Probiotics are known for their health-promoting resources and are considered as beneficial microorganisms. The current study focuses on the isolation, and on a complete in vitro and in vivo characterization, of yeast and lactic acid bacteria acquired from traditional homemade kefir in order to assess their potentiality as probiotic candidates. In particular, the isolates Pichia kudriavzevii Y1, Lactococcus lactis subsp. hordniae LAB1 and Lactococcus lactis subsp. lactis LAB2 were subjected to in vitro characterization to evaluate their suitability as probiotics. Resistance to acid and bile salts, auto-aggregation, co-aggregation, hydrophobicity, and biofilm production capability were examined, as well as their antioxidant activity. A safety assessment was also conducted to confirm the non-pathogenic nature of the isolates, with hemolysis assay and antibiotic resistance assessment. Moreover, mortality in the invertebrate model Galleria mellonella was evaluated. Current findings showed that P. kudriavzevii exhibited estimable probiotic properties, placing them as promising candidates for functional foods. Both lactic acid bacteria isolated in this work could be classified as potential probiotics with advantageous traits, including antimicrobial activity against enteric pathogens and good adhesion ability on intestinal cells. This study revealed that homemade kefir could be a beneficial origin of different probiotic microorganisms that may enhance health and wellness.

There is an increasing interest in yeasts isolated from natural sources to be used as probiotics. Saccharomyces-based probiotics have been proposed as a valid alternative to the conventional drug therapy for the prevention and treatment of vulvovaginal candidiasis, also considering the resistance of some Candida strains to many antifungals. Here, we isolated from an artisanal sourdough a new yeast strain which was identified as Kluyveromyces marxianus and assessed its probiotic and safety properties, which resulted in comparable properties to all those exhibited by the commercial probiotic Saccharomyces boulardii. Then, we checked the antagonistic activity of the new isolate against some clinical fluconazole resistant C. albicans strains, showing its ability to inhibit filamentation, biofilm formation, and the adhesion of C. albicans to vaginal epithelial A-431 cells. Also, K. marxianus reduced the cell damage provoked by C. albicans and the expression of SAP2 and SAP6 genes. On the whole, our results enlarge the spectrum of the beneficial properties of the food-grade yeast K. marxianus showing for the first time its biotherapeutic potential against C. albicans.

The opportunistic human fungal pathogen Candida albicans produces and releases into the surrounding medium extracellular vesicles (EVs), which are involved in some processes as communication between fungal cells and host–pathogen interactions during infection. Here, we have conducted the isolation of EVs produced by a clinical isolate of C. albicans during biofilm formation and proved their effect towards the ability of the Gram-negative bacterial pathogen Klebsiella pneumoniae to adhere to HaCaT cells and form a biofilm in vitro. The results represent the first evidence of an antagonistic action of fungal EVs against bacteria.

Nowadays, the spread of foodborne diseases and the growing concerns about antibiotic resistance have shifted the focus of researchers towards the use of substances of natural origin. Phenyllactic acid (PLA), a naturally produced compound, has already demonstrated antimicrobial properties against pathogenic microorganisms and those responsible for food spoilage. This study examines the antibacterial and antibiofilm properties of PLA against foodborne pathogens such as Salmonella enterica Derby and Escherichia coli O26. The study showed that PLA effectively inhibited both biofilm formation and bacterial planktonic growth, with minimal inhibitory concentrations (MICs) ranging from 2 to 2.75 mg mL⁻¹. A dose-dependent inhibition of biofilm formation was observed, reaching approximately 90% for Salmonella strains and 50% for E. coli at 1.5 mg mL⁻¹. The cytotoxicity evaluation on Caco-2 cells showed that PLA was well-tolerated at concentrations up to 2 mg mL⁻¹. PLA’s effectiveness was also demonstrated in real food matrices, where its application in minced beef stored at 4 °C significantly reduced microbial populations, unlike in untreated samples where bacterial counts increased. PLA showed a good ability to inhibit biofilm formation and eradicate a mature biofilm, measuring the total bacterial biofilm biomass. Additionally, PLA was found to be biocompatible in Caco-2 cells, confirming that it poses no health risk at the tested concentrations. The study also observed that PLA reduced bacterial adhesion to intestinal cells, suggesting its potential in preventing intestinal bacterial colonization. These results highlight PLA as a promising natural antimicrobial agent for food preservation, with potential applications in sustainable packaging and controlling microbial contamination in food processing. Future studies should further explore PLA’s long-term stability and its interactions in complex food environments.

Background/Objectives: Extracellular vesicles (EVs) derived from probiotics represent a new and exciting frontier in host-microbe therapeutics. These nanoscale carriers are not merely cellular byproducts but are sophisticated mediators of intercellular communication, capable of modulating immune responses, reducing inflammation, and inhibiting pathogens through a rich cargo of bioactive molecules. Methods: The EVs isolated from the culture supernatants of the yeast probiotic candidate Pichia kudriavzevii were characterized for their dimensions, protein composition, and targeting both the gut pathogen virulence and the host inflammatory response. Results: The vesicles had a size distribution from 100 to 150 nm, which is consistent with previous reports on fungal EVs. Proteomic analysis of the purified EVs identified a complex array of 189 proteins, hypothesized to be responsible for some of the antimicrobial and immunomodulatory properties observed. Safety was a key consideration, and the isolated EVs demonstrated no cytotoxicity in human Caco-2 cells and no in vivo toxicity in the Galleria mellonella larval model, confirming their potential for safe use. Conclusions: The field is moving towards a new era of “postbiotics,” where cell-free therapies offer a safer, more stable alternative to live probiotics.

Persister cells are a small fraction of the microbial population that survive lethal concentrations of antimicrobial agents. Candida albicans causes vaginal candidiasis, including recurrent vulvovaginal candidiasis, and may survive common antifungal treatments. The triazole VT-1161 is an antifungal agent that specifically targets fungal CYP51, as opposed to the human CYP enzyme. This work illustrates a new role of VT-1161 in eradicating the biofilm created from the persister cells of a primary biofilm of a clinical vaginal isolate of C. albicans. Antifungal activity was determined by the minimum inhibitory concentration (MIC), and the primary biofilm was treated with amphotericin B to obtain persister cells that were able to form a new biofilm. Results obtained using the new azole VT-1161 showed that VT-1161 not only eradicated a secondary biofilm formed from the persister-derived biofilm and counteracted the adhesion of C. albicans in vitro to human cells but also ameliorated C. albicans-induced infection in vivo in Galleria mellonella larvae, suggesting that it could be proposed as an alternative therapeutic strategy for the treatment of recurrent candidiasis.