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.

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.

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.

The Response of Pseudomonas aeruginosa PAO1 to UV-activated Titanium Dioxide/Silica Nanotubes.

Pseudomonas aeruginosa is a bacterium of high clinical and biotechnological importance thanks to its high adaptability to environmental conditions. The increasing incidence of antibiotic-resistant strains has created a need for alternative methods to increase the chance of recovery in infected patients. Various nanomaterials have the potential to be used for this purpose. Therefore, we aimed to study the physiological response of P. aeruginosa PAO1 to titanium dioxide/silica nanotubes. The results suggest that UV light-irradiated nanomaterial triggers strong agglomeration in the studied bacteria that was confirmed by microscopy, spectrophotometry, and flow cytometry. The effect was diminished when the nanomaterial was applied without initial irradiation, with UV light indicating that the creation of reactive oxygen species could play a role in this phenomenon. The nanocomposite also affected biofilm formation ability. Even though the biomass of biofilms was comparable, the viability of cells in biofilms was upregulated in 48-hour biofilms. Furthermore, from six selected genes, the mexA coding efflux pump was upregulated, which could be associated with an interaction with TiO2. The results show that titanium dioxide/silica nanotubes may alter the physiological and metabolic functions of P. aeruginosa PAO1.

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.

Antimicrobial Activity of Al2O3, CuO, Fe3O4, and ZnO Nanoparticles in Scope of Their Further Application in Cement-Based Building Materials.

Nanoparticles were proposed as antibacterial cement admixtures for the production of cement-based composites. Nevertheless, the standards for evaluation of such admixtures still do not indicate which model organisms to use, particularly in regard to the further application of material. Apart from the known toxicity of nanomaterials, in the case of cement-based composites there are limitations associated with the mixing and dispersion of nanomaterials. Therefore, four nanooxides (Al2O3, CuO, Fe3O4, and ZnO) and seven microorganisms were tested to initially evaluate the applicability of nanooxides in relation to their further use in cement-based composites. Studies of nanoparticles included chemical analysis, microbial growth kinetics, 4- and 24 h toxicity, and biofilm formation assay. Nanooxides showed toxicity against microorganisms in the used concentration, although the populations were able to re-grow. Furthermore, the effect of action was variable even between strains from the same genus. The effect of nanoparticles on biofilms depended on the used strain. Gathered results show several problems that can occur while studying nanoparticles for specific further application. Proper protocols for nanomaterial dispersion prior the preparation of cement-based composites, as well as a standardized approach for their testing, are the fundamental issues that have to be resolved to produce efficient composites.

Characterization of Mechanical and Bactericidal Properties of Cement Mortars Containing Waste Glass Aggregate and Nanomaterials.

The recycling of waste glass is a major problem for municipalities worldwide. The problem concerns especially colored waste glass which, due to its low recycling rate as result of high level of impurity, has mostly been dumped into landfills. In recent years, a new use was found for it: instead of creating waste, it can be recycled as an additive in building materials. The aim of the study was to evaluate the possibility of manufacturing sustainable and self-cleaning cement mortars with use of commercially available nanomaterials and brown soda-lime waste glass. Mechanical and bactericidal properties of cement mortars containing brown soda-lime waste glass and commercially available nanomaterials (amorphous nanosilica and cement containing nanocrystalline titanium dioxide) were analyzed in terms of waste glass content and the effectiveness of nanomaterials. Quartz sand is replaced with brown waste glass at ratios of 25%, 50%, 75% and 100% by weight. Study has shown that waste glass can act as a successful replacement for sand (up to 100%) to produce cement mortars while nanosilica is incorporated. Additionally, a positive effect of waste glass aggregate for bactericidal properties of cement mortars was observed.

Effects of rotating magnetic field exposure on the functional parameters of different species of bacteria.

The aim of the present study was to determine the effect of the rotating magnetic field (RMF) on the growth, cell metabolic activity and biofilm formation by S. aureus, E. coli, A. baumannii, P. aeruginosa, S. marcescens, S. mutans, C. sakazakii, K. oxytoca and S. xylosus. Bacteria were exposed to the RMF (RMF magnetic induction B = 25-34 mT, RMF frequency f = 5-50 Hz, time of exposure t = 60 min, temperature of incubation 37 °C). The persistence of the effect of exposure (B = 34 mT, f = 50 Hz, t = 60 min) on bacteria after further incubation (t = 300 min) was also studied. The work showed that exposure to RMF stimulated the investigated parameters of S. aureus, E. coli, S. marcescens, S. mutans, C. sakazakii, K. oxytoca and S. xylosus, however inhibited cell metabolic activity and biofilm formation by A. baumannii and P. aeruginosa. The results obtained in this study proved, that the RMF, depending on its magnetic induction and frequency can modulate functional parameters of different species of bacteria.

Comparative analysis of superantigen genes in Staphylococcus xylosus and Staphylococcus aureus isolates collected from a single mammary quarter of cows with mastitis.

The purpose of this study was to analyze and compare genes encoding superantigens (SAgs) in Staphylococcus xylosus and Staphylococcus aureus isolates collected simultaneously from milk of the same cows with clinical mastitis. Genes encoding staphylococcal enterotoxins and enterotoxin-like proteins (sea-selu), toxic shock syndrome toxin 1 (tst-1) and exfoliative toxins (eta and etd) were investigated. It was found that among 30 isolates of S. xylosus, 16 (53.3%) harbored from 1 to 10 SAg genes. In total, in 16 SAg positive S. xylosus, 11 different enterotoxin genes were detected: sec, sed, seg, seh, sei, selm, seln, selo, selp, ser, selu and one etd gene encoding exfoliative toxin D. The most prevalent genes were ser, selu, and selo. Among all the positive isolates of S. xylosus, a total of 14 different SAg gene combinations were detected. One combination was repeated in 3 isolates, whereas the rest were detected only once. However, in the case of S. aureus all the 30 isolates harbored the same combination of SAg genes: seg, sei, selm, seln, selo and on the basis of PFGE analysis all belonged to the same clonal type. Also noteworthy was the observation that SAg genes detected in S. aureus have also been found in S. xylosus. The findings of this study further extend previous observations that SAg genes are present not only in S. aureus but also in coagulase-negative staphylococci, including S. xylosus. Therefore, taking into account that the SAg genes are encoded on mobile genetic elements it is possible that these genes can be transferred between different species of coexisting staphylococci.

Effects of 50 Hz rotating magnetic field on the viability of Escherichia coli and Staphylococcus aureus.

This study presents results of research on the influence of rotating magnetic field (RMF) of the induction of 30 mT and the frequency of 50 Hz on the growth dynamics and cell metabolic activity of E. coli and S. aureus, depending on the exposure time. The studies showed that the RMF caused an increase in the growth and cell metabolic activity of all the analyzed bacterial strains, especially in the time interval t = 30 to 150 min. However, it was also found that the optical density and cell metabolic activity after exposition to RMF were significantly higher in S. aureus cultures. In turn, the study of growth dynamics, revealed a rapid and a significant decrease in these values from t = 90 min) in the case of E. coli samples. The obtained results prove that RMF (B = 30 mT, f = 50 Hz) has a stimulatory effect on the growth and metabolic activity of E. coli and S. aureus. Furthermore, taking into account the time of exposure, stronger influence of RMF on the viability was observed in S. aureus cultures, which may indicate that this effect depends on the shape of the exposed cells.

Superantigen gene profiles, genetic relatedness and biological activity of exosecretions of Staphylococcus aureus isolates obtained from milk of cows with clinical mastitis.

This study evaluated the superantigen gene profiles, genetic relatedness and biological activity of exosecretions of 50 Staphylococcus aureus isolates obtained from milk of cows with clinical mastitis. Genomic relatedness of S. aureus was determined by pulsed field gel electrophoresis analysis of macro-restricted chromosomes. The presence of genes encoding superantigens was confirmed by multiplex PCR. To study the biological activity of S. aureus exosecretions, the supernatants from bacterial liquid cultures were classified into three groups: those with leukotoxin­like properties, those with superantigen­like properties and those with no particular activity on leukocytes cultured in vitro. It was shown that all analyzed bacterial isolates belonged to the same clonal type and harbored the same combination of superantigen genes, namely sed, selj and ser. However, 22% of all isolates produced factors with superantigen­like and 48% of them with leukotoxin­like activities. Finally, although there were no detectable genetic differences between the analyzed bacterial isolates, the virulence factors secreted by them differed considerably.

Identification and methicillin resistance of coagulase-negative staphylococci isolated from nasal cavity of healthy horses.

The aim of this study was an analysis of the staphylococcal flora of the nasal cavity of 42 healthy horses from 4 farms, along with species identification of CoNS isolates and determination of resistance to 18 antimicrobial agents, particularly phenotypic and genotypic methicillin resistance. From the 81 swabs, 87 staphylococci were isolated. All isolates possessed the gap gene but the coa gene was not detected in any of these isolates. Using PCR-RFLP of the gap gene, 82.8% of CoNS were identified: S. equorum (14.9%), S. warneri (14.9%), S. sciuri (12.6%), S. vitulinus (12.6%), S. xylosus (11.5%), S. felis (5.7%), S. haemolyticus (3.4%), S. simulans (3.4%), S. capitis (1.1%), S. chromogenes (1.1%), and S. cohnii subsp. urealyticus (1.1%). To our knowledge, this was the first isolation of S. felis from a horse. The species identity of the remaining Staphylococcus spp. isolates (17.2%) could not be determined from the gap gene PCR-RFLP analysis and 16S rRNA gene sequencing data. Based on 16S-23S intergenic transcribed spacer PCR, 11 different ITS-PCR profiles were identified for the 87 analyzed isolates. Results of API Staph were consistent with molecular identification of 17 (19.5%) isolates. Resistance was detected to only 1 or 2 of the 18 antimicrobial agents tested in the 17.2% CoNS isolates, including 6.9% MRCoNS. The mecA gene was detected in each of the 5 (5.7%) phenotypically cefoxitin-resistant isolates and in 12 (13.8%) isolates susceptible to cefoxitin. In total, from 12 horses (28.6%), 17 (19.5%) MRCoNS were isolated. The highest percentage of MRCoNS was noted among S. sciuri isolates (100%).

Influence of S. aureus exosecretions on cytokine profile in bovine leukocyte cultures in vitro.

The aim of the research was to evaluate the in vitro effect of Staphylococcus aureus exosecretions on the expression of genes encoding IL-2 and IL-12 and secretion of IFN-γ and TNF-α, in bovine leukocyte cultures in vitro. The research was based on 30 S. aureus isolates collected from milk samples from cows with clinical mastitis. Supernatants prepared from the bacterial liquid cultures, which were used to treat leukocytes, were divided into three groups: one with superantigen-like properties, one with leukotoxic-like properties and the one without superantigen or leukotoxic-like properties. The MNC, PMN and MIX (consisted of MNC and PMN leukocytes) cultures were grown and treated with the supernatants. The work shows that the effect on the cytokine gene expression and cytokine secretion caused by S. aureus exosecretions is mainly due to the presence of virulence factors connected with superantigen-like activity and less with leukotoxic-like activity whereas exosecretions of other activity are not or only slightly involved in this process.

The effect of auto-vaccination therapy on the phenotypic variation of one clonal type of Staphylococcus aureus isolated from cows with mastitis.

The aim of this study was to demonstrate the effect of auto-vaccine therapy on selected properties of Staphylococcus aureus strains, isolated from milk of cows with subclinical mastitis. The experiment was based on auto-vaccines which were prepared from S. aureus strains isolated from 16 cows. S. aureus strains isolated from cows on the 7th, 21st and 35th day following auto-vaccination, were analyzed phenotypically and genotypically. The isolated strains represented 17 biotypes all belonging to one clonal type. Increases of new biotypes of S. aureus were detected on the 35th day of therapy. Among 48 re-isolated strains, 18.75% (9/48) revealed single and 12.50% (6/48) multiple phenotypical changes. The present study demonstrated that during auto-vaccine therapy, S. aureus strains can change phenotypically, pointing out the necessity for using precise diagnostic methods, that would make possible a better assessment of the used therapy.

Secretory virulence factors produced by Staphylococcus aureus isolates obtained from mastitic bovine milk--effect on bovine polymorphonuclear neutrophils.

The aim of the research was to test whether exogenic virulence factors secreted by Staphylococcus aureus isolates are involved in mechanisms that allow the bacteria to modulate and evade phagocytosis by bovine polymorphonuclear neutrophils. The research was based on the comparison of the effects of supernatants, prepared from cultures of 30 S. aureus isolates, on the functional properties of bovine neutrophils in vitro. S. aureus isolates were collected from milk samples from cows with clinical mastitis. Supernatants, which were used to treat leukocytes, were prepared from 18 h S. aureus cultures. Exogenic virulence factors secreted by S. aureus isolates significantly influenced the phagocytosis parameters evaluated. Depending on their leukotoxic or superantigenic properties, supernatants could affect the ingestion process, and also showed an influence on the digestion efficiency and phagocytosis carried out by bovine polymorphonuclear neutrophils in vitro.

[The characteristics of staphylococcal enterotoxins produced by strains isolated from mastitic cows, including epidemiological aspects]. / Charakterystyka enterotoksyn gronkowcowych wytwarzanych przez szczepy izolowane od krów z zapaleniem gruczolu mlekowego (mastitis), z uwzglednieniem elementów epidemiologicznych.

Staphylococcal enterotoxins (SEs) are big heterogenic group of exotoxins, rather differential in respect of their nucleotide and amino-acid homology, as well as the location of their genes, molecular weight and iso-electric point value. SEs were identified in 1959 as the extra-cellular proteins produced by some Staphylococcus aureus strains. These enterotoxins are known as the pyrogenic toxins and this group contains also other staphylococcal toxins (staphylococcal toxic-shock syndrome toxin--TSST-1, A and B exfoliative toxins and streptococcal scarlet fever toxin). Twenty one serological types of staphylococcal enterotoxins are distinguished. All of them are structurally and functionally similar to the toxic shock syndrome toxin (TSST-1). SEs are thermo-stabile proteins, resistant to many proteolytic enzymes (pepsin, trypsine, chymotrypsine, renine and papain), but this resistance depends on the temperature and pH. Staphylococcal enterotoxin-encoding genes are located as well in the chromosomal DNA, as on the pathogenicity island, in phages, transposones and plasmids. Enterotoxins are staphylococcal virulence factors responsible for food poisonings in humans. These proteins are also isolated from cows with mastitis. In various countries, the percentage of enterotoxin-producing S. aureus strains ranges from 5 to 60%, depending on the enterotoxine type. The variability and prevalence of enterotoxins produced by staphylococci isolated from mastitic cows is very important clinical and epidemiological problem. The analysis of enterotoxins interrelations, their structure, properties and occurrence, will provide better revealing their role in the emerging, development and spreading of human and animal diseases. Classical enterotoxins, as well as the new types of these proteins, are variable element of staphylococcal virulence that connects the occurence of mastitis with human food poisonings.