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 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.

Extracellular DNA secreted in yeast cultures is metabolism-specific and inhibits cell proliferation.

Extracellular DNA (exDNA) can be actively released by living cells and different putative functions have been attributed to it. Further, homologous exDNA has been reported to exert species-specific inhibitory effects on several organisms. Here, we demonstrate by different experimental evidence, including 1H-NMR metabolomic fingerprint, that the growth rate decline in Saccharomyces cerevisiae fed-batch cultures is determined by the accumulation of exDNA in the medium. Sequencing of such secreted exDNA represents a portion of the entire genome, showing a great similarity with extrachromosomal circular DNA (eccDNA) already reported inside yeast cells. The recovered DNA molecules were mostly single strands and specifically associated to the yeast metabolism displayed during cell growth. Flow cytometric analysis showed that the observed growth inhibition by exDNA corresponded to an arrest in the S phase of the cell cycle. These unprecedented findings open a new scenario on the functional role of exDNA produced by living cells.

A Re-Purposing Strategy: Sub-Lethal Concentrations of an Eicosanoid Derived from the Omega-3-Polyunsaturated Fatty Acid Resolvin D1 Affect Dual Species Biofilms.

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.

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.

VT-1161-A Tetrazole for Management of Mono- and Dual-Species Biofilms.

VT-1161 is a novel tetrazole antifungal agent with high specificity for fungal CYP51 (compared to human CYP enzymes) which has been proven to have fewer adverse effects and drug-drug interaction profiles due to fewer off-target inhibitors. In this study, we evaluated the anti-biofilm potential of VT-1161 against mono- and dual-species biofilms of Candida albicans, Klebsiella pneumoniae and Staphylococcus aureus. VT-1161 inhibited planktonic growth of all three strains, with an MIC value of 2 µg mL-1 for C. albicans and 0.5 µg mL-1 for K. pneumoniae and S. aureus, and killed 99.9% of the microbial populations, indicating a cytocidal action. Additionally, VT-1161 showed an excellent anti-biofilm action, since it inhibited mono-microbial biofilms by 80% at 0.5 µg mL-1, and dual-species biofilms of C. albicans/K. pneumoniae and C. albicans/S. aureus by 90% at the same concentration. Additionally, the eradication of mature biofilms after 24 h of VT-1161 exposure was excellent, reaching 90% at 2 µg mL-1 for both mono- and dual-species biofilms. In such mixed biofilms, the use of VT-1161 was revealed to be an alternative treatment because it was able to reduce the number of cells of each species during both inhibition and eradication. Since long-term therapy is necessary for most fungal biofilm infections due to their recurrence and obstinacy, VT-1161 showed low cytotoxicity against normal human cell lines and also against the invertebrate model Caenorhabditis elegans. Considering the excellent anti-biofilm potential and its GRAS (generally recognized as safe) status, VT-1161 may find use in the prevention or therapeutic treatment of mono- or poly-microbial biofilms.

Undesired Effect of Vancomycin Prolonged Treatment: Enhanced Biofilm Production of the Nosocomial Pathogen Candida auris.

Fungal infections are often consequent to prolonged antibiotic treatments. Vancomycin (Van) is the first-choice antibiotic in the treatment of Staphylococcus aureus infections associated with colonization of catheter surfaces. We demonstrate the direct effect of Van in promoting the formation of the biofilm of the emergent yeast pathogen Candida auris, developed in the conventional polystyrene microwell plate model, as well as on silicone surfaces (22 and 28% increase in total biomass, respectively) and on an S. aures biofilm, residual after vancomycin treatment, where C. auris achieved 99% of the mixed biofilm population. The effect of Van was assessed also in vivo, in the Galleria mellonella infection model, which showed higher mortality when infected with the yeast pathogen in the presence of the antibiotic. This evidence enhances awareness of the potential risk associated with prolonged antibiotic use in promoting fungal infections.

The Arylamidine T-2307 as a Novel Treatment for the Prevention and Eradication of Candida tropicalis Biofilms.

Candida tropicalis is an emerging pathogen with a high mortality rate due to its virulence factors, including biofilm formation, that has important repercussions on the public health system. The ability of C. tropicalis to form biofilms, which are potentially more resistant to antifungal drugs and the consequent increasing antimicrobial resistance, highlights an urgent need for the development of novel antifungal. The present study analyzed the antibiofilm capacity of the arylamidine T-2307 on two strains of Candida tropicalis. Antimicrobial activity and time-killing assays were performed to evaluate the anticandidal effects of T-2307, the antibiofilm ability on biomass inhibition and eradication was evaluated by the crystal violet (CV) method. Furthermore, in Galleria mellonella infected larvae an increased survival after pre-and post- treatment with T-2307 was observed. The MTT test was used to determine the viability of immortalized human prostate epithelial cells (PNT1A) after exposure to different concentrations of T-2307. Levels of interleukin IL-4, IL-8, IL-10 were quantified after Candida infection of PNT1A cells and treatment. Active doses of T-2307 did not affect the viability of PNT1A cells, and drug concentrations of 0.005 or 0.01 µg mL-1 inhibited the secretion of inflammatory cytokines. Taken together, these results provide new information on T-2307, indicating this drug as a new and promising alternative therapeutic option for the treatment of Candida infections.

Effect of Myrtenol and Its Synergistic Interactions with Antimicrobial Drugs in the Inhibition of Single and Mixed Biofilms of Candida auris and Klebsiella pneumoniae.

The increased incidence of mixed infections requires that the scientific community develop novel antimicrobial molecules. Essential oils and their bioactive pure compounds have been found to exhibit a wide range of remarkable biological activities and are attracting more and more attention. Therefore, the aim of this study was to evaluate myrtenol (MYR), one of the constituents commonly found in some essential oils, for its potential to inhibit biofilms alone and in combination with antimicrobial drugs against Candida auris/Klebsiella pneumoniae single and mixed biofilms. The antimicrobial activity of MYR was evaluated by determining bactericidal/fungicidal concentrations (MIC), and biofilm formation at sub-MICs was analyzed in a 96-well microtiter plate by crystal violet, XTT reduction assay, and CFU counts. The synergistic interaction between MYR and antimicrobial drugs was evaluated by the checkerboard method. The study found that MYR exhibited antimicrobial activity at high concentrations while showing efficient antibiofilm activity against single and dual biofilms. To understand the underlying mechanism by which MYR promotes single/mixed-species biofilm inhibition, we observed a significant downregulation in the expression of mrkA, FKS1, ERG11, and ALS5 genes, which are associated with bacterial motility, adhesion, and biofilm formation as well as increased ROS production, which can play an important role in the inhibition of biofilm formation. In addition, the checkerboard microdilution assay showed that MYR was strongly synergistic with both caspofungin (CAS) and meropenem (MEM) in inhibiting the growth of Candida auris/Klebsiella pneumoniae-mixed biofilms. Furthermore, the tested concentrations showed an absence of toxicity for both mammalian cells in the in vitro and in vivo Galleria mellonella models. Thus, MYR could be considered as a potential agent for the management of polymicrobial biofilms.

Competitiveness during Dual-Species Biofilm Formation of Fusarium oxysporum and Candida albicans and a Novel Treatment Strategy.

During an infection, a single or multispecies biofilm can develop. Infections caused by non-dermatophyte molds, such as Fusarium spp. and yeasts, such as Candida spp., are particularly difficult to treat due to the formation of a mixed biofilm of the two species. Fusarium oxysporum is responsible for approximately 20% of human fusariosis, while Candida albicans is responsible for superficial mucosal and dermal infections and for disseminated bloodstream infections with a mortality rate above 40%. This study aims to investigate the interactions between C. albicans and F. oxysporum dual-species biofilm, considering variable formation conditions. Further, the ability of the WMR peptide, a modified version of myxinidin, to eradicate the mixed biofilm when used alone or in combination with fluconazole (FLC) was tested, and the efficacy of the combination of WMR and FLC at low doses was assessed, as well as its effect on the expression of some biofilm-related adhesin and hyphal regulatory genes. Finally, in order to confirm our findings in vivo and explore the synergistic effect of the two drugs, we utilized the Galleria mellonella infection model. We concluded that C. albicans negatively affects F. oxysporum growth in mixed biofilms. Combinatorial treatment by WMR and FLC significantly reduced the biomass and viability of both species in mature mixed biofilms, and these effects coincided with the reduced expression of biofilm-related genes in both fungi. Our results were confirmed in vivo since the synergistic antifungal activity of WMR and FLC increased the survival of infected larvae and reduced tissue invasion. These findings highlight the importance of drug combinations as an alternative treatment for C. albicans and F. oxysporum mixed biofilms.

WMR Peptide as Antifungal and Antibiofilm against Albicans and Non-Albicans Candida Species: Shreds of Evidence on the Mechanism of Action.

Candida species are the most common fungal pathogens infecting humans and can cause severe illnesses in immunocompromised individuals. The increased resistance of Candida to traditional antifungal drugs represents a great challenge in clinical settings. Therefore, novel approaches to overcome antifungal resistance are desired. Here, we investigated the use of an antimicrobial peptide WMR against Candida albicans and non-albicans Candida species in vitro and in vivo. Results showed a WMR antifungal activity on all Candida planktonic cells at concentrations between 25 µM to >50 µM and exhibited activity at sub-MIC concentrations to inhibit biofilm formation and eradicate mature biofilm. Furthermore, in vitro antifungal effects of WMR were confirmed in vivo as demonstrated by a prolonged survival rate of larvae infected by Candida species when the peptide was administered before or after infection. Additional experiments to unravel the antifungal mechanism were performed on C. albicans and C. parapsilosis. The time-killing curves showed their antifungal activity, which was further confirmed by the induced intracellular and mitochondrial reactive oxygen species accumulation; WMR significantly suppressed drug efflux, down-regulating the drug transporter encoding genes CDR1. Moreover, the ability of WMR to penetrate within the cells was demonstrated by confocal laser scanning microscopy. These findings provide novel insights for the antifungal mechanism of WMR against Candida albicans and non-albicans, providing fascinating scenarios for the identification of new potential antifungal targets.

An Integrated Analysis of Intracellular Metabolites and Virulence Gene Expression during Biofilm Development of a Clinical Isolate of Candida tropicalis on Distinct Surfaces.

Emergence of Candida tropicalis, which causes potential life-threatening invasive candidiasis, is often associated with colonization of medical devices as biofilm. Biofilm plays an important role in the virulence of the pathogen because of its complex structure, which provides resistance to conventional antimicrobials. In this study, the metabolic response of a clinical strain of C. tropicalis colonizing three distinct surfaces (polytetrafluoroethylene (PTFE), polystyrene, and polycarbonate) as well as the expression of virulence and stress related genes (ALS3, Hsp21, SAP1, SAP2, SAP3, and CYR1), were explored. Our results showed that lesser biofilm was developed on PTFE compared to polystyrene and polycarbonate. GS-MS metabolic analysis identified a total of 36 metabolites in the intracellular extract of cells grown on polystyrene, polycarbonate, and PTFE, essentially belonging to central carbon metabolism, amino acids, and lipids metabolism. The metabolic analysis showed that saturated and unsaturated fatty acids are preferentially produced during biofilm development on polycarbonate, whereas trehalose and vitamin B6, known as cellular protectors against a variety of stressors, were characteristic of biofilm on PTFE. The results of the transcriptomic analysis consider the different degrees of colonization of the three substrates, being CYR1, which encodes the component of signaling pathway of hyphal formation-cAMP-PKA, downregulated in PTFE biofilm compared to polycarbonate or polystyrene biofilms, while Hsp21 was upregulated in concomitance with the potential unfavorable conditions for biofilm formation on PTFE. Overall, this work provides new insights into the knowledge of C. tropicalis biofilm development on surfaces of medical relevance in the perspective of improving the management of Candida infections.

A cytofluorimetric analysis of a Saccharomyces cerevisiae population cultured in a fed-batch bioreactor.

The yeast Saccharomyces cerevisiae is a reference model system and one of the widely used microorganisms in many biotechnological processes. In industrial yeast applications, combined strategies aim to maximize biomass/product yield, with the fed-batch culture being one of the most frequently used. Flow cytometry (FCM) is widely applied in biotechnological processes and represents a key methodology to monitor cell population dynamics. We propose here an application of FCM in the analysis of yeast cell cycle along the time course of a typical S. cerevisiae fed-batch culture. We used two different dyes, SYTOX Green and SYBR Green, with the aim to better define each stage of cell cycle during S. cerevisiae fed-batch culture. The results provide novel insights in the use of FCM cell cycle analysis for the real-time monitoring of S. cerevisiae bioprocesses.

GC-MS-Based Metabolomics Study of Single- and Dual-Species Biofilms of Candida albicans and Klebsiella pneumoniae.

Candida albicans and Klebsiella pneumoniae frequently co-exist within the human host as a complex biofilm community. These pathogens are of interest because their association is also related to significantly increased morbidity and mortality in hospitalized patients. With the aim of highlighting metabolic shifts occurring in the dual-species biofilm, an untargeted GC-MS-based metabolomics approach was applied to single and mixed biofilms of C. albicans and K. pneumoniae. Metabolomic results showed that among the extracellular metabolites identified, approximately 40 compounds had significantly changed relative abundance, mainly involving central carbon, amino acid, vitamin, and secondary metabolisms, such as serine, leucine, arabitol, phosphate, vitamin B6, cyclo-(Phe-Pro), trehalose, and nicotinic acid. The results were related to the strict interactions between the two species and the different microbial composition in the early and mature biofilms.

The Membranotropic Peptide gH625 to Combat Mixed Candida albicans/Klebsiella pneumoniae Biofilm: Correlation between In Vitro Anti-Biofilm Activity and In Vivo Antimicrobial Protection.

The antibiofilm activity of a gH625 analogue was investigated to determine the in vitro inhibition and eradication of a dual-species biofilm of Candida albicans and Klebsiella pneumoniae, two leading opportunistic pathogens responsible for several resistant infections. The possibility of effectively exploiting this peptide as an alternative anti-biofilm strategy in vivo was assessed by the investigation of its efficacy on the Galleria mellonella larvae model. Results on larvae survival demonstrate a prophylactic efficacy of the peptide towards the infection of each single microorganism but mainly towards the co-infection. The expression of biofilm-related genes in vivo showed a possible synergy in virulence when these two species co-exist in the host, which was effectively prevented by the peptide. These findings provide novel insights into the treatment of medically relevant bacterial-fungal interaction.

Activity of Free and Liposome-Encapsulated Essential Oil from Lavandula angustifolia against Persister-Derived Biofilm of Candida auris.

The high virulence of Candida auris, a pathogen fungus considered as a global threat for public health, is due to its peculiar traits such as its intrinsic resistance to conventional antifungals. Its biofilm lifestyle certainly promotes the prolonged survival of C. auris after disinfection or antifungal treatments. In this work, for the first time, we detected persister cells in a biofilm of C. auris in a microwell plate model, following caspofungin treatment. Furthermore, we showed how persisters can progressively develop a new biofilm in situ, mimicking the re-colonization of a surface which may be responsible for recalcitrant infections. Plant-derived compounds, such as essential oils, may represent a valid alternative to combat fungal infections. Here, Lavandula angustifolia essential oil, as free or encapsulated in liposomes, was used to eradicate primary and persister-derived biofilms of C. auris, confirming the great potential of alternative compounds against emergent fungal pathogens. As in other Candida species, the action of essential oils against C. auris involves ROS production and affects the expression of some biofilm-related genes.

A General Process-Based Model for Describing the Metabolic Shift in Microbial Cell Cultures.

The metabolic shift between respiration and fermentation at high glucose concentration is a widespread phenomenon in microbial world, and it is relevant for the biotechnological exploitation of microbial cell factories, affecting the achievement of high-cell-densities in bioreactors. Starting from a model already developed for the yeast Saccharomyces cerevisiae, based on the System Dynamics approach, a general process-based model for two prokaryotic species of biotechnological interest, such as Escherichia coli and Bacillus subtilis, is proposed. The model is based on the main assumption that glycolytic intermediates act as central catabolic hub regulating the shift between respiratory and fermentative pathways. Furthermore, the description of a mixed fermentation with secondary by-products, characteristic of bacterial metabolism, is explicitly considered. The model also represents the inhibitory effect on growth and metabolism of self-produced toxic compounds relevant in assessing the late phases of high-cell density culture. Model simulations reproduced data from experiments reported in the literature with different strains of non-recombinant and recombinant E. coli and B. subtilis cultured in both batch and fed-batch reactors. The proposed model, based on simple biological assumptions, is able to describe the main dynamics of two microbial species of relevant biotechnological interest. It demonstrates that a reductionist System Dynamics approach to formulate simplified macro-kinetic models can provide a robust representation of cell growth and accumulation in the medium of fermentation by-products.

Eradication of Candida albicans persister cell biofilm by the membranotropic peptide gH625.

Biofilm formation poses an important clinical trouble due to resistance to antimicrobial agents; therefore, there is an urgent demand for new antibiofilm strategies that focus on the use of alternative compounds also in combination with conventional drugs. Drug-tolerant persisters are present in Candida albicans biofilms and are detected following treatment with high doses of amphotericin B. In this study, persisters were found in biofilms treated with amphotericin B of two clinical isolate strains, and were capable to form a new biofilm in situ. We investigated the possibility of eradicating persister-derived biofilms from these two Candida albicans strains, using the peptide gH625 analogue (gH625-M). Confocal microscopy studies allowed us to characterize the persister-derived biofilm and understand the mechanism of interaction of gH625-M with the biofilm. These findings confirm that persisters may be responsible for Candida biofilm survival, and prove that gH625-M was very effective in eradicating persister-derived biofilms both alone and in combination with conventional antifungals, mainly strengthening the antibiofilm activity of fluconazole and 5-flucytosine. Our strategy advances our insights into the development of effective antibiofilm therapeutic approaches.

The Role of DNA in the Extracellular Environment: A Focus on NETs, RETs and Biofilms.

The capacity to actively release genetic material into the extracellular environment has been reported for bacteria, archaea, fungi, and in general, for microbial communities, but it is also described in the context of multicellular organisms, animals and plants. This material is often present in matrices that locate outside the cells. Extracellular matrices have important roles in defense response and disease in microbes, animal and plants cells, appearing as barrier against pathogen invasion or for their recognition. Specifically, neutrophils extracellular traps (NETs) in animals and root extracellular traps (RETs) in plants, are recognized to be important players in immunity. A growing amount of evidence revealed that the extracellular DNA, in these contexts, plays an active role in the defense action. Moreover, the protective role of extracellular DNA against antimicrobials and mechanical stress also appears to be confirmed in bacterial biofilms. In parallel, recent efforts highlighted different roles of self (homologous) and non-self (heterologous) extracellular DNA, paving the way to discussions on its role as a "Damage-associated molecular pattern" (DAMP). We here provide an evolutionary overview on extracellular DNA in extracellular matrices like RETs, NETs, and microbial biofilms, discussing on its roles and inferring on possible novel functionalities.