The Bead Assay for Biofilms: A Quick, Easy and Robust Method for Testing Disinfectants

Bacteria live primarily in microbial communities (biofilms), where they exhibit considerably higher biocide tolerance than their planktonic counterparts. Current standardized efficacy testing protocols of disinfectants, however, employ predominantly planktonic bacteria. In order to test the efficacy of biocides on biofilms in a standardized manner, a new assay was developed and optimized for easy-handling, quickness, low running costs, and above all—repeatability. In this assay, 5 mm glass- or polytetrafluoroethylene beads in 24 well microtiter plates served as substrate for Pseudomonas aeruginosa biofilms. After optimizing result-relevant steps, the actual performance of the assay was explored by treating P. aeruginosa biofilms with glutaraldehyde, isopropanol, or peracetic acid in predefined concentrations. The aspired 5 log10 reduction in CFU counts was achieved by glutaraldehyde at 5% (30 min), and by peracetic acid at 0.3% (10 min). In contrast, 80% isopropanol (30 min) failed to meet the reduction goal. However, the main accomplishment of this study was to unveil the potential of the array itself; most noteworthy here, a reliable repeatability of the results. The new bead assay for biofilms is a robust, quick and cost-effective method for assessing the efficacy of biocides against biofilms

Intracellular membranes of bacterial endospores are reservoirs for spore core membrane expansion during spore germination

Bacterial endospores are formed by certain bacteria, such as Bacillus subtilis or the pathogenic Bacillus anthracis and Clostridioides difficile, to allow survival in environmental conditions which are lethal to vegetative bacteria. The spores possess a particular architecture and molecular inventory which endow them with a remarkable resistance against desiccation, heat and radiation. Another remarkable spore feature is their rapid return to vegetative growth during spore germination and outgrowth. The underlying processes of this latter physiological and morphological transformation involve a number of different events, some of which are mechanistically not entirely understood. One of these events is the expansion of the central spore core, which contains the DNA, RNA and most spore enzymes. To date, it has been unclear how the ~1.3- to 1.6-fold expansion of the core membrane surface area that accompanies core expansion takes place, since this occurs in the absence of significant if any ATP synthesis. In the current work, we demonstrate the presence of intracellular membrane structures in spores located just below the core membrane. During spore germination these internal core membranes disappear when the core size increases, suggesting that they are integrated into the core membrane to allow core expansion. These intracellular membranes are most probably present as more or less compressed vesicles or tubules within the dormant spore core. Investigations of spores from different species suggest that these intracellular membrane structures below the core membrane are a general feature of endospore forming bacteria.Peer Reviewe

Dormant Bacillus spores protect their DNA in crystalline nucleoids against environmental stress

Bacterial spores of the genera Bacillus and Clostridium are extremely resistant against desiccation, heat and radiation and involved in the spread and pathogenicity of health relevant species such as Bacillus anthracis (anthrax) or Clostridium botulinum. While the resistance of spores is very well documented, underlying mechanisms are not fully understood. In this study we show, by cryo-electron microscopy of vitreous sections and particular resin thin section electron microscopy, that dormant Bacillus spores possess highly ordered crystalline core structures, which contain the DNA, but only if small acid soluble proteins (SASPs) are present. We found those core structures in spores of all Bacillus species investigated, including spores of anthrax. Similar core structures were detected in Geobacillus and Clostridium species which suggest that highly ordered, at least partially crystalline core regions represent a general feature of bacterial endospores. The crystalline core structures disintegrate in a period during spore germination, when resistance against most stresses is lost. Our results suggest that the DNA is tightly packed into a crystalline nucleoid by binding SASPs, which stabilizes DNA fibrils and protects them against modification. Thus, the crystalline nucleoid seems to be the structural and functional correlate for the remarkable stability of the DNA in bacterial endospores

Comparison of the Declared Nutrient Content of Plant-Based Meat Substitutes and Corresponding Meat Products and Sausages in Germany

Plant-based meat substitutes (PBMS) are becoming increasingly popular due to growing concerns about health, animal welfare, and environmental issues associated with animal-based foods. The aim of this study was to compare the declared energy and nutrient contents of PBMS with corresponding meat products and sausages available on the German market. Mandatory nutrition labelling data of 424 PBMS and 1026 meat products and sausages, surveyed in 2021 and 2020, respectively, as part of the German national monitoring of packaged food were used to test for differences in energy and nutrient contents. Principal component analysis (PCA) was used to describe characteristics in the energy and nutrient contents. The comparison showed that most of the PBMS subcategories had significantly lower contents of fat and saturated fat but higher contents of carbohydrate and sugar than corresponding meat subcategories. For salt, the only striking difference was that PBMS salamis had lower salt content than meat salamis. Overall, the PCA revealed protein as a main characteristic for most PBMS categories, with the protein content being equivalent to or, in most protein-based PBMS, even higher than in the corresponding meat products. The wide nutrient content ranges within subcategories, especially for salt, reveal the need and potential for reformulation

Comparison of the bead assay, the MBEC^™ assay and the CDC biofilm reactor.

<p>Comparison of the bead assay, the MBEC^™ assay and the CDC biofilm reactor.</p

Assay standardization.

<p>(a) CFU counts of 3 and 5 mm glass beads. (b) CFU counts of 5 mm glass and polytetrafluoroethylene (PTFE) beads. (c) CFU counts of 5 mm glass beads cultivated on shakers with 8 and 25 mm orbit (The horizontal lines through the data points represent mean and standard deviation).</p

Microscopic characterization.

<p>(a) SEM image: overview on a glass bead after 24 h cultivation with <i>P</i>. <i>aeruginosa</i>. (b) The bead surface is evenly covered with biofilm. (c) The bacteria are densely arranged in a monolayer. (d) Overview on a glass bead after the biofilm had been removed by sonication. (e, f) The bead surface is virtually empty, except for some residual debris. (g) CLSM image: The sugar-matrix of the <i>P</i>. <i>aeruginosa</i> biofilm was stained with Concanavalin A (assigned color: magenta), and the bacteria with Syto60 (assigned color: green). (h) LIVE/DEAD^® staining of the biofilm on a glass bead. (i) LIVE/DEAD^® staining after the biofilm had been removed from the bead by sonication.</p

Testing for repeatability.

<p>CFU counts of three independent experiments with untreated biofilm on glass beads. (The horizontal lines through the data points represent mean and standard deviation).</p

Reduction of planktonic bacteria and biofilm in comparison.

<p>(a) CFU counts after 30 min glutaraldehyde treatment of planktonic bacteria of <i>P</i>. <i>aeruginosa</i>. (b) Reduction after 30 min glutaraldehyde treatment of <i>P</i>. <i>aeruginosa</i> biofilm and planktonic bacteria (<b>O</b>: biofilm (mean of three independent experiments); <b>Δ</b>: planktonic (mean of three independent experiments); dashed line: 5log₁₀ reduction goal).</p

Disinfectant efficacy testing on biofilm.

<p>(a) CFU counts after 30 min isopropanol treatment of <i>P</i>. <i>aeruginosa</i> biofilm. (b) CFU counts after 10 min peracetic acid treatment of <i>P</i>. <i>aeruginosa</i> biofilm. (c) CFU counts after 30 min glutaraldehyde treatment of <i>P</i>. <i>aeruginosa</i> biofilm. (The horizontal lines through the data points represent mean and standard deviation).</p