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International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens
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Padrig B. Flynn a , Sarah Higginbotham a , Nid’a H. Alshraiedeh a,b , Sean P. Gorman a , William G. Graham c , Brendan F. Gilmore a,∗ a
School of Pharmacy, Queen’s University of Belfast, Belfast BT9 7BL, UK Faculty of Pharmacy, Jordan University of Science and Technology, Irbid 22110, Jordan c Centre for Plasma Physics, Queen’s University of Belfast, Belfast BT7 1NN, UK b
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Article history: Received 3 November 2014 Accepted 25 February 2015
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Keywords: Atmospheric pressure plasma Non-thermal plasma Biocide ESKAPE pathogens Biofilm Plasma medicine
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1. Introduction
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The emergence of multidrug-resistant pathogens within the clinical environment is presenting a mounting problem in hospitals worldwide. The ‘ESKAPE’ pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) have been highlighted as a group of causative organisms in a majority of nosocomial infections, presenting a serious health risk due to widespread antimicrobial resistance. The stagnating pipeline of new antibiotics requires alternative approaches to the control and treatment of nosocomial infections. Atmospheric pressure nonthermal plasma (APNTP) is attracting growing interest as an alternative infection control approach within the clinical setting. This study presents a comprehensive bactericidal assessment of an in-house-designed APNTP jet both against biofilms and planktonic bacteria of the ESKAPE pathogens. Standard plate counts and the XTT metabolic assay were used to evaluate the antibacterial effect of APNTP, with both methods demonstrating comparable eradication times. APNTP exhibited rapid antimicrobial activity against all of the ESKAPE pathogens in the planktonic mode of growth and provided efficient and complete eradication of ESKAPE pathogens in the biofilm mode of growth within 360 s, with the exception of A. baumannii where a >4 log reduction in biofilm viability was observed. This demonstrates its effectiveness as a bactericidal treatment against these pathogens and further highlights its potential application in the clinical environment for the control of highly antimicrobial-resistant pathogens. © 2015 Published by Elsevier B.V.
The ‘ESKAPE’ pathogens, first classified by the Infectious Diseases Society of America (IDSA), refer to Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. They have been highlighted as a group of pathogens frequently associated with nosocomial infections and that represent new paradigms in pathogenesis, transmission and resistance, resulting in the potential to pose a serious threat to public health [1]. This group of pathogens is responsible for the most problematic of hospitalacquired infections (HAIs) owing to their ability to ‘escape’ the antimicrobial action of antibiotics [1]. According to the World Health Organization (WHO), HAIs have been estimated to affect 4.5 million and 1.7 million people annually in Europe and the USA,
∗ Corresponding author. Tel.: +44 2890972305; fax: +44 28 9024 7794. E-mail address: [email protected] (B.F. Gilmore).
respectively, with billions of dollars spent on the incurred costs [2,3]. As well as the financial burden imposed by HAIs, there are serious consequences for the patient, including increased length of stay in the care facility, increased morbidity and, in an increasing number of cases, increased mortality [2]. The ESKAPE pathogens, in particular, are increasingly implicated in nosocomial infections and are especially problematic to critical care patients, usually occurring as biofilm infections [4]. Biofilms represent ca. 80% of all microbial infections [5]. The US Centers for Disease Control and Prevention (CDC) has estimated that 1.7 million HAIs annually in the USA are due to biofilm-associated infections, resulting in healthcare costs of up to US$11 billion [6]. Biofilm formation by micro-organisms represents a significant virulence attribute both in acute and chronic infections. It is accepted that biofilms are the predominant phenotype of most bacteria both in natural and clinical settings, with the planktonic phenotype existing only transiently [7]. One of the most serious threats to global health is the spread of antibiotic-resistant bacteria, caused in part by inappropriate
http://dx.doi.org/10.1016/j.ijantimicag.2015.02.026 0924-8579/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Flynn PB, et al. Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2015.02.026
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Fig. 1. (a) Design image of plasma jet encased within acrylic plastic and (b) photograph of plasma jet in operation.
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antibiotic use and control. Despite the urgent need for new antimicrobial agents, many pharmaceutical companies have withdrawn from the arena in recent years, resulting in stagnation of the antibiotic development pipeline [8]. New and innovative strategies for infection control and treatment are required alongside drug therapy in order to combat the growing problem of multidrugresistant bacteria and nosocomial infections. One strategy that has increasing potential as a non-systemic treatment and disinfection option is atmospheric pressure non-thermal plasma (APNTP) [9]. Plasma is regarded as the fourth state of matter and can be described as an ionised gas. It consists of photons, electrons, ions, atoms, free radicals and electromagnetic fields. Non-thermal plasmas exist in a state of non-equilibrium. This non-equilibrium state refers to the temperature of the neutral gas particles and ions being close to the ambient room temperature while the electron temperature is much higher. The greater mass of the gas particles compared with the electrons produces an atmospheric plasma at a tolerable temperature. Plasma at atmospheric pressure can be formed when energy generated from an electric current ionises a gas such as argon or a helium–oxygen admixture. APNTP devices are simple to set up, reducing the costs and time associated with low-pressure plasmas [10]. They can generate a rich, dry chemistry in air at ambient temperature. This complex chemical environment comprises of a mixture of reactive agents, such as reactive oxygen and nitrogen species (RONS), ultraviolet light and charged particles, all of which contribute to the antibacterial properties of plasma [11]. APNTP has received much attention as a potential approach to controlling microbial organisms [11,12] and is central to an emerging scientific field known as plasma medicine. Different configurations of non-thermal plasma devices have been effectively shown in biomedical applications, including chronic infected wound treatments [13] and cancer treatment [14]. Its potential as an antimicrobial approach is now widely established. However, despite the explosion of interest in this field as well as the serious threat posed by the ESKAPE pathogens, there have been no studies assessing its efficacy against this collection of micro-organisms in different phenotypes (planktonic/biofilm). The current study represents the first report of the in vitro antimicrobial efficacy of an in-house-designed APNTP source against the ESKAPE pathogens both in planktonic and biofilm modes of growth.
2. Materials and methods
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The following microbial strains were used in this study: E. faecium DSM 25390; A. baumannii NCTC 13304; and P. aeruginosa PA14. S. aureus, K. pneumoniae and extended-spectrum -lactamase (ESBL)-producing Enterobacter cloacae (used as a model organism for Enterobacter spp.) were clinical isolates obtained from Antrim Area Hospital, Northern Health and Social Care Trust (Co. Antrim, UK). All strains were stored at −20 ◦ C in MicrobankTM vials (ProLab Diagnostics, Cheshire, UK) and were subcultured overnight at 37 ◦ C on Mueller–Hinton agar (MHA) (Oxoid Ltd., Basingstoke, UK) unless stated otherwise. 2.2. Plasma source A previously described [5,15] in-house-designed plasma source (Fig. 1) was employed for this study. Specifically, the jet consists of a quartz dielectric tube with an inner diameter of 4 mm and an outer diameter of 6 mm. Two copper electrodes, 2 mm wide and 25 mm apart, encircle the quartz tube. The downstream electrode, which is 5 mm from the end of the tube, is powered by a high-voltage power supply (Haiden PHK-2K; Haiden Laboratory Inc., Hyogo, Japan). The jet is operated at a frequency of 20 kHz and voltage amplitude of 6 kV; the upstream electrode is grounded. The plasma jet is operated with an admixture of helium (99.5%) and oxygen (0.5%) with a total flow rate of 2 standard litres per min (SLM). These conditions allow for a long, intense and luminous plume to form in the air as seen in Fig. 1b. The temperature of this plume, reported as rotational temperature, is 39 ◦ C [5]. The plasma jet configuration is encased in solid acrylic tubing, a deviation from the previously reported design [5,15]. 2.3. Treatment of bacterial biofilms with atmospheric pressure non-thermal plasma To evaluate the antimicrobial effect of the plasma jet against biofilms of ESKAPE pathogens, overnight cultures of S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa and E. cloacae were adjusted to an optical density at 550 nm (OD550 ) equivalent to ca. 107 CFU/mL in Mueller–Hinton broth (MHB) (Oxoid Ltd.). Then,
Please cite this article in press as: Flynn PB, et al. Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2015.02.026
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150 L aliquots of the standardised bacterial suspensions were transferred to the wells of a Nunc 96-well plate (Fisher Scientific UK Ltd., Loughborough, UK) that was incubated for 24 h at 37 ◦ C within a humidified compartment in a Gallenkamp Orbital Incubator (AGB Scientific Ltd., Dublin, Ireland) at 100 rpm. After 24 h, the liquid phase was discarded and the wells were rinsed twice with 200 L of phosphate-buffered saline (PBS) to remove unattached and loosely adhered cells. Overnight cultures of E. faecium, grown on anaerobic blood agar (Oxoid Ltd.) with 5% defibrinated horse blood (TCS Biosciences Ltd., Buckingham, UK), were adjusted to an OD550 equivalent to 107 CFU/mL in MHB supplemented with 1% glucose (Sigma-Aldrich Company Ltd., Poole, UK). Then, 150 L aliquots of the standardised suspension were transferred to the wells of a 96-well plate that was incubated for 48 h on an orbital shaker (100 rpm) at 37 ◦ C in anaerobic conditions using the Modular Atmosphere Controlled System (Davidson and Hardy Ltd., Belfast, UK). The liquid phase was discarded after 48 h and the wells were rinsed twice with 200 L of PBS to remove unattached and loosely adhered cells. Biofilms were exposed to the plasma at a separation distance of 15 mm between the end of the plasma jet nozzle and the bottom of the wells of the 96-well plate and were exposed to the plasma effluent for times of 0, 15, 30, 45, 60, 120, 240 and 360 s. Samples were exposed in triplicate for each time. As a negative control, the effect of gas-only treatment (where no power is applied to the electrodes) was also investigated. Following exposure to APNTP or gas only, 200 L of PBS was added to each well and the plate was sonicated for 15 min to dislodge and re-suspend biofilm cells. 2.4. Treatment of planktonic bacteria with atmospheric pressure non-thermal plasma Overnight cultures (as described above) were diluted in PBS to an OD550 equivalent to 108 CFU/mL. Then, 20 L aliquots of each standardised bacterial suspension were exposed in lids of microcentrifuge tubes to the plasma plume at a distance of 15 mm from sample to nozzle. The samples were treated for a maximum of 240 s with the same time intervals as described for biofilms. Treatments were repeated in triplicate for each time point. Following exposure to APNTP, samples were transferred to 180 L of PBS for determination of viable counts. 2.5. Determination of cell viability by XTT metabolic assay The XTT assay (Sigma-Aldrich Company Ltd.) was performed as previously described [15]. In brief, 50 L of treated planktonic or biofilm bacterial suspensions were transferred to the wells of a 96-well plate containing 50 L of MHB and 20 L of XTT stock solution. Control wells contained 50 L of PBS in place of bacterial suspensions. Plates were incubated at 37 ◦ C in an orbital incubator for 5–7 h. The resulting colour change was read using a microplate reader (BioTek EL808; BioTek Instruments Ltd., Potton, UK) at 450 nm. Survival fractions were calculated by dividing the OD value of each well corresponding to a biofilm/planktonic treatment time by the mean OD of the unexposed control. 2.6. Determination of cell viability by standard plate count method Serial 10-fold dilutions of planktonic and sonicated biofilm suspensions were prepared and three 20 L drops from each dilution were plated onto MHA for standard plate counts. Plates were incubated aerobically at 37 ◦ C and were observed after 24 h and 48 h for viable colonies. Plates with no colonies grown after 48 h, indicating complete bacterial eradication, were left for a further 2 days
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to check for re-growth of dormant cells. Plate counts were used to calculate log surviving cells and survival fraction of each bacterial strain. 2.7. Statistical analysis Statistical analyses were performed using Prism 6.0 software (GraphPad Software Inc., San Diego, CA). The P-value was derived from the paired t-test and a P-value of 4 log reduction of A. baumannii biofilm in 360 s, indicating biofilm formation as an important factor to consider when investigating A. baumannii tolerance to APNTP. Pseudomonas aeruginosa PA14 biofilm eradication was achieved within 240 s of exposure. This pathogen is an extremely virulent strain of P. aeruginosa containing two pathogenicity islands that are not present in the PAO1 strain [23]. This raises an interesting possibility that there may be, in addition to phenotypic susceptibility, strain-specific susceptibility profiles to non-thermal plasma exposure. Whilst it took 10 min to eradicate a 48-h-old PAO1 biofilm [5], only 4 min were required for the 24-h-old PA14 biofilm in
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A. baumannii biofilms proved the least susceptible of the ESKAPE pathogens to APNTP. A >4 log reduction was achieved after 360 s of plasma exposure. A similar profile of resistance in A. baumannii has previously been described with different atmospheric plasma sources. A study by Cahill et al. using plasma generated from compressed air at a higher flow rate of 12 SLM reported that A. baumannii was the most resistant organism to plasma exposure, with a 1.7 log reduction on a powdered steel surface [19]. A. baumannii, an intrinsically multidrug-resistant pathogen, also displays environmental resistance to heat, disinfection and desiccation [20]. It has been reported to survive on inanimate surfaces for as long as 5 months [21]. These tenacious environmental characteristics have been shown to be greater when A. baumannii grows as a biofilm
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Fig. 5. Biofilm survival fractions of the standard plate count and XTT assays: (a) Enterococcus faecium DSM 25390; (b) Staphylococcus aureus; (c) Klebsiella pneumoniae; (d) Acinetobacter baumannii NCTC 13304; (e) Pseudomonas aeruginosa PA14; and (f) Enterobacter cloacae. Solid lines represent survival fraction of log survival (CFU/well) and dashed lines represent survival fraction from XTT assay. Bars represent the standard error of the mean.
Please cite this article in press as: Flynn PB, et al. Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2015.02.026
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Fig. 6. Planktonic survival fractions of the standard plate count and XTT assays: (a) Enterococcus faecium DSM 25390; (b) Staphylococcus aureus, (c) Klebsiella pneumoniae; (d) Acinetobacter baumannii NCTC 13304; (e) Pseudomonas aeruginosa PA14; and (f) Enterobacter cloacae. Solid lines represent survival fraction of log survival (CFU/well) and dashed lines represent survival fraction from XTT assay. Bars represent the standard error of the mean.
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this study. Acknowledging the different biofilm growth times as a potential explanation for increased eradication times, it must also be taken into account that PA14 is a ladS mutant that is associated with repression of biofilm formation when considering the difference in plasma susceptibility [23]. Specifically, the ladS gene is important for polysaccharide biogenesis, thus implicating the importance of EPS in reasoning the varied susceptibilities of biofilms. S. aureus was the most resistant of the clinical isolates with regard to both biofilm and planktonic susceptibility to plasma exposure. A number of recent studies have examined the antimicrobial efficacy of a range of APNTP sources against S. aureus [12,24]. Alkawareek et al. [12] reported complete killing of S. aureus NCTC 10788 biofilms (grown on pegs of the MBEC device) within 60 s, a shorter eradication time than that reported for the clinical isolate used in this study (360 s). Whilst the greater surface area of the S. aureus biofilm exposed in this study compared with that described by Alkawareek et al. [12] should be taken into account when comparing strain-specific variability, it is also consistent with variable susceptibilities exhibited between PA14 and PAO1 biofilms, indicating strain-to-strain variability to APNTP. Varied susceptibility to plasma has also previously been reported with different strains of S. aureus [25], indicating a caveat when assessing the susceptibility of micro-organisms to APNTP, and such strain-to-strain variation and surface conditions may make predictions of exposure times necessary to achieve eradication difficult. The effect of organic matter present on surfaces and in wounds on bactericidal efficacy must be considered. These difficulties may be overcome by greater eradication times or examining strategies to optimise this approach. APNTP has been shown to have an effect on the cell surface and membrane [15], with the Gram-positive cell wall thought to provide some protection from electrostatic forces and reactive species produced by the plasma [26]. This may go some way to explaining the tolerance of S. aureus compared with the planktonic Gram-negative organisms. However, when considering the production of reactive oxygen species by APNTP such as superoxide, hydroxyl radicals and hydrogen peroxide, and that E. faecium is Gram-positive and did not display similar tolerance, it is apparent that other factors are at play. Staphyloxanthin is a carotenoid
responsible for the golden yellow/orange colour of S. aureus. It is a membrane-bound antioxidant and the presence of staphyloxanthin in S. aureus has been demonstrated to increase resistance to hydrogen peroxide, superoxide and hydroxyl radicals [27]. This may contribute to its relative tolerance to non-thermal plasma exposure compared with the other organisms studied. Antioxidants have been demonstrated to increase bacterial plasma tolerance [28]. 4.2. Evaluation of bacterial viability The ability of non-thermal plasma exposure to induce a viablebut-non-culturable (VBNC) state in a range of micro-organisms has been reported recently [29], indicating the necessity for validation of colony counting methods with a suitable metabolic reporter. Consequently, the XTT metabolic assay was performed alongside standard colony enumeration post-exposure. Inference of viability is made from the rate at which XTT is reduced to form an orange/red formazan derivative. Figs. 5 and 6 show the XTT survival fractions compared with the standard plate count. The results are comparable and demonstrate that eradication times reported correlated with no survival using the metabolic XTT assay. However, there are discrepancies at shorter plasma exposure times (relative to the reported eradication times). For E. faecium, S. aureus and K. pneumonia (Fig. 5), following 30 s of exposure XTT survival fractions were 0.66, 0.68 and 0.55, respectively, compared with standard plate count survival fractions at the same time point of 0.49, 0.2 and 0.2. It is conceivable that complex physiological changes are occurring due to interactions of RONS or electrostatic forces with the cell wall or membrane affecting the electron transport chain and XTT metabolism. The cell surface has been shown to be a primary target for APNTP-mediated bactericidal activity [15]. Fig. 6 shows that the survival fractions of the standard plate count at shorter exposure times are greater than those reported by the XTT assay for E. faecium, S. aureus and P. aeruginosa. It may be the case that planktonic cells suffered more damage leading to a lag time in XTT metabolism. The fact that no neutralisation step (aside from dilution) was carried out post-exposure is unlikely to account for differences between XTT and plate counts as the pH of PBS has
Please cite this article in press as: Flynn PB, et al. Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2015.02.026
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been shown to remain unchanged with no long-term antimicrobial effect seen with plasma-treated PBS [30]. XTT assay plates were also incubated overnight after the initial read to ensure no growth compared with the negative control. This demonstrates the complexity of interpreting how plasma exposure affects bacterial cells at shorter exposure times and requires further work in order to elucidate how the complex chemistry of APNTP affects bacteria in biofilms and planktonic cultures, respectively, upon short exposure times.
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The bactericidal efficacy of an in-house-designed APNTP jet, operating a He/O2 admixture (99.5%:0.5%) was assessed against clinical and type strains of the ESKAPE pathogens both in planktonic and biofilm modes of growth. Of the six ESKAPE strains tested, A. baumannii proved the most resistant, exhibiting a >4 log reduction in biofilm viability after 360 s, a time shown to be capable of bringing about biofilm eradication in the remaining ESKAPE pathogens evaluated. K. pneumoniae was the most susceptible biofilm to APNTP, and E. cloacae was the most susceptible planktonic bacteria to APNTP. In general, biofilms were more tolerant than planktonic bacteria. Interestingly, planktonic A. baumannii eradication times were comparable with other ESKAPE pathogens (E. faecium, S. aureus and K. pneumoniae), indicating significant phenotypic resistance to APNTP exposure associated with biofilm formation in A. baumannii. The XTT viability assay validated the results from the standard plate count. The current simple configuration is amenable to a number of topical applications, such as treatment of infected wounds and some in dwelling medical devices. This study describes a systematic approach to assessing the efficacy of an in-house-designed plasma jet against a notorious group of pathogens that are at the forefront of antimicrobial resistance and the source of the majority of HAIs. APNTP is an emerging technology whose bactericidal application represents an innovative approach to infection and contamination control.
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Funding
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The authors are grateful to the following funders for support of 435 Q4 this work: The Department of Employment and Learning Northern 436 Q5 Ireland; The Society for Applied Microbiology through a Research 437 Development Fund (2013); and Invest NI through Proof of Principle 438 and Proof of Concept grants [PoC402/2014]. 434
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International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens
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Padrig B. Flynn a , Sarah Higginbotham a , Nid’a H. Alshraiedeh a,b , Sean P. Gorman a , William G. Graham c , Brendan F. Gilmore a,∗ a
School of Pharmacy, Queen’s University of Belfast, Belfast BT9 7BL, UK Faculty of Pharmacy, Jordan University of Science and Technology, Irbid 22110, Jordan c Centre for Plasma Physics, Queen’s University of Belfast, Belfast BT7 1NN, UK b
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Article history: Received 3 November 2014 Accepted 25 February 2015
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Keywords: Atmospheric pressure plasma Non-thermal plasma Biocide ESKAPE pathogens Biofilm Plasma medicine
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1. Introduction
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The emergence of multidrug-resistant pathogens within the clinical environment is presenting a mounting problem in hospitals worldwide. The ‘ESKAPE’ pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) have been highlighted as a group of causative organisms in a majority of nosocomial infections, presenting a serious health risk due to widespread antimicrobial resistance. The stagnating pipeline of new antibiotics requires alternative approaches to the control and treatment of nosocomial infections. Atmospheric pressure nonthermal plasma (APNTP) is attracting growing interest as an alternative infection control approach within the clinical setting. This study presents a comprehensive bactericidal assessment of an in-house-designed APNTP jet both against biofilms and planktonic bacteria of the ESKAPE pathogens. Standard plate counts and the XTT metabolic assay were used to evaluate the antibacterial effect of APNTP, with both methods demonstrating comparable eradication times. APNTP exhibited rapid antimicrobial activity against all of the ESKAPE pathogens in the planktonic mode of growth and provided efficient and complete eradication of ESKAPE pathogens in the biofilm mode of growth within 360 s, with the exception of A. baumannii where a >4 log reduction in biofilm viability was observed. This demonstrates its effectiveness as a bactericidal treatment against these pathogens and further highlights its potential application in the clinical environment for the control of highly antimicrobial-resistant pathogens. © 2015 Published by Elsevier B.V.
The ‘ESKAPE’ pathogens, first classified by the Infectious Diseases Society of America (IDSA), refer to Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. They have been highlighted as a group of pathogens frequently associated with nosocomial infections and that represent new paradigms in pathogenesis, transmission and resistance, resulting in the potential to pose a serious threat to public health [1]. This group of pathogens is responsible for the most problematic of hospitalacquired infections (HAIs) owing to their ability to ‘escape’ the antimicrobial action of antibiotics [1]. According to the World Health Organization (WHO), HAIs have been estimated to affect 4.5 million and 1.7 million people annually in Europe and the USA,
∗ Corresponding author. Tel.: +44 2890972305; fax: +44 28 9024 7794. E-mail address: [email protected] (B.F. Gilmore).
respectively, with billions of dollars spent on the incurred costs [2,3]. As well as the financial burden imposed by HAIs, there are serious consequences for the patient, including increased length of stay in the care facility, increased morbidity and, in an increasing number of cases, increased mortality [2]. The ESKAPE pathogens, in particular, are increasingly implicated in nosocomial infections and are especially problematic to critical care patients, usually occurring as biofilm infections [4]. Biofilms represent ca. 80% of all microbial infections [5]. The US Centers for Disease Control and Prevention (CDC) has estimated that 1.7 million HAIs annually in the USA are due to biofilm-associated infections, resulting in healthcare costs of up to US$11 billion [6]. Biofilm formation by micro-organisms represents a significant virulence attribute both in acute and chronic infections. It is accepted that biofilms are the predominant phenotype of most bacteria both in natural and clinical settings, with the planktonic phenotype existing only transiently [7]. One of the most serious threats to global health is the spread of antibiotic-resistant bacteria, caused in part by inappropriate
http://dx.doi.org/10.1016/j.ijantimicag.2015.02.026 0924-8579/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Flynn PB, et al. Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2015.02.026
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Fig. 1. (a) Design image of plasma jet encased within acrylic plastic and (b) photograph of plasma jet in operation.
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antibiotic use and control. Despite the urgent need for new antimicrobial agents, many pharmaceutical companies have withdrawn from the arena in recent years, resulting in stagnation of the antibiotic development pipeline [8]. New and innovative strategies for infection control and treatment are required alongside drug therapy in order to combat the growing problem of multidrugresistant bacteria and nosocomial infections. One strategy that has increasing potential as a non-systemic treatment and disinfection option is atmospheric pressure non-thermal plasma (APNTP) [9]. Plasma is regarded as the fourth state of matter and can be described as an ionised gas. It consists of photons, electrons, ions, atoms, free radicals and electromagnetic fields. Non-thermal plasmas exist in a state of non-equilibrium. This non-equilibrium state refers to the temperature of the neutral gas particles and ions being close to the ambient room temperature while the electron temperature is much higher. The greater mass of the gas particles compared with the electrons produces an atmospheric plasma at a tolerable temperature. Plasma at atmospheric pressure can be formed when energy generated from an electric current ionises a gas such as argon or a helium–oxygen admixture. APNTP devices are simple to set up, reducing the costs and time associated with low-pressure plasmas [10]. They can generate a rich, dry chemistry in air at ambient temperature. This complex chemical environment comprises of a mixture of reactive agents, such as reactive oxygen and nitrogen species (RONS), ultraviolet light and charged particles, all of which contribute to the antibacterial properties of plasma [11]. APNTP has received much attention as a potential approach to controlling microbial organisms [11,12] and is central to an emerging scientific field known as plasma medicine. Different configurations of non-thermal plasma devices have been effectively shown in biomedical applications, including chronic infected wound treatments [13] and cancer treatment [14]. Its potential as an antimicrobial approach is now widely established. However, despite the explosion of interest in this field as well as the serious threat posed by the ESKAPE pathogens, there have been no studies assessing its efficacy against this collection of micro-organisms in different phenotypes (planktonic/biofilm). The current study represents the first report of the in vitro antimicrobial efficacy of an in-house-designed APNTP source against the ESKAPE pathogens both in planktonic and biofilm modes of growth.
2. Materials and methods
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The following microbial strains were used in this study: E. faecium DSM 25390; A. baumannii NCTC 13304; and P. aeruginosa PA14. S. aureus, K. pneumoniae and extended-spectrum -lactamase (ESBL)-producing Enterobacter cloacae (used as a model organism for Enterobacter spp.) were clinical isolates obtained from Antrim Area Hospital, Northern Health and Social Care Trust (Co. Antrim, UK). All strains were stored at −20 ◦ C in MicrobankTM vials (ProLab Diagnostics, Cheshire, UK) and were subcultured overnight at 37 ◦ C on Mueller–Hinton agar (MHA) (Oxoid Ltd., Basingstoke, UK) unless stated otherwise. 2.2. Plasma source A previously described [5,15] in-house-designed plasma source (Fig. 1) was employed for this study. Specifically, the jet consists of a quartz dielectric tube with an inner diameter of 4 mm and an outer diameter of 6 mm. Two copper electrodes, 2 mm wide and 25 mm apart, encircle the quartz tube. The downstream electrode, which is 5 mm from the end of the tube, is powered by a high-voltage power supply (Haiden PHK-2K; Haiden Laboratory Inc., Hyogo, Japan). The jet is operated at a frequency of 20 kHz and voltage amplitude of 6 kV; the upstream electrode is grounded. The plasma jet is operated with an admixture of helium (99.5%) and oxygen (0.5%) with a total flow rate of 2 standard litres per min (SLM). These conditions allow for a long, intense and luminous plume to form in the air as seen in Fig. 1b. The temperature of this plume, reported as rotational temperature, is 39 ◦ C [5]. The plasma jet configuration is encased in solid acrylic tubing, a deviation from the previously reported design [5,15]. 2.3. Treatment of bacterial biofilms with atmospheric pressure non-thermal plasma To evaluate the antimicrobial effect of the plasma jet against biofilms of ESKAPE pathogens, overnight cultures of S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa and E. cloacae were adjusted to an optical density at 550 nm (OD550 ) equivalent to ca. 107 CFU/mL in Mueller–Hinton broth (MHB) (Oxoid Ltd.). Then,
Please cite this article in press as: Flynn PB, et al. Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2015.02.026
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150 L aliquots of the standardised bacterial suspensions were transferred to the wells of a Nunc 96-well plate (Fisher Scientific UK Ltd., Loughborough, UK) that was incubated for 24 h at 37 ◦ C within a humidified compartment in a Gallenkamp Orbital Incubator (AGB Scientific Ltd., Dublin, Ireland) at 100 rpm. After 24 h, the liquid phase was discarded and the wells were rinsed twice with 200 L of phosphate-buffered saline (PBS) to remove unattached and loosely adhered cells. Overnight cultures of E. faecium, grown on anaerobic blood agar (Oxoid Ltd.) with 5% defibrinated horse blood (TCS Biosciences Ltd., Buckingham, UK), were adjusted to an OD550 equivalent to 107 CFU/mL in MHB supplemented with 1% glucose (Sigma-Aldrich Company Ltd., Poole, UK). Then, 150 L aliquots of the standardised suspension were transferred to the wells of a 96-well plate that was incubated for 48 h on an orbital shaker (100 rpm) at 37 ◦ C in anaerobic conditions using the Modular Atmosphere Controlled System (Davidson and Hardy Ltd., Belfast, UK). The liquid phase was discarded after 48 h and the wells were rinsed twice with 200 L of PBS to remove unattached and loosely adhered cells. Biofilms were exposed to the plasma at a separation distance of 15 mm between the end of the plasma jet nozzle and the bottom of the wells of the 96-well plate and were exposed to the plasma effluent for times of 0, 15, 30, 45, 60, 120, 240 and 360 s. Samples were exposed in triplicate for each time. As a negative control, the effect of gas-only treatment (where no power is applied to the electrodes) was also investigated. Following exposure to APNTP or gas only, 200 L of PBS was added to each well and the plate was sonicated for 15 min to dislodge and re-suspend biofilm cells. 2.4. Treatment of planktonic bacteria with atmospheric pressure non-thermal plasma Overnight cultures (as described above) were diluted in PBS to an OD550 equivalent to 108 CFU/mL. Then, 20 L aliquots of each standardised bacterial suspension were exposed in lids of microcentrifuge tubes to the plasma plume at a distance of 15 mm from sample to nozzle. The samples were treated for a maximum of 240 s with the same time intervals as described for biofilms. Treatments were repeated in triplicate for each time point. Following exposure to APNTP, samples were transferred to 180 L of PBS for determination of viable counts. 2.5. Determination of cell viability by XTT metabolic assay The XTT assay (Sigma-Aldrich Company Ltd.) was performed as previously described [15]. In brief, 50 L of treated planktonic or biofilm bacterial suspensions were transferred to the wells of a 96-well plate containing 50 L of MHB and 20 L of XTT stock solution. Control wells contained 50 L of PBS in place of bacterial suspensions. Plates were incubated at 37 ◦ C in an orbital incubator for 5–7 h. The resulting colour change was read using a microplate reader (BioTek EL808; BioTek Instruments Ltd., Potton, UK) at 450 nm. Survival fractions were calculated by dividing the OD value of each well corresponding to a biofilm/planktonic treatment time by the mean OD of the unexposed control. 2.6. Determination of cell viability by standard plate count method Serial 10-fold dilutions of planktonic and sonicated biofilm suspensions were prepared and three 20 L drops from each dilution were plated onto MHA for standard plate counts. Plates were incubated aerobically at 37 ◦ C and were observed after 24 h and 48 h for viable colonies. Plates with no colonies grown after 48 h, indicating complete bacterial eradication, were left for a further 2 days
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to check for re-growth of dormant cells. Plate counts were used to calculate log surviving cells and survival fraction of each bacterial strain. 2.7. Statistical analysis Statistical analyses were performed using Prism 6.0 software (GraphPad Software Inc., San Diego, CA). The P-value was derived from the paired t-test and a P-value of 4 log reduction of A. baumannii biofilm in 360 s, indicating biofilm formation as an important factor to consider when investigating A. baumannii tolerance to APNTP. Pseudomonas aeruginosa PA14 biofilm eradication was achieved within 240 s of exposure. This pathogen is an extremely virulent strain of P. aeruginosa containing two pathogenicity islands that are not present in the PAO1 strain [23]. This raises an interesting possibility that there may be, in addition to phenotypic susceptibility, strain-specific susceptibility profiles to non-thermal plasma exposure. Whilst it took 10 min to eradicate a 48-h-old PAO1 biofilm [5], only 4 min were required for the 24-h-old PA14 biofilm in
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A. baumannii biofilms proved the least susceptible of the ESKAPE pathogens to APNTP. A >4 log reduction was achieved after 360 s of plasma exposure. A similar profile of resistance in A. baumannii has previously been described with different atmospheric plasma sources. A study by Cahill et al. using plasma generated from compressed air at a higher flow rate of 12 SLM reported that A. baumannii was the most resistant organism to plasma exposure, with a 1.7 log reduction on a powdered steel surface [19]. A. baumannii, an intrinsically multidrug-resistant pathogen, also displays environmental resistance to heat, disinfection and desiccation [20]. It has been reported to survive on inanimate surfaces for as long as 5 months [21]. These tenacious environmental characteristics have been shown to be greater when A. baumannii grows as a biofilm
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Fig. 5. Biofilm survival fractions of the standard plate count and XTT assays: (a) Enterococcus faecium DSM 25390; (b) Staphylococcus aureus; (c) Klebsiella pneumoniae; (d) Acinetobacter baumannii NCTC 13304; (e) Pseudomonas aeruginosa PA14; and (f) Enterobacter cloacae. Solid lines represent survival fraction of log survival (CFU/well) and dashed lines represent survival fraction from XTT assay. Bars represent the standard error of the mean.
Please cite this article in press as: Flynn PB, et al. Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2015.02.026
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Fig. 6. Planktonic survival fractions of the standard plate count and XTT assays: (a) Enterococcus faecium DSM 25390; (b) Staphylococcus aureus, (c) Klebsiella pneumoniae; (d) Acinetobacter baumannii NCTC 13304; (e) Pseudomonas aeruginosa PA14; and (f) Enterobacter cloacae. Solid lines represent survival fraction of log survival (CFU/well) and dashed lines represent survival fraction from XTT assay. Bars represent the standard error of the mean.
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this study. Acknowledging the different biofilm growth times as a potential explanation for increased eradication times, it must also be taken into account that PA14 is a ladS mutant that is associated with repression of biofilm formation when considering the difference in plasma susceptibility [23]. Specifically, the ladS gene is important for polysaccharide biogenesis, thus implicating the importance of EPS in reasoning the varied susceptibilities of biofilms. S. aureus was the most resistant of the clinical isolates with regard to both biofilm and planktonic susceptibility to plasma exposure. A number of recent studies have examined the antimicrobial efficacy of a range of APNTP sources against S. aureus [12,24]. Alkawareek et al. [12] reported complete killing of S. aureus NCTC 10788 biofilms (grown on pegs of the MBEC device) within 60 s, a shorter eradication time than that reported for the clinical isolate used in this study (360 s). Whilst the greater surface area of the S. aureus biofilm exposed in this study compared with that described by Alkawareek et al. [12] should be taken into account when comparing strain-specific variability, it is also consistent with variable susceptibilities exhibited between PA14 and PAO1 biofilms, indicating strain-to-strain variability to APNTP. Varied susceptibility to plasma has also previously been reported with different strains of S. aureus [25], indicating a caveat when assessing the susceptibility of micro-organisms to APNTP, and such strain-to-strain variation and surface conditions may make predictions of exposure times necessary to achieve eradication difficult. The effect of organic matter present on surfaces and in wounds on bactericidal efficacy must be considered. These difficulties may be overcome by greater eradication times or examining strategies to optimise this approach. APNTP has been shown to have an effect on the cell surface and membrane [15], with the Gram-positive cell wall thought to provide some protection from electrostatic forces and reactive species produced by the plasma [26]. This may go some way to explaining the tolerance of S. aureus compared with the planktonic Gram-negative organisms. However, when considering the production of reactive oxygen species by APNTP such as superoxide, hydroxyl radicals and hydrogen peroxide, and that E. faecium is Gram-positive and did not display similar tolerance, it is apparent that other factors are at play. Staphyloxanthin is a carotenoid
responsible for the golden yellow/orange colour of S. aureus. It is a membrane-bound antioxidant and the presence of staphyloxanthin in S. aureus has been demonstrated to increase resistance to hydrogen peroxide, superoxide and hydroxyl radicals [27]. This may contribute to its relative tolerance to non-thermal plasma exposure compared with the other organisms studied. Antioxidants have been demonstrated to increase bacterial plasma tolerance [28]. 4.2. Evaluation of bacterial viability The ability of non-thermal plasma exposure to induce a viablebut-non-culturable (VBNC) state in a range of micro-organisms has been reported recently [29], indicating the necessity for validation of colony counting methods with a suitable metabolic reporter. Consequently, the XTT metabolic assay was performed alongside standard colony enumeration post-exposure. Inference of viability is made from the rate at which XTT is reduced to form an orange/red formazan derivative. Figs. 5 and 6 show the XTT survival fractions compared with the standard plate count. The results are comparable and demonstrate that eradication times reported correlated with no survival using the metabolic XTT assay. However, there are discrepancies at shorter plasma exposure times (relative to the reported eradication times). For E. faecium, S. aureus and K. pneumonia (Fig. 5), following 30 s of exposure XTT survival fractions were 0.66, 0.68 and 0.55, respectively, compared with standard plate count survival fractions at the same time point of 0.49, 0.2 and 0.2. It is conceivable that complex physiological changes are occurring due to interactions of RONS or electrostatic forces with the cell wall or membrane affecting the electron transport chain and XTT metabolism. The cell surface has been shown to be a primary target for APNTP-mediated bactericidal activity [15]. Fig. 6 shows that the survival fractions of the standard plate count at shorter exposure times are greater than those reported by the XTT assay for E. faecium, S. aureus and P. aeruginosa. It may be the case that planktonic cells suffered more damage leading to a lag time in XTT metabolism. The fact that no neutralisation step (aside from dilution) was carried out post-exposure is unlikely to account for differences between XTT and plate counts as the pH of PBS has
Please cite this article in press as: Flynn PB, et al. Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2015.02.026
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been shown to remain unchanged with no long-term antimicrobial effect seen with plasma-treated PBS [30]. XTT assay plates were also incubated overnight after the initial read to ensure no growth compared with the negative control. This demonstrates the complexity of interpreting how plasma exposure affects bacterial cells at shorter exposure times and requires further work in order to elucidate how the complex chemistry of APNTP affects bacteria in biofilms and planktonic cultures, respectively, upon short exposure times.
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The bactericidal efficacy of an in-house-designed APNTP jet, operating a He/O2 admixture (99.5%:0.5%) was assessed against clinical and type strains of the ESKAPE pathogens both in planktonic and biofilm modes of growth. Of the six ESKAPE strains tested, A. baumannii proved the most resistant, exhibiting a >4 log reduction in biofilm viability after 360 s, a time shown to be capable of bringing about biofilm eradication in the remaining ESKAPE pathogens evaluated. K. pneumoniae was the most susceptible biofilm to APNTP, and E. cloacae was the most susceptible planktonic bacteria to APNTP. In general, biofilms were more tolerant than planktonic bacteria. Interestingly, planktonic A. baumannii eradication times were comparable with other ESKAPE pathogens (E. faecium, S. aureus and K. pneumoniae), indicating significant phenotypic resistance to APNTP exposure associated with biofilm formation in A. baumannii. The XTT viability assay validated the results from the standard plate count. The current simple configuration is amenable to a number of topical applications, such as treatment of infected wounds and some in dwelling medical devices. This study describes a systematic approach to assessing the efficacy of an in-house-designed plasma jet against a notorious group of pathogens that are at the forefront of antimicrobial resistance and the source of the majority of HAIs. APNTP is an emerging technology whose bactericidal application represents an innovative approach to infection and contamination control.
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Funding
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The authors are grateful to the following funders for support of 435 Q4 this work: The Department of Employment and Learning Northern 436 Q5 Ireland; The Society for Applied Microbiology through a Research 437 Development Fund (2013); and Invest NI through Proof of Principle 438 and Proof of Concept grants [PoC402/2014]. 434
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Please cite this article in press as: Flynn PB, et al. Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2015.02.026
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