Epiphytic Yeasts from South Romania for Preventing Food Microbial Contamination.

Epiphytic yeasts represent an important source for the development of novel strategies aiming to combat food microbial contamination. The present study deals with the characterization of nine yeast strains belonging to Starmerella, Candida, Metschinikowia, Lachancea, Kodamaea and Pichia genera, isolated from the surface of plants from the Botanical Garden "Dimitrie Brandza" (Bucharest, Romania) for use as antimicrobial and probiotic agents. The tests involved the determination of the safe status, cell growth under stress conditions, and activity against pathogenic Candida and bacteria strains, respectively, as well as phytopathogenic filamentous fungi and lipolytic activity. None of the nine strains showed all the characteristics for virulence and pathogenicity, with the rare positive results being explained rather by their adaptability to the habitats of origin. The strains Lachancea thermotolerans CMGB-ST12 and Kodamaea ohmeri CMGB-ST19 grew at 37 °C; Metschnikowia reukaufii CMGB-ST21.2, M. reukaufii CMGB-ST.8.1 and M. reukaufii CMGB ST10 grew in the presence of 10% NaCl, while L. thermotolerans CMGB-ST12 and K. ohmeri CMGB-ST19 tolerated both acidic and alkaline pH values well (3.0 to 12.0). The studied yeast strains showed good antimicrobial activity against Candida krusei, Candida albicans and Gram-negative bacterial strains, with K. ohmeri CMGB-ST19 and Pichia membranaefaciens CMGB-ST53 inhibiting up to 100% the development of filamentous fungi. All the strains produced lipases for tributyrin hydrolysis, the best producer being Starmerella bombi CMGB-ST1, and only Candida magnoliae CMGB-ST8.2 tested positive against other probiotic yeasts. Overall, our nine yeast strains show high potential for industrial applications, for obtaining probiotic products and for preventing the development of a wide range of microbial food contaminants.

Phenotypic and Genotypic Characterization of Resistance and Virulence Markers in Candida spp. Isolated from Community-Acquired Infections in Bucharest, and the Impact of AgNPs on the Highly Resistant Isolates.

BACKGROUND: This study aimed to determine, at the phenotypic and molecular levels, resistance and virulence markers in Candida spp. isolated from community-acquired infections in Bucharest outpatients during 2021, and to demonstrate the efficiency of alternative solutions against them based on silver nanoparticles (AgNPs). METHODS: A total of 62 Candida spp. strains were isolated from dermatomycoses and identified using chromogenic culture media and MALDI-TOF MS, and then investigated for their antimicrobial resistance and virulence markers (VMs), as well as for metabolic enzymes using enzymatic tests for the expression of soluble virulence factors, their biofilm formation and adherence capacity on HeLa cells, and PCR assays for the detection of virulence markers and the antimicrobial activity of alternative solutions based on AgNPs. RESULTS: Of the total of 62 strains, 45.16% were Candida parapsilosis; 29.03% Candida albicans; 9.67% Candida guilliermondii; 3.22% Candida lusitaniae, Candia pararugosa, and Candida tropicalis; and 1.66% Candida kefyr, Candida famata, Candida haemulonii, and Candida metapsilosis. Aesculin hydrolysis, caseinase, and amylase production were detected in the analyzed strains. The strains exhibited different indices of adherence to HeLa cells and were positive in decreasing frequency order for the LIP1, HWP1, and ALS1,3 genes (C. tropicalis/C. albicans). An inhibitory effect on microbial growth, adherence capacity, and on the production of virulence factors was obtained using AgNPs. CONCLUSIONS: The obtained results in C. albicans and Candida non-albicans circulating in Bucharest outpatients were characterized by moderate-to-high potential to produce VMs, necessitating epidemiological surveillance measures to minimize the chances of severe invasive infections.

Molecular Basis of Yeasts Antimicrobial Activity-Developing Innovative Strategies for Biomedicine and Biocontrol.

In the context of the growing concern regarding the appearance and spread of emerging pathogens with high resistance to chemically synthetized biocides, the development of new agents for crops and human protection has become an emergency. In this context, the yeasts present a huge potential as eco-friendly agents due to their widespread nature in various habitats and to their wide range of antagonistic mechanisms. The present review focuses on some of the major yeast antimicrobial mechanisms, their molecular basis and practical applications in biocontrol and biomedicine. The synthesis of killer toxins, encoded by dsRNA virus-like particles, dsDNA plasmids or chromosomal genes, is encountered in a wide range of yeast species from nature and industry and can affect the development of phytopathogenic fungi and other yeast strains, as well as human pathogenic bacteria. The group of the "red yeasts" is gaining more interest over the last years, not only as natural producers of carotenoids and rhodotorulic acid with active role in cell protection against the oxidative stress, but also due to their ability to inhibit the growth of pathogenic yeasts, fungi and bacteria using these compounds and the mechanism of competition for nutritive substrate. Finally, the biosurfactants produced by yeasts characterized by high stability, specificity and biodegrability have proven abilities to inhibit phytopathogenic fungi growth and mycelia formation and to act as efficient antibacterial and antibiofilm formation agents for biomedicine. In conclusion, the antimicrobial activity of yeasts represents a direction of research with numerous possibilities of bioeconomic valorization as innovative strategies to combat pathogenic microorganisms.

The Antimicrobial Potency of Mesoporous Silica Nanoparticles Loaded with Melissa officinalis Extract.

Melissa officinalis is an important medicinal plant that is used and studied intensively due to its numerous pharmacological effects. This plant has numerous active compounds with biomedical potential; some are volatile, while others are sensitive to heat or oxygen. Therefore, to increase stability and prolong biological activities, the natural extract can be loaded into various nanostructured systems. In this study, different loading systems were obtained from mesoporous silica, like Mobile Composition of Matter family (MCM) with a hexagonal (MCM-41) or cubic (MCM-48) pore structure, simple or functionalized with amino groups (using 3-aminopropyl) such as triethoxysilane (APTES). Thus, the four materials were characterized from morphological and structural points of view by scanning electron microscopy, a BET analysis with adsorption-desorption isotherms, Fourier-transform infrared spectroscopy (FTIR) and a thermogravimetric analysis coupled with differential scanning calorimetry. Natural extract from Melissa officinalis was concentrated and analyzed by High-Performance Liquid Chromatography to identify the polyphenolic compounds. The obtained materials were tested against Gram-negative bacteria and yeasts and against both reference strains and clinical strains belonging to Gram-positive bacteria that were previously isolated from intra-hospital infections. The highest antimicrobial efficiency was found against Gram-positive and fungal strains. Good activity was also recorded against methicillin-resistant S. aureus, the Melissa officinalis extract inhibiting the production of various virulence factors.

Phenotypic and Genotypic Characterization of Recently Isolated Multidrug-Resistant Acinetobacter baumannii Clinical and Aquatic Strains and Demonstration of Silver Nanoparticle Potency.

This study aims to demonstrate the effectiveness of silver nanoparticles (Ag NPs) on multidrug-resistant (MDR) Acinetobacter baumannii (AB) strains isolated from the clinical and aquatic environment. Three types of Ag NPs were investigated for their antimicrobial, antibiofilm, and antivirulence properties on a total number of 132 AB strains isolated in the same temporal sequence from intra-hospital infections (IHIs), wastewater (WW), and surface water (SW) samples between 2019 and 2022 from different Romanian locations and characterized at the phenotypic and genotypic levels. The comparative analysis of the antimicrobial resistance (AR) profiles according to the isolation source and the geographical location demonstrated a decrease in MDR level in AB recovered from WW samples in 2022 from north-eastern/central/southern regions (N-E/C-W/analyzed strains S): 87.5/60/32.5%. The AB strains were lecithinase, caseinase, amylase, and lipase producers, had variable biofilm formation ability, and belonged to six genotypes associated with the presence of different virulence genes (ompA, csuE, bap, and bfmS). The Ag NPs synthesized with the solvothermal method exhibited an inhibitory effect on microbial growth, the adherence capacity to the inert substratum, and on the production of soluble virulence factors. We report here the first description of a powerful antibacterial agent against MDR AB strains circulating between hospitals and anthropically polluted water in Romania.

Current Insights in Fungal Importance-A Comprehensive Review.

Besides plants and animals, the Fungi kingdom describes several species characterized by various forms and applications. They can be found in all habitats and play an essential role in the excellent functioning of the ecosystem, for example, as decomposers of plant material for the cycling of carbon and nutrients or as symbionts of plants. Furthermore, fungi have been used in many sectors for centuries, from producing food, beverages, and medications. Recently, they have gained significant recognition for protecting the environment, agriculture, and several industrial applications. The current article intends to review the beneficial roles of fungi used for a vast range of applications, such as the production of several enzymes and pigments, applications regarding food and pharmaceutical industries, the environment, and research domains, as well as the negative impacts of fungi (secondary metabolites production, etiological agents of diseases in plants, animals, and humans, as well as deteriogenic agents).

Bioremediation of Wastewater Using Yeast Strains: An Assessment of Contaminant Removal Efficiency.

The main goal of wastewater treatment is to significantly reduce organic compounds, micronutrients (nitrogen and phosphorus) and heavy metals and other contaminants (pathogens, pharmaceuticals and industrial chemicals). In this work, the efficiency of removing different contaminants (COD, NO3-, NO2-, NH4+, PO43-, SO42-, Pb2+, Cd2+) from synthetic wastewater was tested using five different yeast strains: Kluyveromyces marxianus CMGBP16 (P1), Saccharomyces cerevisiae S228C (P2), Saccharomyces cerevisiae CM6B70 (P3), Saccharomyces cerevisiae CMGB234 (P4) and Pichia anomala CMGB88 (P5). The results showed a removal efficiency of up to 70% of COD, 97% of nitrate, 80% of nitrite, 93% of phosphate and 70% of sulfate ions for synthetic wastewater contaminated with Pb2+ (43 mg/L) and Cd2+ ions (39 mg/L). In contrast, the results showed an increase in ammonium ions, especially in the presence of Pb2+ ions. The yeast strains showed a high capacity to reduce Pb2+ (up to 96%) and Cd2+ (up to 40%) ions compared to the initial concentrations. In presence of a crude biosurfactant, the removal efficiency increased up to 99% for Pb2+ and 56% for Cd2+ simultaneously with an increase in yeast biomass of up to 11 times. The results, which were obtained in the absence of aeration and in neutral pH conditions, proved a high potential for practical applications in the biotreatment of the wastewater and the recovery of Pb and Cd ions, with a high benefit-cost ratio.

Eco-Friendly Solution Based on Rosmarinus officinalis Hydro-Alcoholic Extract to Prevent Biodeterioration of Cultural Heritage Objects and Buildings.

Biodeterioration of cultural heritage is caused by different organisms capable of inducing complex alteration processes. The present study aimed to evaluate the efficiency of Rosmarinus officinalis hydro-alcoholic extract to inhibit the growth of deteriogenic microbial strains. For this, the physico-chemical characterization of the vegetal extract by UHPLC-MS/MS, its antimicrobial and antibiofilm activity on a representative number of biodeteriogenic microbial strains, as well as the antioxidant activity determined by DPPH, CUPRAC, FRAP, TEAC methods, were performed. The extract had a total phenol content of 15.62 ± 0.97 mg GAE/mL of which approximately 8.53% were flavonoids. The polyphenolic profile included carnosic acid, carnosol, rosmarinic acid and hesperidin as major components. The extract exhibited good and wide spectrum antimicrobial activity, with low MIC (minimal inhibitory concentration) values against fungal strains such as Aspergillus clavatus (MIC = 1.2 mg/mL) and bacterial strains such as Arthrobacter globiformis (MIC = 0.78 mg/mL) or Bacillus cereus (MIC = 1.56 mg/mL). The rosemary extract inhibited the adherence capacity to the inert substrate of Penicillium chrysogenum strains isolated from wooden objects or textiles and B. thuringiensis strains. A potential mechanism of R. officinalis antimicrobial activity could be represented by the release of nitric oxide (NO), a universal signalling molecule for stress management. Moreover, the treatment of microbial cultures with subinhibitory concentrations has modulated the production of microbial enzymes and organic acids involved in biodeterioration, with the effect depending on the studied microbial strain, isolation source and the tested soluble factor. This paper reports for the first time the potential of R. officinalis hydro-alcoholic extract for the development of eco-friendly solutions dedicated to the conservation/safeguarding of tangible cultural heritage.

Molecular and Physiological Diversity of Indigenous Yeasts Isolated from Spontaneously Fermented Wine Wort from Ilfov County, Romania.

(1) Background: Wine yeast research offers the possibility of isolating new strains with distinct metabolic properties due to the geographical location of the vineyard and the processes used in winemaking. Our study deals with the isolation and identification of six yeasts from spontaneously fermented wine wort from Romania and their characterization as new potential starter culture for traditional beverages, for food industry or biomedicine. (2) Materials and methods: The isolates were identified using conventional taxonomy tests, phenotypic phylogeny analysis (Biolog YT), MALDI-TOF mass spectrometry, PCR-RFLP, and sequencing of the ITS1-5,8S-ITS2 rDNA region. The capacity of the yeasts to grow under thermal, ionic, and osmotic stress was determined. The safe status was confirmed by testing virulence and pathogenicity factors. Assays were performed in order to evaluate the growth inhibition of Candida strains and determine the antimicrobial mechanism of action. (3) Results and discussions: The yeast isolates were identified as belonging to the Metschinikowia, Hanseniaspora, Torulaspora, Pichia, and Saccharomyces genera. All the isolates were able to develop under the tested stress conditions and were confirmed as safe. With the exception of S. cerevisiae CMGB-MS1-1, all the isolates showed good antimicrobial activity based on competition for iron ions or production of killer toxins. (4) Conclusions: The results revealed the resistance of our yeasts to environmental conditions related to industrial and biomedical applications and their high potential as starter cultures and biocontrol agents, respectively.

Demonstration of Allium sativum Extract Inhibitory Effect on Biodeteriogenic Microbial Strain Growth, Biofilm Development, and Enzymatic and Organic Acid Production.

To the best of our knowledge, this is the first study demonstrating the efficiency of Allium sativum hydro-alcoholic extract (ASE) againstFigure growth, biofilm development, and soluble factor production of more than 200 biodeteriogenic microbial strains isolated from cultural heritage objects and buildings. The plant extract composition and antioxidant activities were determined spectrophotometrically and by HPLC-MS. The bioevaluation consisted of the qualitative (adapted diffusion method) and the quantitative evaluation of the inhibitory effect on planktonic growth (microdilution method), biofilm formation (violet crystal microtiter method), and production of microbial enzymes and organic acids. The garlic extract efficiency was correlated with microbial strain taxonomy and isolation source (the fungal strains isolated from paintings and paper and bacteria from wood, paper, and textiles were the most susceptible). The garlic extract contained thiosulfinate (307.66 ± 0.043 µM/g), flavonoids (64.33 ± 7.69 µg QE/g), and polyphenols (0.95 ± 0.011 mg GAE/g) as major compounds and demonstrated the highest efficiency against the Aspergillus versicolor (MIC 3.12-6.25 mg/mL), A. ochraceus (MIC: 3.12 mg/mL), Penicillium expansum (MIC 6.25-12.5 mg/mL), and A. niger (MIC 3.12-50 mg/mL) strains. The extract inhibited the adherence capacity (IIBG% 95.08-44.62%) and the production of cellulase, organic acids, and esterase. This eco-friendly solution shows promising potential for the conservation and safeguarding of tangible cultural heritage, successfully combating the biodeteriogenic microorganisms without undesirable side effects for the natural ecosystems.