Modification of bacterial cells for in vivo remotely guided systems.

It was shown recently that bacterial strains, which can act specifically against malignant cells, can be used efficiently in cancer therapy. Many appropriate bacterial strains are either pathogenic or invasive and there is a substantial shortage of methods with which to monitor in vivo the distribution of bacteria used in this way. Here, it is proposed to use a Layer-by-Layer (LbL) approach that can encapsulate individual bacterial cells with fluorescently labeled polyelectrolytes (PE)s and magnetite nanoparticles (NP)s. The NP enable remote direction in vivo to the site in question and the labeled shells in the far-red emission spectra allow non-invasive monitoring of the distribution of bacteria in the body. The magnetic entrapment of the modified bacteria causes the local concentration of the bacteria to increase by a factor of at least 5. The PEs create a strong barrier, and it has been shown in vitro experiments that the division time of bacterial cells coated in this way can be regulated, resulting in control of their invasion into tissues. That animals used in the study survived and did not suffer septic shock, which can be attributed to PE capsules that prevent release of endotoxins from bacterial cells.

Isolation, Identification, and Selection of Bacteria With Proof-of-Concept for Bioaugmentation of Whitewater From Wood-Free Paper Mills.

In the wood-free paper industry, whitewater is usually a mixture of additives for paper production. We are currently lacking an efficient, cost-effective purification technology for their removal. In closed whitewater cycles the additives accumulate, causing adverse production problems, such as the formation of slime and pitch. The aim of our study was to find an effective bio-based strategy for whitewater treatment using a selection of indigenous bacterial isolates. We first obtained a large collection of bacterial isolates and then tested them individually by simple plate and spectrophotometric methods for their ability to degrade the papermaking additives, i.e., carbohydrates, resin acids, alkyl ketene dimers, polyvinyl alcohol, latex, and azo and fluorescent dyes. We examined correlation between carbon source use, genera, and inoculum source of isolates using two multivariate methods: principal component analysis and FreeViz projection. Of the 318 bacterial isolates, we selected a consortium of four strains (Xanthomonadales bacterium sp. CST37-CF, Sphingomonas sp. BLA14-CF, Cellulosimicrobium sp. AKD4-BF and Aeromonas sp. RES19-BTP) that degrade the entire spectrum of tested additives by means of dissolved organic carbon measurements. A proof-of-concept study on a pilot scale was then performed by immobilizing the artificial consortium of the four strains and inserting them into a 33-liter, tubular flow-through reactor with a retention time of < 15 h. The consortium caused an 88% reduction in the COD of the whitewater, even after 21 days.

Thickness of Polyelectrolyte Layers of Separately Confined Bacteria Alters Key Physiological Parameters on a Single Cell Level.

Confinement of bacterial cells in a matrix or in capsules is an integral part of many biotechnological applications. Here, the well-known layer-by-layer method of deposition of a polyelectrolyte film a few nanometers in thickness to confine separated bacterial cells in permeable and physically durable shells has been examined. Due to the physical properties of such a confinement, we found that this method enables investigation of effects of physical barriers against mass gain and cell division. Using the method of time-lapse confocal microscopy, we observed a prolonged lag phase, dependent on the number of polyelectrolyte layers. In the confinement, both the GFP fluorescent signal from the leaking T7 promoter and the cell size were increased by factors of more than five and two, respectively. This creates a paradigm shift that enables use of mechanical entrapment for control of bacterial cell physiology and opens possibilities of controlling the division rate as well as gene expression. These effects can be attributed to the perturbation of the sensing of the cell size, which results in disproportional synthesis of a cell envelope impinging the intracellular material and compels cells to grow rapidly. In addition, the charged surface of cells enables prolonged intercellular physical interaction and results in spherically shaped microcolonies.

Medium-Dependent Antibacterial Properties and Bacterial Filtration Ability of Reduced Graphene Oxide.

Toxicity of reduced graphene oxide (rGO) has been a topic of multiple studies and was shown to depend on a variety of characteristics of rGO and biological objects of interest. In this paper, we demonstrate that when studying the same dispersions of rGO and fluorescent Escherichia coli (E. coli) bacteria, the outcome of nanotoxicity experiments also depends on the type of culture medium. We show that rGO inhibits the growth of bacteria in a nutrition medium but shows little effect on the behavior of E. coli in a physiological saline solution. The observed effects of rGO on E. coli in different media could be at least partially rationalized through the adsorption of bacteria and nutrients on the dispersed rGO sheets, which is likely mediated via hydrogen bonding. We also found that the interaction between rGO and E. coli is medium-dependent, and in physiological saline solutions they form stable flocculate structures that were not observed in nutrition media. Furthermore, the aggregation of rGO and E. coli in saline media was observed regardless of whether the bacteria were alive or dead. Filtration of the aggregate suspensions led to nearly complete removal of bacteria from filtered liquids, which highlights the potential of rGO for the filtration and separation of biological contaminants, regardless of whether they include live or dead microorganisms.

Effect of GO on bacterial cells: Role of the medium type and electrostatic interactions.

Graphene and graphene oxide (GO) both being two-dimensional materials are gaining popularity among researchers as a promising nanomaterial for various medical and biological applications. The aim of this study is to elucidate the influence of nanostructured GO sheets on viability of a model species of gram-negative E. coli bacteria transformed with pRSET-emGFP plasmid in in vitro experiments. It was shown that GO at concentrations between 0.0025 and 2.5 g/l in growth medium inhibits growth of bacterial colonies, while in physiological saline solution (PS) this effect decreases dramatically to the point of complete disappearance. It was shown that in order to obtain a pronounced antibacterial effect one needs to introduce high concentrations of GO into the media (up to 2.5 g/l), which can be important for development of antibacterial materials for biomedical applications. Some of the obtained data provide clear evidence to electrostatic nature of interaction between bacterial and GO sheets. A number of previous papers suggested the process of biofilms formation by bacteria as the primary reason for aggregation between graphene-like materials and bacterial cells. However, formation of flocculent structures consisting of GO and dead bacteria and accompanied with decrease in zeta-potential of particles in the suspension to 18 mV proves that electrostatic interactions play the major role in aggregation. The obtained data can be used for employing GO and similar materials in new systems for water-purification from biological contaminants. Besides, our results stress the importance of accounting for the conditions in which goods and coatings containing graphene-like materials as an antibacterial agent are used, as well as unification of the experimental conditions.