An investigation of the biological effects of non-thermal energy technologies in prokaryotic and eukaryotic cells

O'Brien, Rebecca

Abstract

With the increasing use of antimicrobial products, particularly in the healthcare sector and agri-food industry, there has been a concomitant rise in the prevalence of antimicrobial-resistant (AMR) and multi-drug resistant (MDR) microorganisms. The involvement of pathogenic MDR strains in healthcare-associated infections (HAI) poses serious challenges in terms of treatment options, increasing pressure on the drive to discover effective alternative antimicrobial strategies. To this end, non-thermal plasma (NTP) technologies and electrochemically-activated solutions (ECAS) may provide viable means of helping to control the spread of drug-resistant pathogens.

The effects of a novel Radio Frequency – Microwave (RF-MW) NTP system against planktonic microbes and single-species bacterial biofilms were investigated, using short-contact treatments of 30 – 180 seconds on non-porous surfaces, using Staphylococcus aureus and Pseudomonas aeruginosa as target organisms. Against dried surface-associated microbial loads, significant reductions in viable bioburden were achieved in both species, reaching a maximal 3.1 Log10 reduction in S. aureus, and 4.6 Log10 reduction in P. aeruginosa following treatments of 180s. Treatments with an ECAS solution of 200ppm FAC produced superior antimicrobial effects in parallel surface-decontamination tests, surpassing a reduction of 6 Log10 at contact times of 30 - 300 seconds. Direct NTP treatments applied against single-species S. aureus and P. aeruginosa biofilms for either 60 or 120 seconds showed a much lower efficacy, achieving a maximal reduction of <1.0 Log10 in S. aureus and up to approximately 1.4 Log10 in P. aeruginosa.

Plasma-activated solutions (PAS) were produced using the MW-RF NTP system, and two Surface Barrier Discharge (SBD) plasma systems, and were applied to planktonic microbial loads, and to S. aureus and P. aeruginosa biofilms, to determine the efficacy of indirect NTP treatment. Although moderate inhibition was noted using SBD-plasma-activated water (PAW) against surface-associated S. aureus, these effects were not reproducible with repeat testing, indicating a decline in the concentration of key reactive species responsible for the antimicrobial activity of PAW during the storage period. Similarly, despite showing a significant effect initially, SBD-PAW showed a decline in antimicrobial activity against P. aeruginosa in suspension tests when repeated after a period of refrigerated storage of the PAW. No effects were seen with PAW treatment of biofilms for up to 300 seconds. In contrast, ECAS achieved significant and reproducible reductions of up to 2.5 Log10 in P. aeruginosa biofilms.

Cytotoxicity testing performed on immortalised human epithelial cell lines H103 and A375 demonstrated sensitivity to all antimicrobial treatments investigated, including direct and indirect NTP application. Aqueous antimicrobial solutions were investigated in dose-response studies, from which IC50 values were derived for both cell lines. The 50% inhibitory dose thresholds for ECAS, and the topical antiseptic povidone-iodine (PVP-I) appeared at concentrations 10 – 100-fold below those seen to exert effective antimicrobial activity against planktonic microbial loads.

The effects of RF-MW NTP plasma-activated culture medium (PAM) and PBS (pPBS) on H103 and A375 cell cultures were explored by monitoring cellular proliferation over 72 hours, using live-cell imaging and analysis of confluence. Both plasma-activated solutions appeared to reduce the viable population, but did not produce complete growth inhibition in either cell line. PAM produced a significant growth deficit reaching up to 13.64% in A375 when applied in complete medium, whilst pPBS treatment appeared to suppress proliferation more markedly in serum-free medium, eliciting a maximal effect of up to 13.32% confluence deficit at 72h, which was not significant. In H103, the effects of both PAM and pPBS were greater in serum-free medium, with maximal growth deficits of 21.34% and 18.52% seen, respectively (both p>0.05), at 72h. PAM treatment significantly limited H103 growth in complete medium, by up to 11.59% at 72h (p<0.05).

This research has explored the biological effects of two relatively novel non-thermal energy technologies upon prokaryotic and eukaryotic cells, using various treatment methods. Although some promising antimicrobial efficacy was seen, significant cytotoxic effects was exerted upon both eukaryotic cell lines, indicating limitations in terms of the biocompatibility of these treatments.

• Thesis Type: Thesis
• Deposit Date: Apr 4, 2024
• Publicly Available Date: Feb 25, 2025
• Public URL: https://uwe-repository.worktribe.com/output/11877600
• Award Date: Feb 25, 2025