Minimum Inhibitory Concentrations Of Amphotericin Research Articles

Antifungal susceptibility of clinically isolated invasive Candida tropicalis in East China from 2017 to 2021

Objective: To explore the epidemiological characteristics of sample distribution and antifungal susceptibilities of clinically invasive C. tropicalis isolates from 2017 to 2021 in East China. Methods: Using a retrospective analysis, the East China Invasive Fungal Infection Group (ECIFIG) collected C. tropicalis clinically isolated from 32 hospitals in East China between January 2017 and December 2021. The identification results of the strains were reviewed using mass spectrometry by the central laboratory of the Shanghai East Hospital. The minimum inhibitory concentrations (MICs) of the strains against fluconazole (FLU), voriconazole (VOR), itraconazole (ITR), Posaconazole (POS), isavuconazole (ISA), anidulafungin (ANI), caspofungin (CAS), micafungin (MICA) and 5-fluorocytosine (FCT) were tested by the ThermoFisher CMC1JHY colorimetric microdilution method. The MIC of amphotericin B (AMB) was tested by the broth microdilution method. The MIC results were analyzed based on the clinical breakpoints and epidemiological cutoff values (ECV) published by the Clinical and Laboratory Standards Institute (CLSI) M27 Ed3 and M57 Ed4 documents. Data analysis was conducted using the Kruskal-Wallis test and paired t-test. Results: In total, 305 C. tropicalis isolates were collected. There were 38.0% (116/305) strains isolated from blood, 11.5% (35/305) ascites, 8.9% (27/305) catheter and 8.9% (27/305) drainage fluid. The resistance rate of C. tropicalis to FLU was 32.5%, to VOR was 28.5%, and the cross-resistance rate to FLU and VOR was 28.5%. The wild-type proportions for ITR and POS were 79.3% and 29.2% respectively. There was no significant difference in resistance rates, MIC50, and MIC90 of FLU and VOR, or in the wild-type rates of ITR and POS over five years. More than 95.0% of the isolates were susceptible to echinocandins. However, one strain was identified as being multi-drug resistant. In azole antifungals, voriconazole, itraconazole, posaconazole, and isavuconazole have similar GM MIC values. The GM MIC of fluconazole is significantly higher than that of itraconazole (t=9.95, P<0.05), posaconazole (t=9.99, P<0.05), and voriconazole (t=10.01, P<0.05), Meanwhile, among echinocandins, the GM MIC of ANI was comparable to that of CAS (t=1.17, P>0.05), both of which were significantly higher than MICA (t=11.56, P<0.05; t=4.15, P<0.05). Conclusion: The clinical isolates of C. tropicalis in East China from 2017 to 2021 were relatively susceptible to echinocandins. However, there was consistently high resistance to fluconazole and voriconazole. More intensive efforts should be made on the monitoring of drug resistance in C. tropicalis.

Gradient concentration strip–specific epidemiological cut-off values of antifungal drugs in various yeast species and five prevalent Aspergillus species complexes

ObjectivesTo determine the epidemiological cut-off values (ECVs) of ten antifungal agents in a wide range of yeasts and Aspergillus spp. using gradient concentration strips. MethodsThe minimum inhibitory concentrations for amphotericin B, anidulafungin, caspofungin, micafungin, flucytosine, fluconazole, itraconazole, isavuconazole, posaconazole, and voriconazole, determined with gradient concentration strips at 35 French microbiology laboratories between 2002 and 2020, were retrospectively collected. Then, the ECVs were calculated using the iterative method and a cut-off value of 97.5%. ResultsMinimum inhibitory concentrations were available for 17 653 clinical isolates. In total, 48 ECVs (including 32 new ECVs) were determined: 29 ECVs for frequent yeast species (e.g. Candida albicans and itraconazole/flucytosine, and Candida glabrata species complex [SC] and flucytosine) and rare yeast species (e.g. Candida dubliniensis, Candida inconspicua, Saccharomyces cerevisiae, and Cryptococcus neoformans) and 19 ECVs for Aspergillusflavus SC, Aspergillusfumigatus SC, Aspergillusnidulans SC, Aspergillusniger SC, and Aspergillusterreus SC. ConclusionsThese ECVs can be added to the already available gradient concentration strip–specific ECVs to facilitate minimum inhibitory concentration interpretation and streamline the identification of nonwild type isolates.

Clinical utility of antifungal susceptibility testing in patients with fungal rhinosinusitis

PurposeTo determine the association between antifungal susceptibility test (AFST) results and in vivo therapeutic response in Indian patients with fungal rhinosinusitis. MethodsThe clinicoradiological, fungal culture, AFST, histopathology results and outcomes of 48 patients with fungal rhinosinusitis seen between 20132015 were analysed. Minimum inhibitory concentration (MIC) determination was performed for amphotericin B, itraconazole, voriconazole and posaconazole. ResultsForty patients had invasive and 8 had non-invasive fungal sinusitis. Rhizopus and Aspergillus species which comprised 46.9% each of isolates were mostly associated with acute invasive fungal rhinosinusitis and chronic granulomatous fungal rhinosinusitis respectively. All patients with non-invasive fungal rhinosinusitis had Aspergillus isolates.The Geometric Mean (GM) MIC for R. arrhizus of amphotericin B and posaconazole was 0.51 mcg/mL and 3.08 mcg/mL respectively and for A. flavus species for amphotericin B and voriconazole values were 1.41mcg/mL and 0.35 mcg/mL respectively.In patients with Aspergillus infections, while there was no association of MICs for azoles and outcome (p = 1), a strong association was noted between azole therapy and a good outcome (p = 0.003). In patients with Rhizopus infections, no association was found between MICs for amphotericin B and outcome (p = 1) and because of therapeutic complications, no association was found between amphotericin B therapy and outcome (p = 1). ConclusionNo significant association exists between in vitro (AFST) and in vivo responses despite low GM MICs for the drugs used in Aspergillus and Rhizopus infections. Therapeutic complications following conventional amphotericin B therapy confounds analysis. Clinical responses suggest that azoles are the drug of choice for Aspergillus infections.

Effect of phenotypic switching on the susceptibility of Candida krusei towards amphotericin B, nystatin and Piper betle aqueous extract

Candida krusei (C. krusei) is associated with oral candidiasis, particularly in immunecompromised patients. The objective of the study was to determine the effect of phenotypic switching to the susceptibility of C. krusei towards amphotericin B, nystatin and aqueous extract of Piper betle (P. betle). To induce phenotypic switching, C. krusei was inoculated in yeast extract peptone dextrose (YEPD) broth supplemented with 5 mg mL-1 phloxine B andincubated for five hours at 25 °C. Later, 100 µL of the suspension was inoculated on YEPD agar supplemented with 5 mg mL-1 phloxine B and incubated for five days at 25 °C. Disc diffusion and minimum inhibitory concentration (MIC) assays were conducted to determine the susceptibility of C. krusei. The results showed that all C. krusei switched generations were susceptible towards amphotericin B and nystatin with the 3rd and 4th generations significantly more susceptible than the un-switched, respectively (P&lt;0.05). All C. krusei switched generations were also observed to be susceptible towards the aqueous extract of P. betle. The MIC of amphotericin B, nystatin and P. betle were determined at 10 µg mL-1, 10 µg mL-1 and 12.5 mg mL-1, respectively for all generations of C. krusei. In conclusion, the susceptibility of C. krusei was phenotypically switched generation dependent towards amphotericin B, nystatin, and Piper betle aqueous extract.

A fatal invasive Scedosporium apiospermum pulmonary infection in an adult patient with malignant lung adenocarcinoma.

Background and Purpose: Scedosporium apiospermum complex as a ubiquitous environmental mold is increasingly reported to cause an invasive fungal infection in immunosuppressive hosts. Herein, we present the case of an immunosuppressive 54 - year-old man who developed S. apiospermum complex lung infection and pulmonary adenocarcinoma.Case report: The patient had some complaints of dyspnea and cough during a neutropenic episode. The computed tomography (CT) scan of the patient revealed pleural effusion. After culturing the pleural fluid sample, the fungus was identified by microscopic examination and ITS sequencing. In addition, antifungal susceptibility testing was performed using the M38-A2 microdilution method. The minimum inhibitory concentrations of amphotericin B, voriconazole, posaconazole, and caspofungin were obtained as > 64, 0.06, 0.06, and 0.03 µg/mL, respectively. Voriconazole (administered in two doses of 6 mg/kg and a maximum of 250 mg) was preferred for treatment. The patient received antifungal treatment for 2 months; however, he was lost to follow-up.Conclusion: Scedosporium apiospermum complex should be considered a cause of systemic fungal infections in neutropenic patients. Furthermore, the determination of the in vitro antifungal susceptibilities of clinical strains may contribute to the development of therapeutic approaches.

A Case of Chromoblastomycosis Caused by Fonsecaea pedrosoi Successfully Treated by Oral Itraconazole Together with Terbinafine.

Patients with chromoblastomycosis (CBM) usually have a history of local skin damage related to outdoor activities, mainly manifested as chronic refractory proliferative pathologic changes. We report a case of a 56-year-old man with CBM, identified as Fonsecaea pedrosoi infection by fungal culture and gene sequencing. This patient was successfully treated with a regimen of oral itraconazole (ITZ) and terbinafine lasting 7 months. Through in vitro drug sensitivity tests, minimum inhibitory concentrations of amphotericin, ITZ, and terbinafine were 1 μg/ml, 0.25 μg/ml, and 1 μg/ml, respectively. In this case, terbinafine was found to be more effective than ITZ.

Candidaemia in a Tertiary Care Centre of Developing Country: Monitoring Possible Change in Spectrum of Agents and Antifungal Susceptibility

AbstractPurpose: Candidaemia is a major cause of morbidity and mortality of hospitalised patients, especially in developing countries. This study was conducted to monitor any change in species distribution and antifungal susceptibility pattern of Candida species causing candidaemia over the last 20 years. Materials and Methods: The candidaemia cases reported during January 1999 and December 2018 at our centre were reviewed. The yeasts were identified by phenotypic characters (during 1999–2014) and matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) (during 2014–2018). Antifungal susceptibility testing (AFST) was performed in accordance with the Clinical and Laboratory Standards Institute guidelines. Results: A total of 602,963 blood samples from patients with suspected sepsis were processed. Candidaemia was diagnosed in 7927 (1.31%) cases. The frequency of cases rose significantly (P = 0.000) in the last quarter of the study. Candida tropicalis (40.1%) was the most common species, followed by Candida albicans (15.2%), Wickerhamomyces anomalus (13.1%), Candida krusei (6.6%), Candida parapsilosis (4.7%) and others. Rare species such as Candida auris, Candida lambica, Candida orthopsilosis, Candida vishwanathii were identified after the introduction of MALDI-TOF. The minimum inhibitory concentrations of amphotericin B rose significantly from the first to last quarter (0.5%–4.9%). Fluconazole resistance was fairly constant at 7.4%–8.8%. Conclusion: Local epidemiology of candidaemia at our centre was distinct regarding prevalence and change of spectrum of species. The identification of rare species was possible after the introduction of MALDI-TOF. With the emergence of multidrug-resistant C. auris and resistance in other species, routine AFST has become imperative.

Surveillance for azoles resistance in Aspergillus spp. highlights a high number of amphotericin B-resistant isolates.

Aspergillus spp. are the most common invasive mould infection and are responsible for high mortality. Aspergillus fumigatus is currently of interest because resistance to azole antifungals has emerged. The Campinas University Hospital (HC-UNICAMP) receives high-risk patients susceptible to opportunistic infections but there have been no reports of resistant A.fumigatus. This study aimed to assess the susceptibility profile of Aspergillus isolates, specifically looking for azole resistance. ITS and β-tubulin DNA sequencing was performed on 228 sequential clinical isolates. Broth microdilution susceptibility testing was performed for all isolates. A.fumigatus represented 74% of the isolates followed by Aspergillus flavus (12%). Nine A.fumigatus isolates from 9 different patients showed high MIC values to at least 1 azole, but cyp51A polymorphisms were detected in only 6 isolates and none correlated with known resistance mutations. The most troubling observation was that the minimum inhibitory concentration for amphotericin B was elevated (≥2mgL-1 ) in 87% of patients with A.flavus isolates and 43% with Aspergillusfumigatus isolates. Given that amphotericin B is used to treat azole-resistant infections, these data highlight the need for continuous surveillance in Aspergillus for all antifungal resistance to implement correct treatment strategies for the management of these pathogens.

In vitro susceptibility of Cuban Aspergillus spp. strains of clinical and environmental origin

The behavior of antifungal susceptibility of Aspergillus spp. in Cuba remains unknown. The antifungals recommended to treat aspergillosis are amphotericin B, itraconazole, voriconazole and echinocandins. The influence of the environment may set off the emergence of drug-resistance in these microorganisms. To evaluate in vitro susceptibility of Aspergillus spp. strains to amphotericin B, itraconazole and voriconazol, and the relationship between susceptibility patterns and their origin. Minimum inhibitory concentrations of amphotericin B, itraconazole and voriconazole were determined for 60 Aspergillus spp. strains of clinical and environmental origin using the M38-A2 method of the Clinical and Laboratory Standards Institute. We found 21 amphotericin B resistant strains (mainly from clinical samples and hospital environments), as well as three itraconazole resistant strains (from non-hospital outdoor and indoor environments). No voriconazole resistance was found. No relationship was found between strain origin and susceptibility. Results suggest the possible existence of environmental factors or interactions with resistant genotypes which may give rise to resistant phenotypes in our country. This is the first report of in vitro Aspergillus spp. resistant strains in Cuba. These studies should be broadened and include molecular and phylogenetic analyses.

Pathogenic potential of environmental resident fungi from ornithogenic soils of Antarctica

We assessed the diversity of cultivable fungi in the ornithogenic soil nests of bird species like Phalacrocorax atriceps, Macronectes giganteus, Pygoscelis antarcticus, and Pygoscelis papua in the Antarctic islands. From 481 fungi isolated at 15 °C, only 50 displayed growth at 37 °C, and were identified as 14 species of 15 genera. Aspergillus fumigatus, Penicillium chrysogenum, and Rhodotorula mucilaginosa were the most abundant species obtained. Fifty taxa grew at 40 °C; displayed haemolytic and phospholipase activities; produced tiny spores, capsule, and melanin; showed growth at different pH; and showed resistance to amphotericin B. Interestingly, the minimum inhibitory concentration of amphotericin B increased by 5–10 fold for some A. fumigatus isolates after phagocytosis by amoeba. Our results show relations among fungal community compositions present in Antarctic ornithogenic soil and their pathogenic risk to humans in vitro. As the Antarctica Peninsula is a major region of the planet affected by global climate changes, our results, though preliminary, raise concerns about the dispersal of potential pathogenic microbes present in Antarctic substrates by wild birds, which can fly great distances and spread potential pathogens mainly to South America and Oceania.

Relationship between intracranial pressure and antifungal agents levels in the CSF of patients with cryptococcal meningitis.

The purpose of this study was to evaluate the influence of intracranial hypertension in the cerebrospinal fluid (CSF) levels of amphotericin B and fluconazole levels of patients with cryptococcal meningitis. CSF samples and intracranial pressure were obtained by means of routine punctures performed at days 1, 7, and 14 of therapy, respectively. Amphotericin B and fluconazole CSF levels were measured by HPLC method as previously described. The minimum inhibitory concentration for amphotericin B, fluconazole, 5΄flucytosine, and voriconazole of each Cryptococcus isolate was performed according to CLSI. The predominant Cryptococcus species found was C. neoformans, and the major underlying condition was AIDS. Only one CSF sample had a detectable level for amphotericin B during the 14 days of therapy. Fluconazole CSF levels progressively increased from day 1 to day 14 of therapy for most cases. Fluconazole levels in the CSF were above the minimum inhibitory concentrations (MICs) for Cryptococcus during the initial 14 days of antifungal therapy. Variations of intracranial pressure did not affect amphotericin B and fluconazole levels in the CSF. The generalized estimating correlation (GEE) and Spearman correlation test (SCT) showed no significant correlation between the amphotericin B or fluconazole concentrations in the CSF and intracranial pressure (P = .953 and P = .093, respectively for GEE test and P = .477 and P = .847, respectively, for SCT). Combination therapy of amphotericin B with fluconazole was effective in 60% of the patients considering CSF cultures were negative in 9 of 15 patients after 14 days of therapy. Further studies are necessary to evaluate the role of intracranial hypertension on the therapeutic efficacy of different antifungal agents in patients with cryptococcal meningitis.

Antifungal susceptibility of Sporothrix schenckii complex biofilms.

Sporotrichosis, caused by species of Sporothrix schenckii complex, is the most prevalent subcutaneous mycosis in many areas of Latin America. The aim of this study was to evaluate the ability of Sporothrix spp. to form biofilms in vitro and to characterize the growth kinetics, morphology, and antifungal susceptibility of biofilms against classical antifungals. We investigated the ability of strains to produce biofilms in vitro and determined the effects of exposure to amphotericin B, itraconazole, caspofungin, ketoconazole, voriconazole, and fluconazole at minimum inhibitory concentration (MIC) against planktonic form and at 10× MIC and 50× MIC on the biomass and metabolic activity of these biofilms. Biofilm structure was analyzed by optical microscopy using Congo-red staining, confocal and scanning electron microscopy. Strains were classified for biofilm-forming ability, through the analysis of absorbance of crystal violet retained by biomass of mature biofilms. We found that all S. brasiliensis (n = 10), S. schenckii sensu stricto (n = 2), S. globosa (n = 2), and S. mexicana (n = 4) strains were strong biofilm-producers. The analyzed biofilms had dense network of hyphae and conidia immersed in extracellular matrix, with presence of water channels. Antifungal drugs at the three tested concentrations showed different effects on biomass and metabolic activity of biofilms. However, the best inhibitory response was observed with 50× MIC of amphotericin B and caspofungin, which reduced these parameters. Furthermore, high drug concentrations, especially amphotericin B and caspofungin, showed antifungal activity against these biofilms, probably because they damaged the architecture and extracellular matrix, allowing diffusion of the drugs.

In Vitro Low Responsiveness of Leishmania donovani Towards Sodium Antimony Gluconate

Kala-azar has been uprising concomitantly with drug-resistant strains of the causatinve agent, particularly in the neighbouring India. The actual perspective of drug resistance in Leishmania donovani in Bangladesh is yet to be explored. So, this prospective study, as a preliminary one, was done to observe in vitro drug responsiveness against Sodium Antimony Gluconate (SAG) and Amphotericin B of 41 strains of L. donovani isolated from Kala-azar cases. The cases (n=41) were selected from 45 clinically suspected febrile patients those who were positive for Kala-azar by immunochromatographic test (ICT). The selected cases were subsequently confirmed as Kala-azar by detection of Leishmania Donovan (LD) bodies from bone marrow aspirates (n=38) by microscopy and/or showing promastigotes in modified McNeal, Nicole and Novy (NNN) media (n=41). Minimum Inhibitory Concentrations (MICs) of SAG and Amphotericin B were seen in relation with history of previous SAG therapy of the patients. Among 08 strains with previous SAG therapy, MICs of SAG were 500 µg in 05 (62.5%) and 250 µg in 03 (37.5%) cases. In remaining 33 strains with no previous SAG therapy, MIC of the drug was 250 µg. In all 41 strains, MIC of Amphotericin B was 05 µg irrespective of the history of previous SAG therapy. The study revealed that strains of L. donovani with low responsiveness to standard dose of pentavalent antimonials have been started to appear in our community that needs further study at community level in a larger population.Bangladesh J Med Microbiol 2008; 02 (01): 8-12DOI: http://dx.doi.org/10.3329/bjmm.v2i1.21782

In vitro activities of five antifungal agents against 199 clinical and environmental isolates of Aspergillus flavus, an opportunistic fungal pathogen

Aspergillus flavus is the second leading cause of invasive and non-invasive aspergillosis, as well as the most common cause of fungal sinusitis, cutaneous infections, and endophthalmitis in tropical countries. Since resistance to antifungal agents has been observed in patients, susceptibility testing is helpful in defining the activity spectrum of antifungals and determining the appropriate drug for treatment. A collection of 199 clinical and environmental strains of Aspergillus flavus consisted of clinical (n=171) and environmental (n=28) were verified by DNA sequencing of the partial b-tubulin gene. MICs of amphotericin B, itraconazole, voriconazole, posaconazole, and MEC of caspofungin were determined in accordance with the Clinical and Laboratory Standards Institute M38-A2 document. Caspofungin, followed by posaconazole, exhibited the lowest minimum inhibitory concentrations (MIC). All isolates had caspofungin MEC90 (0.063μg/ml) lower than the epidemiologic cutoff values, and 3.5% of the isolates had amphotericin B MIC higher than the epidemiologic cutoff values. However, their clinical effectiveness in the treatment of A.flavus infection remains to be determined.

Time-Kill Kinetics and In Vitro Antifungal Susceptibility of Non-fumigatus Aspergillus Species Isolated from Patients with Ocular Mycoses.

Aspergillus species can cause ocular morbidity and blindness, and thus, appropriate antifungal therapy is needed. We investigated the in vitro activity of itraconazole, voriconazole, posaconazole, caspofungin, anidulafungin, and amphotericin B against 14 Aspergillus isolates obtained from patients with ocular mycoses, using the CLSI reference broth microdilution methodology. In addition, time-kill assays were performed, exposing each isolate separately to 1-, 4-, and 16-fold concentrations above the minimum inhibitory concentration (MIC) of each antifungal agent. A sigmoid maximum-effect (Emax) model was used to fit the time-kill curve data. The drug effect was further evaluated by measuring an increase/decrease in the killing rate of the tested isolates. The MICs of amphotericin B, itraconazole, voriconazole, and posaconazole were 0.5–1.0, 1.0, 0.5–1.0, and 0.25 µg/ml for A. brasiliensis, A. niger, and A. tubingensis isolates, respectively, and 2.0–4.0, 0.5, 1.0 for A. flavus, and 0.12–0.25 µg/ml for A. nomius isolates, respectively. A. calidoustus had the highest MIC range for the azoles (4.0–16.0 µg/ml) among all isolates tested. The minimum effective concentrations of caspofungin and anidulafungin were ≤0.03–0.5 µg/ml and ≤0.03 µg/ml for all isolates, respectively. Posaconazole demonstrated maximal killing rates (Emax = 0.63 h−1, r2 = 0.71) against 14 ocular Aspergillus isolates, followed by amphotericin B (Emax = 0.39 h−1, r2 = 0.87), voriconazole (Emax = 0.35 h−1, r2 = 0.098), and itraconazole (Emax = 0.01 h−1, r2 = 0.98). Overall, the antifungal susceptibility of the non-fumigatusAspergillus isolates tested was species and antifungal agent dependent. Analysis of the kinetic growth assays, along with consideration of the killing rates, revealed that posaconazole was the most effective antifungal against all of the isolates.

Evidence of Fluconazole-Resistant Candida Species in Tortoises and Sea Turtles.

The aim of this study was to evaluate the antifungal susceptibility of Candida spp. recovered from tortoises (Chelonoidis spp.) and sea turtles (Chelonia mydas, Caretta caretta, Lepidochelys olivacea, Eretmochelys imbricata). For this purpose, material from the oral cavity and cloaca of 77 animals (60 tortoises and 17 sea turtles) was collected. The collected specimens were seeded on 2% Sabouraud dextrose agar with chloramphenicol, and the identification was carried out by morphological and biochemical methods. Sixty-six isolates were recovered from tortoises, out of which 27 were C. tropicalis, 27 C. famata, 7 C. albicans, 4 C. guilliermondii and 1 C. intermedia, whereas 12 strains were obtained from sea turtles, which were identified as Candida parapsilosis (n = 4), Candida guilliermondii (n = 4), Candida tropicalis (n = 2), Candida albicans (n = 1) and Candida intermedia (n = 1). The minimum inhibitory concentrations for amphotericin B, itraconazole and fluconazole ranged from 0.03125 to 0.5, 0.03125 to >16 and 0.125 to >64, respectively. Overall, 19 azole-resistant strains (14 C. tropicalis and 5 C. albicans) were found. Thus, this study shows that Testudines carry azole-resistant Candida spp.

Phenotypic switching of Candida guilliermondii is associated with pseudohyphae formation and antifungal resistance.

Switching between two cell types in fungi is called phenotypic switching, and it is commonly observed in pathogenic yeast. Candida lusitaniae undergoes antifungal resistance-associated phenotypic switching and results in three colony colors: light brown, brown and dark brown. In this study, we included C. lusitaniae as control. This study had two objectives. First, we wanted to evaluate whether also a prevalent human pathogen C. guilliermondii can undergo phenotypic switching. Second, our aim was to determine whether switching can change yeasts susceptibility to antifungals. Yeast suspension (1 × 10(3)-5 × 10(3) c.f.u./ml) was plated on the YPD medium containing 1 mM CuSO4. Colonies exhibiting the original and variant phenotypes were counted and converted to percentage of the population. Minimum inhibitory concentrations of amphotericin B, formic acid and acetic acid for the cells from random colonies of the different phenotypes were determined by microdilution method. After 5 days of incubation, C. guilliermondii switched spontaneously and reversibly among two phenotypes distinguishable on CuSO4 containing agar, white and dark brown. Phenotypes occurred with greater frequency (10(-1)-10(-2)) than spontaneous mutations and were reversible, fulfilling the two phenotypic switching criteria. The study showed that phenotypic switching was associated with filamentation and affected antifungal resistance. Resistance to amphotericin B increased tenfold and was associated with C. lusitaniae dark brown phenotype. C. guilliermondii colonies with brown phenotype displayed 20 and 2 times higher resistance to amphotericin B and acetic acid, respectively.

Anti-Candida albicans effectiveness of citral and investigation of mode of action

Context: Candidiasis is a mycosis caused by Candida species, which is of clinical importance due to the increase in resistant yeasts. Candida infection has been a serious health problem due to the inappropriate use of antibiotics. Therefore, it is necessary to study molecules with an antifungal action. Citral is a monoterpene with known pharmacological properties, including antimicrobial action.Objective: The aim of this work was to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of citral and the probable mode of action.Materials and methods: The MIC of citral was determined by the broth microdilution method using Sabouraud dextrose medium. Additionally, the interference of citral in cell wall (sorbitol assay) and the binding of citral to ergosterol and cholesterol were studied, carried out by broth microdilution method.Results: The MIC and MFC of citral were 512 and 1024 µg/mL, respectively. The MIC of amphotericin B was 1 µg/mL. The mechanism of action did not involve either the cell wall or ergosterol. However, the presence of cholesterol increased the MIC of citral to 1024 µg/mL, indicating there is some interaction between citral and cholesterol. Amphotericin B was used as the positive control, and it showed a high MIC in the presence of ergosterol (32 µg/mL), while in the presence of cholesterol MIC increased to 4 µg/mL.Conclusion: Citral inhibits the growth of C. albicans. The probable mechanism of action did not involve the cell wall or ergosterol. Citral is able to interact with cholesterol. More studies are necessary to describe their effects completely.

Minimum inhibitory concentrations of amphotericin B, azoles and caspofungin againstCandidaspecies are reduced by farnesol

The objective of this study was to evaluate the antifungal activity of farnesol and its interaction with traditional antifungals against drug-resistant strains of Candida species. To do so, we studied the minimum in vitro inhibitory concentration (MIC) of amphotericin B (AMB), fluconazole (FLC), itraconazole (ITC), caspofungin (CAS) and farnesol against 45 isolates of Candida spp., i.e., 24 C. albicans, 16 C. parapsilosis and 5 C. tropicalis through the use of the broth microdilution method. Then, the isolates were tested with the combination of farnesol plus drugs to which they were previously found to be resistant. Additionally, the strains were pre-incubated at sub-inhibitory farnesol concentrations and their antifungal susceptibilities were re-evaluated. We found the MIC values for farnesol varied from 4.68-150 µM for Candida spp., with 19 isolates having a MIC > 1 mg/l, 18 a MIC ≥ 64 mg/l, 35 having a MIC ≥ 1 mg/l and 6 isolates a MIC ≥ 2 mg/l or were resistant to AMB, FLC, ITC and CAS, respectively. Significant MIC reductions were observed when farnesol and antifungal drugs were combined (P < 0.05) and when Candida strains were incubated with farnesol (P < 0.05). We conclude that the in vitro effects of farnesol improved the activity of traditional antifungals to which the Candida spp. isolates were resistant. These results support further investigation of the role of farnesol in the balance of the sterol biosynthetic pathway and how it interferes with cell viability.

Minimum inhibitory concentrations of amphotericin B against Candida krusei isolates from a French teaching hospital laboratory: a retrospective study over 8 years

Recent studies have shown decreased susceptibility of Candida krusei to amphotericin B (AmB), in addition to its inherent resistance to fluconazole. The susceptibility of C. krusei to AmB was studied in the Parasitology-Mycology laboratory of Grenoble Teaching Hospital, France. Between 2003 and 2011, we analysed 200 C. krusei isolates from 130 patients. The isolates were mainly collected in intensive care, cardio-thoracic and cancer/haematology units. Minimum inhibitory concentrations (MICs) were determined by the E-test method. The modal MIC was 0.5 μg ml(-1); the MIC(50) and MIC(90) (MICs encompassing 50% and 90% of all isolates tested, respectively) were 0.5 μg ml(-1) and 1 μg ml(-1). The Cuzick's and Kendall's tests showed a significant increase in MIC values between 2003 and 2011 (P = 0.001 and P ≤ 0.001, respectively), regardless of age or gender. No statistical difference was reached with these tests when the first 100 or 50 data were excluded. Despite the increase observed in the first period of the study, our results confirm the low AmB MICs reported in previous studies. However, some authors have recently reported much higher MICs. This discrepancy cannot be explained by method biases and could reflect C. krusei epidemiological differences among populations.