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The antimicrobial activity of atmospheric pressure non-thermal plasma has been exhaustively characterised, however elucidation of the interactions between biomolecules produced and utilised by bacteria and short plasma exposures are required for optimisation and clinical translation of cold plasma technology. This study characterizes the effects of non-thermal plasma exposure on acyl homoserine lactone (AHL)-dependent quorum sensing (QS). Plasma exposure of AHLs reduced the ability of such molecules to elicit a QS response in bacterial reporter strains in a dose-dependent manner. Short exposures (30-60 s) produce of a series of secondary compounds capable of eliciting a QS response, followed by the complete loss of AHL-dependent signalling following longer exposures. UPLC-MS analysis confirmed the time-dependent degradation of AHL molecules and their conversion into a series of by-products. FT-IR analysis of plasma-exposed AHLs highlighted the appearance of an OH group. In vivo assessment of the exposure of AHLs to plasma was examined using a standard in vivo model. Lettuce leaves injected with the rhlI/lasI mutant PAO-MW1 alongside plasma treated N-butyrylhomoserine lactone and n-(3-oxo-dodecanoyl)-homoserine lactone, exhibited marked attenuation of virulence. This study highlights the capacity of atmospheric pressure non-thermal plasma to modify and degrade AHL autoinducers thereby attenuating QS-dependent virulence in P. aeruginosa.The ability to generate chemically rich plasmas at or near ambient temperature and their demonstrated ability to influence biological processes has led to increasing interest in their use in a range of clinical interventions, and to the emergence of the nascent field of plasma medicine. The term 'plasma medicine' describes the emerging multidisciplinary field of study that examines the applicability and use of atmospheric pressure non-thermal plasmas in biomedical applications and more specifically in the treatment of viable tissue. The potential uses of atmospheric plasmas in biomedical applications include skin decontamination 1 , wound treatments 2,3 , surface disinfection 4,5 and invasive surgical treatments of tumours 6,7 . The physical and reactive chemistry of atmospheric pressure non-thermal plasma is derived from the production of an electric field capable of ionising air or a carrier gas such as helium/argon at atmospheric pressure. This ionisation process leads to the formation of photons, electrons, reactive species, electromagnetic energy and charged particles at a low temperature (sub 60 °C) 8 . Ambient temperatures are possible due to the inefficient transfer of energy between gas particles and electrons 9 . The efficacy of non-thermal plasma in proposed applications ranging from treatment of solid tumours, modification of cell proliferation and migration for wound healing, sterilisation and blood coagulation relies on the synergistic action of the localised generation of reactive oxygen and nitrogen species (RONS), electromagnetic energy, charged particles and UV li...

Clostridium difficile is a spore forming bacterium and the leading cause of colitis and antibiotic associated diarrhoea in the developed world. Spores produced by C. difficile are robust and can remain viable for months, leading to prolonged healthcare-associated outbreaks with high mortality. Exposure of C. difficile spores to a novel, non-thermal atmospheric pressure gas plasma was assessed. Factors affecting sporicidal efficacy, including percentage of oxygen in the helium carrier gas admixture, and the effect on spores from different strains representing the five evolutionary C. difficile clades was investigated. Strains from different clades displayed varying resistance to cold plasma. Strain R20291, representing the globally epidemic ribotype 027 type, was the most resistant. However all tested strains displayed a ~3 log reduction in viable spore counts after plasma treatment for 5 minutes. Inactivation of a ribotype 078 strain, the most prevalent clinical type seen in Northern Ireland, was further assessed with respect to surface decontamination, pH, and hydrogen peroxide concentration. Environmental factors affected plasma activity, with dry spores without the presence of organic matter being most susceptible. This study demonstrates that cold atmospheric plasma can effectively inactivate C. difficile spores, and highlights factors that can affect sporicidal activity.

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Bacterial biofilms are surface adhered communities of bacteria encased within a protective extracellular polymeric matrix. These heterogeneous microbial communities are characterized by elevated tolerance to antimicrobial agents, host immune clearance mechanisms and physical disinfection approaches. Atmospheric pressure non-thermal plasmas have proven to be highly effective in the eradication of bacteria and fungi in both planktonic and biofilm modes of growth at low temperatures, making it a promising approach for surface decontamination of both biotic and abiotic surfaces alike. In addition, non-thermal plasmas as a putative non-antibiotic approach to controlling infectious microorganisms, holds significant promise as an antibiotic alternative infection control strategy, with demonstrated efficacy against antibiotic resistant microorganisms. This topical review introduces the reader to key concepts in biofilm tolerance mechanisms relevant to treatment and control of these surface adhered bacterial communities with cold plasmas. In addition, the ability of plasma-derived active species to interact with both biofilm extracellular matrix components and bacterial cellular targets will be discussed in order to elucidate the mechanisms of antimicrobial and antibiofilm action. By understanding these fundamental interactions, plasma sources may be precisely tailored for antimicrobial applications, specifically for biofilm control where bacterial and fungal physiology (and sensitivity to physical and chemical decontamination) is markedly different from that of their planktonic, or free swimming, counterparts. Recently, novel roles for reactive oxygen and nitrogen species in the activity of conventional antibiotics have been proposed. This extends the possibility that plasmas may enhance the activity of conventional antibiotics and biocides in controlling these highly tolerant microbial populations. Lessons from classical biofilm microbiology can be usefully translated and applied to the design of plasma-based approaches aimed at biofilm control, while potential for tolerance and persistence to plasma in bacterial communities will be reviewed.

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Acinetobacter baumannii is an intrinsically multidrug‐resistant pathogen that, when existing as a biofilm, confers increased environmental tolerance to desiccation, nutrient starvation as well as increased tolerance to antimicrobials. Outbreaks of A. baumannii infections within the clinical setting are often associated with the biofilm phenotype. This study investigates the role of biofilm biomass in A. baumannii susceptibility to exposure to a kilohertz‐driven, in‐house–designed, cold plasma jet, through the examination of cold plasma treatment efficacy in A. baumannii biofilms grown over various times for up to 72 h. For biofilms grown for 24, 48 and 72 h, D values were 19·32 ± 2·71, 29·18 ± 3·15 and 24·70 ± 3·07 s respectively. Monitoring A. baumannii biofilm biomass over these time periods revealed that the greatest biomass was observed at 48 h with the lowest biofilm biomass at 24 h growth. Enumeration of viable biofilm colony counts at each time point was comparable. Scanning electron microscopy images of plasma‐treated biofilms revealed extensive surface damage of A. baumannii cells. These results describe the role of biomass in mediating A. baumannii biofilm susceptibility to cold plasma treatment, implicating the biofilm matrix as a protective barrier to the antimicrobial effects of cold plasma. Significance and Impact of the Study Acinetobacter baumannii biofilm formation results in increased environmental and antimicrobial tolerance and resistance compared to the planktonic phenotype. Cold plasma technology is increasingly investigated as a new tool for decontamination of biofilm‐contaminated surfaces, especially those found in the clinical setting. This new technology presents a promising approach to the remediation of surfaces contaminated by biofilms. This study identifies the role played by A. baumannii biofilm biomass in mediating tolerance and susceptibility to cold plasma treatment. This work demonstrates that increased biofilm biomass reduces the efficacy of antimicrobial species generated by cold plasma, resulting in greater tolerance to plasma exposure.