multijet cold plasma for surface decontamination 1183


TheMRSAstrain 43300 and the extended-spectrum β-lactamase– producing E. coli (ESBLEC) strain CL2 are clinical strains from our collection. The VRE strain was provided by the Beaumont Hospital Microbiology Department (Dublin, Ireland) and A. baumannii strain 19606 was obtained from the American Type Culture Collection (ATCC). Before the experiments, bacteria were stored at −20°C in cryovial pre- servation beads (Microbank, Pro-Lab Diagnostics, Merseyside, UK) and were revived on either Columbia blood agar plates (Oxoid, Basingstoke, UK) for MRSA and A. baumannii,on Mueller-Hinton agar plates (Fluka, Sigma-Aldrich, Ireland) for ESBLEC, or trypticase soy broth agar plates (Oxoid) for VRE. Bacterial cultures were grown aerobically overnight for 16–18 hours at 37°C with rotation in trypticase soy broth supplemented with 5% sodium chloride for MRSA and VRE, brain heart infusion broth for A. baumannii, or Mueller- Hinton broth for the ESBLEC.


Test Surface Preparation


The test surfaces used in this study were 5-cm2 sections of polyurethane mattress (Meditec Medical, Dublin, Ireland) used in hospitals and provided by Beaumont Hospital (Dublin, Ireland) and stainless-steel sections of the same size. Before use, both surface sections were autoclaved and confirmed microbe free. The sections were then aseptically transferred to sterile Petri dishes and placed under UVL for 30 minutes to ensure they were microbe free before use in the experiments.


Preparation of Inocula With and Without Protein


A volume of 25mL of the appropriate broth was inoculated from an overnight culture plate. Overnight cultures were centrifuged for 10 minutes at 15,500×g (11,000 rpm) (Eppendorf centrifuge 5804R), washed 3 times with phosphate-buffered saline (PBS), and adjusted to a 4 McFarland standard (approximately 9 log10 colony-forming units per milliliter [CFU/mL]) with PBS. For experiments with protein, a 5 McFarland standard (approximately 9.2 log10 CFU/mL)


was used, and 300 μL of a 30% in 0.85% sodium chloride solution of HSA (Sigma-Aldrich) was added to 2.7mL of the McFarland suspension.11 Each test surface was inoculated with a 100-µL suspension spread evenly across the entire surface and allowed to air dry.


Multijet Cold Plasma System


The multijet CAPP prototype (Figure 1) was developed in the National Centre for Plasma Science Technology at Dublin City University. Each plasma jet was driven by a separate sinusoidal, high-voltage power supply. The drive frequency used was 8 kHz, the amplitude of the voltage was approximately 1. 5 kV, and each supply consumed an average of 15W. The operating gas used was dry air, which flowed into the device at 13 L/min. The 9 jets (Figure 1) covered a surface area of approximately 7cm2 (ie, greater than the test surfaces). The plume-contacting surface temperature did not exceed 45°C, and the distance between the plume and the test surface was approximately 1cm. The artificially inoculated test surfaces were placed under the jets for periods of 15, 20, 30 and 45 seconds, respectively, to determine the efficacy of the system. All experiments were carried out three times in duplicate (ie, six sets of results).


Bacterial Recovery and Enumeration


Both test and control (nontreated) surfaces were swabbed using flocked eSwabs (Copan, Italy). Swabs were placed into Falcon round-bottom tubes (BD Bioscience, UK) with 3mL of PBS, were briefly vortexed, and were cultured onto Columbia blood agar plates for enumeration of MRSA and A. baumannii, ESBL brilliance agar plates (Oxoid) for enumeration of ESBEC and VRE brilliance agar plates (Oxoid) for enumeration of VRE. When needed, 1 and 10 serial dilutions were performed to determine a total viable count (TVC, the number of coun- table CFUs per milliliter of the sample on the plate [range, 30–300 CFUs]) after plasma treatment. Before exposure to the multijet CAPP, bacterial enumeration was conducted to


figure 1. Multijet cold-plasma prototype with electrodes, air inlet, and direction-of-jet array.


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