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.

The fungal species Candida parapsilosis and the bacterial species Staphylococcus aureus may be responsible for hospital-acquired infections in patients undergoing invasive medical interventions or surgical procedures and often coinfect critically ill patients in complicating polymicrobial biofilms. The efficacy of the re-purposing therapy has recently been reported as an alternative to be used. PUFAs (polyunsaturated fatty acids) may be used alone or in combination with currently available traditional antimicrobials to prevent and manage various infections overcoming antimicrobial resistance. The objectives of the study were to evaluate the effects of Resolvin D1 (RvD1) as an antimicrobial on S. aureus and C. parapsilosis, as well as the activity against the mixed biofilm of the same two species. Microdilution assays and time–kill growth curves revealed bacterial and fungal inhibition at minimum concentration values between 5 and 10 μg mL⁻¹. In single-species structures, an inhibition of 55% and 42% was reported for S. aureus and C. parapsilosis, respectively. Moreover, RvD1 demonstrated an eradication capacity of 60% and 80% for single- and mixed-species biofilms, respectively. In association with the inhibition activity, a downregulation of genes involved in biofilm formation as well as ROS accumulation was observed. Eradication capability was confirmed also on mature mixed biofilm grown on silicone platelets as shown by scanning electron microscopy (SEM). In conclusion, RvD1 was efficient against mono and polymicrobial biofilms in vitro, being a promising alternative for the treatment of mixed bacterial/fungal infections.

Candida species constitute the most common cause of fungal infections in humans; the emergence of resistance and biofilm formation by Candida species further threaten the limited availability of antifungal agents. Over the past decade, C. auris has caused significant outbreaks worldwide and has emerged as a human pathogenic fungus that causes diseases ranging from superficial to life-threatening disseminated infections. Despite the recent advances in antifungal research, the mechanisms of drug resistance in C. auris remain poorly understood even as its ability to form biofilms poses a significant therapeutic challenge. The purpose of this research was to elucidate the fungal properties of Sphaeropsidin A (SphA), a secondary metabolite derived from Diplodia fungi, with a specific focus on its efficacy against C. auris. This study revealed that SphA and its liposomal encapsulated (SphA-L) form are fungistatic with time-kill kinetics highlighting their efficacy and significantly inhibited the formation of C. auris biofilms. Our investigation into the antifungal mechanism of this drug revealed notable alterations in ROS production and the disruption of the Candida cell cycle. Our findings show that SphA-L impairs key pathogenic traits of C. auris, such as its ability to adhere to human epithelial cell lines, while exhibiting no harmful effects on human cells, highlighting its potential as a future therapeutic agent. In Caenorhabditis elegans infection models, both ShpA and SphA-L displayed effective antifungal activity, significantly reducing the C. auris fungal load and improving nematode survival rates, underscoring their promise as antifungal candidates. Overall, the potent antifungal effects of SphA and SphA-L against C. auris encourage further research.

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.