Antibacterial activity of blue light against nosocomial wound pathogens growing planktonically and as mature biofilms

The blue wavelengths within the visible light spectrum are intrinisically antimicrobial and can photodynamically inactivate the cells of a wide spectrum of bacteria (Gram positive and negative) and fungi. Furthermore, blue light is equally effective against both drug-sensitive and -resistant members of target species and is less detrimental to mammalian cells than is UV radiation. Blue light is currently used for treating acnes vulgaris and Helicobacter pylori infections; the utility for decontamination and treatment of wound infections is in its infancy. Furthermore, limited studies have been performed on bacterial biofilms, the key growth mode of bacteria involved in clinical infections. Here we report the findings of a multicenter in vitro study performed to assess the antimicrobial activity of 400-nm blue light against bacteria in both planktonic and biofilm growth modes. Blue light was tested against a panel of 34 bacterial isolates (clinical and type strains) comprising Acinetobacter baumannii, Enterobacter cloacae, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Klebsiella pneumoniae, and Elizabethkingia meningoseptica. All planktonic-phase bacteria were susceptible to blue light treatment, with the majority (71%) demonstrating a ≥ 5-log10 decrease in viability after 15 to 30 min of exposure (54 J/cm2 to 108 J/cm2). Bacterial biofilms were also highly susceptible to blue light, with significant reduction in seeding observed for all isolates at all levels of exposure. These results warrant further investigation of blue light as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications

Bacillus subtilis Spore Resistance towards Low Pressure Plasma Sterilization

1. Non-thermal Plasmas for Sterilization 1.1 Motivation for Search of Alternative Sterilization Methods Microbial contamination on surfaces is a recurring problem within health, pharmaceutical and food industry sectors (Abreu et al., 2013, Rutala and Weber, 2013). Surface sterilization is a crucial step to ensure sterility of food processing equipment, minimize spread of pathogens and prevent the transmission of nosocomial infections (Williams et al., 2009). Common sterilization procedures that are widely used for microbial inactivation include heat treatment (dry and moist heat), chemical disinfection with solutions or gases (H2O2, ethylene oxide), filtration, and exposure to ionizing radiation (reviewed in Rutala and Weber, 2013). While generally effective, most conventional methods suffer from significant drawbacks and restrictions. Currently employed procedures can introduce considerable damage to the treated material due to the exposure to elevated temperatures or aggressive chemicals and often pose a risk to the operator, rendering these methods suboptimal in many applications (Yardimci and Setlow, 2010). These limitations have motivated the search for alternative sterilization methods. Plasma sterilization is a promising alternative to conventional sterilization methods as it offers rapid and efficient microbial inactivation, while being gentle to sensitive and heatlabile materials and very safe to the operating staff. Over the last decade, the application of non-thermal plasmas has gained wide attention in biomedical and nutritional research, as well as in space-flight applications (De Geyter and Morent, 2012, Shimizu et al., 2014; Lerouge, 2000)

Bacillus subtilis\textit {Bacillus subtilis} spore resistance towards low pressure plasma sterilization

Plasmasterilisation im Niederdruck gilt als vielversprechende Alternative für die Behandlung von hitzeempfindlichen Plastiken und korrosionsanfälligen Materialen. Bakterielle Sporen der Gattung $\it Bacillus$ werden oft als biologischer Indikator für Sterilisationsprozesse eingesetzt. In dieser Arbeit wird die Anwendbarkeit von Niederdruckplasma als schnelle und effiziente Methode zur verbesserten Inaktivierung von Sporen dargestellt. Weiterhin werden der Sporenmantel, Dipicolinsäure, der niedrige Wassergehalt, und die Anlagerung von $\alpha$/$\beta$-SASPs als wichtige Aspekte, die zur Sporenresistenz gegenüber der Sterilisation mit Niederdruckplasma beitragen, identifiziert. Niederdruckplasma induziert signifikante Oberflächenmodifikationen im Sporenmantel, sowie Photoprodukte und Strangbrüche im Erbgut, wobei das Sporenphotoprodukt am häufigsten gebildet wurde. Viele verschiedene DNA Reparaturmechanismen spielen eine zentrale Rolle für das Überleben von $\textit {B. subtilis}$ Sporen nach Plasmabehandlungen.Low pressure plasma sterilization is a promising alternative for the sterilization of heat-labile plastics and materials prone to corrosion. Plasma is an ionized gas comprising various reactive components. Bacterial spores of the genus $\it Bacillus$ are frequently used as biological indicator to verify the functionality of a sterilization process. In this work the applicability of low pressure plasma sterilization for enhanced spore inactivation is demonstrated. The spore coat, accumulation of dipicolinic acid, and genome saturation with $\alpha$/$\beta$-type SASPs are identified as crucial factors providing spore resistance towards low pressure plasma discharges. Low pressure plasma induces significant surface modifications on the spore surface as well as photoproducts and strand breaks in the spore genome. The formation of the spore photoproduct was favored. Several general and spore-specific DNA repair mechanisms play a central role in spore survival after plasma sterilization

Translating physics to microbiology: spore resistance to terrestrial and extraterrestrial extremes

Spore-forming bacteria are of particular concern in the context of planetary protection because their tough endospores are capable of withstanding certain sterilization procedures as well as harsh environments. Spores of Bacillus subtilis have been shown to be suitable dosimeters for probing extreme terrestrial and extraterrestrial environmental conditions in astrobiological and environmental studies. During dormancy spores are metabolically inactive; thus substantial DNA, protein, tRNA and ribosome damage can accumulate while the spores are incapable of repairing and/or degrading damaged DNA and proteins. Consequently, damage to essential components of spores poses a unique problem, since damage repair does not occur until the processes of spore revival. Spores appear to have two possible ways to minimize deleterious effects of environmental extremes: (i) by protecting dormant spore macromolecules (in particular the spore DNA) from damage in the first place and (ii) by ensuring repair of damage during spore outgrowth. In our research, we used spores of different genotypes of B. subtilis to study the effects of various extraterrestrial conditions (e.g., planetary conditions as present on Mars or low Earth orbit (LEO)) for astrobiological purposes. Spores of wild-type and mutant B. subtilis strains lacking various structural components were exposed to simulated Martian atmospheric, galactic cosmic and UV irradiation conditions. Spore survival was strongly dependent on the functionality of all of the structural components, with small acid-soluble spore proteins, coat layers, and dipicolinic acid (DPA) as key protectants. In addition, the interaction of several DNA repair mechanisms (e.g., non-homologous end joining (NHEJ) and spore photoproduct (SP) lyase) was identified as crucial for surviving environmental extremes in space or Martian surface (i.e., exposure to solar UV and galactic cosmic radiation. The ultimate goal is to obtain a complete model describing spore persistence and longevity in harsh environments

Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair

Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5'-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2'- deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway.We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs

“Sticky little devils” … improving planetary protection forward decontamination strategies – studies on the spore resistance to low-pressure plasma sterilization & persistence on metallic copper surfaces

Microbial contamination arising from spacecraft exploration harbors the distinct potential to impact the development and integrity of life-detection missions on planetary bodies such as Mars and Europa. Such missions are subjected to strict regulations. In the context of the planetary protection guidelines, established by the Committee of Space Research (COSPAR) in 1967, it is essential to reduce or eliminate the biological burden on flight hardware prior to launch in order to prevent cross contamination of celestial bodies with environmental or human-associated microorganisms. Depending on type of mission and planetary body, specific planetary protection guidelines are required to clean and sterilize a spacecraft or its components to avoid contamination from terrestrial organisms. The search for extraterrestrial life will rely heavily on validated cleaning and bioreduction strategies to ensure that terrestrial microbial contamination does not compromise the scientific integrity of such missions

Simulated Space Radiation: Impact of Four Different Types of High-Dose Ionizing Radiation on the Lichen Xanthoria elegans

This study addresses the viability of the lichen Xanthoria elegans after high-dose ionizing irradiation in the frame of the STARLIFE campaign. The first set of experiments was intended to resemble several types of galactic cosmic radiation (GCR) as present beyond the magnetic shield of Earth. In the second set of experiments, γ radiation up to 113 kGy was applied to test the limit of lichen resistance to ionizing radiation. Entire thalli of Xanthoria elegans were irradiated in the anhydrobiotic state. After STARLIFE 1, the metabolic activity of both symbionts was quantified by live/dead staining with confocal laser scanning microscopy. The photosynthetic activity was measured after the respective irradiation to assess the ability of the symbiotic green algae to restore photosynthesis after irradiation. The STARLIFE campaign complements the results of the LIFE experiments at the EXPOSE-E facility on the International Space Station by testing the model organism Xanthoria elegans on its resistance to hazardous radiation that might be accumulated during long-term space exposure. In addition, the photosynthetic activity of metabolically active lichen was investigated after X-ray irradiation up to 100 Gy (3.3 Gy/min). Since previous astrobiological experiments were mostly performed with anhydrobiotic lichen, these experiments will broaden our knowledge on the correlation of physiological state and astrobiological stressors

Utilization of Low-Pressure Plasma to Inactivate Bacterial Spores on Stainless Steel Screws

A special focus area of planetary protection is the monitoring, control, and reduction of microbial contaminations that are detected on spacecraft components and hardware during and after assembly. In this study, wildtype spores of Bacillus pumilus SAFR-032 (a persistent spacecraft assembly facility isolate) and the laboratory model organism B. subtilis 168 were used to study the effects of low-pressure plasma, with hydrogen alone and in combination with oxygen and evaporated hydrogen peroxide as a process gas, on spore survival, which was determined by a colony formation assay. Spores of B. pumilus SAFR-032 and B. subtilis 168 were deposited with an aseptic technique onto the surface of stainless steel screws to simulate a spore-contaminated spacecraft hardware component, and were subsequently exposed to different plasmas and hydrogen peroxide conditions in a very high frequency capacitively coupled plasma reactor (VHF-CCP) to reduce the spore burden. Spores of the spacecraft isolate B. pumilus SAFR-032 were significantly more resistant to plasma treatment than spores of B. subtilis 168. The use of low-pressure plasma with an additional treatment of evaporated hydrogen peroxide also led to an enhanced spore inactivation that surpassed either single treatment when applied alone, which indicates the potential application of this method as a fast and suitable way to reduce spore-contaminated spacecraft hardware components for planetary protection purposes

Resistance of lichens to simulated galactic cosmic radiation: limits of survival capacity and biosignature detection

Space constitutes an extremely harmful environment for survival of terrestrial organisms. Amongst extremophiles on Earth, lichens are one of the most resistant organisms to harsh terrestrial environments, as well as some species of microorganisms, such as bacteria (Moeller et al., 2010), criptoendolithic cyanobacteria and lithic fungi (de los Ríos et al. 2004). To study the survival capacity of lichens to the harmful radiation environment of space, we have selected the lichen Circinaria gyrosa, an astrobiological model defined by its high capacity of resistance to space conditions (De la Torre et al. 2010) and to a simulated Mars environment (Sanchez et al., 2012). Samples were irradiated with four types of space-relevant ionizing radiation in the STARLIFE campaign: helium and iron ion doses (up to 2,000 Gy), X-ray doses (up to 5,000 Gy) and ultra-high γ-ray doses (from 6 to 113 kGy). Results on resistance of C. gyrosa to space-relevant ionizing radiation and its post-irradiation viability were obtained by: (i) chlorophyll a fluorescence of photosystem II (PS II); (ii) epifluorescence microscopy; (iii) confocal laser-scanning microscopy (CLSM), and (iv) field emission scanning electron microscopy (FESEM). Results of photosynthetic activity and epifluorescence showed no significant changes on the viability of C. gyrosa with increasing doses of helium and iron ions as well as X-rays. In contrast, γ-irradiation elicited significant dose-correlated effects as revealed by all applied techniques. Relevant is the presence of whewellite-like crystals, detected by FESEM on C. gyrosa thalli after high irradiation doses, which has been also identified in previous Mars simulation studies (Böttcher et al., 2014). These studies contribute to the better understanding of the adaptability of extremophile organisms to harsh environments, as well as to estimate the habitability of a planet’s surface, like Mars; they will be important for planning experiments on the search of life in the universe, and as contribution of lithopanspermia, the theory that supports the interplanetary transfer of rock inhabiting life by means of meteorites (Mileikovsky et al., 2000). References Böttger U, Meessen J, Martinez-Frias J, Hübers H-W, Rull F, Sánchez FJ, de la Torre R, de Vera J-P. 2014. International Journal of Astrobiology 13: 19–27. de la Torre R, Sancho LG, Horneck G, de los Ríos A, Wierzchos J, Olsson-Francis K, et al. 2010. Icarus 208: 735-748. de los Ríos A, Wierzchos J, Sancho LG, Ascaso C. 2004. FEMS Microbiology Ecology 50: 143–152. Mileikovsky C, Cucinotta F, Wilson JW, Gladman B, Horneck G, Lindegren L, Melosh J, Rickman H, Valtonen M, Zheng JQ. 2000. Icarus 145, 391-427. Moeller R, Rohde M, Reitz G. 2010. Icarus 206: 783–786. Sánchez FJ, Mateo-Martí E, Raggio J, Meeßen J, Martínez-Frías J, Sancho LG, et al. 2012. Planetary and Space Science 72: 102–110.AZ Miller acknowledges the support from the Marie Skłodowska-Curie actions (PIEF-GA2012-328689).Peer Reviewe

VUV absorption spectroscopy of bacterial spores and DNA components

Low-pressure plasmas can be used to inactivate bacterial spores and sterilize goods for medical and pharmaceutical applications. A crucial factor are damages induced by UV and VUV radiation emitted by the plasma. To analyze inactivation processes and protection strategies of spores, absorption spectra of two B. subtilis strains are measured. The results indicate, that the inner and outer coat of the spore significantly contribute to the absorption of UV-C and also of the VUV, protecting the spore against radiation based damages. As the sample preparation can significantly influence the absorption spectra due to salt residues, the cleaning procedure and sample deposition is tested for its reproducibility by measuring DNA oligomers and pUC18 plasmid DNA. The measurements are compared and discussed with results from the literature, showing a strong decrease of the salt content enabling the detection of absorption structures in the samples