Heteroaggregation of virions and microplastics reduces the number of active bacteriophages in aqueous environments.

The objective of this study is to explore the effects of microplastics on the viability of the bacteriophages in an aqueous environment. Bacteriophages (phages), that is, viruses of bacteria, are essential in homeostasis. It is estimated that phages cause up to 40% of the death of all bacteria daily. Any factor affecting phage activity is vital for the whole food chain and the ecology of numerous niches. We hypothesize that the number of active phages decreases due to the virions' adsorption on microplastic particles or by the released leachables from additives used in the production of plastic, for example, stabilizers, plasticizers, colorants, and reinforcements. We exposed three diverse phages, namely, T4 (tailed), MS2 (icosahedral), and M13 (filamentous), to 1 mg/mL suspension of 12 industrial-grade plastics [acrylonitrile butadiene styrene, high-impact polystyrene, poly-ε-caproamide, polycarbonate, polyethylene, polyethylene terephthalate, poly(methyl methacrylate), polypropylene, polystyrene, polytetrafluoroethylene, polyurethane, and polyvinyl chloride] shredded to obtain microparticles of radius ranging from 2 to 50 µm. The effect of leachables was measured upon exposure of phages not to particles themselves but to the buffer preincubated with microplastics. A double-overlay plaque counting method was used to assess phage titers. We employed a classical linear regression model to verify which physicochemical parameters (65 variables were tested) govern the decrease of phage titers. The key finding is that adsorption mechanisms result in up to complete scavenging of virions, whereas leachables deactivate up to 50% of phages. This study reveals microplastic pollution's plausible and unforeseen ecotoxicological effect causing phage deactivation. Moreover, phage transmission through adsorption can alter the balance of the food chain in the new environment. The effect depends mainly on the zeta potentials of the polymers and the phage type.

Peptide-Grafted Nontoxic Cyclodextrins and Nanoparticles against Bacteriophage Infections.

One of the biggest threats for bacteria-based bioreactors in the biotechnology industry is infections caused by bacterial viruses called bacteriophages. More than 70% of companies admitted to encountering this problem. Despite phage infections being such a dangerous and widespread risk, to date, there are no effective methods to avoid them. Here we present a peptide-grafted compounds that irreversibly deactivate bacteriophages and remain safe for bacteria and mammalian cells. The active compounds consist of a core (cyclodextrin or gold nanoparticle) coated with a hydrophobic chain terminated with a peptide selective for bacteriophages. Such peptides were selected via a phage display technique. This approach enables irreversible deactivation of the wide range of T-like phages (including the most dangerous in phage infections, phage T1) at 37 °C in 1 h. We show that our compounds can be used directly inside the environment of the bioreactor, but they are also a safe additive to stocks of antibiotics and expression inducers (such as isopropyl ß-d-1-thiogalactopyranoside, i.e., IPTG) that cannot be autoclaved and are a common source of phage infections.

The Effect of Zero-Valent Iron Nanoparticles (nZVI) on Bacteriophages.

Bacteriophages are viruses that attack and usually kill bacteria. Their appearance in the industrial facilities using bacteria to produce active compounds (e.g., drugs, food, cosmetics, etc.) causes considerable financial losses. Instances of bacteriophage resistance towards disinfectants and decontamination procedures (such as thermal inactivation and photocatalysis) have been reported. There is a pressing need to explore new ways of phage inactivation that are environmentally neutral, inexpensive, and more efficient. Here, we study the effect of zero-valent iron nanoparticles (nZVI) on four different bacteriophages (T4, T7, MS2, M13). The reduction of plaque-forming units (PFU) per mL varies from greater than 7log to around 0.5log depending on bacteriophages (M13 and T7, respectively). A comparison of the importance of oxidation of nZVI versus the release of Fe2+/Fe3+ ions is shown. The mechanism of action is proposed in connection to redox reactions, adsorption of virions on nZVI, and the effect of released iron ions. The nZVI constitutes a critical addition to available antiphagents (i.e., anti-bacteriophage agents).

Adsorption of bacteriophages on polypropylene labware affects the reproducibility of phage research.

Hydrophobicity is one of the most critical factors governing the adsorption of molecules and objects, such as virions, on surfaces. Even moderate change of wetting angle of plastic surfaces causes a drastic decrease ranging from 2 to 5 logs of the viruses (e.g., T4 phage) in the suspension due to adsorption on polymer vials' walls. The effect varies immensely in seemingly identical containers but purchased from different vendors. Comparison of glass, polyethylene, polypropylene, and polystyrene containers revealed a threshold in the wetting angle of around 95°: virions adsorb on the surface of more hydrophobic containers, while in more hydrophilic vials, phage suspensions are stable. The polypropylene surface of the Eppendorf-type and Falcon-type can accommodate from around 108 PFU/ml to around 1010 PFU/ml from the suspension. The adsorption onto the container's wall might result in complete scavenging of virions from the bulk. We developed two methods to overcome this issue. The addition of surfactant Tween20 and/or plasma treatment provides a remedy by modulating surface wettability and inhibiting virions' adsorption. Plastic containers are essential consumables in the daily use of many bio-laboratories. Thus, this is important not only for phage-related research (e.g., the use of phage therapies as an alternative for antibiotics) but also for data comparison and reproducibility in the field of biochemistry and virology.

Engineering Protein Nanoparticles Out from Components of the Human Microbiome.

Nanoscale protein materials are highly convenient as vehicles for targeted drug delivery because of their structural and functional versatility. Selective binding to specific cell surface receptors and penetration into target cells require the use of targeting peptides. Such homing stretches should be incorporated to larger proteins that do not interact with body components, to prevent undesired drug release into nontarget organs. Because of their low interactivity with human body components and their tolerated immunogenicity, proteins derived from the human microbiome are appealing and fully biocompatible building blocks for the biofabrication of nonreactive, inert protein materials within the nanoscale. Several phage and phage-like bacterial proteins with natural structural roles are produced in Escherichia coli as polyhistidine-tagged recombinant proteins, looking for their organization as discrete, nanoscale particulate materials. While all of them self-assemble in a variety of sizes, the stability of the resulting constructs at 37 °C is found to be severely compromised. However, the fine adjustment of temperature and Zn2+ concentration allows the formation of robust nanomaterials, fully stable in complex media and under physiological conditions. Then, microbiome-derived proteins show promise for the regulatable construction of scaffold protein nanomaterials, which can be tailored and strengthened by simple physicochemical approaches.

Efficiency of induction of Shiga-toxin lambdoid prophages in Escherichia coli due to oxidative and antibiotic stress depends on the combination of prophage and the bacterial strain.

In the study presented here, we tested, how large a fraction of lysogenic culture was undergoing filamentation, which could indicate triggering of the SOS response or SOS-independent prophage induction that is also known to cause cell filamentation. Here, antibiotic stress was triggered by adding mitomycin C and oxidative stress was induced by hydrogen peroxide. Observation of bacterial cells under an optical microscope revealed more filamenting cells for lysogenic Escherichia coli than for strains not carrying a prophage. Moreover, the amount of filamenting cells depended not only on the stress agents used and the type of the prophage, but also on the host. During induction of the 933W prophage, the resulting phage titer and the amount of elongating cells were different when using E. coli O157:H7 EDL933 clinical isolate and the E. coli MG1655 laboratory strain. The amount of filamenting cells correlates well with the observed phage titers.

Parallel splitting solvers for the isogeometric analysis of the Cahn-Hilliard equation.

Modeling tumor growth in biological systems is a challenging problem with important consequences for diagnosis and treatment of various forms of cancer. This growth process requires large simulation complexity due to evolving biological and chemical processes in living tissue and interactions of cellular and vascular constituents in living organisms. Herein, we describe with a phase-field model, namely the Cahn-Hilliard equation the intricate interactions between the tumors and their host tissue. The spatial discretization uses highly-continuous isogeometric elements. For fast simulation of the time-dependent Cahn-Hilliard equation, we employ an alternating directions implicit methodology. Thus, we reduce the original problems to Kronecker products of 1 D matrices that can be factorized in a linear computational cost. The implementation enables parallel multi-core simulations and shows good scalability on shared-memory multi-core machines. Combined with the high accuracy of isogeometric elements, our method shows high efficiency in solving the Cahn-Hilliard equation on tensor-product meshes.

Phenotypic plasticity of Escherichia coli upon exposure to physical stress induced by ZnO nanorods.

Evolution of bacteria to selective chemical pressure (e.g. antibiotics) is well studied in contrast to the influence of physical stressors. Here we show that instantaneous physical stress in a homogeneous environment (without concentration gradient) induces fast adaptation of Escherichia coli. We exposed E. coli to a large number of collisions of around 105 per bacterium per second with sharp ZnO nanorods. The pressure exerted on the bacterial cell wall was up to 10 GPa and induced phenotype changes. The bacteria's shape became more spherical, the density of their periplasm increased by around 15% and the average thickness of the cell wall by 30%. Such E. coli cells appeared almost as Gram-positive bacteria in the standard Gram staining. Additionally, we observed a combination of changes occurring at the genomic level (mutations identified in form of single nucleotide polymorphisms) and down-regulation of expression of 61 genes encoding proteins involved in ß-oxidation of fatty acids, glycolysis, the citric acid cycle, as well as uptake of amino acids and enzyme cofactors. Thus, we show that bacteria undergo phenotypic changes upon instantaneous, acute physical stress without any obviously available time for gradual adaptation.

Correction: Sequence, genome organization, annotation and proteomics of the thermophilic, 47.7-kb Geobacillus stearothermophilus bacteriophage TP-84 and its classification in the new Tp84virus genus.

[This corrects the article DOI: 10.1371/journal.pone.0195449.].

Sequence, genome organization, annotation and proteomics of the thermophilic, 47.7-kb Geobacillus stearothermophilus bacteriophage TP-84 and its classification in the new Tp84virus genus.

Bacteriophage TP-84 is a well-characterized bacteriophage of historical interest. It is a member of the Siphoviridae, and infects a number of thermophilic Geobacillus (Bacillus) stearothermophilus strains. Its' 47.7-kbp double-stranded DNA genome revealed the presence of 81 coding sequences (CDSs) coding for polypeptides of 4 kDa or larger. Interestingly, all CDSs are oriented in the same direction, pointing to a dominant transcription direction of one DNA strand. Based on a homology search, a hypothetical function could be assigned to 31 CDSs. No RNA or DNA polymerase-coding genes were found on the bacteriophage genome indicating that TP-84 relies on the host's transcriptional and replication enzymes. The TP84 genome is tightly packed with CDSs, typically spaced by several-to-tens of bp or often overlapping. The genome contains five putative promoter-like sequences showing similarity to the host promoter consensus sequence and allowing for a 2-bp mismatch. In addition, ten putative rho-independent terminators were detected. Because the genome sequence shows essentially no similarity to any previously characterised bacteriophage, TP-84 should be considered a new species in an undefined genus within the Siphoviridae family. Thus a taxonomic proposal of a new Tp84virus genus has been accepted by the International Committee on Taxonomy of Viruses. The bioinformatics genome analysis was verified by confirmation of 33 TP-84 proteins, which included: a) cloning of a selected CDS in Escherichia coli, coding for a DNA single-stranded binding protein (SSB; gene TP84_63), b) purification and functional assays of the recombinant TP-84 SSB, which has been shown to improve PCR reactions, c) mass spectrometric (MS) analysis of TP-84 bacteriophage capsid proteins, d) purification of TP-84 endolysin activity, e) MS analysis of the host cells from infection time course.

First insight into microbial community composition in a phosphogypsum waste heap soil.

The aim of this study was to investigate the soil microbial communities of a phosphogypsum waste heap. The soil microbial community structures can differ over time, as they are affected by the changing environmental conditions caused by a long-term exposure to different kinds of pollutions, like is the case of soil in the post-production waste area in Wislinka (in the northern part of Poland) currently undergoing restoration. Our analyses indicated that the most abundant phyla were Proteobacteria, Acidobacteria, and Actinobacteria, and generally such an abundance is common for most of the studied soils. The most dominant class were Alphaproteobacteria, with their participation in 33.46% of the total reads. Among this class, the most numbered order was Sphingomonadales, whereas among this order the Sphingomonadaceae family was the most abundant one. The Sphingomonadaceae family is currently in the center of interest of many researchers, due to the ability of some of its members to utilize a wide range of naturally occurring organic compounds and many types of environmental contaminants. This kind of knowledge about microbial populations can support efforts in bioremediation and can improve monitoring changes in the contaminated environments.

Dense Layer of Bacteriophages Ordered in Alternating Electric Field and Immobilized by Surface Chemical Modification as Sensing Element for Bacteria Detection.

Faster and more sensitive environmental monitoring should be developed to face the worldwide problem of bacterial infections. To remedy this issue, we demonstrate a bacteria-sensing element that utilizes dense and ordered layers of bacteriophages specific to the given bacteria strain. We combine (1) the chemical modification of a surface to increase the surface coverage of bacteriophages (2) with an alternating electric field to greatly increase the number of properly oriented bacteriophages at the surface. Usually, in sensing elements, a random orientation of bacteriophages results in steric hindrance, which results in no more than a few percent of all receptors being available. An increased number of properly ordered phages results in the optimal performance of phage receptors, manifesting in up to a 64-fold increase in sensitivity and a limit of detection as low as 100 CFU mL-1. Our sensing elements can be applied for selective, sensitive, and fast (15 min) bacterial detection. A well-studied pair T4 bacteriophage-bacteria Escherichia coli, was used as a model; however, the method could be adapted to prepare bacteriophage-based sensors for detection of a variety of bacterial strains.

Bacteriophages as Factories for Eu2O3 Nanoparticle Synthesis.

The use of phage display to identify peptides with an ability to bind and synthesize Eu2O3 nanoparticles is demonstrated in this report. This is the first report of modified phages specifically binding a lanthanide. The peptides exposed on virions revealed very strong binding to Eu2O3 nanoparticles and the ability to catalyze Eu2O3 nanoparticles' formation from Eu(OH)3 and Eu(NO3)3 solutions. The luminescence emission spectrum of Eu3+ ions indicated that these ions existed mostly in sites deviated from the inversion symmetry in crystalline Eu2O3 aggregates and gelatinous Eu(OH)3 precipitate. The ability of phage-displayed peptides to catalyze formation of Eu2O3 nanoparticles provides a useful tool for a low-cost and effective synthesis of lanthanide nanoparticles, which serve as attractive biomedical sensors or fluorescent labels, among their other applications.

The choice of the DNA extraction method may influence the outcome of the soil microbial community structure analysis.

Metagenomics approaches and recent improvements in the next-generation sequencing methods, have become a method of choice in establishing a microbial population structure. Many commercial soil DNA extraction kits are available and due to their efficiency they are replacing traditional extraction protocols. However, differences in the physicochemical properties of soil samples require optimization of DNA extraction techniques for each sample separately. The aim of this study was to compare the efficiency, quality, and diversity of genetic material extracted with the use of commonly used kits. The comparative analysis of microbial community composition, displayed differences in microbial community structure depending on which kit was used. Statistical analysis indicated significant differences in recovery of the genetic material for 24 out of 32 analyzed phyla, and the most pronounced differences were seen for Actinobacteria. Also, diversity indexes and reproducibility of DNA extraction with the use of a given kit, varied among the tested methods. As the extraction protocol may influence the apparent structure of a microbial population, at the beginning of each project many extraction kits should be tested in order to choose one that would yield the most representative results and present the closest view to the actual structure of microbial population.

Bacteriophage-Based Bioconjugates as a Flow Cytometry Probe for Fast Bacteria Detection.

Robust detection of bacteria can significantly reduce risks of nosocomial infections, which are a serious problem even in developed countries (4.1 million cases each year in Europe). Here we demonstrate utilization of novel multifunctional bioconjugates as specific probes for bacteria detection. Bifunctional magnetic-fluorescent microparticles are coupled with bacteriophages. The T4 bacteriophage, due to its natural affinity to bacterial receptors, namely, OmpC and LPS, enables specific and efficient detection of Escherichia coli bacteria. Prepared probes are cheap, accessible (even in nonbiological laboratories), as well as versatile and easily tunable for different bacteria species. The magnetic properties of the bioconjugates facilitate the separation of captured target bacteria from other components of complex samples and other bacteria strains. Fluorescence enables simple analysis. We chose flow cytometry as the detection method as it is fast and widely used for biotests. The capture efficiency of the prepared bioconjugates is close to 100% in the range of bacteria concentrations from tens to around 105 CFU/mL. The limit of detection is restricted by flow cytometry capabilities and in our case was around 104 CFU/mL.

Modified Filamentous Bacteriophage as a Scaffold for Carbon Nanofiber.

With the advent of nanotechnology, carbon nanomaterials such as carbon nanofibers (CNF) have aroused substantial interest in various research fields, including energy storage and sensing. Further improvement of their properties might be achieved via the application of viral particles such as bacteriophages. In this report, we present a filamentous M13 bacteriophage with a point mutation in gene VII (pVII-mutant-M13) that selectively binds to the carbon nanofibers to form 3D structures. The phage-display technique was utilized for the selection of the pVII-mutant-M13 phage from the phage display peptide library. The properties of this phage make it a prospective candidate for a scaffold material for CNFs. The results for binding of CNF by mutant phage were compared with those for maternal bacteriophage (pVII-M13). The efficiency of binding between pVII-mutant-M13 and CNF is about 2 orders of magnitude higher compared to that of the pVII-M13. Binding affinity between pVII-mutant-M13 and CNF was also characterized using atomic force microscopy, scanning electron microscopy, and transmission electron microscopy, which confirmed the specificity of the interaction of the phage pVII-mutant-M13 and the CNF; the binding occurs via the phage's ending, where the mutated pVII protein is located. No similar behavior has been observed for other carbon nanomaterials such as graphite, reduced graphene oxide, single-walled carbon nanotubes, and multiwalled carbon nanotubes. Infrared spectra confirmed differences in the interaction with CNF between the pVII-mutant-M13 and the pVII-M13. Basing on conducted research, we hypothesize that the interactions are noncovalent in nature, with π-π interactions playing the dominant role. Herein, the new bioconjugate material is introduced.

The Arctic Soil Bacterial Communities in the Vicinity of a Little Auk Colony.

Due to deposition of birds' guano, eggshells or feathers, the vicinity of a large seabirds' breeding colony is expected to have a substantial impact on the soil's physicochemical features as well as on diversity of vegetation and the soil invertebrates. Consequently, due to changing physicochemical features the structure of bacterial communities might fluctuate in different soil environments. The aim of this study was to investigate the bacterial assemblages in the Arctic soil within the area of a birds' colony and in a control sample from a topographically similar location but situated away from the colony's impact area. A high number of OTUs found in both areas indicates a highly complex microbial populations structure. The most abundant phyla in both of the tested samples were: Proteobacteria, Acidobacteria, Actinobacteria, and Chloroflexi, with different proportions in the total share. Despite differences in the physicochemical soil characteristics, the soil microbial community structures at the phylum level were similar to some extent in the two samples. The only share that was significantly higher in the control area when compared to the sample obtained within the birds' colony, belonged to the Actinobacteria phylum. Moreover, when analyzing the class level for each phylum, several differences between the samples were observed. Furthermore, lower proportions of Proteobacteria and Acidobacteria were observed in the soil sample under the influence of the bird's colony, which most probably could be linked to higher nitrogen concentrations in that sample.

Phage-Directed Synthesis of Photoluminescent Zinc Oxide Nanoparticles under Benign Conditions.

Biological systems, especially bacteriophages and peptides, are an attractive green alternative to other known methods of nanoparticle synthesis. In this work, for the first time, bacteriophages were employed to synthesize a specific peptide, capable of producing nanoparticles (NPs). Derivatives of M13 bacteriophage exposing a ZnO-binding peptide (TMGANLGLKWPV) on either pIII or pVIII phage coat protein were constructed and used as a biotemplate. The exposition of the ZnO-binding peptide, synthesized by phages during their propagation in bacteria, on M13 virions provided a groundwork for growing ZnO nanostructures. Depending on the recombinant phage type used (M13-pIII-ZnO or M13-pVIII-ZnO), well separated ZnO NPs or complex 3D structures of ZnO NPs of ca. 20-40 nm were synthesized at room temperature. The synthesized ZnO nanoparticles served as a luminescent material that emitted light near the short wavelength end of the visible region (at ca. 400 nm). The next very low intensity emission band at 530 nm demonstrated that the ZnO material obtained is characterized by a low concentration of surface defects.

New Insights into the Microbiota of the Svalbard Reindeer Rangifer tarandus platyrhynchus.

Svalbard reindeer (Rangifer tarandus platyrhynchus) is a non-migratory subspecies of reindeer inhabiting the high-arctic archipelago of Svalbard. In contrast to other Rangifer tarandus subspecies, Svalbard reindeer graze exclusively on natural sources of food and have no chance of ingestion of any crops. We report the use of a non-invasive method for analysis of fecal microbiome by means of sequencing the 16S rDNA extracted from the fecal microbiota of R. tarandus platyrhynchus from a small, isolated population in Hornsund, South Spitsbergen National Park. Analyses of all samples showed that 99% of the total reads were represented by Bacteria. Taxonomy-based analysis showed that fecal bacterial communities consisted of 14 phyla. The most abundant phyla across the population were Firmicutes and Bacteroidetes, and those phyla jointly accounted for more than 95% of total bacterial sequences (ranging between 90.14 and 98.19%). Specifically, Firmicutes comprised 56.53% (42.98-63.64%) and Bacteroidetes comprised 39.17% (34.56-47.16%) of the total reads. The remaining 5% of the population reads comprised of Tenericutes, Cyanobacteria, TM7, Actinobacteria, Proteobacteria, Verrucomicrobia, Elusimicrobia, Planctomycetes, Fibrobacteres, Spirochaetes, Chloroflexi, and Deferribacteres. Differences in the fecal bacteria composition between particular reindeer were not statistically significant which may reflect the restricted location and similar diet of all members of the local population.

Isolation and Characterization of Phages Infecting Bacillus subtilis.

Bacteriophages have been suggested as an alternative approach to reduce the amount of pathogens in various applications. Bacteriophages of various specificity and virulence were isolated as a means of controlling food-borne pathogens. We studied the interaction of bacteriophages with Bacillus species, which are very often persistent in industrial applications such as food production due to their antibiotic resistance and spore formation. A comparative study using electron microscopy, PFGE, and SDS-PAGE as well as determination of host range, pH and temperature resistance, adsorption rate, latent time, and phage burst size was performed on three phages of the Myoviridae family and one phage of the Siphoviridae family which infected Bacillus subtilis strains. The phages are morphologically different and characterized by icosahedral heads and contractile (SIOΦ, SUBω, and SPOσ phages) or noncontractile (ARπ phage) tails. The genomes of SIOΦ and SUBω are composed of 154 kb. The capsid of SIOΦ is composed of four proteins. Bacteriophages SPOσ and ARπ have genome sizes of 25 kbp and 40 kbp, respectively. Both phages as well as SUBω phage have 14 proteins in their capsids. Phages SIOΦ and SPOσ are resistant to high temperatures and to the acid (4.0) and alkaline (9.0 and 10.0) pH.