"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

Structural analysis and resistance properties of Bacillus subtilis biofilms under simulated microgravity

Starting with mission Apollo 16, the Gram positive bacterium Bacillus subtilis has been used in multitude of space experiments. Investigating the influence of extreme space conditions like radiation, vacuum or microgravity, experiments with model organisms like B. subtilis, which forms highly resistant endospores and biofilms, enlighten our understanding regarding survivability, resistance and potential virulence in unfavourable habitats. Biofilms are organized in a complex self-produced extracellular polymeric matrix commonly composed of polysaccharides, proteins and nucleic acids. Building a biofilm protects the individual cell against shear forces, chemicals (e.g. antibiotics or disinfectants), temperature changes and water as well as nutrient depletion (Vlamakis et al., 2013, Cairns et al., 2014). The intrinsic resistance of biofilms is challenging, not only in industry and medicine, but it can be problematic during spaceflight conditions, especially for the crew as well as for the spacecraft. In particular, long term missions with complex cooling systems, water supply and heat pipes may be vulnerable to biofilm colonisation. In our work, we used a biofilm-forming B. subtilis strain and a biofilm-matrix deficient mutant to study the impact of reduced gravity on maturated biofilms. Our major research goal is to compare biofilm formation in simulated microgravity (μg, using a fast-rotating 2D clinostat) to terrestrial gravity (1g) conditions by using different microscopic techniques. White light profilometry, scanning and transmission electron microscopy (SEM, TEM) and confocal laser scanning microscopy (CLSM) were used to analyse biofilms regarding their topology and structure, respectively. Furthermore we conducted a multitude of different survival experiments to evaluate changes and resemblances due to the impact of microgravity. First results show qualitative architectural differences between simulated microgravity and 1g in cross-sections, but no significant qualitative variations in biofilm surface topography. Biofilms grown under simulated microgravity seem to exhibit similar extreme resistances to environmental changes, compared to 1g-controls, when exposed to space-like conditions

Biofilm formation under simulated microgravity - a Bacillus subtilis case study

Starting with mission Apollo 16, the Gram-positive bacterium Bacillus subtilis has been used in multitude of space experiments. Investigating the influence of extreme space conditions like radiation, vacuum or microgravity, experiments with model organisms like B. subtilis, which forms highly resistant endospores and biofilms, enlighten our understanding regarding survivability, resistance and potential virulence in unfavourable habitats. Biofilms are organized in a complex self-produced extracellular polymeric matrix commonly composed of polysaccharides, proteins and nucleic acids. Building a biofilm protects the individual cell against shear forces, chemicals (e.g. antibiotics or disinfectants), temperature changes and water as well as nutrient depletion (Vlamakis et al., 2013, Cairns et al., 2014). The intrinsic resistance of biofilms is challenging, not only in industry and medicine, but it can be problematic during spaceflight conditions, especially for the crew as well as for the spacecraft. In particular, long term missions with complex cooling systems, water supply and heat pipes may be vulnerable to biofilm colonisation. In our work, we used a biofilm-forming B. subtilis strain and a biofilmmatrix deficient mutant to study the impact of reduced gravity on maturated biofilms. Our research aim is to compare biofilm formation in simulated microgravity (μg, using a fast-rotating 2D clinostat) to terrestrial gravity (1g) conditions by using different microscopic techniques. White light profilometry, scanning and transmission electron microscopy (SEM, TEM) and confocal laser scanning microscopy (CLSM) were used to analyse biofilms regarding their topology and structure, respectively. Different types of survival experiments were conducted to evaluate changes and resemblances due to the impact of microgravity. Our results show qualitative architectural differences between simulated microgravity and 1g in cross-sections, but no significant qualitative variations in biofilm surface topography. Our results show qualitative architectural differences in cross-sections of biofilms grown in simulated microgravity and 1g

Why is it important to correctly classify and report SARS-CoV-2 infections and COVID-19 deaths?

BACKGROUND : In a public health emergency such as the coronavirus disease 2019 (COVID-19) pandemic, mortality surveillance is crucial, as it guides the public health response and serves as a measure of its effectiveness. It is therefore critical that deaths are uniformly and accurately classified, calculated and reported. OBJECTIVE : To discuss the importance of making accurate COVID-19 diagnoses and correctly classifying COVID-19 deaths, drawing on the experience of the misclassification of HIV/AIDS deaths in South Africa (SA). METHOD : We performed an electronic literature search on PubMed, Google Scholar and EBSCOhost to identify studies with key words such as coronavirus, COVID-19, SARS-CoV-2 and mortality or death. Relevant studies were reviewed for suitability and used in the development of the argument. RESULTS : In SA, from 6 May to 30 June 2020, an excess of 6 849 deaths were reported from natural causes among people >1 year old when using a revised base accounting for lower mortality during lockdown. Over this same period, the SA government reported 2 504 COVID-19 related deaths. Both overestimation and underestimation of deaths have significant implications for health policy and resource allocation, as was evident in SA when the misclassification of deaths during the period of HIV denialism led directly to fewer resources being spent on dealing with the prevention and treatment of HIV, which allowed the disease to spiral out of control. CONCLUSION : To ensure an appropriate public health response to COVID-19, accurate data on SARS-CoV-2 infections and COVID-19-related deaths are needed. The SA National Department of Health should develop clear guidelines on classification of deaths, and share this information with all those responsible for certifying deaths.http://www.samj.org.za/index.php/samjam2021Family MedicineImmunolog

Biofilm formation and sporulation of Bacillus subtilis under simulated microgravity

Since humans are travelling into the outer atmospheres of our planet and beyond, a new environmental habitat was established. Knowing that life - possibly since its first appearance on Earth is adapted to terrestrial gravity, the impact of microgravity on prokaryotes and humans remains mostly unclear. Investigating the influence of extreme conditions like those in space, Bacillus subtilis was chosen as model organism. Non-domesticated strains, such as NCIB 3610 have the ability to form biofilms as well as highly resistant endospores. Since it is known that planktonic life is the exception, biofilms are considered as predominant way of living (Moons et al., 2009). Building a biofilm protects the individual cell against shear forces, chemicals (e.g. antibiotics or disinfectants), temperature changes and water as well as nutrient depletion (Vlamakis et al., 2013, Cairns et al., 2014). The intrinsic resistance of biofilms is a problem, not only in industry and medicine, but it can be problematic during spaceflight conditions, especially for the crew as well as for the spacecraft. In particular, long term missions with complex cooling systems, water supply and heat pipes may be vulnerable to biofilm colonisation. In our work, we used a rotating 2D-clinostat to determine the effect of simulated microgravity (sim.-μg) and terrestrial gravity (1g) on B. subtilis biofilms and spores by using different microscopic techniques. White light profilometry, scanning and transmission electron microscopy (SEM, TEM) and confocal laser scanning microscopy (CLSM) were used to analyse biofilms regarding their topology and inner structure, respectively. First results show qualitative architectural differences between simulated microgravity and 1g in cross-sections, but no significant qualitative variations in biofilm surface topography. In order to test the resistance of spores grown under simulated gravity, germination assays, as well as survival assays were used. First results revealed that spores grown under the influence of sim.-μg had an increased spontaneous germination rate compared to spores grown at 1g

Influence of simulated microgravity on B. subtilis biofilms

Bacillus subtilis is one of the most studied Gram positive model organisms. Since mission Apollo 16, B. subtilis has been used for a multitude of space experiments. Investigating the influence of extreme conditions like those in space, non-domesticated strains, such as NCIB 3610 are of special interest regarding their ability to form biofilms. Since it is known that planktonic life is the exception, biofilms are considered as predominant way of living (Moons et al., 2009). Biofilms are organized in a complex self-produced extracellular polymeric matrix commonly composed of polysaccharides, proteins and nucleic acids. Building a biofilm protects the individual cell against shear forces, chemicals (e.g. antibiotics or disinfectants), temperature changes and water as well as nutrient depletion (Vlamakis et al., 2013, Cairns et al., 2014). The intrinsic resistance of biofilms is a problem, not only in industry and medicine, but it can be problematic under spaceflight conditions. Especially the loss of gravity coupled with changed levels of radiation might influence the resistance and therefore the virulence of bacterial biofilms. This can possibly evoke problems for the crew as well as for the spacecraft. In particular, long term missions with complex cooling systems, water supply and heat pipes may be vulnerable to biofilm colonisation. In our work, we used the biofilm-forming wildtype strain NCIB 3610 and a biofilm-matrix deficient mutant (deletion of 15-gene exopolysaccharide operon, epsA-O) to study the impact of reduced gravity on maturated biofilms. Our major research goal is to compare biofilm formation in simulated microgravity (using a 2D clinostat) to terrestrial gravity (1g) conditions by using different microscopic techniques. White light profilometry, scanning and transmission electron microscopy (SEM, TEM) and confocal laser scanning microscopy (CLSM) were used to analyse biofilms regarding their topology and inner structure, respectively. First results show qualitative architectural differences between simulated microgravity and 1g in cross-sections, but no significant qualitative variations in biofilm surface topography

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth

Fill Me App: An Interactive Mobile Game Application for Children with Autism

FillMeApp is an interactive mobile game application which is a supplementary learning material intended for children with Autism that helps them motivate in their learning process. This game application is focus mainly on Science basically on identifying the human’s body parts. Accumulating the best time for focus monitoring, eye-catching graphics, simple level of exercises, video tutorial and background music that coincide with the current educational teachings are among the primary features that this application has to offer. The researchers analyzed the results of the test survey and proved that the application is user friendly, interactive, the logic of the game is understandable and would learn to use this application very useful in their learning. Based also from the researchers testing and result on the students motivational rating from the teacher, before, the students with Autisms’ motivation level were LOW but after the game application was deployed and tested their motivation status begin to grow and become HIGH

DMPK and Metabolism Studies of Nucleoside Phosphoramidates Including INX-08189, A Novel Double Pro-drug and Clinical Candidate for Hepatitis C Virus Therapy

Hepatitis C Virus (HCV) infection is a serious health problem that leads to chronic liver disease, such as cirrhosis and hepatocellular carcinoma, in an estimated 2–15% of the world's population. A collaboration between Inhibitex and the University of Caridiff in Wales has produced a novel double pro-drug approach to the anti-HCV agent 2′-β-C-methylguanosine. A phosphoramidate (ProTide) motif and a C6-methoxy base pro-drug moiety are combined to generate lipophilic prodrugs of the monophosphate of the guanine nucleoside. Extensive DMPK studies in multiple species which supported the selection of the lead compound will be discussed. Details of the pre-clinical development of INX-08189 including radiolabeled metabolism studies will be described. INX-08189 has completed investigational new drug enabling studies and has been progressed into human clinical trials for the treatment of chronic HCV infection