Endophytic fungal diversity and bioactive potentials: investigating antimicrobial and antioxidant activities.

This study investigates the colonization of endophytic fungi in nettle leaf tissues and evaluates their antibacterial and antioxidant activities. Using an inverted optical microscope, extensive fungal colonization was observed in all leaf parts, with hyphae prevalent in epidermal cells, parenchyma cells, and vascular tissues. 144 endophytic fungal isolates were isolated from 800 leaf fragments, indicating an 18% retention rate. ANOVA analysis revealed significant differences (p < 0. 001) in colonization frequencies among 20 subjects, with subject 3 showing the highest frequency (40%) and subject 11 the lowest (2. 5%). Ethyl acetate extracts of the three most abundant endophytic fungi demonstrated notable antibacterial activity against both Gram-positive and Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Inhibition zones ranged from 9. 5 to 15. 16 mm, with minimum inhibitory concentrations (MICs) between 0. 19 to 25 mg/mL. Alternaria sp. exhibited the highest antimicrobial activity against MRSA. Antioxidant activity was assessed using the DPPH radical scavenging test and FRAP method. All extracts showed substantial free radical scavenging properties, with IC50 values close to those of standards like BHT. Alternaria sp. had the highest antioxidant activity, followed by Epicocum sp. and Ulocladium sp. The FRAP method confirmed high reducing potential, with Alternaria sp. again exhibiting the highest activity. These findings highlight the potential of endophytic fungi in nettle leaves as sources of antimicrobial and antioxidant agents, with significant implications for pharmaceutical and biotechnological applications.

Enhancing Antibiotic Efficacy and Combating Biofilm Formation: Evaluating the Synergistic Potential of Origanum vulgare Essential Oil against Multidrug-Resistant Gram-Negative Bacteria.

Multidrug-resistant (MDR) Gram-negative bacteria remain a global public health issue due to the barrier imposed by their outer membrane and their propensity to form biofilms. It is becoming imperative to develop new antibacterial strategies. In this context, this study aims to evaluate the antibacterial efficacy of Origanum vulgare essential oil (OEO), alone and in combination with antibiotics, as well as its antibiofilm action against multidrug-resistant Gram-negative strains. OEO components were identified by gas chromatography-mass spectrometry (GC-MS), and antibacterial activity was assessed using the agar diffusion test and the microdilution method. Interactions between OEO and antibiotics were examined using the checkerboard method, while antibiofilm activity was analyzed using the crystal violet assay. Chemical analysis revealed that carvacrol was the major compound in OEO (61.51%). This essential oil demonstrated activity against all the tested strains, with inhibition zone diameters (IZDs) reaching 32.3 ± 1.5 mm. The combination of OEO with different antibiotics produced synergistic and additive effects, leading to a reduction of up to 98.44% in minimum inhibitory concentrations (MICs). In addition, this essential oil demonstrated an ability to inhibit and even eradicate biofilm formation. These results suggest that OEO could be exploited in the development of new molecules, combining its metabolites with antibiotics.

Designing a multiepitope mRNA-based vaccine against enterotoxigenic Escherichia coli infection in calves: Immunoinformatics and molecular modeling approach.

Background: Escherichia coli is one of the serious pathogens causing various infections in the animal field, such as neonatal calf diarrhea, which is responsible for mortality associated with diarrhea during the first days of life. Aim: Current work is aimed at designing an effective and safe multiepitope vaccine candidate against E. coli infection in calves based on the fimbrial protein K99 of Enterotoxigenic E. coli (ETEC) and Immuno-informatics. Methods: A conserved sequence of K99 protein was generated, and then highly antigenic, nonallergic, and overlapped epitopes were used to construct a multiepitope vaccine. Five THL, six MHC II, and four beta cell epitopes were targeted to create the candidate. The candidate vaccine was produced utilizing 15 epitopes and three types of linkers, two types of untranslated region (UTR) human hemoglobin subunit beta (HBB), UTR beta-globin (Rabb), and RpfE protein as an immunomodulation adjuvant. Results: Immuno-informatics analysis of the constructed protein showed that the protein was antigenic (antigenic score of 0.8841), stable, nonallergen, and soluble. Furthermore, the Immuno-informatics and physiochemical analysis of the constructed protein showed a stable, nonallergic, soluble, hydrophilic, and acidic PI (isoelectric point). of 9.34. Docking of the candidate vaccine with the toll-like receptor TLR3 was performed, and results showed a strong interaction between the immune receptor and the vaccine. Finally, the expression efficiency of the construct in E. coli was estimated via computational cloning of the vaccine sequence into Pet28a. Conclusion: Results of immunoinformatics and in silico approaches reveal that the designed vaccine is antigenic, stable, and able to bind to the immune cell receptors. Our results interpret the proposed multiepitope mRNA vaccine as a good preventive option against E. coli infection in calves.

Chemical Composition of Ailanthus altissima (Mill.) Swingle Methanolic Leaf Extracts and Assessment of Their Antibacterial Activity through Oxidative Stress Induction.

The present study was conducted to investigate the chemical composition of Ailanthus altissima (Mill.) Swingle methanolic leaf extracts from geographically distinct regions and to assess their antimicrobial properties along with their ability to induce oxidative stress. The HPLC-DAD analysis revealed the presence of phenolic acids and flavonoids including chlorogenic acid, gallic acid, synapic acid, p-coumaric acid, apigenin, hyperoside, isoamnétine-3-O-beta-D-glucotrioside, quercetin, and isoquercetin in various amounts depending on the origin of tested extracts. The assessment of antibacterial activity showed the effectiveness of the A. altissima extracts particularly against Gram-positive bacteria, with inhibition zone diameters reaching 14 ± 1 mm and minimum inhibitory concentrations ranging from 4 to 72.2 mg/mL. These bioactive substances also exhibited strong antibiofilm activity with an eradication percentage reaching 67.07%. Furthermore, they increased ROS production to levels two to five times higher than the control group, altered the membrane integrity and caused lipid peroxidation with MDA production exceeding 2.5 µmol/mg protein in the Gram-positive and Gram-negative strains. A decrease in the levels of the antioxidant enzymes SOD and CAT was also observed, indicating an impairment of the bacterial response to the oxidative stress caused by the tested extracts. These findings highlight the antibacterial properties of A. altissima leaf extracts depending on their origins and promote their exploitation and application in the agro-food and pharmaceutical sectors.

Effect of Achillea fragrantissima Extract on Excision Wound Biofilms of MRSA and Pseudomonas aeruginosa in Diabetic Mice.

Achillea fragrantissima, a desert plant commonly known as yarrow, is traditionally used as an antimicrobial agent in folklore medicine in Saudi Arabia. The current study was undertaken to determine its antibiofilm activity against methicillin-resistant Staphylococcus aureus (MRSA) and multi-drug-resistant Pseudomonas aeruginosa (MDR-P. aeruginosa) using in vitro and in vivo studies. A biofilm model induced through an excision wound in diabetic mice was used to evaluate its effect in vivo. The skin irritation and cytotoxic effects of the extract were determined using mice and HaCaT cell lines, respectively. The Achillea fragrantissima methanolic extract was analyzed with LC-MS to detect different phytoconstituents, which revealed the presence of 47 different phytoconstituents. The extract inhibited the growth of both tested pathogens in vitro. It also increased the healing of biofilm-formed excision wounds, demonstrating its antibiofilm, antimicrobial, and wound-healing action in vivo. The effect of the extract was concentration-dependent, and its activity was stronger against MRSA than MDR-P. aeruginosa. The extract formulation was devoid of a skin irritation effect in vivo and cytotoxic effect on HaCaT cell lines in vitro.

Myriad applications of bacteriophages beyond phage therapy.

Bacteriophages are the most abundant biological entity on the planet, having pivotal roles in bacterial ecology, animal and plant health, and in the biogeochemical cycles. Although, in principle, phages are simple entities that replicate at the expense of their bacterial hosts, due the importance of bacteria in all aspects of nature, they have the potential to influence and modify diverse processes, either in subtle or profound ways. Traditionally, the main application of bacteriophages is phage therapy, which is their utilization to combat and help to clear bacterial infections, from enteric diseases, to skin infections, chronic infections, sepsis, etc. Nevertheless, phages can also be potentially used for several other tasks, including food preservation, disinfection of surfaces, treatment of several dysbioses, and modulation of microbiomes. Phages may also be used as tools for the treatment of non-bacterial infections and pest control in agriculture; moreover, they can be used to decrease bacterial virulence and antibiotic resistance and even to combat global warming. In this review manuscript we discuss these possible applications and promote their implementation.

A Simple In-Vivo Method for Evaluation of Antibiofilm and Wound Healing Activity Using Excision Wound Model in Diabetic Swiss Albino Mice.

The study developed a simple and inexpensive method to induce biofilm formation in-vivo for the evaluation of the antibiofilm activity of pharmacological agents using Swiss albino mice. Animals were made diabetic using streptozocin and nicotinamide. A cover slip containing preformed biofilm along with MRSA culture was introduced into the excision wound in these animals. The method was effective in developing biofilm on the coverslip after 24 h incubation in MRSA broth which was confirmed by microscopic examination and a crystal violet assay. Application of preformed biofilm along with microbial culture induced a profound infection with biofilm formation on excision wounds in 72 h. This was confirmed by macroscopic, histological, and bacterial load determination. Mupirocin, a known antibacterial agent effective against MRSA was used to demonstrate antibiofilm activity. Mupirocin was able to completely heal the excised wounds in 19 to 21 days while in the base-treated group, healing took place between 30 and 35 days. The method described is robust and can be reproduced easily without the use of transgenic animals and sophisticated methods such as confocal microscopy.

Antibacterial Activity of Syzygium aromaticum (Clove) Bud Oil and Its Interaction with Imipenem in Controlling Wound Infections in Rats Caused by Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of infection worldwide. Clove oil's ability to inhibit the growth of MRSA was studied through in vitro and in vivo studies. The phytochemical components of clove oil were determined through gas chromatography-mass spectrometry (GC-MS) analysis. The antibacterial effects of clove oil and its interaction with imipenem were determined by studying MIC, MBC, and FIC indices in vitro. The in vivo wound-healing effect of the clove oil and infection control were determined using excision wound model rats. The GC-MS analysis of clove oil revealed the presence of 16 volatile compounds. Clove oil showed a good antibacterial effect in vitro but no interaction was observed with imipenem. Clove bud oil alone or in combination with imipenem healed wounds faster and reduced the microbial load in wounds. The findings of this study confirmed the antibacterial activity of clove oil in vitro and in vivo and demonstrated its interaction with imipenem.

Non-canonical Staphylococcus aureus pathogenicity island repression.

Mobile genetic elements control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here, we describe an unprecedented repression-derepression mechanism involved in the transfer of Staphylococcus aureus pathogenicity islands (SaPIs). Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor StlSaPI1 presents a unique tetrameric conformation never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of StlSaPI1, preventing the binding of the repressor to its cognate StlSaPI1 sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterize a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature.

Hijacking the Hijackers: Escherichia coli Pathogenicity Islands Redirect Helper Phage Packaging for Their Own Benefit.

Phage-inducible chromosomal islands (PICIs) represent a novel and universal class of mobile genetic elements, which have broad impact on bacterial virulence. In spite of their relevance, how the Gram-negative PICIs hijack the phage machinery for their own specific packaging and how they block phage reproduction remains to be determined. Using genetic and structural analyses, we solve the mystery here by showing that the Gram-negative PICIs encode a protein that simultaneously performs these processes. This protein, which we have named Rpp (for redirecting phage packaging), interacts with the phage terminase small subunit, forming a heterocomplex. This complex is unable to recognize the phage DNA, blocking phage packaging, but specifically binds to the PICI genome, promoting PICI packaging. Our studies reveal the mechanism of action that allows PICI dissemination in nature, introducing a new paradigm in the understanding of the biology of pathogenicity islands and therefore of bacterial pathogen evolution.