The use of plant extracts and bacteriophages as an alternative therapy approach in combatting bacterial infections: the study of lytic phages and Stevia rebaudiana.

Introduction: In the light of the problem of antibiotic resistance, the use of combined alternative therapies in combatting bacteria-related disorders has gained popularity. Bacteriophages are one element implemented in new combination therapy. Stevia rebaudiana is known to have antimicrobial activity and regarded as potentially having a synergistic effect with bacteriophages. Therefore, possible interactions of lytic bacteriophages (MS2, T4 and Phi6) with acetone and methanol S. rebaudiana extracts (SRa and SRm) in the bacterial environment were examined. Material and Methods: The interactions were tested using a microdilution method, phage-extract co-incubation assay, static interaction (synography) and dynamic growth profile experiments in a bioreactor. Results: The interactions of the tested factors in a static environment differed from those in a dynamic environment. Dynamic conditions altered the effect of the extracts in a concentration-dependent manner. How different the effect of the SRa extract was to that of the SRm extract on bacterial growth in a dynamic environment depended on the species of the phage and bacterial host. The greatest differences were observed for E. coli strains and their phages, whereas Pseudomonas syringae and the Phi6 phage reacted very similarly to both extracts. Differences also emerged for the same extract in different E. coli strains and their phages. Conclusion: Every extract type should be tested on a case-by-case basis and experiment outcomes should not be generalised before gathering data. Moreover, many varied experiments should be performed, especially when examining such multifactorial mixtures. The tested mixtures could potentially be used in multidrug-resistant bacterial infection treatments.

Nanoparticles Influence Lytic Phage T4-like Performance In Vitro.

Little is known about interactions of non-filamentous, complex-structured lytic phages and free, non-ordered nanoparticles. Emerging questions about their possible bio-sanitization co-applications or predictions of possible contact effects in the environment require testing. Therefore, we revealed the influence of various nanoparticles (NPs; SiO2, TiO2-SiO2, TiO2, Fe3O4, Fe3O4-SiO2 and SiO2-Fe3O4-TiO2) on a T4-like phage. In great detail, we investigated phage plaque-forming ability, phage lytic performance, phage progeny burst times and titers by the eclipse phase determinations. Additionally, it was proved that TEM micrographs and results of NP zeta potentials (ZP) were crucial to explain the obtained microbiological data. We propose that the mere presence of the nanoparticle charge is not sufficient for the phage to attach specifically to the NPs, consequently influencing the phage performance. The zeta potential values in the NPs are of the greatest influence. The threshold values were established at ZP < −35 (mV) for phage tail binding, and ZP > 35 (mV) for phage head binding. When NPs do not meet these requirements, phage−nanoparticle physical interaction becomes nonspecific. We also showed that NPs altered the phage lytic activity, regardless of the used NP concentration. Most of the tested nanoparticles positively influenced the phage lytic performance, except for SiO2 and Fe3O4-SiO2, with a ZP lower than −35 (mV), binding with the phage infective part­the tail.

Polyethylene Films Containing Plant Extracts in the Polymer Matrix as Antibacterial and Antiviral Materials.

Low density polyethylene (LDPE) films covered with active coatings containing mixtures of rosemary, raspberry, and pomegranate CO2 extracts were found to be active against selected bacterial strains that may extend the shelf life of food products. The coatings also offer antiviral activity, due to their influence on the activity of Φ6 bacteriophage, selected as a surrogate for SARS-CoV-2 particles. The mixture of these extracts could be incorporated into a polymer matrix to obtain a foil with antibacterial and antiviral properties. The initial goal of this work was to obtain active LDPE films containing a mixture of CO2 extracts of the aforementioned plants, incorporated into an LDPE matrix via an extrusion process. The second aim of this study was to demonstrate the antibacterial properties of the active films against Gram-positive and Gram-negative bacteria, and to determine the antiviral effect of the modified material on Φ6 bacteriophage. In addition, an analysis was made on the influence of the active mixture on the polymer physicochemical features, e.g., mechanical and thermal properties, as well as its color and transparency. The results of this research indicated that the LDPE film containing a mixture of raspberry, rosemary, and pomegranate CO2 extracts incorporated into an LDPE matrix inhibited the growth of Staphylococcus aureus. This film was also found to be active against Bacillus subtilis. This modified film did not inhibit the growth of Escherichia coli and Pseudomonas syringae cells; however, their number decreased significantly. The LDPE active film was also found to be active against Φ6 particles, meaning that the film had antiviral properties. The incorporation of the mixture of CO2 extracts into the polymer matrix affected its mechanical properties. It was observed that parameters describing mechanical properties decreased, although did not affect the transition of LDPE significantly. Additionally, the modified film exhibited barrier properties towards UV radiation. Modified PE/CO2 extracts films could be applied as a functional food packaging material with antibacterial and antiviral properties.

Development of an Autochthonous Microbial Consortium for Enhanced Bioremediation of PAH-Contaminated Soil.

The main objectives of this study were to isolate bacteria from soil chronically contaminated with polycyclic aromatic hydrocarbons (PAHs), develop an autochthonous microbial consortium, and evaluate its ability to degrade PAHs in their native contaminated soil. Strains with the best bioremediation potential were selected during the multi-stage isolation process. Moreover, to choose bacteria with the highest bioremediation potential, the presence of PAH-degrading genes (pahE) was confirmed and the following tests were performed: tolerance to heavy metals, antagonistic behavior, phytotoxicity, and antimicrobial susceptibility. In vitro degradation of hydrocarbons led to the reduction of the total PAH content by 93.5% after the first day of incubation and by 99.22% after the eighth day. Bioremediation experiment conducted in situ in the contaminated area resulted in the average reduction of the total PAH concentration by 33.3% after 5 months and by over 72% after 13 months, compared to the concentration recorded before the intervention. Therefore, this study implicates that the development of an autochthonous microbial consortium isolated from long-term PAH-contaminated soil has the potential to enhance the bioremediation process.

Double-Layer Agar (DLA) Modifications for the First Step of the Phage-Antibiotic Synergy (PAS) Identification.

The research carried out so far for phage-antibiotic synergy (PAS) differs as regards the technique of modifying the double-layer agar (DLA) method to show the PAS effect on Petri plates, which may contribute to non-uniform research results. Therefore, there is a need to unify the method to effectively detect the PAS effect, at its most basic in vitro test. In this study, bacteriophage T45 and 43 antibiotics belonging to different antibiotic classes were used. Seven different DLA method modifications were tested, in terms of antibiotic addition placement and presence or absence of the base agar. The overall number of phage plaques per plate mainly depended on the antibiotic used. Differences in plaque quantity depended on the type of the DLA method modification. The largest total number of plaques was obtained by the addition of an antibiotic to a bottom agar with the presence of a top agar. This indicates that even though an antibiotic could manifest the PAS effect by a standard disk method, it would be worth examining if the effect is equally satisfactory when applying antibiotics directly into the agar, with regards to using the same bacteriophage and bacterial host.

Evaluation of nutritional value and microbiological safety in commercial dog food.

In addition to properly balancing nutritional value in accordance with the needs of a dog, estimating the microbiological quality of dog food is crucial in providing healthy and safe foods. The aim of this study was to examine the quality of dry food for adult dogs, with particular reference to: (1) evaluating the nutritional value and compliance with nutritional guidelines for dogs, (2) comparing the nutritional value of dog foods, with particular emphasis on the division into cereal and cereal-free foods, and (3) evaluating their microbiological safety. All thirty-six evaluated dry dog foods met the minimum European Pet Food Industry FEDIAF requirement for total protein and fat content. The total aerobic microbial count in the analyzed dry dog foods ranged from 2.7 × 102 to above 3.0 × 107 cfu/g. In five (14%) dog foods the presence of staphylococci was detected; however, coagulase positive Staphylococcus (CPS) was not found. Mold presence was reported in one cereal-free dog food and in six cereal foods. In none of the analyzed foods Enterobacteriaceae were found, including coliforms, Escherichia coli and Salmonella spp. Bacteria of the genus Listeria and Clostridium as well as yeasts were also not detected. In conclusion, the evaluated dry dog foods had varied microbiological quality. The detected number of microorganisms may have some implications for long-term consumption of contaminated food. The lack of European Commission standards regarding the permissible amounts of microorganisms in pet food may result in insufficient quality control of these products.

Packaging Covered with Antiviral and Antibacterial Coatings Based on ZnO Nanoparticles Supplemented with Geraniol and Carvacrol.

The purpose of the study was to obtain an external coating based on nanoparticles of ZnO, carvacrol, and geraniol that could be active against viruses such as SARS-Co-V2. Additionally, the synergistic effect of the chosen substances in coatings was analyzed. The goal of the study was to measure the possible antibacterial activity of the coatings obtained. Testing antiviral activity with human pathogen viruses, such as SARS-Co-V2, requires immense safety measures. Bacteriophages such as phi 6 phage represent good surrogates for the study of airborne viruses. The results of the study indicated that the ZC1 and ZG1 coatings containing an increased amount of geraniol or carvacrol and a very small amount of nanoZnO were found to be active against Gram-positive and Gram-negative bacteria. It is also important that a synergistic effect between these active substances was noted. This explains why polyethylene (PE) films covered with the ZC1 or ZG1 coatings (as internal coatings) were found to be the best packaging materials to extend the quality and freshness of food products. The same coatings may be used as the external coatings with antiviral properties. The ZC1 and ZG1 coatings showed moderate activity against the phi 6 phage that has been selected as a surrogate for viruses such as coronaviruses. It can be assumed that coatings ZG1 and ZC1 will also be active against SARS-CoV-2 that is transmitted via respiratory droplets.

Potential Use of Silica Nanoparticles for the Microbial Stabilisation of Wine: An In Vitro Study Using Oenococcus oeni as a Model.

The emerging trend towards the reduction of SO2 in winemaking has created a need to look for alternative methods to ensure the protection of wine against the growth of undesired species of microorganisms and to safely remove wine microorganisms. This study describes the possible application of silica nanospheres as a wine stabilisation agent, with Oenococcus oeni (DSM7008) as a model strain. The experiment was conducted firstly on model solutions of phosphate-buffered saline and 1% glucose. Their neutralising effect was tested under stirring with the addition of SiO2 (0.1, 0.25, and 0.5 mg/mL). Overall, the highest concentration of nanospheres under continuous stirring resulted in the greatest decrease in cell counts. Transmission electron microscope (TEM) and scanning electron microscopy (SEM) analyses showed extensive damage to the bacterial cells after stirring with silica nanomaterials. Then, the neutralising effect of 0.5 mg/mL SiO2 was tested in young red wine under stirring, where cell counts were reduced by over 50%. The obtained results suggest that silica nanospheres can serve as an alternative way to reduce or substitute the use of sulphur dioxide in the microbial stabilisation of wine. In addition, further aspects of following investigations should focus on the protection against enzymatic and chemical oxidation of wine.