Bacteriophage Phi Research Articles

How to validate UV-C based air cleaners using viruses containing aerosols in a test room.

UV-C based air cleaners may reduce the transmission of infectious diseases. However, microbiological validation is necessary to quantify their efficiency. In this study, the stability of aerosolized bacteriophages for validation purposes was investigated in a test room, before an UV-C based air cleaner was exemplarily evaluated regarding the inactivation of airborne bacteriophages. The bacteriophage Phi6 was selected as virus surrogate and aerosolized in a room of 30 m³ volume. The recovery of infectious bacteriophages was first analyzed under variation of the relative humidity (20-55% RH) and sampling time. The aerosol studies showed that a low humidity between 20% RH and 30% RH provides a high and stable recovery of bacteriophages Phi6 over 1h. However, with increasing humidity, the number of infectious airborne bacteriophages Phi6 decreased significantly. At 50% RH, the recovery of Phi6 was 4 orders of magnitude lower compared to 20% RH. The validation of an UV-C based air cleaner was then demonstrated in the test room whereat the decline of infectious airborne bacteriophages was recorded over time. The non-enveloped bacteriophage MS2 was used as a reference. The validation results were significantly different for Phi6 when the humidity in the test room was either 40% RH or 30% RH whereas comparable results were obtained for MS2 at both humidities. A rising humidity in the test room caused a significant decline in the recovery of infectious airborne bacteriophages Phi6. The result of a quantitative validation of UV-C based air cleaners may therefore be affected by the respective humidity.

Synergistic Integration of α-Ag2WO4 into PLA/PBAT for the Development of Electrospun Membranes: Advancing Structural Integrity and Antimicrobial Efficacy.

The rising resistance of various pathogens and the demand for materials that prevent infections drive the need to develop broad-spectrum antimicrobial membranes capable of combating a range of microorganisms, thereby enhancing safety in biomedical and industrial applications. Herein, we introduce a simple and efficient technique to engineer membranes composed of polylactic acid (PLA) and polybutylene adipate terephthalate (PBAT) biopolymers and α-Ag2WO4 particles using an electrospinning technique. The corresponding structural, thermal, mechanical, and antimicrobial properties were characterized. X-ray diffraction (XRD) patterns confirmed the integration of crystalline α-Ag2WO4 within the polymer matrix. Scanning electron microscopy (SEM) and Raman confocal microscopy revealed uniformly dispersed α-Ag2WO4 particles in the electrospun fibers, influencing their diameter and surface roughness. Thermal analysis indicated adjustments in the thermal stability and crystallinity of the composites with an increasing α-Ag2WO4 content. Dynamic mechanical analysis (DMA) highlighted variations in storage modulus and glass transition temperatures due to interactions between α-Ag2WO4 and polymer chains, with tensile tests showing an increase in elastic modulus and ultimate tensile strength as the α-Ag2WO4 content increased. Antimicrobial assessments revealed that PLA/PBAT membranes with α-Ag2WO4 showed pronounced antibacterial activity, forming inhibition halos across all samples against Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and Mycobacterium smegmatis (a surrogate for Mycobacterium tuberculosis). These membranes also exhibited potent antiviral activity against bacteriophage phi 6, a surrogate for SARS-CoV-2, suggesting potential applications in combating infections caused by enveloped viruses. The antimicrobial activities are attributed to the generation of reactive oxygen species (ROS) and the controlled release of Ag+ ions. This work underscores the multifaceted capabilities of α-Ag2WO4-enhanced PLA/PBAT membranes in combating bacterial and viral growth, where both durability and microbial resistance are critical. Taken together, our findings provide a solution for obtaining advanced materials to be applied in a wide range of industrial applications, such as filtration systems, food preservation, antimicrobial coatings, protective textiles, and cleaning products.

Immuno-RCA for highly sensitive detection of the antigen-antibody complex in the blood group antigen model

The problem of detecting tiny quantities of analytes by immunochemical methods has been tried for a long time. One approach is to use nucleic acid amplification methods to amplify the signal from a single antigen-antibody interaction. An amplification method suitable for microarrays is the rolling circle amplification reaction. The principle of the method is usage of a conjugate of a detecting antibody with a primer and subsequent isothermal amplification. The generation of a huge single-stranded reaction product starts after adding the necessary components for amplification reaction: circular oligonucleotides, which serves as a template for amplification and phage phi29 polymerase with the other components. This reaction product consists of a lot of repeats of a nucleotide sequence, that is complementary to the circular template. The fluorescent DNA probe can hybridize to each repeat on the product molecule, resulting in a significantly higher level of fluorescence than with fluorescently labeled antibody or streptavidin development systems. In addition, the reaction product remains immobilized on the surface, allowing usage of this approach for the detection of antigen-antibody interactions in other solid-phase analysis systems, such as microarrays. A common problem with such approaches is the nonspecific sorption of components of the immunochemical reaction or amplification reaction, leading to a high background. It is obvious that no matter how highly sensitive the analysis is in theory, a high background will reduce the entire potential of the method to nothing. Herein, we have developed a method that makes it possible to detect small amounts of antibodies to glycans in blood serum and in swabs from tumor cells in a microarray format using a model of blood group antigens. It was possible to obtain a 30 to 70-fold increase of fluorescence level from a specific interaction compared to the use of fluorescently labeled streptavidin. The method we are developing is promising, as it allows us to significantly increase the signal from the specific antigen/antibody interaction in the glycochip format, which will make it possible to detect antibodies to glycans in samples with a very low concentrations of antibodies, for example, in washes from tumor cells.

Darwinian Evolution of Self-Replicating DNA in a Synthetic Protocell

Replication, heredity, and evolution are characteristic of Life. We and others have postulated that the reconstruction of a synthetic living system in the laboratory will be contingent on the development of a genetic self-replicator capable of undergoing Darwinian evolution. Although DNA-based life dominates, the in vitro reconstitution of an evolving DNA self-replicator has remained challenging. We hereby emulate in liposome compartments the principles according to which life propagates information and evolves. Using two different experimental configurations supporting intermittent or semi-continuous evolution (i.e., with or without DNA extraction, PCR, and re-encapsulation), we demonstrate sustainable replication of a linear DNA template – encoding the DNA polymerase and terminal protein from the Phi29 bacteriophage – expressed in the ‘protein synthesis using recombinant elements’ (PURE) system. The self-replicator can survive across multiple rounds of replication-coupled transcription-translation reactions in liposomes and, within only ten evolution rounds, accumulates mutations conferring a selection advantage. Combined data from next-generation sequencing with reverse engineering of some of the enriched mutations reveal nontrivial and context-dependent effects of the introduced mutations. The present results are foundational to build up genetic complexity in an evolving synthetic cell, as well as to study evolutionary processes in a minimal cell-free system.

Antiviral respiratory masks with plasma-functionalized polypropylene textiles for optimal adsorption of antiviral substance

During the COVID-19 pandemic, face masks were the first line of defense against the spread of infection. However, infectious viruses may remain on medical textiles, potentially serving as an additional source of infection. Due to their chemical inertness, many textiles cannot be enhanced with antiviral functionalities. Through treatment with low-pressure gaseous plasma, we have activated the surface of a medical-grade melt-blown, non-woven polypropylene textile so that it can absorb sodium dodecyl sulfate, an antimicrobial surfactant. Within two hours of contact time, the functionalized textile has been able to inactivate over 7 log10 PFU mL−1 of bacteriophage phi6, a surrogate of enveloped viruses such as SARS-CoV-2, and it has retained its antiviral properties for over 100 days. The functionalized material has not disrupted facial mask filtration efficiency or breathability. In addition, the in vitro biocompatibility testing in accordance with ISO 10993-5 for testing of medical devices has demonstrated that the selected formulation causes no adverse effects on the mouse fibroblast cell line L-929. With the treatment processes that have been completed within seconds, the method seems to have great potential to produce antiviral textiles against future outbreaks.

Bacteriophages Phi 8 and Phi 12 host infection are inhibited by OMVs and LPS purified from P. pseudoalcaligenes strain: East River Isolate A.

Most phage families studied to date use a tailed appendage, composed of a multitude of proteins, for cellular recognition, membrane penetration, and genome injection. This contrasts with members of the Cystoviridae family which possess a phospholipid membrane bilayer with embedded proteins responsible for cellular recognition and membrane fusion. Thus, the Cystoviridae are akin to enveloped viruses which also use protein complexes embedded into their membrane for cellular recognition and membrane fusion. Examples of such viruses include the Retroviridae, Coronoviridae, Herpesviridae , and Orthomyxoviridae families. The binding specifics of Cystoviridae to the host outer membrane are unknown. Using Cystoviridae -OMV interaction we began to uncover the host requirements for binding Cystoviridae . The results presented determine that only lipid A and the core polysaccharide of LPS are required for Cystoviridae outer membrane binding.

Calcium alginate films loaded with copper-molybdenum oxide nanoparticles for antimicrobial applications

The rise in petroleum-based plastic waste, driven by increased consumption from delivery services and e-commerce, necessitates the search for sustainable alternatives like alginate-based materials to mitigate environmental risks. However, alginate, a biopolymer, has limited physico-chemical properties, lacks antibacterial capacity, and exhibits low antiviral activity. To address these issues, this study synthesized Cu3Mo2O9 semiconductor nanoparticles (CMO) and integrated them into alginate films crosslinked with Ca2+, producing advanced nanocomposite films with varying nanoparticle concentrations (0–10 wt%). CMO nanoparticles release Cu2+ and Mo6+ ions and generate reactive oxygen species (ROS), even in the dark. Results show that CMO weakly interacts with alginate blocks, causing slight structural changes but not significantly altering mechanical or thermal properties. CMO released Cu2+ (150–250 ppb at 24 h) and Mo6+ ions (5–8 ppm at 24 h), with Cu2+ undergoing cation exchange with Ca2+ crosslinking ions. Antibacterial tests demonstrated the composites' effectiveness against Gram-negative Pseudomonas aeruginosa (97.30 % and 91.36 % growth inhibition at 5 % and 10 % CMO), Gram-positive methicillin-resistant Staphylococcus aureus (87.26 % and 96.82 % inhibition at 5 % and 10 % CMO), and Mycobacterium smegmatis (99.83 % and 99.97 % inhibition at 5 % and 10 % CMO). These composites also showed antiviral activity against bacteriophage phi 6 (99.81 % and 92.66 % inactivation at 5 % and 10 % CMO) and bacteriophage MS2 (92.32 % and 92.93 % inactivation at 5 % and 10 % CMO). The primary mechanism is attributed to the release of Cu2+ cations and ROS generation. These nanocomposite films exhibit broad-spectrum antimicrobial activity, offering potential industrial applications in packaging and antimicrobial surfaces.

Engineering psychrophilic polymerase for nanopore long-read sequencing.

Unveiling the potential application of psychrophilic polymerases as candidates for polymerase-nanopore long-read sequencing presents a departure from conventional choices such as thermophilic Bacillus stearothermophilus (Bst) renowned for its limitation in temperature and mesophilic Bacillus subtilis phage (phi29) polymerases for limitations in strong exonuclease activity and weak salt tolerance. Exploiting the PB-Bst fusion DNA polymerases from Psychrobacillus (PB) and Bacillus stearothermophilus (Bst), our structural and biochemical analysis reveal a remarkable enhancement in salt tolerance and a concurrent reduction in exonuclease activity, achieved through targeted substitution of a pivotal functional domain. The sulfolobus 7-kDa protein (Sso7d) emerges as a standout fusion domain, imparting significant improvements in PB-Bst processivity. Notably, this study elucidates additional functional sites regulating exonuclease activity (Asp43 and Glu45) and processivity using artificial nucleotides (Glu266, Gln283, Leu334, Glu335, Ser426, and Asp430). By disclosing the intricate dynamics in exonuclease activity, strand displacement, and artificial nucleotide-based processivity at specific functional sites, our findings not only advance the fundamental understanding of psychrophilic polymerases but also provide novel insights into polymerase engineering.

Multi-factor normalisation of viral counts from wastewater improves the detection accuracy of viral disease in the community

The detection of viruses (e.g. SARS-CoV-2, norovirus) in wastewater represents an effective way to monitor the prevalence of these pathogens circulating within the community. However, accurate quantification of viral concentrations in wastewater, proportional to human input, is constrained by a range of uncertainties, including (i) dilution within the sewer network, (ii) degradation of viral RNA during wastewater transit, (iii) catchment population and facility use, (iv) efficiency of viral concentration and extraction from wastewater, and (v) inhibition of amplification during the RT-qPCR step. Here, we address these uncertainties by investigating several potential normalisation factors including the concentration of ammonium and orthophosphate. A faecal indicator virus (crAssphage), and the recovery of the process-control viruses (murine norovirus and bacteriophage Phi6), used for quality control during the RT-qPCR step, were also considered. We found that multi-factor normalisation of SARS-CoV-2 RT-qPCR data was optimal using a combination of crAssphage, process-control virus recovery, and concentration efficiency to improve prediction accuracy relative to clinical test data. Using multi-normalised SARS-CoV-2 RT-qPCR data, we found a lasso regression model with random forest modelled residuals lowers the prediction error of positives by 46 %, compared to a single linear regression using raw data. This multi-normalised approach enables more accurate wastewater-based predictions of clinical cases up to five days in advance of clinical data, identifying trends in disease prevalence before clinical testing, and demonstrates the potential to improve viral pathogen detection for a range of currently monitored and emerging diseases.

Effect of Buffer Room Configuration on Isolation of Bacteriophage phi6 and Micrococcus Luteus Emissions

Effect of Buffer Room Configuration on Isolation of Bacteriophage phi6 and Micrococcus Luteus Emissions

Photobiocidal Activity of TiO2/UHMWPE Composite Activated by Reduced Graphene Oxide under White Light.

Herein, we introduce a photobiocidal surface activated by white light. The photobiocidal surface was produced through thermocompressing a mixture of titanium dioxide (TiO2), ultra-high-molecular-weight polyethylene (UHMWPE), and reduced graphene oxide (rGO) powders. A photobiocidal activity was not observed on UHMWPE-TiO2. However, UHMWPE-TiO2@rGO exhibited potent photobiocidal activity (>3-log reduction) against Staphylococcus epidermidis and Escherichia coli bacteria after a 12 h exposure to white light. The activity was even more potent against the phage phi 6 virus, a SARS-CoV-2 surrogate, with a >5-log reduction after 6 h exposure to white light. Our mechanistic studies showed that the UHMWPE-TiO2@rGO was activated only by UV light, which accounts for 0.31% of the light emitted by the white LED lamp, producing reactive oxygen species that are lethal to microbes. This indicates that adding rGO to UHMWPE-TiO2 triggered intense photobiocidal activity even at shallow UV flux levels.

The Influence of Functional Composite Coatings on the Properties of Polyester Films before and after Accelerated UV Aging.

The aim of this study was to cover biopolymeric packaging films based on PLA/PHBV blend with a functional composite coating (to retain their ecological character) and to investigate their antimicrobial properties before and after UV irradiation. As an active coating, the carrier hydroxypropyl methyl cellulose (HPMC), as well as its modified form with Achillea millefolium L., Hippophae rhamnoides L., and Hypericum L. extract (E) and a combined system based on the extracts and nano-ZnO (EZ), was used to obtain active formulations. Additionally, film surface morphology (SEM, FTIR-ATR) and color (CIELab scale) analysis of the pre- and post-UV-treatment samples were performed. The results confirmed that the E and EZ-modified films exhibited antibacterial properties, but they were not effective against phage phi6. Q-SUN irradiation led to a decrease in the activity of E coating against Staphylococcus aureus, Pseudomonas syringae, and Candida albicans. In this case, the effectiveness of EZ against C. albicans at 24 h and 72 h UV irradiation decreased. However, the irradiation boosted the antiviral effectiveness of the EZ layer. SEM micrographs of the film surface showed that UV treatment did not significantly influence the native film morphology, but it had an impact on the coated film. FTIR analysis results showed that the coatings based on HPMC altered the IR absorption of the nonpolar groups of the biopolyester material. The applied coatings only marginally affected film color changes and increased their yellowness after UV irradiation, whereas a composite layer of nano-ZnO limited these changes.

Acinetobacter baumannii satellite phage Aci01-2-Phanie depends on a helper myophage for its multiplication.

We report the discovery of a satellite-helper phage system with a novel type of dependence on a tail donor. The Acinetobacter baumannii satellite podovirus Aci01-2-Phanie (short name Phanie) uses a phage phi29-like DNA replication and packaging mode. Its linear 11,885 bp dsDNA genome bears 171 bp inverted terminal repeats (ITR). Phanie is related to phage DU-PP-III from Pectobacterium and to members of the Astrithrvirus from Salmonella enterica. Together, they form a new clade of phages with 27% to 30% identity over the whole genome. Detailed 3D protein structure prediction and mass spectrometry analyses demonstrate that Phanie encodes its capsid structural genes and genes necessary to form a short tail. However, our study reveals that Phanie virions are non-infectious unless they associate with the contractile tail of an unrelated phage, Aci01-1, to produce chimeric myoviruses. Following the coinfection of Phanie with myovirus Aci01-1, hybrid viral particles composed of Phanie capsids and Aci01-1 contractile tails are assembled together with Phanie and Aci01-1 particles.IMPORTANCEThere are few reported cases of satellite-helper phage interactions but many more may be yet undiscovered. Here we describe a new mode of satellite phage dependence on a helper phage. Phanie, like phage phi29, replicates its linear dsDNA by a protein primed-mechanism and protects it inside podovirus-like particles. However, these particles are defective, requiring the acquisition of the tail from a myovirus helper for production of infectious virions. The formation of chimeras between a phi29-like podovirus and a helper contractile tail reveals an unexpected association between very different bacterial viruses.

Wastewater disinfection with photodynamic treatment and evaluation of its ecotoxicological effects

Research has demonstrated the presence of viruses in wastewater (WW), which can remain viable for a long period, posing potential health risks. Conventional WW treatment methods involving UV light, chlorine and ozone efficiently reduce microbial concentrations, however, they produce hazardous byproducts and microbial resistance that are detrimental to human health and the ecosystem. Hence, there is a need for novel disinfection techniques. Antimicrobial Photodynamic Inactivation (PDI) emerges as a promising strategy, utilizing photosensitizers (PS), light, and dioxygen to inactivate viruses. This study aims to assess the efficacy of PDI by testing methylene blue (MB) and the cationic porphyrin TMPyP as PSs, along a low energy consuming white light source (LED) at an irradiance of 50 mW/cm2, for the inactivation of bacteriophage Phi6. Phi6 serves as an enveloped RNA-viruses surrogate model in WW. PDI experiments were conducted in a buffer solution (PBS) and real WW matrices (filtered and non-filtered). Considering the environmental release of the treated effluents, this research also evaluated the ecotoxicity of the resulting solution (post-PDI treatment effluent) on the model organism Daphnia magna, following the Organisation for Economic Cooperation and Development (OECD) immobilization technical 202 guideline. Daphnids were exposed to WW containing the tested PS at different concentrations and dilutions (accounting for the dilution factor during WW release into receiving waters) over 48 h. The results indicate that PDI with MB efficiently inactivated the model virus in the different aqueous matrices, achieving reductions superior to 8 log10 PFU/mL, after treatments of 5 min in PBS and of ca. 90 min in WW. Daphnids survival increased when subjected to the PDI-treated WW with MB, considering the dilution factor. Overall, the effectiveness of PDI in eliminating viruses in WW, the fading of the toxic effects on daphnids after MB’ irradiation and the rapid dilution effect upon WW release in the environment highlight the possibility of using MB in WW PDI-disinfection.

Photostability and photodynamic antimicrobial profile of dye extracts from four (4) plants: prospects for eco-friendly low-cost food disinfection and topical biomedical applications.

In this study, photostability and photodynamic antimicrobial performance of dye extracts from Hibiscus sabdariffa (HS) calyces, Sorghum bicolor (SB) leaf sheaths, Lawsonia inermis (LI) leaves and Curcuma longa (CL) roots were investigated in Acetate-HCl (AH) Buffer (pH 4.6), Tris Base-HCl (TBH) Buffer (pH 8.6), distilled water (dH2O), and Phosphate Buffer Saline (PBS, pH 7.2) using Bacillus subtilis as model for gram positive bacteria, Escherichia coli as model for gram negative bacteria, phage MS2 as model for non-envelope viruses and phage phi6 as model for envelope viruses including SARS CoV-2 which is the causative agent of COVID-19. Our results showed that the photostability of the dye extracts is in the decreasing order of LI > CL > SB > HS. The dye extract-HS is photostable in dH2O but bleaches in buffers-AH, TBH and PBS. The rate of bleaching is higher in AH compared to in TBH and PBS. The bleaching and buffers affected the photodynamic and non-photodynamic antimicrobial activity of the dye extracts. The photodynamic antibacterial activity of the dye extracts is in the decreasing order of CL > HS > LI > SB while the non-photodynamic antibacterial activity is in the decreasing order of LI > CL > HS > SB. The non-photodynamic antiviral activity pattern observed is the same as that of non-photodynamic antibacterial activity observed. However, the photodynamic antiviral activity of the dye extracts is in the decreasing order of CL > LI > HS > SB. Given their performance, the dye extracts maybe mostly suitable for environmental applications including fresh produce and food disinfection, sanitation of hands and contact surfaces where water can serve as diluent for the extracts and the microenvironment is free of salts.

Hybrid Machine Learning and Experimental Studies of Antiviral Potential of Ionic Liquids against P100, MS2, and Phi6.

Viruses are a group of widespread organisms that are often responsible for very dangerous diseases, as most of them follow a mechanism to multiply and infect their hosts as quickly as possible. Pathogen viruses also mutate regularly, with the result that measures to prevent virus transmission and recover from the disease caused are often limited. The development of new substances is very time-consuming and highly budgeted and requires the sacrifice of many living organisms. Computational chemistry methods allow faster analysis at a much lower cost and, most importantly, reduce the number of living organisms sacrificed experimentally to a minimum. Ionic liquids (ILs) are a group of chemical compounds that could potentially find a wide range of applications due to their potential virucidal activity. In our study, we conducted a complex computational analysis to predict the antiviral activity of ionic liquids against three surrogate viruses: two nonenveloped viruses, Listeria monocytogenes phage P100 and Escherichia coli phage MS2, and one enveloped virus, Pseudomonas syringae phage Phi6. Based on experimental data of toxic activity (logEC90), we assigned activity classes to 154 ILs. Prediction models were created and validated according to the Organization for Economic Co-operation and Development (OECD) recommendations using the Classification Tree method. Further, we performed an external validation of our models through virtual screening on a set of 1277 theoretically generated ionic liquids and then selected 10 active ionic liquids, which were synthesized to verify their activity against the analyzed viruses. Our study proved the effectiveness and efficiency of computational methods to predict the antiviral activity of ionic liquids. Thus, computational models are a cost-effective alternative approach compared with time-consuming experimental studies where live animals are involved.

Utilization of Bacteriophage phi6 for the Production of High-Quality Double-Stranded RNA Molecules.

Double-stranded RNA (dsRNA) molecules are mediators of RNA interference (RNAi) in eukaryotic cells. RNAi is a conserved mechanism of post-transcriptional silencing of genes cognate to the sequences of the applied dsRNA. RNAi-based therapeutics for the treatment of rare hereditary diseases have recently emerged, and the first sprayable dsRNA biopesticide has been proposed for registration. The range of applications of dsRNA molecules will likely expand in the future. Therefore, cost-effective methods for the efficient large-scale production of high-quality dsRNA are in demand. Conventional approaches to dsRNA production rely on the chemical or enzymatic synthesis of single-stranded (ss)RNA molecules with a subsequent hybridization of complementary strands. However, the yield of properly annealed biologically active dsRNA molecules is low. As an alternative approach, we have developed methods based on components derived from bacteriophage phi6, a dsRNA virus encoding RNA-dependent RNA polymerase (RdRp). Phi6 RdRp can be harnessed for the enzymatic production of high-quality dsRNA molecules. The isolated RdRp efficiently synthesizes dsRNA in vitro on a heterologous ssRNA template of any length and sequence. To scale up dsRNA production, we have developed an in vivo system where phi6 polymerase complexes produce target dsRNA molecules inside Pseudomonas cells.

Biological Properties of Recently Described Wild Bramble Rubus oklejewiczii against the Species from Similar Niches.

The aim of this study was to compare the biological properties, such as antiviral, antibacterial, and antioxidant, of recently described pentaploid species Rubus oklejewiczii with tetraploid taxa growing in similar habitats including R. plicatus, R. gracilis, and R. wimmerianus. The antiviral potential was analyzed against bacteriophages with different genetic material: phi6 (a surrogate for the SARS-CoV-2 virus), T7, phiX174, and MS2. Antibacterial properties of fruit and leaf extracts were determined against Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Salmonella enterica. The total phenolic content, as well as anthocyanins, ascorbic acid, pH, and antioxidant properties (FRAP and DPPH) were determined. R. oklejewiczii leaf extract was characterized by the weakest antibacterial and antiviral properties, which was closely correlated with the lowest content of polyphenolic compounds and antioxidant properties. The strongest biological properties were observed for R. wimmerianus leaves. Fruit extracts were characterized by lower phenolic content and antioxidant activities than leaves, with the lowest values observed for R. oklejewiczii extract. The antibacterial properties of fruit extracts were strongest for R. gracilis. The strongest antiviral potential was observed for R. oklejewiczii and R. wimmerianus fruit extracts against the bacteriophage phi6, which correlated with the lowest pH and the highest ascorbic acid content. The positive effect of the higher ploidy of R. oklejewiczii for most of the analyzed biological properties was not observed except for the antiviral potential of fruit extract. Due to its large and tasty fruits, this species seems to be very promising for cultivation and attractive for consumers, even though most of its biological properties were not any better compared to other examined tetraploid species.

Identification and characterization of integrated prophages and CRISPR-Cas system in Bacillus subtilis RS10 genome.

Bacteriophages have been extensively investigated due to their prominent role in the virulence and resistance of pathogenic bacteria. However, little attention has been given to the non-pathogenic Bacillus phages, and their role in the ecological bacteria genome is overlooked. In the present study, we characterized two Bacillus phages with a linear DNA genome of 33.6kb with 44.83% GC contents and 129.3kb with 34.70% GC contents. A total of 46 and 175 putative coding DNA sequences (CDS) were identified in prophage 1 (P1) and prophage 2 (P2), respectively, with no tRNA genes. Comparative genome sequence analysis revealed that P1 shares eight CDS with phage Jimmer 2 (NC-041976), and phage Osiris (NC-028969), and six with phage phi CT9441A (NC-029022). On the other hand, P2 showed high similarity with Bacill_SPbeta_NC_001884 and Bacillus phage phi 105. Further, genome analysis indicates several horizontal gene transfer events in both phages during the evolution process. In addition, we detected two CRISPR-Cas systems for the first time in B. subtilis. The identified CRISPR system consists of 24 and 25 direct repeats and integrase coding genes, while the cas gene which encodes Cas protein involved in the cleavage of a target sequence is missing. These findings will expand the current knowledge of soil phages as well as help to develop a new perspective for investigating more ecological phages to understand their role in bacterial communities and diversity.

Chemical inhibition of cell surface modification sensitizes bacteria to phage infection.

Many bacteriophages that infect Gram-positive bacteria rely on the bacterial cell surface polymer wall teichoic acid (WTA) as a receptor. However, some bacteria modulate their cell wall with d-alanine residues, which can disrupt phage adsorption. The prevalence and significance of WTA alanylation as an anti-phage defense is unknown. A chemical inhibitor of WTA d-alanylation could be employed to efficiently screen phage-host combinations for those that exhibit alanylation-dependent infections. Since the incorporation of d-alanine residues into the cell wall requires the activity of d-alanine:alanyl carrier protein ligase (DltA), a DltA inhibitor was employed as this tool. Herein, we found that a chemical probe inhibiting DltA activity impeded bacterial cell wall alanylation and enhanced infectivity of many phages against Bacillus subtilis, including phages Phi29, SPP1, SPO1, SP50, and Goe2. This finding reveals the breadth of immunity conferred by WTA alanylation in B. subtilis, which was previously known to impact only phages Phi29 and SPP1, but not SPO1, SP50, or Goe2. DltA inhibition selectively promoted infection by several phages that bind WTA, having no impact on the flagellotropic phage PBS1. Unexpectedly, DltA inhibition also had no effect on phage SP10, which binds to WTA. This selective chemical tool has the potential to unravel bacteriophage interactions with bacteria, leading to improved phage therapies in the future.