Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens

https://doi.org/10.1016/j.ijantimicag.2015.02.026Get rights and content

Highlights

  • The antimicrobial activity of an atmospheric pressure non-thermal plasma (APNTP) jet against the ESKAPE pathogens is reported.

  • Complete eradication was achieved for all planktonic bacteria in less than 240 seconds.

  • Biofilms of the ESKAPE pathogens were eradicated in under 360 seconds, except A. baumannii biofilms for which a > 4 log reduction was achieved.

  • Rapid antimicrobial activity against all of the ESKAPE pathogens in the planktonic mode of growth was observed.

  • This study validates the potential of non thermal plasmas for the control of highly antimicrobial resistant pathogens.

Abstract

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 non-thermal 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.

Introduction

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 hospital-acquired 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, 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 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 multidrug-resistant 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.

Section snippets

Bacterial strains and growth conditions

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 β-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 Microbank™ vials (Pro-Lab Diagnostics, Cheshire, UK) and were subcultured

Plasma inactivation of biofilms

Biofilms were grown for 24 h (48 h for E. faecium) in the wells of a 96-well plate and were exposed to APNTP for discreet time points up to 360 s. E. faecium required 48 h in anaerobic conditions and the addition of glucose to the medium to optimise biofilm formation. Complete eradication of biofilms, defined as <1 surviving CFU, was achieved within this time for all but one bacterial species. Fig. 2 shows the log survival of biofilm cells following exposure to APNTP. K. pneumoniae was the most

Plasma inactivation of ESKAPE pathogens

Here we report the results of a comprehensive antibacterial/antibiofilm efficacy study designed to evaluate a helium/oxygen atmospheric pressure plasma as a tool to prevent and control infections caused by the ESKAPE pathogens. Defining the bactericidal action of APNTP, an emerging technology for non-invasive infection and contamination control, is an absolute requisite for validation of any proposed non-thermal plasma-based approach for infection control. APNTP was shown to be highly effective

Conclusion

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

Funding

The authors are grateful to the following funders for support of this work: The Department of Employment and Learning Northern Ireland; The Society for Applied Microbiology through a Research Development Fund (2013); and Invest NI through Proof of Principle and Proof of Concept grants [PoC402/2014].

Competing interests

None declared.

Ethical approval

Not required.

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