygen influencing the plasma production itself (‘discharge failure’) through the replacement of electrons with negative ions, thus reducing the overall density of reactive species delivered (Wang and Wan 2009). Overall (as shown in Fig. 1), compared with gas-only control, significant reductions in MS2 bacteriophage survival following exposure to the plasma jet were observed at each oxygen concentration; 0% oxygen: 100% helium (P < 00001), 025% oxygen: 9975% helium (P < 00001), 05% oxygen: 995% helium (P = 00001),075% oxygen: 9925% helium (P = 00009) and 1% oxygen: 99% helium mixtures (P < 00001). Discussion Despite decades of use of biocidal agents for inactivation of viral agents, the precise mechanisms of action of virucidal agents remain poorly understood. Even highly related viruses display wide variation in disinfection kinetics when

% Survival

(e) 100

Figure 1 MS2 bacteriophage percentage survival curves following the exposure to atmospheric pressure, nonthermal plasma jet operated at various oxygen [(a)–(e) 0–1% O2]/helium gas mixture concentrations.

3

Time (min)

80 60 40 20 0 0

1

2

3

4

5

Time (min)

exposed to the same biocidal agents (Halfhide et al. 2008; Wigginton et al. 2012) and the mechanism, type and extent of damage that a given virus can sustain before infectivity is lost are essentially unknown (Wigginton et al. 2012). However, previous studies indicate that exposure to inactivating oxidants and UV cause modifications in viral proteins and nucleic acids (Hotze et al. 2009; Sano et al. 2009; Wigginton et al. 2010, 2012). In this study, the effect of increasing oxygen concentration in the carrier gas mixture on MS2 inactivation, using in an in-house-designed plasma source, was evaluated. The data presented here suggest that an optimum concentration of oxygen in the gas mixture is required for efficient inactivation of MS2 bacteriophage, which once exceeded, results in decreased inactivation rate (as shown in Fig. 2). Inactivation rates for each oxygen concentration were calculated by linear regression of the survival curve, with inactivation rate taken as the negative of the slope of each line. Importantly, the data therefore suggest an important role for ROS in viral inactivation. The addition of oxygen to the helium carrier gas in a plasma discharge is known to produce a high density of ROS, including ozone, atomic oxygen, single delta oxygen, superoxide, peroxide and hydroxyl radicals (Gaunt


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Inactivation rate (min–1)

0·80 0·75 0·70 0·65 0·60 0·55 0·50 0·00

0·25

0·50 0·75 % Oxygen

1·00

Figure 2 Inactivation rate of MS2 bacteriophage upon exposure to plasma operated at different oxygen admixtures.

et al. 2006; Kong et al. 2009; Alkawareek et al. 2012b). These data are therefore in accordance with recent studies on MS2 phage inactivation where singlet oxygen 1O2 was shown to cause inactivation of MS2 phage by impairing genome replication and significant genome decay, as well as minor effects on host binding and genome injection into the host (Wigginton et al. 2012). Production of 1O2 by UVA illumination of nanoparticles aggregates in fullerol (polyhydroxy fullerenes) suspensions was also shown to account for virucidal activity of these suspensions against MS2 bacteriophage, as well as dsDNA phages, T7 and PRD1 (Hotze et al. 2009). Recently, a number of groups have examined atmospheric pressure plasma as a potential approach to controlling viruses, using bacteriophages as surrogates for human viruses (Venezia et al. 2008; Yasuda et al. 2010). Venezia et al. (2008) examined the antimicrobial activity of the PlasmaSol nonthermal plasma sterilizer apparatus (PlasmaSol Corporation, Hoboken, NJ, USA), which consists of a sealable metal chamber into which the humidified gas mixture flows, on wide range of bacteria, spores and viruses. Exposure of 106 PFU ml 1 of temperate k bacteriophage C-17 (ATCC 23724-B1) and lytic bacteriophage (Rambo; Microphage) resulted in at least a 4–6 log10 reduction in viability following 10 min of exposure. Yasuda and co-workers, on the other hand, observed rapid inactivation of k phage using a novel atmospheric dielectric barrier discharge (DBD) reactor, with up to 6 log10 reductions in phage infectivity after 20 s. In both these studies, genome (RNA or DNA) damage was detected, as well as alteration to coat proteins. The results presented here compared favourably with published data for the inactivation of MS2 bacteriophage using chemical biocides, indicating significantly shorter exposure times are required to bring about equivalent reductions in viral survival using nonthermal plasma, compared with a number of chemical disinfectants. For example, an exposure time of 20 min was required to 1424

achieve 395 and 368 log10 reductions in MS2 survival density with 1% v/v peracetic acid and 70% v/v ethanol, respectively, and 10 min of exposure to 05% or 1% glutaraldehyde to achieve a reduction of 4 log10 (Maillard et al. 1994). Similar results were reported for MS2 inactivation by the exposure to proprietary formulations of polyhexamethylene biguanides (Pinto et al. 2010). The potential application of this technology in a range of biomedical and clinical scenarios is extensive, because rapid rates of kill for vegetative bacteria, spores, bacterial biofilms and viruses have been demonstrated. Nonthermal plasma technology also benefits from low operating costs and an absence of harmful residues, often associated with chemical biocides and sterilants. Therefore, a wide range of patient and healthcare facility infection and contamination control applications are envisioned for atmospheric pressure, nonthermal plasmas. Conclusion This study demonstrates the potential of atmospheric pressure, nonthermal plasmas to be used as a rapid, effective method for disinfection of surfaces contaminated with nonenveloped viruses such as MS2 bacteriophages, which are useful surrogates of human enteric viruses such as norovirus, the most common global cause of infectious gastroenteritis. The plasma configuration described here gave significant reductions in viral titre over relatively short exposure times, with inactivation rate shown to be a function of oxygen concentration in the carrier gas mixture. Atmospheric pressure, nonthermal plasma devices may have significant utility in reducing the significant burden of healthcare-associated infections caused by human pathogenic viruses such as norovirus, or indeed for bacteriophage disinfection in fermentation or dairy facilities. Acknowledgements The authors gratefully acknowledge the generous support of the Society for Applied Microbiology through the award of a Research Development Fund grant to BG, which contributed to the development of the plasma device used in this study. Conflict of interest No conflict of interest declared. References Adams, M.D. (1959) Bacteriophages. New York: Interscience Publishers, Inc.



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