Antifungal Combination of Ethyl Acetate Extract of Poincianella pluviosa (DC.) L. P. Queiros Stem Bark With Amphotericin B in Cryptococcus neoformans

Cryptococcus neoformans is the leading cause of cryptococcosis, an invasive and potentially fatal infectious disease. Therapeutic failures are due to the increase in antifungal resistance, the adverse effects of drugs, and the unavailability of therapeutic regimens in low-income countries, which limit the treatment of cryptococcosis, increasing the morbidity and mortality associated with these infections. Thus, new antifungal drugs and innovative strategies for the cryptococcosis treatment are urgently needed. The aim of the present study was to evaluate the effect of ethyl acetate fraction (EAF) of Poincianella pluviosa stem bark on planktonic and biofilm mode of growth of C. neoformans. Furthermore, the interaction between the EAF and amphotericin B (AmB) was evaluated in vitro and in Galleria mellonella infection model. Minimal inhibitory concentrations (MICs) of EAF ranged from 125.0 to >1,000.0 μg/ml and >1,000.0 μg/ml for planktonic and sessile cells, respectively. The combination between EAF and AmB exhibited a synergistic fungicidal activity toward C. neoformans, with a fractional inhibitory concentration index (FICI) ranging from 0.03 to 0.06 and 0.08 to 0.28 for planktonic and sessile cells, respectively. Microscopy analyses of planktonic C. neoformans cells treated with EAF, alone or combined with AmB, revealed morphological and ultrastructural alterations, including loss of integrity of the cell wall and cell membrane detachment, suggesting leakage of intracellular content, reduction of capsule size, and presence of vacuoles. Moreover, EAF alone or combined with AmB prolonged the survival rate of C. neoformans-infected G. mellonella larvae. These findings indicate that P. pluviosa may be an important source of new compounds that can be used as a fungus-specific adjuvant for the treatment of cryptococcosis.

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Dectin-2-Targeted Antifungal Liposomes Exhibit Enhanced Efficacy

mSphere ◽

10.1128/msphere.00715-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 1

Author(s):

Suresh Ambati ◽

Emma C. Ellis ◽

Jianfeng Lin ◽

Xiaorong Lin ◽

Zachary A. Lewis ◽

...

Keyword(s):

Candida Albicans ◽

Aspergillus Fumigatus ◽

Effective Dose ◽

Cryptococcus Neoformans ◽

Amphotericin B ◽

Antifungal Drugs ◽

Extracellular Matrices ◽

Content Type ◽

Order Of Magnitude ◽

Life Threatening

ABSTRACT Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus cause life-threatening candidiasis, cryptococcosis, and aspergillosis, resulting in several hundred thousand deaths annually. The patients at the greatest risk of developing these life-threatening invasive fungal infections have weakened immune systems. The vulnerable population is increasing due to rising numbers of immunocompromised individuals as a result of HIV infection or immunosuppressed individuals receiving anticancer therapies and/or stem cell or organ transplants. While patients are treated with antifungals such as amphotericin B, all antifungals have serious limitations due to lack of sufficient fungicidal effect and/or host toxicity. Even with treatment, 1-year survival rates are low. We explored methods of increasing drug effectiveness by designing fungicide-loaded liposomes specifically targeted to fungal cells. Most pathogenic fungi are encased in cell walls and exopolysaccharide matrices rich in mannans. Dectin-2 is a mammalian innate immune membrane receptor that binds as a dimer to mannans and signals fungal infection. We coated amphotericin-loaded liposomes with monomers of Dectin-2’s mannan-binding domain, sDectin-2. sDectin monomers were free to float in the lipid membrane and form dimers that bind mannan substrates. sDectin-2-coated liposomes bound orders of magnitude more efficiently to the extracellular matrices of several developmental stages of C. albicans, C. neoformans, and A. fumigatus than untargeted control liposomes. Dectin-2-coated amphotericin B-loaded liposomes reduced the growth and viability of all three species more than an order of magnitude more efficiently than untargeted control liposomes and dramatically decreased the effective dose. Future efforts focus on examining pan-antifungal targeted liposomal drugs in animal models of fungal diseases. IMPORTANCE Invasive fungal diseases caused by Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus have mortality rates ranging from 10 to 95%. Individual patient costs may exceed $100,000 in the United States. All antifungals in current use have serious limitations due to host toxicity and/or insufficient fungal cell killing that results in recurrent infections. Few new antifungal drugs have been introduced in the last 2 decades. Hence, there is a critical need for improved antifungal therapeutics. By targeting antifungal-loaded liposomes to α-mannans in the extracellular matrices secreted by these fungi, we dramatically reduced the effective dose of drug. Dectin-2-coated liposomes loaded with amphotericin B bound 50- to 150-fold more strongly to C. albicans, C. neoformans, and A. fumigatus than untargeted liposomes and killed these fungi more than an order of magnitude more efficiently. Targeting drug-loaded liposomes specifically to fungal cells has the potential to greatly enhance the efficacy of most antifungal drugs.

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Darunavir inhibits Cryptococcus neoformans/Cryptococcus gattii species complex growth and increases the susceptibility of biofilms to antifungal drugs

Journal of Medical Microbiology ◽

10.1099/jmm.0.001194 ◽

2020 ◽

Vol 69 (6) ◽

pp. 830-837

Author(s):

Raimunda Sâmia Nogueira Brilhante ◽

José Alexandre Telmos Silva ◽

Géssica dos Santos Araújo ◽

Vandbergue Santos Pereira ◽

Wilker Jose Perez Gotay ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Amphotericin B ◽

Metabolic Activity ◽

Biofilm Formation ◽

Species Complex ◽

Cryptococcus Gattii ◽

Antifungal Drugs ◽

Planktonic Cells ◽

Species Activity ◽

Checkerboard Assay

Introduction. Cryptococcus species are pathogens commonly associated with cases of meningoencephalitis in individuals who are immunosuppressed due to AIDS. Aim. The aim was to evaluate the effects of the antiretroviral darunavir alone or associated with fluconazole, 5-flucytosine and amphotericin B against planktonic cells and biofilms of Cryptococcus species. Methodology. Susceptibility testing of darunavir and the common antifungals against 12 members of the Cryptococcus neoformans/Cryptococcus gattii species complex was evaluated by broth microdilution. The interaction between darunavir and antifungals against planktonic cells was tested by a checkerboard assay. The effects of darunavir against biofilm metabolic activity and biomass were evaluated by the XTT reduction assay and crystal violet staining, respectively. Results. Darunavir combined with amphotericin B showed a synergistic interaction against planktonic cells. No antagonistic interaction was observed between darunavir and the antifungals used. All Cryptococcus species strains were strong biofilm producers. Darunavir alone reduced biofilm metabolic activity and biomass when added during and after biofilm formation (P<0.05). The combination of darunavir with antifungals caused a significant reduction in biofilm metabolic activity and biomass when compared to darunavir alone (P<0.05). Conclusion. Darunavir presents antifungal activity against planktonic cells of Cryptococcus species and synergism with amphotericin B. In addition, darunavir led to reduced biofilm formation and showed activity against mature biofilms of Cryptococcus species. Activity of the antifungals against mature biofilms was enhanced in the presence of darunavir.

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Lactoferrin Is Broadly Active against Yeasts and Highly Synergistic with Amphotericin B

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02284-19 ◽

2020 ◽

Vol 64 (5) ◽

Cited By ~ 5

Author(s):

Kenya E. Fernandes ◽

Kerry Weeks ◽

Dee A. Carter

Keyword(s):

Amphotericin B ◽

Galleria Mellonella ◽

Comprehensive Evaluation ◽

Yeast Species ◽

Phenotypic Diversity ◽

Antifungal Drugs ◽

Fungal Cell ◽

Content Type ◽

Iron Saturation ◽

Capsule Size

ABSTRACT Lactoferrin (LF) is a multifunctional milk protein with antimicrobial activity against a range of pathogens. While numerous studies report that LF is active against fungi, there are considerable differences in the level of antifungal activity and the capacity of LF to interact with other drugs. Here we undertook a comprehensive evaluation of the antifungal spectrum of activity of three defined sources of LF across 22 yeast and 24 mold species and assessed its interactions with six widely used antifungal drugs. LF was broadly and consistently active against all yeast species tested (MICs, 8 to 64 μg/ml), with the extent of activity being strongly affected by iron saturation. LF was synergistic with amphotericin B (AMB) against 19 out of 22 yeast species tested, and synergy was unaffected by iron saturation but was affected by the extent of LF digestion. LF-AMB combination therapy significantly prolonged the survival of Galleria mellonella wax moth larvae infected with Candida albicans or Cryptococcus neoformans and decreased the fungal burden 12- to 25-fold. Evidence that LF directly interacts with the fungal cell surface was seen via scanning electron microscopy, which showed pore formation, hyphal thinning, and major cell collapse in response to LF-AMB synergy. Important virulence mechanisms were disrupted by LF-AMB treatment, which significantly prevented biofilms in C. albicans and C. glabrata, inhibited hyphal development in C. albicans, and reduced cell and capsule size and phenotypic diversity in Cryptococcus. Our results demonstrate the potential of LF-AMB as an antifungal treatment that is broadly synergistic against important yeast pathogens, with the synergy being attributed to the presence of one or more LF peptides.

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Assessment of Candida auris Response to Antifungal Drugs Using Time–Kill Assays and an Animal Model

Open Forum Infectious Diseases ◽

10.1093/ofid/ofx163.003 ◽

2017 ◽

Vol 4 (suppl_1) ◽

pp. S73-S73 ◽

Cited By ~ 1

Author(s):

Ronen Ben-Ami ◽

Liat Ashkenazi ◽

Judith Berman ◽

Nuphar Korolker ◽

Anna Novikov

Keyword(s):

Amphotericin B ◽

Fungicidal Activity ◽

Antifungal Drugs ◽

Rank Test ◽

Infection Model ◽

Candida Auris ◽

Fungistatic Activity ◽

Kill Curve ◽

Time Kill

Abstract Background Candida auris is an emerging nosocomial pathogen that is resistant to Fluconazole and variably susceptible to other systemic drug classes. Treatment with echinocandins has been recommended based on MICs in the susceptible range, but supporting in vivo data is lacking. Methods We tested the MIC of C. auris strains (n = 12) to fluconazole, voriconazole, posaconazole. anidulafungin, amphotericin B and flucytosine. Representative C. auris strains from Israel and South Africa, and a reference C. albicans strain were analysed using time–kill curve assays. Fungicidal activity was defined as reduction of ≥3 log from baseline CFU/ml. Response to caspofungin treatment was assessed in BALB/c mice immunosuppressed with cyclophosphamide and inoculated with 7 × 107C. auris cells by tail vein injection. Mice were treated from day +1 to day +7 with caspofungin (IP) at doses of 1 or 5 mg/kg and compared with sham-treated controls. Survival was assessed daily. Kaplan-Meier survival analyses were performed and treatment arms were compared using the log-rank test. Results Drug susceptibility results (MIC50 and MIC90) were: fluconazole, 64 and 128 mg/l; voriconazole, 0.5 and 24 mg/l; posaconazole, 0.5 and 27 mg/l; anidulafungin, 0.03 and 0.06 mg/l; amphotericin B, 2 and 8 mg/l; flucytosine, 0.3 and 1 mg/l. Time–kill curve analyses showed log reduction from baseline CFU concentration of −3.0 to −2.8 for fluconazole (MIC ×1), 5.6–6.1 for amphotericin B (MIC ×4) and −0.4 to −0.9 for caspofungin (MIC ×16), consistent with fungicidal activity of amphotericin B and weak fungistatic activity of caspofungin. In the mouse model, survival rate was similar with sham treatment (33%) and treatment with caspofungin 1 mg/kg/day (44%) and 5 mg/kg/day (22%), P = 0.7. Conclusion Despite generally low MIC, caspofungin has only mild fungistatic activity on C. auris and no effect on survival in a mouse infection model. Amphotericin B has fungicidal activity against C. auris. Disclosures All authors: No reported disclosures.

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Effect of Amphotericin B on Capsule and Cell Size in Cryptococcus neoformans during Murine Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.10.4358-4361.2005 ◽

2005 ◽

Vol 49 (10) ◽

pp. 4358-4361 ◽

Cited By ~ 8

Author(s):

Oscar Zaragoza ◽

Coralia Mihu ◽

Arturo Casadevall ◽

Joshua D. Nosanchuk

Keyword(s):

Cryptococcus Neoformans ◽

Cell Size ◽

Amphotericin B ◽

Antifungal Drugs ◽

Cellular Morphology ◽

Additional Mechanism ◽

Capsule Size

ABSTRACT Antifungal drugs can affect the cellular morphology of Cryptococcus neoformans in culture, which alters its interactions with phagocytes. We examined the effects of amphotericin B on C. neoformans during murine infection. The antifungal reduced capsule size and serum polysaccharide, which suggests an additional mechanism for amphotericin B's efficacy in cryptococcosis.

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Eugenol and Isoeugenol In Association with Antifungal Against Cryptococcus neoformans

International Journal of Pharmacognosy and Phytochemical Research ◽

10.25258/phyto.v9i4.8133 ◽

2017 ◽

Vol 9 (4) ◽

Author(s):

Pinheiro L. S. ◽

Sousa J. P. ◽

Barreto N. A. ◽

Dantas T B ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Amphotericin B ◽

Inhibitory Concentration ◽

Synergistic Effects ◽

Antagonistic Effect ◽

Antifungal Drugs ◽

Antifungal Effect ◽

Beneficial Effects ◽

Combined Use ◽

Antifungal Effects

The antifungal therapy combined is used in clinical practice of several mycoses as it may increase the efficacy of the treatment. The use of natural products (phytochemicals) in combination with conventional antifungal drugs has been related to beneficial effects, mainly synergistic effects. The aim of this study was to evaluate the effect of the combined use of eugenol / isoeugenol, compounds with recognized antimicrobial activity, in association with antifungal amphotericin B against strains of Cryptococcus neoformans. The combined antifungal effect were be determined from the Fraction Inhibitory Concentration index - checkerboard technique. The results obtained in this study showed that eugenol in combination with amphotericin B had antagonistic effect against the strains of C. neoformans, LM 615 and INCQS 40221 (FIC index 6.0 and 4.0), respectively. The combination of the isoeugenol and amphotericin B also showed antagonistic effects for both the LM 615 strain and INCQS 40221 (FIC index 6.0 and 5.0), respectively. This study contributed to the understanding of the antifungal effects of the association of phenylpropanoids (eugenol / isoeugenol) with amphotericin B. Further studies are needed to evaluate and compare the effects of the association of these phytochemicals with other conventional antifungal drugs used against C. neoformans.

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Serial Passage of Cryptococcus neoformans in Galleria mellonella Results in Increased Capsule and Intracellular Replication in Hemocytes, but Not Increased Resistance to Hydrogen Peroxide

Pathogens ◽

10.3390/pathogens9090732 ◽

2020 ◽

Vol 9 (9) ◽

pp. 732 ◽

Cited By ~ 1

Author(s):

Muhammad Fariz Ali ◽

Stephen M. Tansie ◽

John R. Shahan ◽

Rebecca L. Seipelt-Thiemann ◽

Erin E. McClelland

Keyword(s):

Hydrogen Peroxide ◽

Cryptococcus Neoformans ◽

Galleria Mellonella ◽

Ex Vivo ◽

Histopathological Examination ◽

Serial Passage ◽

Infection Model ◽

Hydrogen Peroxide Production ◽

Species Response

To gain insight into how pathogens adapt to new hosts, Cryptococcus neoformans (H99W) was serially passaged in Galleria mellonella. The phenotypic characteristics of the passaged strain (P15) and H99W were evaluated. P15 grew faster in hemolymph than H99W, in vitro and in vivo, suggesting that adaptation had occurred. However, P15 was more susceptible to hydrogen peroxide in vitro, killed fewer mouse macrophages, and had less fungal burden in human ex vivo macrophages than H99W. Analysis of gene expression changes during Galleria infection showed only a few different genes involved in the reactive oxygen species response. As P15 sheds more GXM than H99W, P15 may have adapted by downregulating hemocyte hydrogen peroxide production, possibly through increased capsular glucuronoxylomannan (GXM) shedding. Hemocytes infected with P15 produced less hydrogen peroxide, and hydrogen peroxide production in response to GXM-shedding mutants was correlated with shed GXM. Histopathological examination of infected larvae showed increased numbers and sizes of immune nodules for P15 compared to H99W, suggesting an enhanced, but functionally defective, response to P15. These results could explain why this infection model does not always correlate with murine models. Overall, C. neoformans’ serial passage in G. mellonella resulted in a better understanding of how this yeast evolves under selection.

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Increased Antifungal Drug Resistance in Clinical Isolates of Cryptococcus neoformans in Uganda

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01299-15 ◽

2015 ◽

Vol 59 (12) ◽

pp. 7197-7204 ◽

Cited By ~ 89

Author(s):

Kyle D. Smith ◽

Beatrice Achan ◽

Kathy Huppler Hullsiek ◽

Tami R. McDonald ◽

Laura H. Okagaki ◽

...

Keyword(s):

Amphotericin B ◽

Low Income ◽

Low Cost ◽

Drug Susceptibility ◽

Antifungal Drug ◽

Clinical Isolates ◽

Hiv Care ◽

Low Income Countries ◽

Broth Microdilution ◽

Content Type

ABSTRACTCryptococcal antigen screening is recommended among people living with AIDS when entering HIV care with a CD4 count of <100 cells/μl, and preemptive fluconazole monotherapy treatment is recommended for those with subclinical cryptococcal antigenemia. Yet, knowledge is limited of current antimicrobial resistance in Africa. We examined antifungal drug susceptibility in 198 clinical isolates collected from Kampala, Uganda, between 2010 and 2014 using the CLSI broth microdilution assay. In comparison with two previous studies from 1998 to 1999 that reported an MIC50of 4 μg/ml and an MIC90of 8 μg/ml prior to widespread human fluconazole and agricultural azole fungicide usage, we report an upward shift in the fluconazole MIC50to 8 μg/ml and an MIC90value of 32 μg/ml, with 31% of isolates with a fluconazole MIC of ≥16 μg/ml. We observed an amphotericin B MIC50of 0.5 μg/ml and an MIC90of 1 μg/ml, of which 99.5% of isolates (197 of 198 isolates) were still susceptible. No correlation between MIC and clinical outcome was observed in the context of amphotericin B and fluconazole combination induction therapy. We also analyzedCryptococcussusceptibility to sertraline, with an MIC50of 4 μg/ml, suggesting that sertraline is a promising oral, low-cost, available, novel medication and a possible alternative to fluconazole. Although the CLSI broth microdilution assay is ideal to standardize results, limit human bias, and increase assay capacity, such assays are often inaccessible in low-income countries. Thus, we also developed and validated an assay that could easily be implemented in a resource-limited setting, with similar susceptibility results (P= 0.52).

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In Vitro and In Vivo Experimental Activities of Antifungal Agents against Fusarium solani

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.5.1256 ◽

1999 ◽

Vol 43 (5) ◽

pp. 1256-1257 ◽

Cited By ~ 37

Author(s):

J. Guarro ◽

I. Pujol ◽

E. Mayayo

Keyword(s):

Drug Therapy ◽

Amphotericin B ◽

Fusarium Solani ◽

Antifungal Agents ◽

Antifungal Drugs ◽

Infection Model ◽

Disseminated Infection

ABSTRACT In the treatment of disseminated Fusarium infections, amphotericin B either alone or in combination with flucytosine and rifampin is the drug therapy most frequently used. The efficacy of these antifungal drugs was evaluated in a murine disseminated-infection model, with five strains of Fusarium solani. All the treatments were clearly ineffective.

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Susceptibility of Cryptococcus neoformans Biofilms to Antifungal Agents In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.3.1021-1033.2006 ◽

2006 ◽

Vol 50 (3) ◽

pp. 1021-1033 ◽

Cited By ~ 109

Author(s):

Luis R. Martinez ◽

Arturo Casadevall

Keyword(s):

Confocal Microscopy ◽

Cryptococcus Neoformans ◽

Amphotericin B ◽

Biofilm Formation ◽

Antifungal Agents ◽

Capsular Polysaccharide ◽

Antifungal Drugs ◽

Enzyme Linked Immunosorbent Assay ◽

Sequence Of Events ◽

Biofilm Development

ABSTRACT Microbial biofilms contribute to virulence and resistance to antibiotics by shielding microbial cells from host defenses and antimicrobial drugs, respectively. Cryptococcus neoformans was demonstrated to form biofilms in polystyrene microtiter plates. The numbers of CFU of disaggregated biofilms, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide reduction, and light and confocal microscopy were used to measure the fungal mass, the metabolic activity, and the appearance of C. neoformans biofilms, respectively. Biofilm development by C. neoformans followed a standard sequence of events: fungal surface attachment, microcolony formation, and matrix production. The susceptibilities of C. neoformans cells of the biofilm and planktonic phenotypes to four antifungal agents were examined. The exposure of C. neoformans cells or preformed cryptococcal biofilms to fluconazole or voriconazole did not result in yeast growth inhibition and did not affect the metabolic activities of the biofilms, respectively. In contrast, both C. neoformans cells and preformed biofilms were susceptible to amphotericin B and caspofungin. However, C. neoformans biofilms were significantly more resistant to amphotericin B and caspofungin than planktonic cells, and their susceptibilities to these drugs were further reduced if cryptococcal cells contained melanin. A spot enzyme-linked immunosorbent assay and light and confocal microscopy were used to investigate how antifungal drugs affected C. neoformans biofilm formation. The mechanism by which amphotericin B and caspofungin interfered with C. neoformans biofilm formation involved capsular polysaccharide release and adherence. Our results suggest that biofilm formation may diminish the efficacies of some antifungal drugs during cryptococcal infection.

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