Search Results (17)

With rising multidrug-resistant yeast pathogens, conventional antifungals are becoming less effective, urging the need for adjuvants that enhance their activity at lower doses. This study evaluated the synergistic activity of antimicrobial peptidomimetics (TM8 and RK758) or colistin sulphate in combination with conventional antifungals against Candida albicans, C. tropicalis, C. parapsilosis, Meyerozyma guilliermondii, Nakaseomyces glabratus, Pichia kudriavzevii and Kluyveromyces marxianus, and Candidozyma auris using the checkerboard microdilution test. RK758 was synergistic with fluconazole in 78% of isolates, with the remaining 22% of isolates still showing partial synergy; it showed synergy with amphotericin B in 56% of isolates, and with caspofungin, 78% of isolates exhibited either synergy or partial synergy. TM8 showed synergy with fluconazole in 44% (with partial synergy in another 44%) of isolates, with amphotericin B in 67% of isolates, and with caspofungin in 44% (with partial synergy in another 44%) of isolates. Colistin with fluconazole or caspofungin exhibited synergy or partial synergy in 56% of the isolates. No antagonism was observed in any of the combinations. Additionally, a time-kill assay further demonstrated synergistic activity between fluconazole and TM8 or RK758. The effects of these peptidomimetics on cell membrane integrity were demonstrated in an ergosterol binding assay, supported by SYTOX Green and cellular leakage assays, both indicating a lytic effect. These results suggest that peptidomimetics can synergise with conventional antifungals, offering a potential strategy for combination therapy against yeast infections. The membrane lytic activity of the peptidomimetics likely plays a role in their synergistic interaction with antifungals, thereby enhancing the antimicrobial activities of both compounds at sub-MIC levels. Full article

Objectives: A simple method for quantifying fluconazole in small blood volumes has been developed using volumetric absorptive microsampling (VAMS^®) technology and isocratic high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. Methods: For sample collection, Mitra^® devices are used to keep the sample volume at 10 µL. For the quantitative determination of fluconazole, the Mitra^® samples are extracted using acetonitrile as the extraction agent, containing 2-(4-chlorophenyl)-1,3-bis(1,2,4-triazol-1-yl)propan-2-ol as the internal standard. A Synergi 4 μm Polar-RP 80 Å (150 × 2 mm) column forms the stationary phase, and a mixture of acetonitrile and phosphate buffer is the mobile phase. The UV detection is set at a wavelength of 210 nm. The therapeutic concentration range of 5 to 160 mg/L is covered, and the linear equation with 1/x² weighting is used to determine unknown samples. This method has been validated according to the current EMA and FDA guidelines for bioanalytical methods. Results: The validation data obtained after analysing whole blood samples (EDTA) showed within- and between-run accuracy between 94.4% and 115% and precision between 0.4% and 9.4%, respectively. A lower limit of quantification (LLOQ) of 5 mg/L was sufficient for therapeutic drug monitoring in paediatric patients receiving fluconazole as antifungal prophylaxis after haematopoietic cell transplantation. Conclusions: So far, 211 samples from 49 patients were successfully analysed, and concentrations between 5.84 mg/L and 107 mg/L were determined for whole blood Mitra^® samples. To our knowledge, this is the first application of VAMS^® technology using simple and cheap HPLC-UV quantification. Full article

The rising antifungal resistance in Nakaseomyces glabratus, especially to azole drugs like fluconazole, itraconazole, and voriconazole, presents a significant clinical challenge. Plant-derived compounds with synergistic antifungal effects offer a promising solution. Fruitless wolfberry bud tea, rich in flavonoids from a Lycium barbarum L. hybrid, shows potential but is underexplored in antifungal therapies. This study assessed FWE’s antifungal efficacy alone and with azoles against resistant N. glabratus isolates, exploring mechanisms like efflux pump inhibition and gene expression changes. A total of 52 clinical isolates were tested. Fruitless wolfberry bud tea was methanol-extracted (FWE) and lyophilized. Antifungal susceptibility was evaluated using broth microdilution, and synergistic effects were analyzed with checkerboard assays. Growth inhibition, rhodamine 6G efflux, and qRT-PCR for resistance-related genes were conducted. FWE demonstrated inhibitory activity with MICs ranging from 16 to 32 μg/mL. When combined with ITR or VRC, synergistic or additive effects were observed, reducing MICs by 2–8-fold. FWE + VRC exhibited synergy (FICI ≤ 0.5) in 50% of isolates, while FWE + ITR showed synergy in 37.5%. Efflux pump activity, measured by rhodamine 6G, significantly decreased in combination groups (11.4–14.6%) compared to monotherapy (17.3–17.5%). qRT-PCR indicated downregulation of CgCDR1, CgERG11, and CgPDR1 in FWE-treated Cg 1 isolate, with greater suppression in combination groups. FWE might boost the bacteriostatic impact of azole antifungal drugs by blocking efflux pumps and altering the expression of resistance genes. This study identifies FWE as a potent adjuvant to overcome cross-resistance, supporting its inclusion in antifungal strategies. Further research to identify bioactive compounds in FWE and in vivo validation is necessary for clinical application. Full article

Motivated by the search for novel antimicrobials against opportunistic resistant pathogens and based on the reported antimicrobial activity of phthalimides, two series of phthalimide and N-phthaloylglycine esters were designed to investigate whether the addition of butyl and aryl groups enhances their antimicrobial properties. Thus, in vitro antimicrobial activity, antifungal mechanism of action, effect combined with Chloramphenicol, in silico/vitro toxicity, and a docking molecular were studied. Phthalimide and N-phthaloylglycine aryl esters were obtained in yields of 75–98%. Phthalimide aryl ester 3b (R = Me) showed the best results against Gram-(+) and Gram-(−) bacteria, S. aureus and P. aeruginosa, respectively, and yeast fungi, C. tropicalis and C. albicans, with MIC values equal to 128 µg·mL⁻¹. Regarding the antifungal mechanism of action on C. albicans, the MIC values of compound 3b changed from 128 to 1024 µg·mL⁻¹ in the presence of ergosterol. Furthermore, compound 3b showed synergy with Chloramphenicol against P. aeruginosa, with a FICI value equal to 0.5. Finally, the four most promising compounds had their in silico/vitro toxicity evaluated, which showed moderate toxicity to non-toxicity on Artemia salina larvae. With the exception of Chloramphenicol, all selected compounds, including Fluconazole, are potentially hepatotoxic, but they were predicted not to cause skin sensitization, suggesting a potential application for topical use. Molecular docking revealed that compound 3b exhibits superior binding affinity and stability with the 50S ribosomal subunit (−92.69 kcal·mol⁻¹) compared to Chloramphenicol, and a unique π–sulfur interaction with CYP51, suggesting its potential as a dual-action antibacterial and antifungal candidate against resistant pathogens. Full article

The rise in non-albicans Candida species, exhibiting unpredictable antifungal resistance, complicates treatment and contributes to the growing threat of invasive, life-threatening infections. This study evaluates the antifungal activity of four benzo[a]phenoxazine derivatives (C34, C35, A42, and A44) against 14 Candida strains following EUCAST standards. Fluconazole interactions are analysed through fractional inhibitory concentration index (FICI) calculation and response surface analysis based on the Bliss model. Macrophage-like J774A.1 cells are used to assess Candida killing in the presence of synergistic compounds. The MIC values against the different strains vary, with C34 showing the strongest activity, followed by C35, while A42 has the highest MIC values, indicating lower efficacy. However, A42 demonstrates the best synergy with fluconazole against fluconazole-resistant Candida strains. Cytotoxicity assays reveal that the chloropropyl group present in C35 and A42 enhances cytocompatibility. Co-culture with macrophages shows significant yeast killing for C. albicans and C. auris when fluconazole and A42 are combined, requiring concentrations 4 and 16 times lower than their MIC values, enhancing antifungal activity. Given fluconazole’s fungistatic nature and the emergence of drug-resistant strains, benzo[a]phenoxazine derivatives’ ability to enhance fluconazole’s efficacy present a promising strategy to address antifungal resistance in critical pathogens. These findings align with global research priorities, offering new potential avenues for developing more effective antifungal therapies. Full article

Plant-derived compounds have proven to be a source of inspiration for new drugs. In this study, piperine isolated from the fruits of Piper nigrum showed anti-Candida activity. Furthermore, the mechanisms of action of piperine and its impact on virulence factors in Candida albicans, which have not been comprehensively understood, were also assessed. Initially, piperine suppressed the hyphal transition in both liquid and solid media, hindered biofilm formation, and resulted in observable cell distortions in scanning electron microscope (SEM) samples, for both fluconazole-sensitive and fluconazole-resistant C. albicans strains. Additionally, the morphogenetic switches triggered by piperine were found to rely on the activity of mutant C. albicans strains. Secondly, piperine treatment increased cell membrane permeability and disrupted mitochondrial membrane potential, as evidenced by propidium iodine and Rhodamine 123 staining, respectively. Moreover, it induced the accumulation of intracellular reactive oxygen species in C. albicans. Synergy was obtained between the piperine and the fluconazole against the fluconazole-sensitive strain. Interestingly, there were no hemolytic effects of piperine, and it resulted in reduced cytotoxicity on fibroblast cells at low concentrations. The results suggest that piperine could have a dual mode of action inhibiting virulence factors and modulating cellular processes, leading to cell death in C. albicans. Full article

The antifungal activity of palindromic peptide RWQWRWQWR and its derivatives was evaluated against clinical isolates of Candida albicans and C. auris. Also, Bidens pilosa ethanolic extracts of leaves and stem were evaluated. Furthermore, combinations of peptide, extract, and/or fluconazole (FLC) were evaluated. The cytotoxicity of peptides and extracts in erythrocytes and fibroblasts was determined. The original palindromic peptide, some derivative peptides, and the ethanolic extract of leaves of B. pilosa exhibited the highest activity in some of the strains evaluated. Synergy was obtained between the peptide and the FLC against C. auris 435. The combination of the extract and the original palindromic peptide against C. albicans SC5314, C. auris 435, and C. auris 537 decreased the minimal inhibitory concentrations (MICs) by a factor of between 4 and 16. These mixtures induced changes in cell morphology, such as deformations on the cell surface. The results suggest that the combination of RWQWRWQWR and B. pilosa extract is an alternative for enhancing antifungal activity and decreasing cytotoxicity and costs and should be considered to be a promising strategy for treating diseases caused by Candida spp. Full article

Infectious diseases caused by various nosocomial microorganisms affect worldwide both immunocompromised and relatively healthy persons. Bacteria and fungi have different tools to evade antimicrobials, such as hydrolysis damaging the drug, efflux systems, and the formation of biofilm that significantly complicates the treatment of the infection. Here, we show that myrtenol potentiates the antimicrobial and biofilm-preventing activity of conventional drugs against S. aureus and C. albicans mono- and dual-species cultures. In our study, the two optical isomers, (−)-myrtenol and (+)-myrtenol, have been tested as either antibacterials, antifungals, or enhancers of conventional drugs. (+)-Myrtenol demonstrated a synergistic effect with amikacin, fluconazole, and benzalkonium chloride on 64–81% of the clinical isolates of S. aureus and C. albicans, including MRSA and fluconazole-resistant fungi, while (−)-myrtenol increased the properties of amikacin and fluconazole to repress biofilm formation in half of the S. aureus and C. albicans isolates. Furthermore, myrtenol was able to potentiate benzalkonium chloride up to sixteen-fold against planktonic cells in an S. aureus–C. albicans mixed culture and repressed the adhesion of S. aureus. The mechanism of both (−)-myrtenol and (+)-myrtenol synergy with conventional drugs was apparently driven by membrane damage since the treatment with both terpenes led to a significant drop in membrane potential similar to the action of benzalkonium chloride. Thus, due to the low toxicity of myrtenol, it seems to be a promising agent to increase the efficiency of the treatment of infections caused by bacteria and be fungi of the genus Candida as well as mixed fungal–bacterial infections, including resistant strains. Full article

The resistance of Candida albicans and other pathogenic yeasts to azole antifungal drugs has increased rapidly in recent years and is a significant problem in clinical therapy. The current state of pharmacological knowledge precludes the withdrawal of azole drugs, as no other active substances have yet been developed that could effectively replace them. Therefore, one of the anti-yeast strategies may be therapies that can rely on the synergistic action of natural compounds and azoles, limiting the use of azole drugs against candidiasis. Synergy assays performed in vitro were used to assess drug interactions Fractional Inhibitory Concentration Index. The synergistic effect of fluconazole (1) and three synthetic lactones identical to those naturally occurring in celery plants—3-n-butylphthalide (2), 3-n-butylidenephthalide (3), 3-n-butyl-4,5,6,7-tetrahydrophthalide (4)—against Candida albicans ATCC 10231, C. albicans ATCC 2091, and C. guilliermondii KKP 3390 was compared with the performance of the individual compounds separately. MIC₉₀ (the amount of fungistatic substance (in µg/mL) inhibiting yeast growth by 90%) was determined as 5.96–6.25 µg/mL for fluconazole (1) and 92–150 µg/mL for lactones 2–4. With the simultaneous administration of fluconazole (1) and one of the lactones 2–4, it was found that they act synergistically, and to achieve the same effect it is sufficient to use 0.58–6.73 µg/mL fluconazole (1) and 1.26–20.18 µg/mL of lactones 2–4. As fluconazole and phthalide lactones show synergy, 11 new fluconazole analogues with lower toxicity and lower inhibitory activity for CYP2C19, CYP1A2, and CYP2C9, were designed after in silico testing. The lipophilicity was also analyzed. A three-carbon alcohol with two rings was preserved. In all compounds 5–15, the 1,2,4-triazole rings were replaced with 1,2,3-triazole or tetrazole rings. The hydroxyl group was free or esterified with phenylacetic acid or thiophene-2-carboxylic acid chlorides or with adipic acid. In structures 11 and 12 the hydroxyl group was replaced with the fragment -CH₂Cl or = CH₂. Additionally, the difluorophenyl ring was replaced with unsubstituted phenyl. The structures of the obtained compounds were determined by ¹H NMR, and ¹³C NMR spectroscopy. Molecular masses were established by GC-MS or elemental analysis. The MIC₅₀ and MIC₉₀ of all compounds 1–15 were determined against Candida albicans ATCC 10231, C. albicans ATCC 2091, AM 38/20, C. guilliermondii KKP 3390, and C. zeylanoides KKP 3528. The MIC₅₀ values for the newly prepared compounds ranged from 38.45 to 260.81 µg/mL. The 90% inhibitory dose was at least twice as high. Large differences in the effect of fluconazole analogues 5–15 on individual strains were observed. A synergistic effect on three strains—Candida albicans ATCC 10231, C. albicans ATCC 2091, C. guilliermondii KKP 339—was observed. Fractional inhibitory concentrations FIC₅₀ and FIC₉₀ were tested for the most active lactone, 3-n-butylphthalide, and seven fluconazole analogues. The strongest synergistic effect was observed for the strain C. albicans ATCC 10231, FIC 0.04–0.48. The growth inhibitory amount of azole is from 25 to 55 µg/mL and from 3.13 to 25.3 µg/mL for 3-n-butylphthalide. Based on biological research, the influence of the structure on the fungistatic activity and the synergistic effect were determined. Full article

Candida auris is considered a serious fungal pathogen frequently exhibiting a high resistance to a wide range of antifungals. In this study, a combination of the quorum-sensing molecule farnesol (FAR) and fluconazole (FLU) was tested on FLU-resistant C. auris isolates (C. auris S and C. auris R) compared to the susceptible C. auris H261. The aim was to assess the possible synergy between FAR and FLU, by reducing the FLU minimal inhibitory concentration, and to determine the mechanism underlying the conjunct effect. The results confirmed a synergic effect between FAR and FLU with a calculated FIC index of 0.75 and 0.4 for C. auris S and C. auris R, respectively. FAR modulates genes involved in azole resistance. When FAR was added to the cells in combination with FLU, a significant decrease in the expression of the CDR1 gene was observed in the resistant C. auris isolates. FAR seems to block the Cdr1 efflux pump triggering a restoration of the intracellular content of FLU. These results were supported by observed increasing accumulation of rhodamine 6G by C. auris cells. Moreover, C. auris treated with FAR showed an ERG11 gene down-regulation. Overall, these results suggest that FAR is an effective modulator of the Cdr1 efflux pump in C. auris and, in combination with FLU, enhances the activity of this azole, which might be a promising strategy to control infections caused by azole-resistant C. auris. Full article

Candida auris, an evolving multidrug-resistant pathogenic yeast, is known for causing severe invasive infections associated with high mortality rates in hospitalized individuals. Distinct from other Candida species, C. auris can persist for longer periods on different surfaces and is resistant to all of the major classes of antifungal drugs. Therefore, there is an urgent need for new antimycotic drugs with improved efficacy and reduced toxicity. The development of new antifungals based on antimicrobial peptides from various sources is considered a promising alternative. In this study, we examined the in vitro anti-yeast activity of the human cathelicidin peptides LL-37 against clinical strains of C. auris alone and in combination with different antifungal drugs by broth microdilution assay. To understand the antifungal mechanism of action, cell envelopes, cell cycle arrest, and effect on oxidative stress enzymes were studied using standard protocols. The minimum inhibitory and fungicidal concentrations of cathelicidin LL-37 ranged from 25–100 and 50–200 µg/mL, respectively. A combination interaction in a 1:1 ratio (cathelicidin LL-37: antifungal drug) resulted in 70% synergy with fluconazole and 100% synergy with amphotericin B and caspofungin. Assessment of the C. auris membrane by using propidium iodide assay after exposure to cathelicidin LL-37 linked membrane permeabilization with inhibition of C. auris cell growth and viability. These results were backed up by scanning electron microscopy studies demonstrating that exposure with cathelicidin LL-37 caused C. auris cells to undergo extensive surface changes. Spectrophotometric analysis revealed that cathelicidin LL-37 caused oxidative stress in C. auris, as is evident from the significant increase in the activity of primary antioxidant enzymes. In addition, cathelicidin LL-37 inhibited the cell cycle and accumulated cells in the S phase. Therefore, these results specify the potential of cathelicidin LL-37 for developing a new and effective anti-Candida agent. Full article

Vulvovaginal candidiasis (VVC) occurs in over 75% of women at least once during their lifetime and is an infection that significantly affects their health. Candida strains resistant to standard azole antifungal therapy and relapses of VVC are more and more common. Hypothetically, biofilm is one of the main reasons of relapses and failure of the therapy. Ultrashort cationic lipopeptides (USCLs) exhibit high antimicrobial activities. Our previous study on USCLs revealed that disulfide cyclization can result in selective antifungal compounds. Therefore, four USCL were selected and their antifungal activity were studied on 62 clinical strains isolated from VVC. The results confirmed previous premises that cyclic analogs have increased selectivity between fungal cells and keratinocytes and improved anticandidal activity compared to their linear analogs against both planktonic and biofilm cultures. On the other hand, linear lipopeptides in combination with fluconazole showed a synergistic effect. It was found that the minimum inhibitory concentrations of the tested compounds in combination with fluconazole were at least four times lower than when used separately. Our results indicate that combination therapy of VVC with USCLs and fluconazole at low non-toxic concentrations can be beneficial owing to the synergistic effect. However, further in vivo studies are needed to confirm this hypothesis. Full article

Background and objectives: This study aimed to evaluate the synergistic antimicrobial activity of extracts obtained from Salvadora persica (Miswak), Commiphora molmol (myrrh) and Azadirachta indica (neem) in combination with commercially available antimicrobial agents: penicillin, tetracycline, ofloxacin and fluconazole on endodontic pathogens such as Enterococcus faecalis, Streptococcus mitis,Actinomyces naeslundii and Candida albicans. Materials and Methods: Microbiological samples from the root canals of the teeth undergoing retreatment were taken using sterile paper points kept at full length in the canal for 30 s. The disc diffusion method was used to check the susceptibility of microbes to the plant extracts and antimicrobials by measuring the diameter of the inhibition zones. Against the microbes, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)/minimum fungicidal concentration (MFC) of the plant extracts were assessed. The fractional inhibitory concentration index (FICI) was used to estimate the synergistic effect of plant extracts combined with antimicrobials against the resistant endodontic microbes. Results: The findings clearly indicate the effectiveness of all the three plant extracts, Commiphora molmol, Azadirachta indica, Salvadora persica, against all the experimental pathogenic microorganisms except for the ineffectiveness of Azadirachta indica, Salvadora persica against Candidaalbicans. Maximum antimicrobial activity was displayed by Azadirachta indica against Enterococcusfaecalis (MIC = 0.09 ± 1.2 mg/mL, MBC = 0.78 ± 1.25 mg/mL) and the minimum antimicrobial activity was displayed by Commiphora molmol against Actinomyces naeslundii (MIC = 12.5 ± 3.25 mg/mL, MBC = 100 ± 3.75 mg/mL). The best synergy was displayed by Commiphora molmol with fluconazole against Candidaalbicans (FICI = 0.45). Conclusions: The current study delineates the variable antimicrobial activity of plant extracts against the experimental endodontic pathogenic microorganisms. Plant extracts in conjunction with various antimicrobials can be valuable aids in combating relatively resistant endodontic microorganisms that have been the cause of worry in recent years, leading to failure even in treatment procedures following all required protocols. Full article

This study was designed to analyze the interaction of 21 antifungal combinations consisting of seven major antifungal agents against 11 echinocandin- susceptible and six-resistant C. glabrata isolates. The combinations were divided into five major groups and were evaluated by checkerboard, disc diffusion, and time-killing assays. Synergy based on the fractional inhibitory concentration index of ≤0.50 was observed in 17.65–29.41% of the cases for caspofungin combinations with azoles or amphotericin B. Amphotericin B combination with azoles induced synergistic interaction in a range of 11.76–29.41%. Azole combinations and 5-flucytosine combinations with azoles or amphotericin B did not show synergistic interactions. None of the 21 combinations showed antagonistic interactions. Interestingly, 90% of the detected synergism was among the echinocandin-resistant isolates. Disk diffusion assays showed that the inhibition zones produced by antifungal combinations were equal to or greater than those produced by single drugs. The time-killing assay showed the synergistic action of caspofungin combination with fluconazole, voriconazole, and posaconazole, and the amphotericin B-5-flucytosine combination. Furthermore, for the first time, this assay confirmed the fungicidal activity of caspofungin-voriconazole and amphotericin B-5-flucytosine combinations. The combination interactions ranged from synergism to indifference and, most importantly, no antagonism was reported and most of the synergistic action was among echinocandin-resistant isolates. Full article

Candida auris is a potential multidrug-resistant pathogen able to persist on indwelling devices as a biofilm, which serve as a source of catheter-associated infections. Neosartorya fischeri antifungal protein 2 (NFAP2) is a cysteine-rich, cationic protein with potent anti-Candida activity. We studied the in vitro activity of NFAP2 alone and in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin against C. auris biofilms. The nature of interactions was assessed utilizing the fractional inhibitory concentration index (FICI), a Bliss independence model, and LIVE/DEAD viability assay. NFAP2 exerted synergy with all tested antifungals with FICIs ranging between 0.312–0.5, 0.155–0.5, 0.037–0.375, 0.064–0.375, and 0.064–0.375 for fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. These results were confirmed using a Bliss model, where NFAP2 produced 17.54 μM²%, 2.16 μM²%, 33.31 μM²%, 10.72 μM²%, and 111.19 μM²% cumulative synergy log volume in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. In addition, biofilms exposed to echinocandins (32 mg/L) showed significant cell death in the presence of NFAP2 (128 mg/L). Our study shows that NFAP2 displays strong potential as a novel antifungal compound in alternative therapies to combat C. auris biofilms. Full article