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.

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.

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.

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.

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.

Enhancing the Potency of Antimicrobial Peptides through Molecular Engineering and Self-Assembly.

Healthcare-associated infections resulting from bacterial attachment and biofilm formation on medical implants are posing significant challenges in particular with the emergence of bacterial resistance to antibiotics. Here, we report the design, synthesis and characterization of self-assembled nanostructures, which integrate on their surface antibacterial peptides. The antibacterial WMR peptide, which is a modification of the native sequence of the myxinidin, a marine peptide isolated from the epidermal mucus of hagfish, was used considering its enhanced activity against Gram-negative bacteria. WMR was linked to a peptide segment of aliphatic residues (AAAAAAA) containing a lipidic tail (C19H38O2) attached to the ε-amino of a terminal lysine to generate a peptide amphiphile (WMR PA). The self-assembly of the WMR PA alone, or combined with coassembling shorter PAs, was studied using spectroscopy and microscopy techniques. The designed PAs were shown to self-assemble into stable nanofiber structures and these nanoassemblies significantly inhibit biofilm formation and eradicate the already formed biofilms of Pseudomonas aeruginosa (Gram-negative bacteria) and Candida albicans (pathogenic fungus) when compared to the native WMR peptide. Our results provide insights into the design of peptide based supramolecular assemblies with antibacterial activity, and establish an innovative strategy to develop self-assembled antimicrobial materials for biomedical applications.

Genotoxicity of gold nanoparticles functionalized with indolicidin towards Saccharomyces cerevisiae.

The toxic effects of gold nanoparticles surface-functionalized with the antimicrobial peptide indolicidin (AuNPs-indolicidin) towards the yeast Saccharomyces cerevisiae, one of the major eukaryotic model organisms, have been evaluated. Growth and survival, genotoxicity, as measured by comet assay, and expression of the YCA1, an apoptosis indicating gene, following 72hr exposure of yeast to AuNPs-indolicidin, and to AuNPs and indolicidin alone have been examined. The gold nanoparticles exerted toxicity with DNA damage, accompanied by reactive oxygen species production (ROS), but they do not inhibit yeast growth and viability. Genotoxicity was less pronounced for surface-functionalized nanoparticles, showing that S. cerevisiae is quite resistant to the complex AuNPs-indolicidin. A progressive reduction of the genotoxic effect was observed along 72hr exposure, presumably due to the activation of DNA repair mechanisms. These findings suggest the occurrence of a physiological protective response of S. cerevisiae towards nanoparticles, thereby providing useful information to the assessment of the environmental impact of metal nanoparticles.

Toxicity Effects of Functionalized Quantum Dots, Gold and Polystyrene Nanoparticles on Target Aquatic Biological Models: A Review.

Nano-based products are widespread in several sectors, including textiles, medical-products, cosmetics, paints and plastics. Nanosafety and safe-by-design are driving nanoparticle (NP) production and applications through NP functionalization (@NPs). Indeed, @NPs frequently present biological effects that differ from the parent material. This paper reviews the impact of quantum dots (QDs), gold nanoparticles (AuNPs), and polystyrene-cored NPs (PSNPs), evidencing the role of NP functionalization in toxicity definition. Key biological models were taken into consideration for NP evaluation: Saccharomyces cerevisiae, fresh- (F) and saltwater (S) microalgae (Raphidocelis subcapitata (F), Scenedesmus obliquus (F) and Chlorella spp. (F), and Phaeodactylum tricornutum (S)), Daphnia magna, and Xenopus laevis. QDs are quite widespread in technological devices, and they are known to induce genotoxicity and oxidative stress that can drastically change according to the coating employed. For example, AuNPs are frequently functionalized with antimicrobial peptides, which is shown to both increase their activity and decrease the relative environmental toxicity. P-NPs are frequently coated with NH2- for cationic and COOH- for anionic surfaces, but when positively charged toxicity effects can be observed. Careful assessment of functionalized and non-functionalized NPs is compulsory to also understand their potential direct and indirect effects when the coating is removed or degraded.

A novel process-based model of microbial growth: self-inhibition in Saccharomyces cerevisiae aerobic fed-batch cultures.

BACKGROUND: Microbial population dynamics in bioreactors depend on both nutrients availability and changes in the growth environment. Research is still ongoing on the optimization of bioreactor yields focusing on the increase of the maximum achievable cell density. RESULTS: A new process-based model is proposed to describe the aerobic growth of Saccharomyces cerevisiae cultured on glucose as carbon and energy source. The model considers the main metabolic routes of glucose assimilation (fermentation to ethanol and respiration) and the occurrence of inhibition due to the accumulation of both ethanol and other self-produced toxic compounds in the medium. Model simulations reproduced data from classic and new experiments of yeast growth in batch and fed-batch cultures. Model and experimental results showed that the growth decline observed in prolonged fed-batch cultures had to be ascribed to self-produced inhibitory compounds other than ethanol. CONCLUSIONS: The presented results clarify the dynamics of microbial growth under different feeding conditions and highlight the relevance of the negative feedback by self-produced inhibitory compounds on the maximum cell densities achieved in a bioreactor.

High cell density culture with S. cerevisiae CEN.PK113-5D for IL-1ß production: optimization, modeling, and physiological aspects.

Saccharomyces cerevisiae CEN.PK113-5D, a strain auxotrophic for uracil belonging to the CEN.PK family of the yeast S. cerevisiae, was cultured in aerated fed-batch reactor as such and once transformed to express human interleukin-1ß (IL-1ß), aiming at obtaining high cell densities and optimizing IL-1ß production. Three different exponentially increasing glucose feeding profiles were tested, all of them "in theory" promoting respiratory metabolism to obtain high biomass/product yield. A non-structured non-segregated model was developed to describe the performance of S. cerevisiae CEN.PK113-5D during the fed-batch process and, in particular, its capability to metabolize simultaneously glucose and ethanol which derived from the precedent batch growth. Our study showed that the proliferative capacity of the yeast population declined along the fed-batch run, as shown by the exponentially decreasing specific growth rates on glucose. Further, a shift towards fermentative metabolism occurred. This shift took place earlier the higher was the feed rate and was more pronounced in the case of the recombinant strain. Determination of some physiological markers (acetate production, intracellular ROS accumulation, catalase activity and cell viability) showed that neither poor oxygenation nor oxidative stress was responsible for the decreased specific growth rate, nor for the shift to fermentative metabolism.

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.

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.

Extracellular Self-DNA Effects on Yeast Cell Cycle and Transcriptome during Batch Growth.

The cell cycle and the transcriptome dynamics of yeast exposed to extracellular self-DNA during an aerobic batch culture on glucose have been investigated using cytofluorimetric and RNA-seq analyses. In parallel, the same study was conducted on yeast cells growing in the presence of (heterologous) nonself-DNA. The self-DNA treatment determined a reduction in the growth rate and a major elongation of the diauxic lag phase, as well as a significant delay in the achievement of the stationary phase. This was associated with significant changes in the cell cycle dynamics, with slower exit from the G0 phase, followed by an increased level of cell percentage in the S phase, during the cultivation. Comparatively, the exposure to heterologous DNA did not affect the growth curve and the cell cycle dynamics. The transcriptomic analysis showed that self-DNA exposure produced a generalized downregulation of transmembrane transport and an upregulation of genes associated with sulfur compounds and the pentose phosphate pathway. Instead, in the case of the nonself treatment, a clear response to nutrient deprivation was detected. Overall, the presented findings represent further insights into the complex functional mechanisms of self-DNA inhibition.

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.

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.

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.

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.

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.