Elsevier

Clinical Plasma Medicine

Volumes 19–20, September–December 2020, 100104
Clinical Plasma Medicine

Cold atmospheric plasma, the removal of blood from steel and its effect on staphylococcal biofilm formation. A pilot study

https://doi.org/10.1016/j.cpme.2020.100104Get rights and content

Abstract

Background

Cold atmospheric plasma (CAP) has antimicrobial properties and may have a role in decontamination. However, CAP can coagulate blood, which could lead to the fixing of blood or proteins and make surface decontamination challenging after blood spillages.

Methods

In vitro assays were performed to assess whether CAP treatment of a surface impaired removal of a blood soil from steel through visual inspection and bicinchoninic acid assays. To assess if remaining blood proteins promoted biofilm formation, crystal violet biomass analysis was performed.

Results

In an in vitro study, CAP treatment did not hinder the removal of blood and pre-treatment decreased protein remaining on the surface. CAP inhibited Staphylococcus aureus biofilm formation on blood-soiled surfaces.

Conclusions

CAP does not enhance the persistence of blood and protein on steel surfaces. Biofilm formation after CAP treatment of blood soiled steel was decreased possibly due to changes in electrostatic charge on surfaces. CAP should be further evaluated as an alternative surface decontaminant.

Introduction

Microbial contamination and subsequent inadequate decontamination of healthcare surfaces and medical devices can contribute to the spread of healthcare-associated infections (HCAI) [1,2]. Both improved cleaning (removal of bioburden) and disinfection (inactivation of microorganisms) are needed to reduce this risk of infection [3,4]. Bacteria on contaminated surfaces often exist in a protective biofilm structure. Bacteria can often form a community or biofilm on surfaces, trapping nutrients and water and allowing bacteria to propagate and survive in otherwise inhospitable environments [5]. Biofilms are traditionally viewed as moist slimy structures such as dental plaque [6] however in more recent time biofilms observed on many hospital surfaces were in fact dry [7,8]. Bacterial biofilms confer a higher resistance to biocides than planktonic cells, sometimes up to 1000 times greater [7]. The increased resistance of biofilms can lead to the greater persistence of pathogens on medical surfaces. Therefore, the inactivation and prevention of the attachment of bacterial biofilms to surfaces by decontamination techniques can greatly reduce the spread of infection.

Often medical devices or hospital surfaces can be sensitive to some disinfectants and even moisture leading to corrosion or discolouration. Certain surfaces such as some rubber used in dental tools, cannot by sterilized through autoclaving and other methods of decontamination must be used [9]. This has led to an interest in alternative approaches to mitigate these factors, one of which may be the dry disinfectant, cold atmospheric plasma (CAP). Plasma is referred to as the fourth state of matter, and is a high-energy gas undergoing excitation and ionisation processes. Laboratory plasma can be excited and sustained by the addition of energy, for instance electrical energy, to an electrode arrangement fed by a gas source [10].

Applications of plasma and in particular non-thermal or low temperature CAP devices have become a burgeoning field of study. The low temperature of the plasma sources for CAP devices operate at below 40 °C. This means that it can be used in some medical applications such as in wound healing and cancer treatment without damaging healthy cells [11,12]. The antimicrobial nature of CAP also means that it could have a role in the bacterial decontamination of hospital surfaces or tools without causing damage to the surface material.

CAP has antimicrobial action against both Gram-positive and Gram-negative bacteria in planktonic and biofilm states [13], [14], [15], [16]. CAP has the benefit of having multifaceted mechanisms of action, which can lead to a broad spectrum bacterial kill, including through the action of reactive oxygen species (ROS), reactive nitrogen species (RNS), charged particles and ultraviolet radiation [13,17,18]. It is thought that CAP inactivates bacteria through cell permeation, DNA denaturation and lysis mediated by the reactive species and particles created [17]. Therefore, the use of CAP systems alongside normal cleaning could be used to reduce surface contamination.

CAP has been investigated for use in wound healing and it was found that plasma treatment can lead to blood coagulation [19]. The rapid blood coagulation caused by CAP has also been examined as a potential method of preventing bleeding Kuo 2009 [20]. The mechanism of action of blood coagulation by CAP treatment is thought to be due to non-calcium dependant blood clotting [21,22]. Although alcohol has potent antimicrobial action, the denaturation and fixation of blood by ethanol is also well known. Ethanol is an effective disinfectant and features in sprays used to disinfect medical equipment. Despite its ability as a disinfectant ethanol has been shown to interfere with routine normal cleaning. Prior et al. [23] showed the strong binding effects of ethanol on stainless steel and fixed blood soil remained visible on steel even after washing. Furthermore, Costa et al. [24] showed that residual blood fixing by ethanol on stainless steel surgical instruments led to an increased protein load and greater difficulty in decontamination. Although the mechanisms of disinfection are different for ethanol and CAP, an investigation of CAP for use in wound healing showed its ability to promote blood coagulation [19]. For CAP to be an effective decontamination tool, it must not interfere with normal cleaning.

We have assessed if CAPs effect on blood could impair the removal of blood soil from stainless steel. To do this we performed in vitro assays to assess whether CAP treatment of a surface impaired removal of a blood soil and if remaining blood proteins promoted biofilm formation.

Section snippets

CAP system

The CAP device used has been previously described [25]. Briefly, the device consisted of a single plasma jet powered from a power supply delivering approximately 30 kVpp at 21–23 kHz, and a gas supply of ambient air at 12 standard litres per minute (SLM). The plasma jet was 30 mm in diameter and the plasma was generated between two stainless steel electrodes, an outer conical shaped ground electrode and inner pin shaped electrode connected to the power supply. Plasma generated within this

Blood and protein fixing

From the visual inspection of the steel coupons, neither direct CAP treatment (coupon E) nor pre-treatment of surfaces with CAP before blood inoculation (coupon F) increased the fixing of blood on stainless steel, compared to washing alone (Fig. 1).

Proteins were not fixed onto the coupon after CAP treatment (Fig. 2). There was a significant decrease in the amount of blood protein on the coupon after CAP pre-treatment (p = 0.0410) (coupon F) and a non-significant decrease (p = 0.4990) in the

Discussion

The purpose of this study was to investigate the effect of CAP on blood soil and whether it would interfere with cleaning or promote biofilm formation. The results obtained indicate that although CAP has the ability to coagulate blood in medical treatment it does not bind or fix it to stainless steel nor prevent its removal through normal cleaning. Our study employed two methods to assess cleaning, a visual inspection and a protein assay to look at blood products which are present but not

Author statement

MF, MB and SK developed the protocol and drafted the manuscript, MF carried out the experiments, and HH and SD supervised the research and reviewed the draft manuscript

Ethical statement

In accordance with Royal College of Surgeon in Ireland guidelines, ethical approval was not needed for this body of work. Research was carried out in full compliance with all regulatory and legal requirements currently in existence and in accordance with the ethical principles that have been outlined in the Declaration of Helsinki.

Financial disclosure

Muireann Fallon is a Strategic Academic Recruitment Scholar (StAR) funded by the Royal College of Surgeons in Ireland. Sarah Kennedy was funded through an Enterprise Ireland grant, CF2015/56701A. Maria Boyle was funded through a Health Research Board grant, HRA-DI-2015-1141.

Declaration of Competing Interest

HH is in receipt of research funds from Astellas (P08100326501) and Pfizer (W1243730) and has recently received a professional fee from Pfizer. No other author has a conflict of interest to declare.

References (29)

  • C.E. Adams et al.

    Examining the association between surface bioburden and frequently touched sites in intensive care

    J. Hosp. Infect.

    (2017)
  • V. Arora

    Cold Atmospheric Plasma (CAP) in dentistry

    Dentistry

    (2013)
  • W.A. Rutala et al.

    Enhanced disinfection leads to reduction of microbial contamination and a decrease in patient colonization and infection

    Infect. Control Hosp. Epidemiol.

    (2018)
  • J.W. Costerton et al.

    Bacterial biofilms: a common cause of persistent infections

    Science

    (1999)
  • View full text