investigating-detrimental-effects-low-pressure-plasma-sterilization

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

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Robert Koch Institute

Chlamydia trachomatis is a medically important human pathogen causing different diseases, including trachoma, the leading cause of preventable blindness in developing countries, and sexually transmitted infections that can lead to infertility and ectopic pregnancies. There is no vaccine against C. trachomatis at present. Broad-spectrum antibiotics are used as standard therapy to treat the infection but have unwanted side effects, such as inducing persistent or recurring infections and affecting the host microbiome, necessitating the development of novel anti-Chlamydia therapies. Here, we describe the establishment of a robust, fast, and simple plaque assay using liquid overlay medium (LOM) for the identification of anti-Chlamydia compounds. Using the LOM plaque assay, we identified nitrobenzoxadiazole (NBD)-labeled 1-O-methyl-ceramide-C16 as a compound that efficiently inhibits C. trachomatis replication without affecting the viability of the host cell. Further detailed analyses indicate that 1-O-methyl-NBD-ceramide-C16 acts outside the inclusion. Thereby, 1-O-methyl-NBD-ceramide-C16 represents a lead compound for the development of novel anti-Chlamydia drugs and furthermore constitutes an agent to illuminate sphingolipid trafficking pathways in Chlamydia infections.

Improved plaque assay identifies a novel anti-Chlamydia ceramide derivative with altered intracellular localization.

Visualization of HIV-1 budding structures.

Plastic cell culture dishes that contain a thin bottom of highest optical quality including an imprinted finder grid (μ-Dish Grid-500) are optimally suited for routine correlative light and electron microscopy using chemical fixation. Such dishes allow high-resolution fluorescence and bright-field imaging using fixed and living cells and are compatible with standard protocols for scanning and transmission electron microscopy. Ease of use during cell culture and imaging, as well as a tight cover render the dishes particularly suitable for working with infectious organisms up to the highest biosafety level. Detailed protocols are provided and demonstrated by showing two examples: monitoring the production of virus-like particles of the Human Endogenous Retrovirus HERV-K(HML-2) by HeLa cells and investigation of Rab11-positive membrane-compartments of HeLa cells after infection with Chlamydia trachomatis.

A simple procedure to analyze positions of interest in infectious cell cultures by correlative light and electron microscopy.

Three-Dimensional Imaging of CD63 Recruitment at the Virological Synapse: HIV-1.

Novel decontamination technologies, including cold low-pressure plasma and blue light (400 nm), are promising alternatives to conventional surface decontamination methods. However, the standardization of the assessment of such sterilization processes remains to be accomplished. Bacterial endospores of the genera Bacillus and Geobacillus are frequently used as biological indicators (BIs) of sterility. Ensuring standardized and reproducible BIs for reliable testing procedures is a significant problem in industrial settings. In this study, an electrically driven spray deposition device was developed, allowing fast, reproducible, and homogeneous preparation of Bacillus subtilis 168 spore monolayers on glass surfaces. A detailed description of the structural design as well as the operating principle of the spraying device is given. The reproducible formation of spore monolayers of up to 5 × 10(7) spores per sample was verified by scanning electron microscopy. Surface inactivation studies revealed that monolayered spores were inactivated by UV-C (254 nm), low-pressure argon plasma (500 W, 10 Pa, 100 standard cubic cm per min), and blue light (400 nm) significantly faster than multilayered spores were. We have thus succeeded in the uniform preparation of reproducible, highly concentrated spore monolayers with the potential to generate BIs for a variety of nonpenetrating surface decontamination techniques.

Improvement of Biological Indicators by Uniformly Distributing Bacillus subtilis Spores in Monolayers To Evaluate Enhanced Spore Decontamination Technologies.

In its natural habitat, the soil bacterium Bacillus subtilis often has to cope with fluctuating osmolality and nutrient availability. Upon nutrient depletion it can form dormant spores, which can revive to form vegetative cells when nutrients become available again. While the effects of salt stress on spore germination have been analyzed previously, detailed knowledge on the salt stress response during the subsequent outgrowth phase is lacking. In this study, we investigated the changes in gene expression during B. subtilis outgrowth in the presence of 1.2 M NaCl using RNA sequencing. In total, 402 different genes were upregulated and 632 genes were downregulated during 90 min of outgrowth in the presence of salt. The salt stress response of outgrowing spores largely resembled the osmospecific response of vegetative cells exposed to sustained high salinity and included strong upregulation of genes involved in osmoprotectant uptake and compatible solute synthesis. The σ(B)-dependent general stress response typically triggered by salt shocks was not induced, whereas the σ(W) regulon appears to play an important role for osmoadaptation of outgrowing spores. Furthermore, high salinity induced many changes in the membrane protein and transporter transcriptome. Overall, salt stress seemed to slow down the complex molecular reorganization processes ("ripening") of outgrowing spores by exerting detrimental effects on vegetative functions such as amino acid metabolism.

Article	Year
Improved plaque assay identifies a novel anti-Chlamydia ceramide derivative with altered intracellular localization. Antimicrobial agents and chemotherapy\| PubMed ID: 25001308	2014
Visualization of HIV-1 budding structures. AIDS research and human retroviruses\| PubMed ID: 25172012	2014
A simple procedure to analyze positions of interest in infectious cell cultures by correlative light and electron microscopy. Methods in cell biology\| PubMed ID: 25287838	2014
Three-Dimensional Imaging of CD63 Recruitment at the Virological Synapse: HIV-1. AIDS research and human retroviruses\| PubMed ID: 25870000	2015
Improvement of Biological Indicators by Uniformly Distributing Bacillus subtilis Spores in Monolayers To Evaluate Enhanced Spore Decontamination Technologies. Applied and environmental microbiology\| PubMed ID: 26801572	2016
Identification of Differentially Expressed Genes during Bacillus subtilis Spore Outgrowth in High-Salinity Environments Using RNA Sequencing. Frontiers in microbiology\| PubMed ID: 27766092	2016

Kazimierz Madela

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