To understand the characteristics of bacterial viability and diversity in landscape waters replenished with reclaimed water, the typical landscape lake using reclaimed water was investigated in this study. Samples were collected from a reclaimed water inlet (P1), a reclaimed water distribution outlet (P2), and a landscape lake replenished by reclaimed water (P3). By means of measuring adenosine triphosphate (ATP), flow cytometry (FCM), and 16S rRNA gene high-throughput sequencing, the bacterial viability and diversity in reclaimed water distribution system and landscape lake were illustrated. The bacterial ATP contents at P1, P2, and P3 were 3.55 ± 1.79 ng/L, 3.31 ± 1.43 ng/L, and 18.97 ± 6.39 μg/L, and the intact bacterial cell concentrations were 5.91 ± 0.52 × 104 cells/mL, 7.95 ± 2.58 × 104 cells/mL, and 5.65 ± 2.10 × 106 cells/mL, respectively. These results indicated a significant increase of bacterial viability in the landscape lake. The Shannon diversity index of 6.535, 7.05, and 6.886 at P1, P2, and P3, respectively, demonstrated no notable change of bacterial diversity from reclaimed water distribution system to landscape lake. However, the relative abundance of Pseudomonas sp. at P3 was significantly higher than that at P1. These findings indicated that viable but non-culturable (VBNC) bacteria could be revived in the landscape lake. The bacterial viability during reclaimed water reuse should deserve special attention.

The mathematical models used in predictive microbiology contain parameters that must be estimated based on experimental data. Due to experimental uncertainty and variability, they cannot be known exactly and must be reported with a measure of uncertainty (usually a standard deviation). In order to increase precision (i.e. reduce the standard deviation), it is usual to add extra sampling points. However, recent studies have shown that precision can also be increased without adding extra sampling points by using Optimal Experiment Design, which applies optimization and information theory to identify the most informative experiment under a set of constraints. Nevertheless, to date, there has been scarce contributions to know a priori whether an experimental design is likely to provide the desired precision in the parameter estimates. In this article, two complementary methodologies to predict the parameter precision for a given experimental design are proposed. Both approaches are based on in silico simulations, so they can be performed before any experimental work. The first one applies Monte Carlo simulations to estimate the standard deviation of the model parameters, whereas the second one applies the properties of the Fisher Information Matrix to estimate the volume of the confidence ellipsoids. The application of these methods to a case study of dynamic microbial inactivation, showing how they can be used to compare experimental designs and assess their precision, is illustrated. The results show that, as expected, the optimal experimental design is more accurate than the uniform design with the same number of data points. Furthermore, it is demonstrated that, for some heating profiles, the uniform design does not ensure that a higher number of sampling points increases precision. Therefore, optimal experimental designs are highly recommended in predictive microbiology.

The growing commercial interest in multi-strain formulations marketed as probiotics has not been accompanied by an equal increase in the evaluation of quality levels of these biotechnological products. The multi-strain product VSL#3 was used as a model to setup a microbiological characterization that could be extended to other formulations with high complexity. Shotgun metagenomics by deep Illumina sequencing was applied to DNA isolated from the commercial VSL#3 product to confirm strains identity safety and composition. Single-cell analysis was used to evaluate the cell viability, and β-galactosidase and urease activity have been used as marker to monitor the reproducibility of the production process. Similarly, these lots were characterized in detail by a metaproteomics approach for which a robust protein extraction protocol was combined with advanced mass spectrometry. The results identified over 1600 protein groups belonging to all strains present in the VSL#3 formulation. Of interest, only 3.2 % proteins showed significant differences mainly related to small variations in strain abundance. The protocols developed in this study addressed several quality criteria that are relevant for marketed multi-strain products and these represent the first efforts to define the quality of complex probiotic formulations such as VSL#3.

The interest towards not-dairy beverages is increasing and almond milk is widespread diffused. The main topic of this paper was a focus on Ultrasound (US) to inhibit Escherichia coli O157:H7 and Listeria monocytogenes. The variables of the treatment (power, duration, and pulse) were combined through a randomized design; the use of DoE theory (Design of Experiments) and its outputs (Pareto chart, 3D plots, desirability profiles) pointed out that the effect of the treatment relies upon the total energy distributed into the system on E. coli O157:H7, as suggested by the significance of interactions of power, pulse and time, while power was the most important factor for L. monocytogenes. A final challenge test was done by using two combinations (H-80% of power, 8 min and pulse at 6 s- for E. coli and F-80%; 2 min; 6 s- for L. monocytogenes) and storing the samples at 4 °C for 2 weeks. This experiment suggests that the treatment could exert a sub-lethal injury on the pathogens, which, combined with the storage under refrigeration, could contribute to increase the shelf life.

The alarming occurrence of antibiotic resistance genes in food production demands continuous monitoring worldwide. One reservoir of resistance genes is thought to be eDNA. There is currently little available information in Europe about either the extracellular DNA distribution of the bacterium or the spread of resistance genes in L. monocytogenes. Therefore, our aims were to give insight into the Listeria monocytogenes resistance situation in the Czech Republic and assess the presence of resistance genes in their extracellular DNA (eDNA). First, susceptibility tests were performed on 49 isolates of L. monocytogenes with selected antibiotics. Next, we tested DNA of suspected isolates for the presence of resistance genes in both planktonic cells and the eDNA of biofilms. Finally, fluorescent confocal microscopy was used to observe the eDNA pattern of selected isolates under conditions that mimicked the food processing environment and the human body. Susceptibility tests found isolates intermediate resistant to chloramphenicol, tetracycline, and ciprofloxacin as well as isolates resistant to ciprofloxacin. For all suspected isolates, PCR confirmed the presence of the gene lde encoding efflux pump in both types of DNA. When the biofilm was observed using confocal laser scanning microscope, the eDNA distribution patterns varied considerably according to the culture conditions. Furthermore, the food and clinical isolates varied in terms of the amount of eDNA detected. The presence of an efflux pump in both types of DNA suggests that the eDNA might serve as a reservoir of resistance genes. Surprising differences were observed in the eDNA pattern. Our results suggest that the current risk of the spread of L. monocytogenes resistance genes is low in the Czech Republic, but they also indicate the need for continuous long-term monitoring of the situation.

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Transcription factors control the expression of genes by binding to specific sites in DNA and repressing or activating transcription in response to stimuli. The lac repressor (LacI) is a well characterized transcription factor that regulates the ability of bacterial cells to uptake and metabolize lactose. Here, we study the intracellular mobility and spatial distribution of LacI in live bacteria using photoactivated localization microscopy combined with single-particle tracking. Since we track single LacI molecules in live cells by stochastically photoactivating and observing fluorescent proteins individually, there are no limitations on the copy number of the protein under study; as a result, we were able to study the behavior of LacI in bacterial strains containing the natural copy numbers (∼40 monomers), as well as in strains with much higher copy numbers due to LacI overexpression. Our results allowed us to determine the relative abundance of specific, near-specific, and non-specific DNA binding modes of LacI in vivo, showing that all these modes are operational inside living cells. Further, we examined the spatial distribution of LacI in live cells, confirming its specific binding to lac operator regions on the chromosome; we also showed that mobile LacI molecules explore the bacterial nucleoid in a way similar to exploration by other DNA-binding proteins. Our work also provides an example of applying tracking photoactivated localization microscopy to studies of low-copy-number proteins in living bacteria.

The current research was focused on the characterization and antimicrobial activity of silver nanoparticles (AgNPs) produced by Bacillus licheniformis NM120-17. The synthesis was initially observed by a colour change from pale yellow to brown which was further confirmed by UV-Vis spectroscopy. The AgNPs were characterized using TEM, EDAX and FTIR. The synthesized nanoparticles were found to be spherical and uniformly distributed with a size in the range of 9-27.5 nm. The antibacterial activities and acting mechanism of AgNPs were studied with respect to Staphylococcus aureus and Escherichia coli by measuring the growth curves, protein and reducing sugar leakage, respiratory chain dehydrogenase activity, as well as the formation of bactericidal reactive oxygen species (ROS). The experimental results indicated that 50 mg/ml AgNPs could completely inhibit the growth of bacterial cells and destroy the permeability of bacterial membranes and depress the activity of some membranous enzymes, which cause bacteria to die eventually. These nontoxic nanomaterials, which can be prepared in a simple and cost-effective manner, may be suitable for the formulation of new types of bactericidal materials.

Bacterial metabolites with communicative functions could provide protection against stress conditions to members of the same species. Yet, information remains limited about protection provided by metabolites in Bacillus cereus and inter-species. This study investigated the effect of extracellular compounds derived from heat shocked (HS) and non-HS cultures of B. cereus and Geobacillus stearothermophilus on the thermotolerance of non-HS vegetative and sporulating B. cereus. Cultures of B. cereus and G. stearothermophilus were subjected to HS (42 or 65 °C respectively for 30 min) or non-HS treatments. Cells and supernatants were separated, mixed in a combined array, and then exposed to 50 °C for 60 min and viable cells determined. For spores, D values (85 and 95 °C) were evaluated after 120 h. In most cases, supernatants from HS B. cereus cultures added to non-HS B. cereus cells caused their thermotolerance to increase (D 50 12.2-51.9) when compared to supernatants from non-HS cultures (D 50 7.4-21.7). While the addition of supernatants from HS and non-HS G. stearothermophilus cultures caused the thermotolerance of non-HS cells from B. cereus to decrease initially (D 50 3.7-7.1), a subsequent increase was detected in most cases (D 50 18-97.7). In most cases, supernatants from sporulating G. stearothermophilus added to sporulating cells of B. cereus caused the thermotolerance of B. cereus 4810 spores to decline, whereas that of B. cereus 14579 increased. This study clearly shows that metabolites in supernatants from either the same or different species (such as G. stearothermophilus) influence the thermotolerance of B. cereus.

We measured the minimum inhibitory concentration (MIC) of the antimicrobial peptide pexiganan acting on Escherichia coli , and found an intrinsic variability in such measurements. These results led to a detailed study of the effect of pexiganan on the growth curve of E. coli, using a plate reader and manual plating (i.e. time-kill curves). The measured growth curves, together with single-cell observations and peptide depletion assays, suggested that addition of a sub-MIC concentration of pexiganan to a population of this bacterium killed a fraction of the cells, reducing peptide activity during the process, while leaving the remaining cells unaffected. This pharmacodynamic hypothesis suggests a considerable inoculum effect, which we quantified. Our results cast doubt on the use of the MIC as \'a measure of the concentration needed for peptide action\' and show how \'coarse-grained\' studies at the population level give vital information for the correct planning and interpretation of MIC measurements.