"Killing them softly" … challenges in the Bacillus subtilis spore inactivation by plasma sterilization

The elimination of bacterial endospores is absolutely essential in numerous fields, ranging from hospital hygiene, the food processing industry, all the way to the space industry. A major goal of space exploration is the search for signatures of life forms and biomolecules on other planetary bodies and moons in our solar system. The transfer of microorganisms or biomolecules of terrestrial origin to critical areas of exploration is of particular risk to impact the development and integrity of life-detection missions.1 Plasma sterilization is a promising alternative to conventional sterilization methods for spaceflight purposes. Due to their extraordinary resistance properties, spores of the Gram-positive bacterium Bacillus subtilis are used as biological indicators for decontamination studies to identify the relevant mechanism that leads to the rapid bacterial inactivation.1,3 Here, we present novel insights into the key factors involved in spore inactivation by low pressure plasma sterilization using a double inductively-coupled plasma reactor. (2,4) In order to standardize the assessment of inactivation efficiencies by plasma discharges, an electrically driven spray deposition device was developed, allowing fast, reproducible, and homogeneous preparation of B. subtilis spore monolayers. We demonstrate that plasma discharges caused significant physical damage to spore surface structures as visualized by atomic force microscopy. A systematic analysis of B. subtilis spores lacking individual coat and crust layers - the first barrier to environmental influences – revealed the coat to be one of the contributing factors in the spore resistance to plasma sterilization. (2-4) Furthermore, we identified spore-specific and general protection mechanisms and DNA repair pathways during spore germination and outgrowth after plasma treatment, leading to a better understanding of the complex molecular mechanisms involved in the inactivation by plasma sterilization processes

Foundations of plasma standards

The field of low-temperature plasmas (LTPs) excels by virtue of its broad intellectual diversity, interdisciplinarity and range of applications. This great diversity also challenges researchers in communicating the outcomes of their investigations, as common practices and expectations for reporting vary widely in the many disciplines that either fall under the LTP umbrella or interact closely with LTP topics. These challenges encompass comparing measurements made in different laboratories, exchanging and sharing computer models, enabling reproducibility in experiments and computations using traceable and transparent methods and data, establishing metrics for reliability, and in translating fundamental findings to practice. In this paper, we address these challenges from the perspective of LTP standards for measurements, diagnostics, computations, reporting and plasma sources. This discussion on standards, or recommended best practices, and in some cases suggestions for standards or best practices, has the goal of improving communication, reproducibility and transparency within the LTP field and fields allied with LTPs. This discussion also acknowledges that standards and best practices, either recommended or at some point enforced, are ultimately a matter of judgment. These standards and recommended practices should not limit innovation nor prevent research breakthroughs from having real-time impact. Ultimately, the goal of our research community is to advance the entire LTP field and the many applications it touches through a shared set of expectations

Chemical fingerprints of cold physical plasmas – an experimental and computational study using cysteine as tracer compound

Reactive oxygen and nitrogen species released by cold physical plasma are being proposed as effectors in various clinical conditions connected to inflammatory processes. As these plasmas can be tailored in a wide range, models to compare and control their biochemical footprint are desired to infer on the molecular mechanisms underlying the observed effects and to enable the discrimination between different plasma sources. Here, an improved model to trace short-lived reactive species is presented. Using FTIR, high-resolution mass spectrometry, and molecular dynamics computational simulation, covalent modifications of cysteine treated with different plasmas were deciphered and the respective product pattern used to generate a fingerprint of each plasma source. Such, our experimental model allows a fast and reliable grading of the chemical potential of plasmas used for medical purposes. Major reaction products were identified to be cysteine sulfonic acid, cystine, and cysteine fragments. Less-abundant products, such as oxidized cystine derivatives or S-nitrosylated cysteines, were unique to different plasma sources or operating conditions. The data collected point at hydroxyl radicals, atomic O, and singlet oxygen as major contributing species that enable an impact on cellular thiol groups when applying cold plasma in vitro or in vivo

Controlling plasma properties under differing degrees of electronegativity using odd harmonic dual frequency excitation

International audienceThe charged particle dynamics in low-pressure oxygen plasmas excited by odd harmonic dual frequency waveforms (low frequency of 13.56 MHz and high frequency of 40.68 MHz) are investigated using a one-dimensional numerical simulation in regimes of both low and high electronegativity. In the low electronegativity regime, the time and space averaged electron and negative ion densities are approximately equal and plasma sustainment is dominated by ionisation at the sheath expansion for all combinations of low and high frequency and the phase shift between them. In the high electronegativity regime, the negative ion density is a factor of 15--20 greater than the low electronegativity cases. In these cases, plasma sustainment is dominated by ionisation inside the bulk plasma and at the collapsing sheath edge when the contribution of the high frequency to the overall voltage waveform is low. As the high frequency component contribution to the waveform increases, sheath expansion ionisation begins to dominate. It is found that the control of the average voltage drop across the plasma sheath and the average ion flux to the powered electrode are similar in both regimes of electronegativity, despite the differing electron dynamics using the considered dual frequency approach. This offers potential for similar control of ion dynamics under a range of process conditions, independent of the electronegativity. This is in contrast to ion control offered by electrically asymmetric waveforms where the relationship between the ion flux and ion bombardment energy is dependent upon the electronegativity

Understanding the molecular mechanisms involved in the spore inactivation by plasma sterilization

Being the most resistant form of a biological system, spores of Bacillus subtilis are very resistant against a broad spectrum of sterilization methods and, therefore, are commonly used as a biological indicator in order to verify the functionality of a sterilization procedure. The process of low-pressure plasma sterilization is a promising alternative to conventional sterilization methods as it is extremely fast, efficient and gentle to heatsensitive materials. Active plasma species contain a high degree of sporicidal UV/VUV-radiation, as well as charged particles and free radicals, which exert detrimental effects on microorganisms. In this study we present novel insights into the key factors involved in spore inactivation by low pressure plasma sterilization using a double inductively-coupled plasma reactor. In order to standardize the assessment of spore inactivation efficiencies by plasma discharges, an electrically operated deposition device was developed, allowing fast, reproducible, and homogeneous preparation of B. subtilis spore in monolayers on surfaces leading to more reliable investigations. We demonstrate that low-pressure plasma discharges of argon and oxygen discharges cause significant physical damage to spore surface structures as visualized by atomic force microscopy. A systematic analysis of B. subtilis spores lacking individual coat and crust layers - the first barrier to environmental influences – revealed the coat to be a major factor in spore resistance towards plasma treatment (Raguse et al., 2016). In order to gain a better understanding of the complex molecular mechanisms involved in the inactivation by plasma sterilization processes, we analyzed plasma-induced DNA lesions in vitro, identified general and spore-specific DNA lesions, and characterized different DNA repair mechanisms during spore revival after plasma treatment

Low pressure plasma inactivation of Bacillus subtilis spores: insights into the mechanisms of spore resistance

Being the most resistant form of a biological system, spores of Bacillus subtilis are very resistant against a broad spectrum of sterilization methods and, therefore, are commonly used as a biological indicator in order to verify the functionality of a sterilization procedure. The process of low-pressure plasma sterilization is a promising alternative to conventional sterilization methods as it is extremely fast, efficient and gentle to heat-sensitive materials. Active plasma species contain a high degree of sporicidal UV/VUV-radiation, as well as charged particles and free radicals, which exert detrimental effects on microorganisms. In this study we present novel insights into the key factors involved in spore inactivation by low pressure plasma sterilization using a double inductively-coupled plasma reactor

Monitoring Plasma Etching of Biomolecules by Imaging Ellipsometry

Low-pressure plasma discharges can be applied to remove various biomolecules from surfaces. However, the knowledge on the interaction between plasma and biomolecules and the kinetics of their removal is still rather poor, which is a major limiting factor for the optimization of this type of plasma treatment. This is, among other reasons, because of the restrictions of currently used techniques for the evaluation of the rates of biomolecule removal during plasma treatment. Therefore, an alternative method based on imaging ellipsometry is applied in this article. It is shown in this study that this method allows reliable semiquantitative comparison of the treatment efficiency of plasma discharges sustained in different gas mixtures.JRC.DDG.I.5-Nanobioscience

Elimination of Pathogenic Biological Residuals by Means of Low-Pressure Inductively Coupled Plasma Discharge

The possibility to use low-pressure, nonequilibrium plasma discharges for decontamination and sterilization of surfaces of medical equipment is nowadays gaining increased attention. The primary interest of this technique is related mainly to its capability to inactivate effectively infectious microorganisms including highly resistant bacterial spores [1-3] without using toxic substances and at low-temperature process conditions required for the treatment of heat degradable materials. Moreover, recent results revealed the ability of plasma discharges to inactivate or also eliminate other kinds of biological pathogens such as bacterial endotoxins [3-5] or proteins [3, 6-8]. Especially, the latter makes the plasma treatment a real alternative to the commonly used decontamination techniques that are in many cases insufficient to assure complete elimination of residual biological contamination.'' Naturally, distinct characteristics of biological pathogens imply different strategies for their destruction: living microorganisms can be readily inactivated by intense UV radiation emitted by plasma discharges [1], in contrast to the elimination of pathogenic biomolecules from surfaces, where physicochemical removal is needed [l0]. However, it is clear that any process that is intended to be effective and universal for complete elimination of biological contamination has to combine both pathways. Recently, it has been suggested that this can be fulfilled by using an inductively coupled plasma (ICP) discharge sustained in a ternary Ar/02/N2 discharge mixture combining high emission of UV radiation in the spectral range suitable for the sterilization of bacterial spores with fast erosion and removal of biomolecules [11, 12]. Nevertheless, the latter has been, up to now, demonstrated solely on bovine serum albumin and no effort has been devoted to test whether it is possible to eliminate a wider range of biological agents, that is, an aspect important for the demonstration of the universality of this sterilization/decontamination approach. Therefore, in this study to fill this experimental gap, the feasibility of low-pressure Ar/02/N2 ICP discharge to remove other biological systems differing significantly in their properties, namely, bacterial spores and biomolecules in general, is investigated. Moreover, to highlight the advantageous properties of Ar/02/N2 mixture, the results are compared directly with the results obtained by using Ar and Ar/Oz plasma treatment.JRC.DG.I.5-Nanobioscience