Single-molecule localisation microscopy: accounting for chance co-localisation between foci in bacterial cells

Using single-molecule fluorescence microscopes, individual biomolecules can be observed within live bacterial cells. Using differently coloured probes, physical associations between two different molecular species can be assessed through co-localisation measurements. However, bacterial cells are finite and small (~ 1 μm) relative to the resolution limit of optical microscopes (~ 0.25 μm). Furthermore, the images produced by optical microscopes are typically two-dimensional projections of three-dimensional objects. These limitations mean that a certain proportion of object pairs (molecules) will inevitably be assigned as being co-localised, even when they are distant at molecular distance scales (nm). What is this proportion? Here, we attack this problem, theoretically and computationally, by creating a model of the co-localisation expected purely due to chance. We thus consider a bacterial cell wherein objects are distributed at random and evaluate the co-localisation in a fashion that emulates an experimental analysis. We consider simplified geometries where we can most transparently investigate the effect of a finite size of the cell and the effect of probing a three-dimensional cell in only two dimensions. Coupling theory to simulations, we also study the co-localisation expected due to chance using parameters relevant to bacterial cells. Overall, we show that the co-localisation expected purely due to chance can be quite substantial and describe the parameters that it depends upon.

Removal of aflatoxin b1 and t-2 toxin by bacteria isolated from commercially available probiotic dairy foods

This study isolated lactic acid bacteria from commercially available probiotic foods to determine their capacity to remove aflatoxin B1 (AFB1) and trichothecene-2 (T-2). The removal rates by original live and heat-treated cells of lactic acid bacteria (LAB) were compared to test the effect of heat treatment on efficacy. LAB is capable to remove up to 46% of AFB1 and up to 45% of T-2 toixn. The toxin removal capability increased as toxin concentration increased despite bacterial cell viability declining. Surprisingly, the denatured LAB removed greater percentages of AFB1 (up to 62%) and T-2 (up to 52%) than live bacterial cells ( P < 0.05), lending support to the hypothesis that there is higher binding of toxins to the cell membrane of nonviable cells. The research provided practical evidences, which suggest that when ingested into the gut biota, LAB could likely reduce absorption of AFB1 and T-2 from contaminated foods.

Measurement of the interconnected turgor pressure and envelope elasticity of live bacterial cells

Explicit expressions are established to extract the turgor pressure and envelope's elastic modulus of a live bacterium from AFM nanoindentation curves. It is found that the two parameters change significantly in different external osmotic conditions.

Packed Like Sardines – How Surface Crowdedness Impacts Accessibility to Peptidoglycan of Staphylococcus aureus

Bacterial cell walls are essential barriers that protect bacteria against the onslaught of potentially lethal molecules from the outside. Small molecule therapeutics, proteins from bacterial foes, and host immune proteins must navigate past a dense layer of bacterial biomacromolecules (e.g., capsular proteins, teichoic acids, and anchored proteins) to reach the peptidoglycan (PG) layer of Gram-positive bacteria. A subclass of molecules (e.g., antibiotics with intracellular targets) must also permeate through the PG (in a molecular sieving manner) to reach the cytoplasmic membrane. In the case of Staphylococcus aureus (S. aureus), teichoic acids are the major biopolymers that decorate bacterial cell surfaces. Despite the biological and therapeutic importance of surface accessibility, systematic analyses in live bacterial cells have been lacking. We describe a novel live cell fluorescence assay that reports on the permeability of molecules to and within the PG scaffold. The assay has robust reproducibility, is readily adoptable to any Gram-positive organism, and is compatible with high-throughput sample processing. Analysis of the factors controlling permeability to S. aureus and the methicillin resistant MRSA revealed that molecular flexibility plays a central role in molecular permeability. Moreover, teichoic acids impeded permeability of molecules of a wide range of sizes and chemical composition.

Electrochemical Evaluation of Bacterial Activity

For the efficient utilization of bacterial bioresources, the quantitative evaluation of metabolic activity in live bacterial cells is required. Using potentiometric measurements, we quantitatively evaluated the electron generation rate of Shewanella oneidensis MR-1 based on individual enzymatic reactions. We evaluated intracellular electron generation in bacterial suspensions supplemented with different carbon sources utilized in the tricarboxylic acid cycle. In bacterial suspensions, ferricyanide was almost completely reduced to ferrocyanide by cell-generated electrons, without an effect on bacterial cell viability. Focusing on this reduction reaction, quantitative evaluations were possible by potentiometry based on the Nernst equation.

Proteomic and Bioinformatic Analysis of Streptococcus suis Human Isolates: Combined Prediction of Potential Vaccine Candidates

Streptococcus suis is a Gram-positive bacterium responsible for major infections in pigs and economic losses in the livestock industry, but also an emerging zoonotic pathogen causing serious diseases in humans. No vaccine is available so far against this microorganism. Conserved surface proteins are among the most promising candidates for new and effective vaccines. Until now, research on this pathogen has focused on swine isolates, but there is a lack of studies to identify and characterize surface proteins from human clinical isolates. In this work, we performed a comparative proteomic analysis of six clinical isolates from human patients, all belonging to the major serotype 2, by “shaving” the live bacterial cells with trypsin, followed by LC-MS/MS analysis. We identified 131 predicted surface proteins and carried out a label-free semi-quantitative analysis of protein abundances within the six strains. Then, we combined our proteomics results with bioinformatic tools to help improving the selection of novel antigens that can enter the pipeline of vaccine candidate testing. Our work is then a complement to the reverse vaccinology concept.

Real-time monitoring and inhibition of the activity of carbapenemase in live bacterial cells: application to screening of β-lactamase inhibitors

Antibiotic resistance mediated by β-lactamases including metallo-β-lactamases (MβLs) has become an emerging threat.

Artificial cell microcapsules containing live bacterial cells and activated charcoal for managing renal failure creatinine: preparation and in-vitro analysis

Activated charcoal was microencapsulated with Lactobacillus acidophilus 314 previously adapted for urea uptake. The creatinine removal capacity of this combination microcapsule was evaluated in-vitro in media simulating the small intestine. Results show that microcapsules containing both activated charcoal and L. acidophilus 314 demonstrated potential for decreasing creatinine. Interestingly, when co-encapsulating both activated charcoal and L. acidophilus 314 a smaller decrease in creatinine was observed than when encapsulating them separately. However, co-encapsulated microcapsules were more stable in various parts of the gastrointestinal system and survived longer in storage. These results suggest the feasibility of using microcapsules containing activated charcoal and probiotic bacteria as oral adjuvants for creatinine removal and provides a theoretical model for the use of these microcapsules to remove any unwanted metabolite.

Dead bacterial absorption of antimicrobial peptides underlies collective tolerance

The collective tolerance towards antimicrobial peptides (APs) is thought to occur primarily through mechanisms associated with live bacterial cells. In contrast to the focus on live cells, we discover that the LL37 antimicrobial peptide kills a subpopulation of Escherichia coli , forming dead cells that absorb the remaining LL37 from the environment. Combining mathematical modelling with population and single-cell experiments, we show that bacteria absorb LL37 at a timing that coincides with the permeabilization of their cytoplasmic membranes. Furthermore, we show that one bacterial strain can absorb LL37 and protect another strain from killing by LL37. Finally, we demonstrate that the absorption of LL37 by dead bacteria can be reduced using a peptide adjuvant. In contrast to the known collective tolerance mechanisms, we show that the absorption of APs by dead bacteria is a dynamic process that leads to emergent population behaviour.