Evaluation of Potential Probiotic Properties and In Vivo Safety of Lactic Acid Bacteria and Yeast Strains Isolated from Traditional Home-Made Kefir.

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.

A metabolomics footprinting approach using GC-MS to study inhibitory effects of the fungal metabolite diplopyrone C against nosocomial pathogen biofilms.

Seen initially as wonder drugs, the widespread and often inappropriate use of antibiotics led to the development of microbial resistances. As a result, a true emergency has arisen, and a significant need has emerged to discover and develop new safe and valuable antibiotics. The captivating chemical structure of the fungal metabolite diplopyrone C has caught our attention as an excellent candidate for a circumstantial study aimed at revealing its antimicrobial and antibiofilm activities. In this work, we describe the full analytical strategy from the isolation/identification to the evaluation of the metabolomics effect on target microorganisms of this fungal metabolite. Our results show interesting antimicrobial and antibiofilm activities of diplopyrone C against two frequently isolated nosocomial pathogens (i.e., the fungus Candida albicans and the gram-negative bacterium Klebsiella pneumoniae). Moreover, a GC-MS based metabolomics footprinting approach gave an insight into the uptake and excretion of metabolites from and into the culture medium as a response to the presence of this active substance. The workflow employed in this study is suitable to exploit natural resources for the search of lead compounds for drug development.

A Preliminary Evaluation on the Antifungal Efficacy of VT-1161 against Persister Candida albicans Cells in Vulvovaginal Candidiasis.

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.

Extracellular Vesicles from a Biofilm of a Clinical Isolate of Candida albicans Negatively Impact on Klebsiella pneumoniae Adherence and Biofilm Formation.

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.

Effect of Escin Alone or in Combination with Antifungal Agents on Resistant Candida glabrata Biofilms: Mechanisms of Action.

Nowadays, the increase in antimicrobial-resistant fungi (AMR) is certainly a major health concern, and the development of alternative therapeutic strategies has become crucial. Natural products have been used to treat various infections, and their chemical properties contribute to the performance of their biological activities, such as antifungal action. The various virulence factors and mechanisms of resistance to antifungals contribute to making Candida glabrata one of the most frequent agents of candidiasis. Here we investigate the in vitro and in vivo activity of ß-escin against Candida glabrata. The ß-escin MICs were determined for a reference strain and two clinical isolates of C. glabrata. Furthermore, growth kinetics assays and biofilm inhibition/eradication assays (crystal violet) were performed. The differences in the expression of some anti-biofilm-associated genes were analyzed during biofilm inhibition treatment so that reactive oxygen species could be detected. The efficacy of ß-escin was evaluated in combination with fluconazole, ketoconazole, and itraconazole. In addition, a Galleria mellonella infection model was used for in vivo treatment assays. Results have shown that ß-escin had no toxicity in vitro or in vivo and was able to inhibit or destroy biofilm formation by downregulating some important genes, inducing ROS activity and affecting the membrane integrity of C. glabrata cells. Furthermore, our study suggests that the combination with azoles can have synergistic effects against C. glabrata biofilm. In summary, the discovery of new antifungal drugs against these resistant fungi is crucial and could potentially lead to the development of future treatment strategies.

Anti-Biofilm Activity of Phenyllactic Acid against Clinical Isolates of Fluconazole-Resistant Candida albicans.

Commonly found colonizing the human microbiota, Candida albicans is a microorganism known for its ability to cause infections, mainly in the vulvovaginal region, and is responsible for 85% to 90% of vulvovaginal candidiasis (VVC) cases. The development of drug resistance in C. albicans isolates after long-term therapy with fluconazole is an important complication to solve and new therapeutic strategies are required to target this organism and its pathogenicity. In the present study, phenyllactic acid (PLA) an important broad-spectrum antimicrobial compound was investigated for its antifungal and antivirulence activities against clinical isolates of C. albicans. Previously characterized strains of C. albicans isolates from women with VVC and C. albicans ATCC90028 were used to evaluate the antimicrobial and time dependent killing assay activity of PLA showing a MIC 7.5 mg mL-1 and a complete reduction of viable Candida cells detected by killing kinetics after 4 h of treatment with PLA. Additionally, PLA significantly reduced the biomass and the metabolic activity of C. albicans biofilms and impaired biofilm formation also with changes in ERG11, ALS3, and HWP1 genes expression as detected by qPCR. PLA eradicated pre-formed biofilms as showed also with confocal laser scanning microscopy (CLSM) observations. Furthermore, the compound prolonged the survival rate of Galleria mellonella infected by C. albicans isolates. These results indicate that PLA is a promising candidate as novel and safe antifungal agents for the treatment of vulvovaginal candidiasis.