Synergistic Effects of Efflux Pump Modulators on the Azole Antifungal Susceptibility of Microsporum canis

The microbiologic and clinical resistance of dermatophytes is seldom reported, and the mechanisms associated with resistance are not well known. This study investigated the effect of efflux pump modulators (EPMs) (i.e., haloperidol HAL and promethazine PTZ) and their inhibiting activity on the minimum inhibitory concentrations of itraconazole (ITZ) and fluconazole (FLZ) against selected M. canis strains. M. canis strains with low (≤?1 μg/ml itraconazole and?<?64 μg/ml fluconazole) and high (>?1 μg/ml itraconazole and?≥?64 μg/ml fluconazole) azole MIC values were tested using Checkerboard microdilution assay. The disk diffusion assay, the minimum fungicidal concentration and the time-kill assay were also performed in order to confirm the results of checkerboard microdilution assay. The MIC values of ITZ and FLZ of M. canis decreased in the presence of subinhibitory concentrations of HAL and PTZ, the latter being more effective with a greater increased susceptibility. Synergism was observed in all strains with high azole MICs (FICI?<?0.5) and no synergism in the strains with low azole MICs. A fungicidal activity was observed after 48 h of incubation when ITZ and FLZ were tested in combination with HAL or PTZ. These results suggest that the drug efflux pumps are involved in the defense mechanisms to azole drugs in M. canis strains. The synergism might be related to an increased expression of efflux pump genes, eventually resulting in azole resistance phenomena. Complementary studies on M. canis resistance are advocated in order to investigate the molecular mechanisms of this phenomenon.

     

Increase in Resistance to Fluconazole and Itraconazole in Trichophyton rubrum Clinical Isolates by Sequential Passages In Vitro under Drug Pressure

Trichophyton rubrum, an anthropophilic dermatophyte fungus, is the predominant causative agent of superficial skin infections in human population. There are only scanty reports on drug susceptibility profiling of T. rubrum. Neither mechanisms for drug resistance development nor correlation between in vitro drug susceptibility and in vivo response to treatment is known for that species. In this study, changes in the in vitro susceptibilities to fluconazole (FLZ) and itraconazole (ITZ) among thirty T. rubrum clinical strains subjected to sequential passages in the presence or absence of the azoles were investigated. Each strain was passaged 12 times at 4-week intervals as three parallel cultures, maintained on a drug-free medium (1), and a medium containing FLZ (2) or ITZ (3) at subinhibitory concentrations. Susceptibility to FLZ and ITZ of the original strain and its 3 subcultures was determined by microdilution method. The MIC values of the two azoles remained unaltered for all T. rubrum strains tested, after 12 passages on a drug-free medium. Among the strains grown with FLZ, an increase in the MICs of FLZ and ITZ was noted in 17 (56.7 %) and 19 (63.3 %) strains, respectively. Increased MICs of ITZ and FLZ were demonstrated for 24 (80 %) and 20 (66.7 %) strains that were propagated with ITZ. The results indicate the capacity of T. rubrum to develop resistance toward the azoles after prolonged exposure to these drugs. Resistance of T. rubrum to azoles plays an important role in therapy failures and consequently contributes to persistence and chronicity of the infections.     

Wild-Type MIC Distributions and Epidemiologic Cutoff Values for Fluconazole and <Emphasis Type="Italic">Candida</Emphasis>: Time for New Clinical Breakpoints?

Antifungal susceptibility testing of Candida against fluconazole has been standardized by both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Both CLSI and EUCAST have developed clinical breakpoint (CBP) criteria for fluconazole, but these differ in both magnitude and target species. Studies using the EUCAST method have also defined wild-type minimum inhibitory concentration (MIC) distributions and epidemiologic cutoff values (ECVs or ECOFFs) for the common species of Candida. The ECVs serve as a sensitive means of discriminating wild-type strains from those with acquired resistance mechanisms and include MICs of 1 μg/mL for C. albicans, 2 μg/mL for C. tropicalis and C. parapsilosis, 32 μg/mL for C. glabrata, and 128 μg/mL for C. krusei. Because the CLSI CBPs may be too insensitive to detect emerging resistance among strains of C. albicans, C. tropicalis, and C. parapsilosis, and bisect the WT MIC distribution of C. glabrata, we sought to establish the wild-type MIC distribution and ECVs for fluconazole and Candida spp. The establishment of the wild-type MIC distributions and ECVs for fluconazole using CLSI methods will be useful in resistance surveillance and may prove to be an important step in the development of species-specific CBPs for this important antifungal agent.     

Definitions and Epidemiology of Candida Species not Susceptible to Echinocandins

Antifungal susceptibility testing of Candida against the echinocandin antifungal agents (anidulafungin [ANF], caspofungin [CSF], micafungin [MCF]) has been standardized by the Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antifungal Testing. The CLSI proposed a single set of clinical breakpoints (CBPs) for all three echinocandins and all species of Candida: susceptible, minimum inhibitory concentration (MIC) ≤ 2 μg/mL; nonsusceptible, MIC > 2 μg/mL. Subsequently, these CBPs have been shown to lack sensitivity in detecting strains of Candida with acquired resistance mechanisms associated with treatment failure. Studies using the CLSI method have defined wild-type (WT) MIC distributions and epidemiologic cutoff values (ECVs) for each echinocandin and the common species of Candida. The ECVs serve as a sensitive means of discriminating WT strains from those with acquired resistance mechanisms. WT MIC distributions revealed ECV ranges of 0.03 to 0.25 μg/mL for all major species except C. parapsilosis (1–4 μg/mL) and C. guilliermondii (4–16 μg/mL). These ECVs reliably differentiate WT strains of each species from non-WT strains containing fks mutations. These data, coupled with additional biochemical, clinical, pharmacokinetic, and pharmacodynamic considerations, have resulted in new CBPs of ≤0.25 μg/mL (susceptible), 0.5 μg/mL (intermediate), and ≥1 μg/mL (resistant) for ANF, CSF, and MCF for C. albicans, C. tropicalis, and C. krusei. For these agents and C. parapsilosis, the new CBPs are ≤2 μg/mL (susceptible), 4 μg/mL (intermediate), and ≥8 μg/mL (resistant). For C. glabrata, the CBPs for ANF and CSF are ≤0.12 μg/mL (susceptible), 0.25 μg/mL (intermediate), and ≥0.5 μg/mL (resistant), whereas those for MCF are ≤0.06 μg/mL, 0.12 μg/mL, and ≥0.25 μg/mL, respectively. Application of both ECVs and the lower species-specific CBPs for the echinocandins has proven useful in both resistance surveillance and clinical care and will serve as an important step in international harmonization of in vitro susceptibility testing of this important antifungal class.     

Comparative Analysis of the Vitek 2 Antifungal Susceptibility System and E-test with the CLSI M27-A3 Broth Microdilution Method for Susceptibility Testing of Indian Clinical Isolates of Cryptococcus neoformans

The emergence of antifungal resistance among Cryptococcus neoformans isolates is a matter of great concern. The Clinical and Laboratory Standards Institute (CLSI) broth microdilution reference method (BMD) for antifungal susceptibility testing of C. neoformans is tedious and time-consuming. Consequently, there is a greater need for a reproducible in vitro susceptibility testing method for use in clinical microbiology laboratories. By random amplified polymorphic DNA analysis, the 62 Indian clinical isolates were characterized as Cryptococcus neoformans var. grubii. We evaluated the susceptibilities of these isolates for amphotericin B (AMB) and fluconazole (FLC) by two commercial techniques, i.e., Vitek 2 and E-test against the CLSI M27-A3 BMD. The essential agreement (EA) between the Vitek 2 and E-test with the reference procedure for FLC was similar (82.2%). For AMB, EA of 92 and 76% was obtained with E-test and Vitek 2. Excellent categorical agreement (CA) (98.3% and 100% by Vitek 2 and E-test, respectively) was obtained for AMB. The CA for FLC was 81 and 77.4% by Vitek 2 and E-test. We conclude that both E-test and Vitek 2 system have acceptable levels of accuracy for susceptibility testing of both the drugs. Both of them could identify fluconazole-resistant strains. Vitek 2 could be used for testing susceptibility of voriconazole and 5-flucytosine also at the same time.     

Evaluation of a new method for antifungal susceptibility testing for azoles

The interpretation of the end points in azole antifungal drug susceptibility testing is challenging, in part due to incomplete growth inhibition of Candida species. Since the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method have limitation with azoles, a new modification of the CLSI microdilution protocol was evaluated. We measure the decrease in growth rate (μ) of exponentially growing cultures in accordance to different azole concentrations at time intervals up to 10 h. Using 15 different Candida strains, an overall agreement within ± 2 dilutions by the CLSI method at 24 h in RPMI and the μ-dependent method for three antifungal agents (fluconazole- itraconazole and voriconazole) was achieved. MIC measurement by the new method was less sensitive to the medium used or the inoculum size applied. The presented data suggested that, measuring the in vitro inhibition kinetics at the logarithmic phase could have advantages for addressing susceptibility testing toward azoles.     

Evaluation of Antifungal Susceptibility Testing with Microdilution and Etest Methods of <Emphasis Type="Italic">Candida</Emphasis> Blood Isolates

Candida species that show an increasing number of clinical and/or microbiological resistance to several antifungals and are the most common agents of invasive fungal infections. The aim of this study was to investigate the in vitro susceptibility of Candida blood isolates to antifungal agents (amphotericin B, fluconazole, itraconazole, and voriconazole) by comparative use of the CLSI reference microdilution method and Etest. Four hundred Candida blood isolates (215 Candida albicans, 185 non-albicans Candida strains) were included in the study. The broth microdilution test was performed according to the CLSI M27 A2 document. Etest was carried out according to the manufacturer’s instructions. The MIC results obtained with reference microdilution were compared with those obtained with the Etest by using percent and categorical agreements. According to MIK₉₀ values, voriconazole was the most active and itraconazole was the least active drug in vitro against all Candida species. Other than voriconazole, statistically significant differences were found when the susceptibility of Candida albicans and non-albicans Candida spp. to amphotericin B, fluconazole, and itraconazole were compared. These antifungal agents were found to be more active to C. albicans. Among the non-albicans Candida species, the lowest MIC values were obtained for Candida parapsilosis isolates. When the standard method was compared with Etest, the total agreement was higher for C. albicans than for non-albicans species, especially for fluconazole and voriconazole. In view of the findings, it was concluded that itraconazole showed the lowest activity against all Candida species. Etest could be an alternative method in assessing the in vitro antifungal susceptibility of Candida spp., but it is more convenient to use the microdilution method for studying in vitro susceptibility of non-albicans species, in particular for those possessing high MIC values against azoles.     

Advances in Antifungal Susceptibility Testing of <Emphasis Type="Italic">Candida</Emphasis>, 2010–2012

Antifungal susceptibility testing of Candida has been standardized and refined and now may play an important role in managing Candida infections. Important new developments include the establishment of species-specific epidemiological cutoff values (ECVs) for the systemically active antifungal agents and both common and uncommon species of Candida. The clinical breakpoints (CBPs) for fluconazole, voriconazole, and the echinocandins have been revised to provide species-specific interpretive criteria for the six most common species that not only are predictive of clinical outcome but also provide a more sensitive means of identifying those strains with acquired or mutational resistance mechanisms. Collaborative work by the CLSI and EUCAST organizations has made major advances in the harmonization of these two international standards. The impact of the recent changes in the CBPs on commercial MIC methods does not appear to be major but additional studies with well defined resistant populations are necessary to confirm the ability of these systems to detect emerging resistance.     

Antibiotic Susceptibility of Helicobacter pylori Clinical Isolates: Comparative Evaluation of Disk-Diffusion and E-Test Methods

Antimicrobial susceptibility of 25 Helicobacter pylori strains isolated from patients with acid peptic diseases were tested for in vitro sensitivity to commonly used antibiotics using disk-diffusion and E-test, methods. All strains tested were susceptible to tetracycline by E-test, with the minimum inhibitory concentration (MIC) values being <0.125 μg/ml for all strains except for 6 (<0.023 μg/ml). However 1 strain was resistant by disk-diffusion method. One strain was resistant to clarithromycin both by disk diffusion and E-test (MIC <48 μg/ml), and 1 strain was resistant only by disk diffusion. Only one strain was resistant to amoxicillin by disk diffusion and E-test (MIC >256 μg/ml). For ciprofloxacin, three strains were resistant by disk diffusion and two by E-test (MIC <32 μg/ml). Sixteen strains were resistant to metronidazole by disk diffusion and E-test (MIC ≥ 8 μg/ml), and 1 was resistant only by E-test (MIC <48 μg/ml). Overall, 64% of the strains were resistant to metronidazole. The MIC for metronidazole was also tested by agar-dilution method, and metronidazole resistant strains had an, MIC >8 μg/ml. The disk-diffusion method showed excellent correlation with E-test results; there was 100% agreement for amoxicillin a other antibiotics showed 90% to 95% accuracy. Disk diffusion is cheaper than E-test (approximately 2.6 cents vs. US$2.60), is easy to perform, and is a reliable method for testing H. pylori susceptibility to antimicrobial agents in the clinical microbiology laboratory.     

In vitro susceptibility of dermatomycoses agents to six antifungal drugs and evaluation by fractional inhibitory concentration index of combined effects of amorolfine and itraconazole in dermatophytes

To investigate the antifungal drug susceptibility of fungi responsible for dermatomycoses, minimum inhibition concentration (MIC) tests were performed in 44 strains of dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum, with six antifungal drugs (amorolfine, terbinafine, butenafine, ketoconazole, itraconazole and bifonazole) by broth microdilution assay according to Clinical Laboratory Standard Institute protocols. Six possible dermatomycosis‐causing non‐dermatophytic fungi were also tested. The two major causes of tinea, T. rubrum and T. mentagrophytes, showed significantly different sensitivities to ketoconazole and bifonazole. Clinically derived dermatophytes were sensitive to the six antifungal drugs tested. However, non‐dermatophytes, especially Fusarium spp., tended to be resistant to these antifungal drugs. In Trichophyton spp., the MICs of non‐azole drugs had narrower distributions than those of azoles. To evaluate the effects of antifungal drug combinations, the fractional inhibitory concentration index was calculated for the combination of amorolfine and itraconazole as representative external and internal drugs for dermatophytes. It was found that this combination had synergistic or additive effects on most dermatophytes, and had no antagonistic effects. The variation in susceptibility of clinically derived fungal isolates indicates that identification of causative fungi is indispensable for appropriately choosing effective antifungal drugs in the early stages of infection. The results of combination assay suggest that multiple drugs with different antifungal mechanisms against growth of dermatophytes should be used to treat refractory dermatomycoses, especially onychomycosis.     

Antifungal Susceptibility Testing of <Emphasis Type="Italic">Candida</Emphasis> and <Emphasis Type="Italic">Cryptococcus</Emphasis> Species and Mechanisms of Resistance: Implications for Clinical Laboratories

Purpose of Review

Resistance to antifungal drugs amongst Candida species is a growing concern, and azole resistance may be emerging in Cryptococcus species. This review provides a contemporary perspective, relevant to the clinical mycology laboratory, of antifungal susceptibility testing of these fungi, focussing on the challenges of phenotypic and genotypic methodologies to detect drug resistance.

Recent Findings

Standardised CLSI and EUCAST broth microdilution (BMD) susceptibility testing methods are the benchmark to determine clinical breakpoints (CBPs) and/or epidemiological cut-off values (ECVs) MICs for Candida and Cryptococcus spp. Commercial methods may be used but caution is required when employing BMD CBPs/ECVs to interpret results. Species-specific CBPs/ECVs for Candida spp. generally correlate well with predicting likelihood of therapeutic failure or of presence of a drug resistance mechanism with the exception of the echinocandins where the presence of specific FKS gene mutations and not the MIC correlates most accurately with clinical outcome. The relationship of presence of one or more mechanisms of azole resistance and drug MICs is uncertain. Next generation sequencing technology is offering insights into the relationships between susceptibility results obtained by phenotypic and genotypic methods. For Cryptococcus spp., CBPs are not established but species- and genetic type-specific EVCs are useful for guiding therapy where clinically indicated. Isolates of genotype VGII appear to exhibit the highest MICs.

Summary

Antifungal susceptibility testing of yeasts is important to detect drug resistance. For Candida spp., MICs have clinical utility for the azoles but detecting echinocandin resistance by genotypic methods is preferred. For Cryptococcus spp., ECVs are useful in guiding therapy.

     

Emerging Resistance to Azoles and Echinocandins: Clinical Relevance and Laboratory Detection

Failure to respond to antifungal therapy could be due to in vitro resistance (intrinsic or developed during therapy) or clinical resistance. In vitro resistance is mostly due to genetic mutations (resistance mechanisms), and it is associated with high minimal inhibitory concentrations (MICs), minimal effective concentrations (MECs), and/or clinical failure. Clinical breakpoints (CBPs) and/or epidemiologic cutoff values (ECVs) are useful to detect the in vitro antifungal resistance when isolates are tested by standardized methods. ECVs are available from the Clinical and Laboratory Standards Institute (CLSI) for Candida spp. versus echinocandins (anidulafungin, caspofungin, and micafungin) and triazoles (fluconazole, posaconazole, and voriconazole). Lately, the CLSI has adjusted to species-specific CBPs for Candida spp. versus fluconazole, similar to those of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and versus echinocandins. However, the available voriconazole EUCAST and CLSI CBPs differ. In the absence of CBPs, EUCAST and CLSI assigned ECVs for various Aspergillus spp. and triazoles. This article reviews emerging resistance, laboratory detection, and clinical relevance as reported in the literature in the past 3 to 4 years.     

Surface plasmon resonance analysis of antifungal azoles binding to CYP3A4 with kinetic resolution of multiple binding orientations

The heme-containing cytochrome P450s (CYPs) are a major enzymatic determinant of drug clearance and drug-drug interactions. The CYP3A4 isoform is inhibited by antifungal imidazoles or triazoles, which form low-spin heme iron complexes via formation of a nitrogen-ferric iron coordinate bond. However, CYP3A4 also slowly oxidizes the antifungal itraconazole (ITZ) at a site that is approximately 25 A from the triazole nitrogens, suggesting that large antifungal azoles can adopt multiple orientations within the CYP3A4 active site. Here, we report a surface plasmon resonance (SPR) analysis with kinetic resolution of two binding modes of ITZ, and the related drug ketoconazole (KTZ). SPR reveals a very slow off-rate for one binding orientation. Multiphasic binding kinetics are observed, and one of the two binding components resolved by curve fitting exhibits "equilibrium overshoot". Preloading of CYP3A4 with the heme ligand imidazole abolishes this component of the antifungal azole binding trajectories, and it eliminates the conspicuously slow off-rate. The fractional populations of CYP3A4 complexes corresponding to different drug orientations can be manipulated by altering the duration of the pulse of drug exposure. UV-vis difference absorbance titrations yield low-spin spectra and K(D) values that are consistent with the high-affinity complex resolved by SPR. These results demonstrate that ITZ and KTZ bind in multiple orientations, including a catalytically productive mode and a slowly dissociating inhibitory mode. Most importantly, they provide the first example of a SPR-based method for the kinetic characterization of binding of a drug to any human CYP, including mechanistic insight not available from other methods.     

EUCAST and CLSI: Working Together Towards a Harmonized Method for Antifungal Susceptibility Testing

The U.S. Clinical and Laboratory Standards Institute (CLSI) and the European Committee of Antimicrobial Susceptibility Testing (AFST-EUCAST) have developed broth microdilution methodologies for testing yeasts and filamentous fungi (molds). The mission of these methodologies is to identify in vitro antifungal resistance, which is accomplished by the use of either clinical breakpoints (CBPs), or to a lesser degree, epidemiologic cutoff values (ECVs). The newly adjusted and species-specific CLSI CBPs for Candida spp. versus fluconazole and voriconazole have ameliorated some of the differences between the two methodologies. In the absence of CBPs for mold testing, CLSI ECVs are available for six Aspergillus species versus the triazoles, caspofungin and amphotericin B. Recently, breakpoints were developed by the EUCAST for certain Aspergillus spp. versus amphotercin B, itraconazole and posaconazole, which to some extent are comparable to ECVs. We summarize these latest accomplishments, which have made possible the harmonization of some susceptibility cutoffs, if not methodologies for some agent/species combinations.     

Toxoplasma gondii: Fluconazole and itraconazole activity against toxoplasmosis in a murine model

Toxoplasma gondii is an important opportunistic pathogen affecting immunocompromised patients with AIDS. Toxoplasmic encephalitis is responsible for high morbidity and mortality. In this study, we investigated the activity of the antifungals fluconazole (FLZ) and itraconazole (ITZ) against T. gondii in mice infected with the Me49 strain. As previously reported for ITZ, FLZ also demonstrated a selective effect against T. gondii in vitro; the IC₅₀ values obtained for FLZ were 8.9 μM and 3.1 μM after 24 h and 48 h of treatment, respectively. A 10-day treatment of mice with orally or intraperitoneally administered 20 mg/kg/day FLZ showed a significant survival difference compared to untreated mice. The administration of 20 mg/kg/day ITZ significantly reduced the brain cyst burden compared to untreated mice but did not exert significant protection against death. The results obtained in this work are rather promising as ITZ and FLZ are safe and low-cost drugs available on the market.     

CLSI M38-A2肉汤稀释法对蝙蝠蛾拟青霉(Paecilomyces hepiali)的药敏检测

[目的]对保健食品生产菌株蝙蝠蛾拟青霉(Paecilomyces hepiali)进行6种常见抗真菌药物:伊曲康唑、酮康唑、伏立康唑、环吡酮胺、5-氟胞嘧啶、氟康唑的药敏检测.[方法]参考美国国家临床实验室标准化委员会CLSI M38-A2《肉汤稀释法抗真菌药敏试验参考方案》.[结果]质控菌株MIC值均在参考范围之内,蝙蝠蛾拟青霉(P.hepiali)对以上6种药物的MIC值分别为1 mg/L、0.5 mg/L、0.25 mg/L、≥128 mg/L、64 mg/L、2 mg/L.[结论]M38-A2方案适合用来测定蝙蝠蛾拟青霉(P.hepiali)对抗真菌药物的敏感性,但获得菌株耐药敏感性的判别标准,仍需进一步的试验数据.     

Efecto inhibitorio del dietilestilbestrol sobre aislamientos clínicos de Candida albicans sensibles y resistentes a los azoles

BackgroundCandida albicans is a microorganism frequently involved in several infections; the patient's oral cavity, caries niches or periodontal disease can sometimes be the reservoir.. The fungal resistance to the available treatments, among other reasons, has led to the search for new antifungal alternatives.AimsTo carry out a comparative study of the in vitro effects of diethylstilboestrol (DES) and fluconazole (FLZ) on the growth of clinical strains of C. albicans.MethodsSeven strains of C. albicans were used: a) one FLZ-sensitive culture collection strain, ATCC 90028 (ATCC); b) four oral isolates from four oncological patients with periodontal disease (period 8, 9, 10, and 11); and c) two oral isolates from an AIDS patient with oropharyngeal candidiasis: one FLZ- sensitive (2-76), and another FLZ- resistant (12-99). The MIC was evaluated by standard spectrophotometric techniques using the CLSI (M27-A3) guidelines. The inhibitory concentration 50% (IC₅₀) was calculated using functional analysis with the Graph Pad software.ResultsDES inhibited the growth of all C. albicans strains, whether sensitive or resistant to FLZ. Experimental data fitted non-linear functions of inhibitor concentration versus response. Minimum inhibitory concentrations (MIC) for DES and FLZ were as follows: 28.18 µg/ml and 4.90 µg/ml (ATCC); 17.16 µg/ml and 3.14 µg/ml (period); 27.64 µg/ml and 4.22 µg/ml (2-76); 6.16 µg/ml and 438.19 µg/ml (12-99), respectively.ConclusionsDES showed antifungal activity on all clinical C. albicans strains isolated from patients with dental and medical diseases. It showed the highest potency on the FLZ-resistant isolate.     

Antifungal and marker effects of Talisia esculenta lectin on Microsporum canis in vitro

Aims: The purpose of this study was to demonstrate the usefulness of lectin obtained from Talisia esculenta (TEL) seeds as a tool to recognize and study Microsporum canis. For this purpose, we investigated the antifungal and marker action of this lectin and the relationship of these effects with the presence of carbohydrates on the structure of this fungus. Methods and Results: The in vitro antifungal activity of TEL was analysed by broth microdilution assay. In addition, TEL was assessed against the arthroconidia present on hairs obtained from infected dogs and cats. The affinity of fluorescein isothiocyanate (FITC)‐labelled TEL for macroconidia and arthroconidia of M. canis was also tested. The effects of TEL on the growth of the M. canis strains began with 0·125 mg ml^?1, and 100% inhibition was obtained with a concentration of 2 mg ml^?1. The addition of carbohydrates, especially N‐acetyl‐glucosamine and d ‐mannose, inhibited these antifungal effects. TEL was able to inhibit the growth of arthroconidial chitin‐rich forms of M. canis obtained from hairs of infected animals and strains cultured in Sabouraud agar. FITC‐labelled TEL efficiently marked macroconidial and arthroconidial forms of M. canis, as shown by fluorescent microscopy. Conclusions: These results show that the inhibitory effects of TEL on M. canis growth may be related to the interaction of lectin with the carbohydrates present at the micro‐organism’s surface, mainly d ‐mannose and N‐acetyl‐glucosamine. Significance and Impact of the Study: Talisia esculenta can be used as an important tool in the biochemical study of M. canis or as a molecule to recognize this dermatophyte in infected tissue.     

Effect of antifungals on itraconazole resistant <Emphasis Type="Italic">Candida glabrata</Emphasis>

In the last decade, infections caused by Candida glabrata have become more serious, particularly due to its decreased susceptibility to azole derivatives and its ability to form biofilm. Here we studied the resistance profile of 42 C. glabrata clinical isolates to different azoles, amphotericin B and echinocandins. This work was also focused on the ability to form biofilm which plays a role in the development of antifungal resistance. The minimal inhibitory concentration testing to antifungal agents was performed according to the CLSI (Clinical and Laboratory Standards Institute) M27-A3 protocol. Quantification of biofilm was done by XTT reduction assay. All C. glabrata clinical isolates were resistant to itraconazole and sixteen also showed resistance to fluconazole. All isolates remained susceptible to voriconazole. Amphotericin B was efficient in a concentration range of 0.125–1 mg/L. The most effective antifungal agents were micafungin and caspofungin with the MIC₁₀₀ values of ≤0.0313–0.125 mg/L. Low concentrations of these agents reduced biofilm formation as well. Our results show that resistance of different C. glabrata strains is azole specific and therefore a single azole resistance cannot be assumed to indicate general azole resistance. Echinocandins proved to have very high efficacy against clinical C. glabrata strains including those with ability to form biofilm.