Candida auris from the Egyptian cobra: Role of snakes as potential reservoirs.

Candida auris represents one of the most urgent threats to public health, although its ecology remains largely unknown. Because amphibians and reptiles may present favorable conditions for C. auris colonization, cloacal and blood samples (n = 68), from several snake species, were cultured and molecularly screened for C. auris using molecular amplification of glycosylphosphatidylinositol protein-encoding genes and ribosomal internal transcribed spacer sequencing. Candida auris was isolated from the cloacal swab of one Egyptian cobra (Naja haje legionis) and molecularly identified in its cloaca and blood. The isolation of C. auris from wild animals is herein reported for the first time, thus suggesting the role that these animals could play as reservoirs of this emerging pathogen. The occurrence of C. auris in blood requires further investigation, although the presence of cationic antimicrobial peptides in the plasma of reptiles could play a role in reducing the vitality of the fungus.

Candida auris represents one of the most urgent threats to public health. In this study, we reported for the first time the isolation of C. auris from snake thus suggesting the role of these animals as reservoirs of this emerging pathogen.

Virulence factors of Malassezia strains isolated from pityriasis versicolor patients and healthy individuals.

Over the last decade, Malassezia species have emerged as increasingly important pathogens associated with a wide range of dermatological disorders and bloodstream infections. The pathogenesis of Malassezia yeasts is not completely clear, but it seems to be strictly related to Malassezia strains and hosts and needs to be better investigated. This study aimed to assess the enzymatic activities, biofilm formation and in vitro antifungal profiles of Malassezia spp. from pityriasis versicolor (PV) and healthy patients. The potential relationship between virulence attributes, the antifungal profiles and the origin of strains was also assessed. A total of 44 Malassezia strains isolated from patients with (n = 31) and without (n = 13) PV were employed to evaluate phospholipase (Pz), lipase (Lz), and hemolytic (Hz) activities and biofilm formation. In addition, in vitro antifungal susceptibility testing was conducted using the CLSI broth microdilution with some modifications. A high percentage of strains produced Pz, Lz, Hz and biofilm regardless of their clinical origin. The highest number of strains producing high enzymatic activities came from PV patients. A correlation between the intensity of hydrolytic activities (Lz and Pz activities) and the Hz activity was detected. Positive associations between Lz and the low fluconazole susceptibility and Hz and biofilm formation were observed. These results suggest that enzyme patterns and biofilm formation along with antifungal profiles inter-play a role in the pathogenicity of Malassezia spp. and might explain the implication of some Malassezia spp. in invasive fungal infections and in the development of inflammation. LAY SUMMARY: There is still little information on the virulence factors of Malassezia spp., despite their implication in severe diseases. Phospholipase, lipase, and hemolytic activities, biofilm formation and decreased antifungal susceptibility seem to contribute to their virulence in susceptible hosts.

Comparison of virulence factors and susceptibility profiles of Malassezia furfur from pityriasis versicolor patients and bloodstream infections of preterm infants.

In spite of the increasing medical interest in Malassezia yeasts, the virulence factors of Malassezia furfur causing bloodstream infections (BSI) were never investigated. Therefore, phospholipase (Pz), lipase (Lz), hemolysin (Hz), biofilm production, and in vitro antifungal susceptibility profiles were evaluated in M. furfur strains, isolated from both pityriasis versicolor (PV) patients (n = 18; Group 1) or from preterm infants BSI (n = 21; Group 2). All the test stains exhibited Pz activity, whereas 92.3% and 97.4% of strains exhibited Lz and Hz activities, respectively. Pz, Lz, and Hz activities were higher (i.e., lower values) within Group 1 strains (i.e., 0.48, 0.40, and 0.77) than those within Group 2 (i.e., 0.54, 0.54, and 0.81). The biofilm production was higher within Malassezia isolates from Group 2 (0.95 ± 0.3) than from Group 1 (0.72 ± 0.4). Itraconazole and posaconazole were the most active drugs against M. furfur, followed by amphotericin B and fluconazole. The minimum inhibitory concentrations (MIC) values varied according to the origin of M. furfur strains being statistically lower in M. furfur from Group 1 than from Group 2. This study suggests that M. furfur strains produce hydrolytic enzymes and biofilm when causing PV and BSI. Data show that the phospholipase activity, biofilm production, and a reduced antifungal susceptibility profile might favor M. furfur BSI, whereas lipase and hemolytic activities might display a synergic role in skin infection.

There is no information on the virulence factors of M. furfur involved in invasive infections. Our data suggest that the phospholipase activity, biofilm production, and a reduced antifungal susceptibility profile might favor M. furfur blood-stream infections.

Wild Boar (Sus scrofa) as Reservoir of Zoonotic Yeasts: Bioindicator of Environmental Quality.

Wildlife animals are recognized as reservoirs for zoonotic fungi and their faeces might play an important role in introducing pathogens into the environment. Thought wild boar (Sus scrofa) population has dramatically increased across Europe, information about their possible role in dissemination of zoonotic pathogenic yeasts in the environment is scant. Therefore, fecal samples (n = 124) from wild boars from Campania region (Southern Italy) were collected and yeasts identified biochemically and molecularly by sequencing of the internal transcribed spacer region and their phylogenetical relationship assessed. The antifungal susceptibility profiles of yeasts were also investigated using AFST-EUCAST method. Yeasts were isolated from 50.1% of the samples with the highest occurrence in samples from the province of Salerno (61.1%). A total of 368 Candida strains belonging to nine species were identified, with Candida albicans (45.7%), followed by Candida krusei (15.2%), Kazachstania slooffiae (9.8%) and Candida parapsilosis (7.6%) as the most prevalent identified species. Among C. albicans four sequence types (i.e., ST1-ST4) were identified with an intraspecific nucleotide difference up to 0.21%. The ML tree grouped all representative sequence types as paraphyletic clades with those of the references yeast species, respectively and supported by high bootstrap values. Fluconazole was the less active drug whereas, posaconazole, voriconazole, and isavuconazole the most active one. No resistance phenomena were observed for C. albicans and high MICs values for 5FC, azoles and echinocandines were registered in non-albicans Candida spp. This study showed, for the first time, the important role of wild boars in dissemination of pathogenic fungi in the environment. The absence of resistance phenomena in the Candida spp. might reflect environmental free from residues of azoles antifungals pollution or chemicals and suggests the role of wild boar as bio indicators of environment quality.

Virulence and in vitro antifungal susceptibility of Candida albicans and Candida catenulata from laying hens.

In spite of evidence that domestic and wild birds may act as carriers of human pathogenic fungi, data on the role of laying hens as reservoirs of drug resistant and virulent yeasts is lacking. Here, we assess several virulence factors (phospholipase and haemolysin activity) and the antifungal susceptibility profiles of 84 Candida albicans and 17 Candida catenulata strains isolated from cloacae (group A), faeces (group B) and eggs (group C) of laying hens. Of these strains, 95% C. albicans and 23% C. catenulata strains displayed phospholipase and haemolytic activities. For C. albicans, the highest values of phospholipase (Pz = 0.62) and haemolytic activities (Hz = 0.49) were recorded among the strains from group C whilst for C. catenulata (Pz = 0.54; Hz = 0.49) among those from group A. High minimum inhibitory concentration (MIC) values for azoles and amphotericin B (AmB) were recorded irrespective of their sources in all C. albicans strains. A total of 22 C. albicans strains were multidrug resistant, displaying resistance to fluconazole, itraconazole (ITZ), voriconazole (VOR) and posaconazole (POS). All C. catenulata strains from group C were resistant to ITZ, POS, micafungin and anidulafungin and susceptible to AmB. In this study, C. albicans and C. catenulata isolated from the cloacae, faeces and eggs of laying hens produced phospholipase and haemolysin and might be multidrug resistant. In the environment (faeces) or in eggs, C. albicans and C. catenulata strains might acquire pathogenic virulence traits and/or show multidrug resistance profiles. Based on these results, breeding and handling of laying hens and/or eggs may have implications for human and animal health.

Conventional therapy and new antifungal drugs against Malassezia infections.

Malassezia yeasts are commensal microorganisms occurring on the skin of humans and animals causing dermatological disorders or systemic infections in severely immunocompromised hosts. Despite attempts to control such yeast infections with topical and systemic antifungals, recurrence of clinical signs of skin infections as well as treatment failure in preventing or treating Malassezia furfur fungemia have been reported most likely due to wrong management of these infections (e.g., due to early termination of treatment) or due to the occurrence of resistant phenomena. Standardized methods for in vitro antifungal susceptibility tests of these yeasts are still lacking, thus resulting in variable susceptibility profiles to azoles among Malassezia spp. and a lack of clinical breakpoints. The inherent limitations to the current pharmacological treatments for Malassezia infections both in humans and animals, stimulated the interest of the scientific community to discover new, effective antifungal drugs or substances to treat these infections. In this review, data about the in vivo and in vitro antifungal activity of the most commonly employed drugs (i.e., azoles, polyenes, allylamines, and echinocandins) against Malassezia yeasts, with a focus on human bloodstream infections, are summarized and their clinical implications are discussed. In addition, the usefulness of alternative compounds is discussed.

Effect of chlorogenic and gallic acids combined with azoles on antifungal susceptibility and virulence of multidrug-resistant Candida spp. and Malassezia furfur isolates.

Chlorogenic acid (CHA) and gallic acid (GA) are safe natural phenolic compounds that are used as enhancers of some drugs in influencing antioxidant, anticancer, and antibacterial activities. Among fungi, Candida spp. and Malassezia spp. are characterized by an increasing prevalence of multidrug resistance phenomena and by a high morbidity and mortality of their infections. No data are available about the efficacy of CHA and GA combined with azoles on the antifungal susceptibility and on the virulence of both fungi. Therefore, their antifungal and antivirulence effects have been tested in combination with fluconazole (FLZ) or ketoconazole (KTZ) on 23 Candida spp. and 8 M. furfur isolates. Broth microdilution chequerboard, time-kill studies, and extracellular enzymes (phospholipase and hemolytic) activities were evaluated, displaying a synergistic antifungal action between CHA or GA and FLZ or KTZ on C. albicans, C. bovina, and C. parapsilosis, and antagonistic antifungal effects on M. furfur and Pichia kudriavzevii (Candida krusei) isolates. The time-kill studies confirmed the chequerboard findings, showing fungicidal inhibitory effect only when the GA was combined with azoles on Candida strains. However, the combination of phenolics with azoles had no effect on the virulence of the tested isolates. Our study indicates that the combination between natural products and conventional drugs could be an efficient strategy for combating azole resistance and for controlling fungistatic effects of azole drugs.

The best type of inoculum for testing the antifungal drug susceptibility of Microsporum canis: In vivo and in vitro results.

BACKGROUND: Data correlating in vitro drug susceptibility of Microsporum canis with clinical outcomes of its infections are lacking as well as the most suitable inoculum and incubation time in broth microdilution assays. OBJECTIVES AND METHODS: Microsporum canis strains were collected from animal hosts that tested positive (Group I; n = 13) and negative (Group II; n = 14) to this pathogen following itraconazole (ITC) therapy. In vitro ITC susceptibility was assessed according to the Clinical Laboratory Standards Institute (CLSI M38-A2) methodology using conidia, hypha-conidia and arthroconidia at 3 and 7 days of incubation in order to assess the most suitable inoculum and incubation time. Successively, ketoconazole (KTC), voriconazole (VRC), terbinafine (TRB), posaconazole (PSZ), fluconazole (FLC) and griseofulvin (GRI) susceptibilities were assessed using the chosen inoculum. RESULTS: The MIC values of ITC after three-day incubation were equal than those recorded after 7-day incubation. Itraconazole MICs were ≤1 µg/mL for strains from Group II and >1 µg/mL for those of Group II only when conidia were used. All strains showed high susceptibility to VRC, POS, TEB and low susceptibility to ITC, KTC, GRI and FLC regardless of the source and incubation time. CONCLUSIONS AND CLINICAL IMPORTANCE: Results suggest that correlation between the in vitro results and clinical outcome was observed only by incubating conidia for 3 days at 30 ± 2°C. These conditions might be most suitable to assess in vitro susceptibility of M. canis and assist in determining the occurrence of drug resistance and cross-resistance phenomena.

Fusarium spp. in Loggerhead Sea Turtles (Caretta caretta): From Colonization to Infection.

With the aim of evaluating the presence of Fusarium spp. in sea turtles with and without lesions and assessing the risk factors favoring colonization and/or infection, 74 loggerhead sea turtles (Caretta caretta) admitted to rescue and rehabilitation clinics in Italy were analyzed. The study compared 31 individuals with no apparent macroscopic lesions and 43 individuals with macroscopic lesions. Shell and skin samples were analyzed using Calcofluor white with 10% potassium hydroxide, standard histopathological examination, and fungal cultures. Fusarium spp. were isolated more frequently from animals with superficial lesions (39%) than from those with no macroscopic lesions (16%). Isolates from animals with superficial lesions were Fusarium solani species complex (FSSC) lineages haplotypes 9, 12, and 27 (unnamed lineages), FSSC-2 (Fusarium keratoplasticum), Fusarium oxysporum (27%), and Fusarium brachygibbosum (3%). In contrast, only F. solani haplotypes 9 and 12 were isolated from animals with no macroscopic lesions. The presence of lesions was identified as a risk factor for the occurrence of Fusarium spp. Of the 74 animals, only 7 (9.5%) scored positive on microscopic examination with Calcofluor, and histological examination of those 7 animals revealed necrosis, inflammatory cells, and fungal hyphae in the carapace and skin. The results of this study suggest that fusariosis should be included in the differential diagnosis of shell and skin lesions in sea turtles. Direct examination using Calcofluor and potassium hydroxide was not useful to diagnose the infection. Histopathological examination and fungal culture should be performed to ensure correct treatment and infection control.

Comparative evaluation of E-test and CLSI methods for Itraconazole, Fluconazole and Ketoconazole susceptibilities of Microsporum canis strains.

The incidence of resistance to antifungal agents for dermatophytes is increasing, but most of the methods currently available to test the antifungal susceptibility of Microsporum canis still require standardization. The aims of this study were: (i) to evaluate the antifungal susceptibility of M. canis strains recovered from animals to ketoconazole (KTZ), fluconazole (FLZ) and itraconazole (ITZ) using a modified CLSI broth microdilution (CLSI M38-A2-BMD) and the E-test® protocols and (ii) to estimate the agreement between the methods. Tentative azole epidemiological cutoff values (ECVs) were also proposed in order to interpret the results of in vitro susceptibility tests and to establish the agreement between the E-test and CLSI BMD methods. A total of forty clinical M. canis strains from animals with skin lesions were tested, and the essential (EA) and categorical agreement (CA) between the two methods were determined. KTZ displayed the lowest MIC values, while ITZ and FLZ the highest. The ECV for KTZ and ITZ were 4 µg/ml, while those of FLZ was 64 µg/ml. Based on ECVs, about 88% of M. canis strains were susceptible to all azoles being a cross-resistance with ITZ-FLZ registered for one strain. A total of five M. canis strains showed MIC > ECV for FLZ using CLSI, while one strain showed MIC > ECV for ITZ using both tests. KTZ, ITZ and FLZ showed EA ranging from 92.5 to 95%, for all azoles and CA > 97% except for FLZ (87.5%). The good CA between the E-test and the CLSI BMD provides evidence of the reliability of the former method to test the antifungal susceptibility of M. canis for ITZ and KTZ and not for FLZ.

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.

Yeasts isolated from cloacal swabs, feces, and eggs of laying hens.

Domestic and wild birds may act as carriers of human pathogenic fungi, although the role of laying hens in spreading yeasts has never been investigated. We evaluated the presence of yeasts in the cloaca (Group I, n = 364), feces (Group II, n = 96), and eggs (Group III, n = 270) of laying hens. The occurrence and the population size of yeasts on the eggshell, as well as in the yolks and albumens, were assessed at the oviposition time and during storage of eggs at 22 ± 1°C and 4 ± 1°C. A statistically higher prevalence and population size of yeasts were recorded in Group I (49.7% and 1.3 × 104 cfu/ml) and II (63.8% and 2.8 × 105 cfu/ml) than in Group III (20.7% and 19.9 cfu/ml). Candida catenulata and Candida albicans were the most frequent species isolated. Candida famata and Trichosporon asteroides were isolated only from the eggshells, whereas Candida catenulata was also isolated from yolks and albumens. During storage, the yeast population size on the shell decreased (from 37.5 to 8.5 cfu/ml) in eggs at 22 ± 1°C and increased (from 4.6 to 35.3 cfu/ml) at 4 ± 1°C. The laying hens harbor potentially pathogenic yeasts in their gastrointestinal tract and are prone to disseminating them in the environment through the feces and eggs. Eggshell contamination might occur during the passage through the cloaca or following deposition whereas yolk and albumen contamination might depend on yeast density on eggshell.

Malassezia ecology, pathophysiology, and treatment.

Malassezia are lipid dependent basidiomycetous yeasts that inhabit the skin and mucosa of humans and other warm-blooded animals, and are a major component of the skin microbiome. They occur as skin commensals, but are also associated with various skin disorders and bloodstream infections. The genus currently comprises 17 species and has recently been assigned its own class, Malasseziomycetes. Importantly, multiple Malassezia species and/or genotypes may cause unique or similar pathologies and vary in their antifungal susceptibility. In addition to culture-based approaches, culture-independent methods have added to our understanding of Malassezia presence and abundance and their relationship to pathogenicity. Moreover, these novel approaches have suggested a much wider-spread presence, including other human body parts and even other ecosystems, but their role in these arenas requires further clarification. With recent successful transformation and genetic engineering of Malassezia, the role of specific genes in pathogenesis can now be studied. We suggest that characterizing the metabolic impact of Malassezia communities rather than species identification is key in elucidation of pathophysiological associations. Finally, the increasing availability of genome sequences may provide key information aiding faster diagnostics, and understanding of the biochemical mechanisms for Malassezia skin adaptation and the design of future drugs.

Blood culture procedures and diagnosis of Malassezia furfur bloodstream infections: Strength and weakness.

The occurrence of Malassezia spp. bloodstream infections (BSIs) in neonatal intensive care unit was evaluated by using pediatric Isolator, BacT/Alert systems and central venous catheter (CVC) culture. The efficacy of BacT/Alert system in detecting Malassezia was assessed by conventional procedures, culturing 1 ml of bottle content before incubation and by studying the survival of Malassezia spp. strains in BacT/Alert bottles. Of the 492 neonates enrolled, blood was collected by pediatric Isolator (290 patients; group I) or by BacT/Alert bottles (202 patients; group II). The survival of Malassezia furfur and Malassezia pachydermatis in BacT/Alert bottles was evaluated by culturing the inoculum suspension (from 106 to 10 colony-forming units, cfu/ml) and assessing the cfu/ml for 15 days. In total, 15 Malassezia BSIs were detected, of which six (2.1%) from both blood and CVC culture in Dixon agar (DixA) in patients belong to group I (blood collected by paediatric Isolator tube) and nine (4.4%) only from CVC culture in DixA in patients of group II (blood collected by BacT/Alert bottle). Only one patient (0.5%) from group II scored positive for M. furfur also by culturing in DixA 1 ml blood content of BacT/Alert bottle before incubation in BacT/Alert system.M. furfur population size in BacT/Alert bottles decreased during the incubation time, whereas that of M. pachydermatis increased. The BacT/Alert system detected M. pachydermatis even at very low concentration (i.e., 10 cfu/ml) but not any positive blood culture for M. furfur. For a correct diagnosis of Malassezia furfur BSI, the blood should be culture in lipid-enriched fungal medium, and the BacT/Alert system implemented by adding lipid substrates to increase the method sensibility. Finally, CVC cultures on lipid-supplemented media may be proposed as a routine procedure to diagnose the Malassezia fungemia.

Transcriptome of larvae representing the Rhipicephalus sanguineus complex.

Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) is possibly the most widespread tick species worldwide, responsible for transmitting several vector-borne pathogens of medical and veterinary importance. Here, we explore the transcriptome of R. sanguineus s.l. larvae (Putignano strain). We sequenced total RNA from R. sanguineus s.l. larvae. A total of 15,566,986 short paired-end reads were de novo-assembled into 33,396 transcripts and then annotated and analyzed. Particular attention was paid to transcripts putatively encoding ATP-binding proteins, due to their importance as mechanisms of detoxification and acaricide resistance. Additionally, microsatellite loci were investigated, as these are useful markers for population genetic studies. The present data and analyses provide a comprehensive transcriptomic resource for R. sanguineus. The results presented here will aid further genetic and genomic studies of this important tick species.

The role of drug efflux pumps in Malassezia pachydermatis and Malassezia furfur defence against azoles.

This study aims to evaluate the effect of efflux pump modulators (EPMs) on the minimal inhibitory concentration (MIC) of fluconazole (FLZ) and voriconazole (VOR) in Malassezia furfur and Malassezia pachydermatis. The in vitro efficacy of azoles, in combination with EPMs (ie haloperidol-HAL, promethazine-PTZ and cyclosporine A-CYS), against 21 M. furfur from bloodstream infection patients and 14 M. pachydermatis from the skin of dogs with dermatitis, was assessed using a broth microdilution chequerboard analysis. Data were analysed using the model-fractional inhibitory concentration index (FICI) method. The MIC of FLZ and VOR of Malassezia spp. decreased in the presence of sub-inhibitory concentrations of HAL and/or PTZ. The synergic effect was observed only in strains with FLZ MIC≥128 µg/mL for M. furfur, FLZ MIC≥64 µg/mL for M. pachydermatis and VOR MIC≥4 µg/mL in both Malassezia spp. These results suggest that the drug efflux pumps are involved as defence mechanisms to azole drugs in Malassezia yeast. The synergism might be related to an increased expression of efflux pump genes, eventually resulting in azole resistance phenomena. Finally, the above FLZ and VOR MIC values might be considered the cut-off to discriminate susceptible and resistant strains.

Chemical Composition, Antibacterial and Antifungal Activities of Crude Dittrichia viscosa (L.) Greuter Leaf Extracts.

The small amount of data regarding the antifungal activity of Dittrichia viscosa (L.) Greuter against dermatophytes, Malassezia spp. and Aspergillus spp., associated with the few comparative studies on the antimicrobial activity of methanolic, ethanolic, and butanolic extracts underpins the study herein presented. The total condensed tannin (TCT), phenol (TPC), flavonoid (TFC), and caffeoylquinic acid (CQC) content of methanol, butanol, and ethanol (80% and 100%) extracts of D. viscosa were assessed and their bactericidal and fungicidal activities were evaluated. The antibacterial, anti-Candida and anti-Malassezia activities were evaluated by using the disk diffusion method, whereas the anti-Microsporum canis and anti-Aspergillus fumigatus activities were assessed by studying the toxicity effect of the extracts on vegetative growth, sporulation and germination. The methanolic extract contained the highest TPC and CQC content. It contains several phytochemicals mainly caffeoylquinic acid derivatives as determined by liquid chromatography with photodiode array and electrospray ionisation mass spectrometric detection (LC/PDA/ESI-MS) analysis. All extracts showed an excellent inhibitory effect against bacteria and Candida spp., whereas methanolic extract exhibited the highest antifungal activities against Malassezia spp., M. canis and A. fumigatus strains. The results clearly showed that all extracts, in particular the methanolic extract, might be excellent antimicrobial drugs for treating infections that are life threatening (i.e., Malassezia) or infections that require mandatory treatments (i.e., M. canis or A. fumigatus).

Species Distribution and In Vitro Azole Susceptibility of Aspergillus Section Nigri Isolates from Clinical and Environmental Settings.

Aspergillus section Nigri includes species of interest for animal and human health, although studies on species distribution are limited to human cases. Data on the antifungal susceptibilities and the molecular mechanism of triazole resistance in strains belonging to this section are scant. Forty-two black Aspergillus strains from human patients (16 isolates), animals (14 isolates), and the environment (12 isolates) were molecularly characterized and their in vitro triazole susceptibilities investigated. Aspergillus tubingensis was isolated from humans, animals, and environmental settings, whereas Aspergillus awamori and Aspergillus niger were isolated exclusively from humans. Phylogenetic analyses of ß-tubulin and calmodulin gene sequences were concordant in differentiating A. tubingensis from A. awamori and A. niger Voriconazole and posaconazole (PSZ) were the most active triazoles. One A. tubingensis strain was resistant to itraconazole and PSZ and one A. niger strain to PSZ. Sequence analysis of the cyp51A gene revealed different sequence types within a species, and A. tubingensis strains were also phylogenetically distinct from A. awamori/A. niger strains according to the strain origin and susceptibility profile. Genetic analysis of the cyp51A sequences suggests that two nonsynonymous mutations resulting in amino acid substitutions in the CYP51A protein (changes of L to R at position 21 [L21R] and of Q to R at position 228 [Q228R]) might be involved in azole resistance. Though azole resistance in black Aspergillus isolates from animals and rural environments does not represent a threat to public health in Southern Italy, the use of triazoles in the clinical setting needs to better monitored. The cyp51A sequence is useful for the molecular identification of black Aspergillus, and point mutations in protein sequences could be responsible for azole resistance phenomena.

Azole susceptibility of Malassezia pachydermatis and Malassezia furfur and tentative epidemiological cut-off values.

This study aims to determine the minimal inhibitory concentration (MIC) distribution and the epidemiological cut-off values (ECVs) of Malassezia pachydermatis and Malassezia furfur isolates for fluconazole (FLZ), itraconazole (ITZ), posaconazole (POS), and voriconazole (VOR). A total of 62 M. pachydermatis strains from dogs with dermatitis and 78 M. furfur strains from humans with bloodstream infections (BSI) were tested by a modified broth microdilution Clinical and Laboratory Standards Institute (CLSI) method. ITZ and POS displayed lower MICs than VOR and FLZ, regardless of the Malassezia species. The MIC data for azoles of M. pachydermatis were four two-fold dilutions lower than those of M. furfur. Based on the ECVs, about 94% of Malassezia strains might be categorized within susceptible population for all azoles, except for FLZ, and azole cross-resistance was detected in association with FLZ in M. pachydermatis but not in M. furfur.The study proposes, for the first time, tentative azole ECVs for M. pachydermatis and M. furfur for monitoring the emergence of isolates with decreased susceptibilities and shows that the azole MIC distribution varied according to the Malassezia species tested, thus suggesting the usefulness of determining the susceptibility profile for effective treatment of each species.

In vitro activity of two amphotericin B formulations against Malassezia furfur strains recovered from patients with bloodstream infections.

Although guidelines for the treatment of Malassezia furfur fungemia are not yet defined, clinical data suggest that amphotericin B (AmB) is effective for treating systemic infections. In the absence of clinical breakpoints for Malassezia yeasts, epidemiological cut-off values (ECVs) are useful to discriminate between isolates with and without drug resistance. This study aimed to compare the distribution of minimal inhibitory concentration (MIC) and the ECVs for AmB of both deoxycholate (d-AmB) and liposomal (l-AmB) formulations of M. furfur isolates. The 84 M. furfur strains analyzed, which included 56 from blood, sterile sites and catheters, and 28 from skin, were isolated from patients with bloodstream infections. MICs were determined by the modified broth microdilution method of the Clinical and Laboratory Standards Institute (CLSI). The l-AmB MIC and the ECVs were two-fold lower than those of d-AmB and a lower l-AmB mean MIC value was found for blood isolates than from skin. The ECVs for l-AmB and d-AmB were 8 mg/l and 32 mg/l, respectively. Three strains (3.6%) showed l-AmB MIC higher than ECV (MIC > 8 mg/l) of which two were isolated from the catheter tip of patients treated with micafugin, l-Amb and fluconazole, and one from skin. The results showed that the l-AmB might be employed for assessing the in vitro antifungal susceptibility of M. furfur by a modified CLSI protocol and that ECVs might be useful for detecting the emergence of resistance.