Ana Cecilia Mesa-Arango

It only takes one to do many jobs: Amphotericin B as antifungal and immunomodulatory drug

“Amphotericin B acts through pore formation at the cell membrane after binding to ergosterol” is an accepted dogma about the action mechanism of this antifungal, and this sentence is widely found in the literature. But after 60 years of investigation, the action mechanism of Amphotericin B is not fully elucidated. Amphotericin B is a polyene substance that is one of the most effective drugs for the treatment of fungal and parasite infections. As stated above, the first mechanism of action described was pore formation after binding to the ergosterol present in the membrane. But it has also been demonstrated that AmB induces oxidative damage in the cells. Moreover, amphotericin B modulates the immune system, and this activity has been related to the protective effect of the molecule, but also to its toxicity in the host. This review tries to provide a general overview of the main aspects of this molecule, and highlight the multiple effects that this molecule has on both the fungal and host cells.

The non-mammalian host Galleria mellonella can be used to study the virulence of the fungal pathogen Candida tropicalis and the efficacy of antifungal drugs during infection by this pathogenic yeast

Although Candida tropicalis is a frequent cause of invasive fungal diseases, its interaction with the host remains poorly studied. Galleria mellonella is a Lepidoptera model which offers a useful tool to study virulence of different microorganisms and drug efficacy. In this work we investigated the virulence of C. tropicalis in G. mellonella at different temperatures and the efficacy of antifungal drugs in this infection model. When larvae were infected with yeast inocula suspensions of different concentrations (4 × 10(6), 2 × 10(6), 10(6) and 5 × 10(5) cells/larva), we observed a dose-dependent effect on the killing of the insect (50% survival ranging from 1.4 ± 0.8 to 8.8 ± 1.2 days with the higher and lower inocula, respectively). Candida tropicalis killed G. mellonella larvae at both 30°C and 37°C, although at 37°C the virulence was more evident. Haemocytes phagocytosed C. tropicalis cells after 2 hours of infection, although the phagocytosis rate was lower when compared with other fungal pathogens, such as Cryptococcus neoformans. Moreover, the haemocyte density in the haemolymph decreased during infection and the yeast formed pseudohyphae in G. mellonella. The efficacy of amphotericin B, caspofungin, fluconazole and voriconazole was tested at different concentrations, and a protective effect was observed with all the drugs at concentrations equivalent to therapeutic dose. Fungal burden increased in infected larvae during time of infection and amphotericin B and fluconazole reduced the number of colony-forming units in the worms. Moreover, antifungal treatment was associated with the presence of cell aggregates around infected areas. We conclude that G. mellonella offers a simple and feasible model to study C. tropicalis virulence and drug efficacy.

Antifungal efficacy during Candida krusei infection in non-conventional models correlates with the yeast in vitro susceptibility profile.

The incidence of opportunistic fungal infections has increased in recent decades due to the growing proportion of immunocompromised patients in our society. Candida krusei has been described as a causative agent of disseminated fungal infections in susceptible patients. Although its prevalence remains low among yeast infections (2–5%), its intrinsic resistance to fluconazole makes this yeast important from epidemiologic aspects. Non mammalian organisms are feasible models to study fungal virulence and drug efficacy. In this work we have used the lepidopteran Galleria mellonella and the nematode Caenorhabditis elegans as models to assess antifungal efficacy during infection by C. krusei. This yeast killed G. mellonella at 25, 30 and 37°C and reduced haemocytic density. Infected larvae melanized in a dose-dependent manner. Fluconazole did not protect against C. krusei infection, in contrast to amphotericin B, voriconazole or caspofungin. However, the doses of these antifungals required to obtain larvae protection were always higher during C. krusei infection than during C. albicans infection. Similar results were found in the model host C. elegans. Our work demonstrates that non mammalian models are useful tools to investigate in vivo antifungal efficacy and virulence of C. krusei.

Citral and carvone chemotypes from the essential oils of Colombian Lippia alba (Mill.) N.E. Brown: composition, cytotoxicity and antifungal activity.

Two essential oils of Lippia alba (Mill.) N.E. Brown (Verbenacea), the carvone and citral chemotypes and 15 of their compounds were evaluated to determine cytotoxicity and antifungal activity. Cytotoxicity assays for both the citral and carvone chemotypes were carried out with tetrazolium-dye, which showed a dose-dependent cytotoxic effect against HeLa cells. Interestingly, this effect on the evaluated cells (HeLa and the non-tumoural cell line, Vero) was lower than that of commercial citral alone. Commercial citral showed the highest cytotoxic activity on HeLa cells. The antifungal activity was evaluated against Candida parapsilosis, Candida krusei, Aspergillus flavus and Aspergillus fumigatus strains following the standard protocols, Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing and CLSI M38-A. Results demonstrated that the most active essential oil was the citral chemotype, with geometric means-minimal inhibitory concentration (GM-MIC) values of 78.7 and 270.8 microg/mL for A. fumigatus and C. krusei, respectively. Commercial citral showed an antifungal activity similar to that of the citral chemotype (GM-MIC values of 62.5 microg/mL for A. fumigatus and 39.7 microg/mL for C. krusei). Although the citronellal and geraniol were found in lower concentrations in the citral chemotype, they had significant antifungal activity, with GM-MIC values of 49.6 microg/mL for C. krusei and 176.8 microg/mL for A. fumigatus.

Synthesis and biological evaluation of dehydroabietic acid derivatives

A series of C18-oxygenated derivatives of dehydroabietic acid were synthesized from commercial abietic acid and evaluated for their cytotoxic, antimycotic, and antiviral activities.

Susceptibility profile of clinical isolates of non-Cryptococcus neoformans/non-Cryptococcus gattii Cryptococcus species and literature review.

The in vitro susceptibility profile of 24 clinical isolates of non-Cryptococcus neoformans/non-Cryptococcus gattii Cryptococcus species was analysed. In addition, the susceptibility results of 98 other strains from seven different reports were reviewed. The latter included studies which used antifungal susceptibility testing reference procedures or commercial methods which exhibited high correlation rates with the reference procedures. A total of 122 isolates were analysed (57 Cryptococcus albidus, 39 Cryptococcus laurentii, ten Cryptococcus uniguttulatus, ten Cryptococcus humicola, four Cryptococcus curvatus, and two Cryptococcus luteolus). Amphotericin B was in vitro the most active compound against all species, while flucytosine and candins were inactive. Fluconazole exhibited a limited in vitro activity, particularly against C. albidus, C. uniguttulatus and C. laurentii. Voriconazole, itraconazole and posaconazole were active against most of isolates, but we found significant rates of decreased susceptibility. Identification and susceptibility testing of Cryptococcus spp. should be performed on a routine basis in view of their unpredictable susceptibility profiles.

Amphotericin B mediates killing in Cryptococcus neoformans through the induction of a strong oxidative burst

We studied the effects of Amphotericin B (AmB) on Cryptococcus neoformans using different viability methods (CFUs enumeration, XTT assay and propidium iodide permeability). After 1h of incubation, there were no viable colonies when the cells were exposed to AmB concentrations ≥ 1 mg/L. In the same conditions, the cells did not become permeable to propidium iodide, a phenomenon that was not observed until 3h of incubation. When viability was measured in parallel using XTT assay, a result consistent with the CFUs was obtained, although we also observed a paradoxical effect in which at high AmB concentrations, a higher XTT reduction was measured than at intermediate AmB concentrations. This paradoxical effect was not observed after 3h of incubation with AmB, and lack of XTT reduction was observed at AmB concentrations higher than 1mg/L. When stained with dihydrofluorescein, AmB induced a strong intracellular oxidative burst. Consistent with oxidative damage, AmB induced protein carbonylation. Our results indicate that in C. neoformans, Amphotericin B causes intracellular damage mediated through the production of free radicals before damage on the cell membrane, measured by propidium iodide uptake.

The Production of Reactive Oxygen Species Is a Universal Action Mechanism of Amphotericin B against Pathogenic Yeasts and Contributes to the Fungicidal Effect of This Drug

ABSTRACT Amphotericin B (AMB) is an antifungal drug that binds to ergosterol and forms pores at the cell membrane, causing the loss of ions. In addition, AMB induces the accumulation of reactive oxygen species (ROS), and although these molecules have multiple deleterious effects on fungal cells, their specific role in the action mechanism of AMB remains unknown. In this work, we studied the role of ROS in the action mechanism of AMB. We determined the intracellular induction of ROS in 44 isolates of different pathogenic yeast species (Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Cryptococcus neoformans, and Cryptococcus gattii). We also characterized the production of ROS in AMB-resistant isolates. We found that AMB induces the formation of ROS in all the species tested. The inhibition of the mitochondrial respiratory chain by rotenone blocked the induction of ROS by AMB and provided protection from the killing action of the antifungal. Moreover, this phenomenon was absent in strains that displayed resistance to AMB. These strains showed an alteration in the respiration rate and mitochondrial membrane potential and also had higher catalase activity than that of the AMB-susceptible strains. Consistently, AMB failed to induce protein carbonylation in the resistant strains. Our data demonstrate that the production of ROS by AMB is a universal and important action mechanism that is correlated with the fungicidal effect and might explain the low rate of resistance to the molecule. Finally, these data provide an opportunity to design new strategies to improve the efficacy of this antifungal.

In vitro antifungal activity and cytotoxic effect of essential oils and extracts of medicinal and aromatic plants against Candida krusei and Aspergillus fumigatus

As plantas sao geralmente utilizadas na medicina tradicional como agentes antimicrobianos e seus oleos essenciais e extratos foram conhecidos por possuir atividade antifungica. O objetivo deste estudo foi avaliar in vitro a atividade de 32 oleos essenciais e 29 extratos contra Candida krusei e Aspergillus fumigatus, bem como o efeito citotoxico em celulas Vero. A curva do tempo-morte e a interacao entre antifungicos e Chenopodium ambrosioidese do extrato de Myrcia cucullata mostraram atividade antifungica contra C. krusei (geometric means of the minimal inhibitory concentration [GM-MIC] 7,82 e 31,25 µg/mL, respectivamente). Lippia citriodora foi ativa contra C. krusei e A. fumigatus (GM-CIM = 99,21 µg/mL e 62,5 µg/mL, respectivamente). Os testes de tempo-morte feitos com oleo de C. ambrosioides mostraram atividade fungicida em 4x MIC. A interacao do oleo C. ambrosioides com itraconazol e anfotericina B foi testada pela tecnica de xadrez. Nenhuma interacao foi detectada pela combinacao do oleo C. ambrosioides com anfotericina B e itraconazol (intervalo fractional inhibitory index [FICI] = 1,03-1,06 e 1,03-1,00, respectivamente). Os ensaios de citotoxicidade para todas as amostras foram realizadas com MTT. Apenas os oleos Hedyosmun sp. e L. dulcis foram citotoxicos.

Process Analysis of Variables for Standardization of Antifungal Susceptibility Testing of Nonfermentative Yeasts

ABSTRACT Nonfermentative yeasts, such as Cryptococcus spp., have emerged as fungal pathogens during the last few years. However, standard methods to measure their antifungal susceptibility (antifungal susceptibility testing [AST]) are not completely reliable due to the impaired growth of these yeasts in standard media. In this work, we have compared the growth kinetics and the antifungal susceptibilities of representative species of nonfermentative yeasts such as Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus albidus, Rhodotorula spp., Yarrowia lipolytica, Geotrichum spp., and Trichosporon spp. The effect of the growth medium (RPMI medium versus yeast nitrogen base [YNB]), glucose concentration (0.2% versus 2%), nitrogen source (ammonium sulfate), temperature (30°C versus 35°C), shaking, and inoculum size (103, 104, and 105 cells) were analyzed. The growth rate, lag phase, and maximum optical density were obtained from each growth experiment, and after multivariate analysis, YNB-based media demonstrated a significant improvement in the growth of yeasts. Shaking, an inoculum size of 105 CFU/ml, and incubation at 30°C also improved the growth kinetics of organisms. Supplementation with ammonium sulfate and with 2% glucose did not have any effect on growth. We also tested the antifungal susceptibilities of all the isolates by the reference methods of the CLSI and EUCAST, the EUCAST method with shaking, YNB under static conditions, and YNB with shaking. MIC values obtained under different conditions showed high percentages of agreement and significant correlation coefficient values between them. MIC value determinations according to CLSI and EUCAST standards were rather complicated, since more than half of isolates tested showed a limited growth index, hampering endpoint determinations. We conclude that AST conditions including YNB as an assay medium, agitation of the plates, reading after 48 h of incubation, an inoculum size of 105 CFU/ml, and incubation at 30°C made MIC determinations easier without an overestimation of MIC values.