Patrícia Campi Santos

In vitro antifungal susceptibility testing of filamentous fungi with Sensititre Yeast OneTM

Sensititre® is a colorimetric microdilution method for in vitro antifungal susceptibility testing based on the M27‐A document (National Committee for Clinical Laboratory Standards) for yeasts. Difference between both methods is the presence of Alamar‐blue® and RPMI 1640 (glucose 2%) as culture medium. Antifungal susceptibility to amphotericin B, fluconazole, itraconazole, ketoconazole and flucytosine, 100 opportunistic filamentous fungi (Aspergillus spp., Fusarium spp., Scedosporium spp.) obtained from pathological samples was determined by the Sensititre method. Induction to conidium and sporangiospore formation at 35°C was used to get inoculum and plates were covered by 1 ml of saline and suspensions were made by gently probing by a sterile loop. Optical densities of the conidial suspensions were adjusted to 80–82% transmittance for Aspergillus spp. and 68–70% for the rest of strains tested. Final inoculum concentration size was 0.4 × 104–5 × 104 CFU ml−1. Readings were made at 72 h of incubation at 35°C; amphotericin B and itraconazole was active against Aspergillus fumigatus with CMI90 1 and 0.5 μg ml−1, respectively, opposite to Scedosporium prolificans and Scedosporium apiospermum. As it was expected, a CMI90 of 256 μg ml−1 for fluconazole and CMI90 for flucytosine amounting to 64 g ml−1 were obtained. Sensititre Yeast OneTM is a useful method and an alternative to reference methods to determine antifungal susceptibility of filamentous fungi for clinical laboratory routine. Correlation with microdilution results is studied. New triazole derivatives should be included as soon as their clinical use will be feasible.

MIF induces osteoclast differentiation and contributes to progression of periodontal disease in mice

Periodontal disease (PD) is a chronic inflammatory and alveolar bone destructive disease triggered by microorganisms from the oral biofilm. Oral inoculation of mice with the periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) induces marked alveolar bone loss and local production of inflammatory mediators, including Macrophage Migration Inhibitory Factor (MIF). The role of MIF for alveolar bone resorption during PD is not known. In the present study, experimental PD was induced in BALB/c wild-type mice (WT) and MIF knockout mice (MIF⁻/⁻) through oral inoculation of Aa. Despite enhanced number of bacteria, MIF⁻/⁻ mice had reduced infiltration of TRAP-positive cells and reduced alveolar bone loss. This was associated with decreased neutrophil accumulation and increased levels of IL-10 in periodontal tissues. TNF-α production was similar in both groups. In vitro, LPS from Aa enhanced osteoclastic activity in a MIF-dependent manner. In conclusion, MIF has role in controlling bacterial growth in the context of PD but contributes more significantly to the progression of bone loss during PD by directly affecting differentiation and activity of osteoclasts.

Fluconazole Alters the Polysaccharide Capsule of Cryptococcus gattii and Leads to Distinct Behaviors in Murine Cryptococcosis

Cryptococcus gattii is an emergent human pathogen. Fluconazole is commonly used for treatment of cryptococcosis, but the emergence of less susceptible strains to this azole is a global problem and also the data regarding fluconazole-resistant cryptococcosis are scarce. We evaluate the influence of fluconazole on murine cryptococcosis and whether this azole alters the polysaccharide (PS) from cryptococcal cells. L27/01 strain of C. gattii was cultivated in high fluconazole concentrations and developed decreased drug susceptibility. This phenotype was named L27/01F, that was less virulent than L27/01 in mice. The physical, structural and electrophoretic properties of the PS capsule of L27/01F were altered by fluconazole. L27/01F presented lower antiphagocytic properties and reduced survival inside macrophages. The L27/01F did not affect the central nervous system, while the effect in brain caused by L27/01 strain began after only 12 hours. Mice infected with L27/01F presented lower production of the pro-inflammatory cytokines, with increased cellular recruitment in the lungs and severe pulmonary disease. The behavioral alterations were affected by L27/01, but no effects were detected after infection with L27/01F. Our results suggest that stress to fluconazole alters the capsule of C. gattii and influences the clinical manifestations of cryptococcosis.

Identification and characterization of Tc1/mariner-like DNA transposons in genomes of the pathogenic fungi of the Paracoccidioides species complex

BackgroundParacoccidioides brasiliensis (Eukaryota, Fungi, Ascomycota) is a thermodimorphic fungus, the etiological agent of paracoccidioidomycosis, the most important systemic mycoses in Latin America. Three isolates corresponding to distinct phylogenetic lineages of the Paracoccidioides species complex had their genomes sequenced. In this study the identification and characterization of class II transposable elements in the genomes of these fungi was carried out.ResultsA genomic survey for DNA transposons in the sequence assemblies of Paracoccidioides, a genus recently proposed to encompass species P. brasiliensis (harboring phylogenetic lineages S1, PS2, PS3) and P. lutzii (Pb01-like isolates), has been completed. Eight new Tc1/mariner families, referred to as Trem (Tr ansposable e lement m ariner), labeled A through H were identified. Elements from each family have 65-80% sequence similarity with other Tc1/mariner elements. They are flanked by 2-bp TA target site duplications and different termini. Encoded DDD-transposases, some of which have complete ORFs, indicated that they could be functionally active. The distribution of Trem elements varied between the genomic sequences characterized as belonging to P. brasiliensis (S1 and PS2) and P. lutzii. TremC and H elements would have been present in a hypothetical ancestor common to P. brasiliensis and P. lutzii, while TremA, B and F elements were either acquired by P. brasiliensis or lost by P. lutzii after speciation. Although TremD and TremE share about 70% similarity, they are specific to P. brasiliensis and P. lutzii, respectively. This suggests that these elements could either have been present in a hypothetical common ancestor and have evolved divergently after the split between P. brasiliensis and P. Lutzii, or have been independently acquired by horizontal transfer.ConclusionsNew families of Tc1/mariner DNA transposons in the genomic assemblies of the Paracoccidioides species complex are described. Families were distinguished based on significant BLAST identities between transposases and/or TIRs. The expansion of Trem in a putative ancestor common to the species P. brasiliensis and P. lutzii would have given origin to TremC and TremH, while other elements could have been acquired or lost after speciation had occurred. The results may contribute to our understanding of the organization and architecture of genomes in the genus Paracoccidioides.

Influence of Different Media, Incubation Times, and Temperatures for Determining the MICs of Seven Antifungal Agents against Paracoccidioides brasiliensis by Microdilution

ABSTRACT MIC assays with Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, had been conducted with variable protocols, employing both macrodilution and microdilution tests and including differences in inoculum preparation, media used, incubation periods, and temperatures. Twenty-one clinical and environmental isolates of Paracoccidioides were tested using amphotericin B, itraconazole, ketoconazole, fluconazole, sulfamethoxazole, sulfamethoxazole-trimethoprim, and terbinafine, according to the National Committee for Clinical Laboratory Standards (National Committee for Clinical Laboratory Standards, document M27-A2, 2002), with modifications such as three medium formulations (RPMI 1640 medium, McVeigh and Morton [MVM] medium, and modified Mueller-Hinton [MMH] medium), two incubation temperatures (room temperature [25 to 28°C] and 37°C), and three incubation periods (7, 10, and 15 days). The antifungal activities were also classified as fungicidal or fungistatic. The best results were obtained after 15 days of incubation, which was chosen as the standard incubation time. The MICs for most individual isolates grown for the same length of time at the same temperature varied with the different media used (P < 0.05). Of the isolates, 81% showed transition from the yeast to the mycelial form in RPMI 1640 medium at 37°C, independent of the presence of antifungals. MMH medium appears to be a suitable medium for susceptibility testing of antifungal drugs with P. brasiliensis, except for sulfamethoxazole and the combination of sulfamethoxazole-trimethoprim, for which the MVM medium yielded better results. The incubation temperature influenced the MICs, with, in general, higher MICs at 25°C (mycelial form) than at 37°C (P < 0.05). Based on our results, we tentatively propose a microdilution assay protocol for susceptibility testing of antifungal drugs against Paracoccidioides.

Ciclopiroxolamine: in vitro antifungal activity against clinical yeast isolates

The in vitro susceptibility of 225 clinical isolates of yeasts to ciclopiroxolamine (CPO) was compared with that of clotrimazole, econazole, ketoconazole, miconazole, tioconazole, fluconazole, itraconazole and nystatin using a standardized agar diffusion method (NeoSensitabs). Two hundred and eight strains of yeasts comprising 16 species of Candida and 22 strains belonging to other yeast genera were tested. One strain (0.4%) was resistant, four strains (1.8%) of intermediate susceptibility and 220 strains (97.3%) susceptible to CPO. More strains were susceptible to CPO than to the other antifungals studied. Susceptibility patterns of antifungal agents were not linked to species. The in vitro antifungal susceptibility profile of CPO was better than topical azole derivatives or fluconazole and itraconazole against a wide variety of clinically important yeasts.

In Vitro Antifungal Activity of Sertaconazole Nitrate Against Recent Isolates of Onychomycosis Causative Agents

Topical antifungal treatment of superficial mycoses can have high relapse rates and recurrence after stopping treatment 1. Onychomycosis is the most common nail disorder in adults, accounting for up to 50% of all nail diseases, specially in adults and the elderly 2-6. Candida infections account for 5-10% (Candida albicans and Candida parapsilosis) of all cases of onychomycosis and about 5% of cases are due to non-dermatophyte molds. The most commonly isolated dermatophyte is Trichophyton rubrum, with Scopulariopsis brevicaulis being the most common non-dermatophyte 2-7. Epidemiological data indicate a high rate of clinical failure, over 25%, due to problems of nail penetration and retention times of active antifungal concentrations. Antifungal treatment compliance is low (<51%) and many patients require oral therapy in cases of previous clinical failure or extended affected areas involving nail matrix. The aim of this study was to assess the in vitro antifungal activity of sertaconazole nitrate against clinical isolates of onychomycosis-causing agents and to compare this activity to those of amorolfine, cyclopiroxolamine, bifonazole, fluconazole and terbinafine. Sertaconazole, an azole derivative, acts by inhibiting ergosterol biosynthesis and damaging cell integrity 8. It has a broad spectrum of activity against yeasts, dermatophytes as well as Gram-positive bacteria 1,2-15. The 100 clinical isolates of yeasts, dermatophytes and opportunistic molds from patients with onychomycosis recruited at some health centers were tested in duplicate with a standardized method of microdilution in RPMI 1640 21-23. The clinical isolates were the following: C. parapsilosis (30), T. rubrum (29), S. brevicaulis (10), C. albicans (5), C. guilliermondii (4), T. mentagrophytes (4), C. famata (3), C. glabrata (3), C. tropicalis (3), C. intermedia (1), C. krusei (3), Cryptococcus laurentii (2), C. lusitaniae (1), Fusarium sp. (1) and Rhodotorula minuta (1). Drug concentrations ranged between 0.016 μg/ml to 16 μg/ml for all antifungal drugs except fluconazole (minimum inhibitory concentration (MIC) range from 0.25 to 256 μg/ml). MIC ranges and geometric means were calculated for each species-drug combination. MIC50 and MIC90 were considered as the concentrations of antifungal agent that were able to inhibit 50% and 90% of the isolates, respectively, and were determined for species including more than 10 isolates. Sertaconazole (STZ) antifungal activity (MIC 0.28 μg/ml) was higher than those obtained for terbinafine (TRB), amorolfine (AMR), cyclopiroxolamine (CPO), bifonazole (BFZ) and fluconazole (FLZ) (Table 1). Antifungal susceptibility patterns of reference strains and quality control strains were in agreement with those previously described 16-18. STZ (MIC 0.31 μg/ml) (Table 1) was the most active antifungal agent against yeast isolates, followed by FLZ, CPO, TRB, AMR and BFZ. The antifungal activity against yeasts was species-dependent for all antifungal drugs tested. STZ was especially active against C. albicans (MIC 0.153 μg/ml), and C. krusei (MIC 0.03 μg/ml). A statistically significant difference (Student’s t test, p<0.001), was found in favor of STZ compared to BFZ, FLZ, AMR and CPO. The rank order of antifungal potency against yeasts was STZ > FLZ ≈ CPO > TRB > AMR > BFZ. A detailed analysis of pathogenic groups revealed a greater antifungal activity of most drugs against dermatophytes, TRB and STZ being the most active. STZ activity was also in the same range as TRB and AMR and was higher than other azole derivatives for T. rubrum (MIC 0.063 μg/ml). The rank order of antifungal activity against dermatophytes was AMR ≈ TRB ≈ STZ > BFZ ≈ CPO > FLZ. Susceptibility patterns of opportunistic filamentous fungi were similar for STZ, BFZ and TRB. STZ (MIC 6.02 μg/ml), CPO (MIC 3.31 μg/ml) and AMR (MIC 0.55 μg/ml) were more active than BFZ and TRB. There was a statistically difference between STZ and TRB and FLZ (p<0.001) in favor of STZ against opportunistic filamentous fungi. The rank order of activity against non-dermatophyte molds was AMR ≈ CPO > STZ > TRB ≈ BFZ > FLZ. Despite many advances in antifungal drug development and therapy, onychomycosis remains difficult REPRINT

The absence of microbiota delays the inflammatory response to Cryptococcus gattii.

The inflammatory response plays a crucial role in infectious diseases, and the intestinal microbiota is linked to maturation of the immune system. However, the association between microbiota and the response against fungal infections has not been elucidated. Our aim was to evaluate the influence of microbiota on Cryptococcus gattii infection. Germ-free (GF), conventional (CV), conventionalized (CVN-mice that received feces from conventional animals), and LPS-stimulated mice were infected with C. gattii. GF mice were more susceptible to infection, showing lower survival, higher fungal burden in the lungs and brain, increased behavioral changes, reduced levels of IFN-γ, IL-1β and IL-17, and lower NFκBp65 phosphorylation compared to CV mice. Low expression of inflammatory cytokines was associated with smaller yeast cells and polysaccharide capsules (the main virulence factor of C. gattii) in the lungs, and less tissue damage. Furthermore, macrophages from GF mice showed reduced ability to engulf, produce ROS, and kill C. gattii. Restoration of microbiota (CVN mice) or LPS administration made GF mice more responsive to infection, which was associated with increased survival and higher levels of inflammatory mediators. This study is the first to demonstrate the influence of microbiota in the host response against C. gattii.

Antifungal activity of 6-quinolinyl N-oxide chalcones against Paracoccidioides

BACKGROUND Chalcones are an important class of natural compounds that have been widely applied as synthons in synthetic organic chemistry and possess diverse and interesting biological properties. METHODS We conducted tests with the synthetic substances 6-quinolinyl N-oxide chalcones 4c and 4e to determine their antifungal activity against several isolates of Paracoccidioides spp. and their activity in a murine model. We also determined whether the chalcones interacted with other drugs or interfered with the morphology of Paracoccidioides brasiliensis (Pb18) yeast cells. RESULTS We verified that the substances were active against Paracoccidioides spp., but we did not show an interaction with the drugs tested when only the fractional inhibitory concentration index values were considered individually. We observed that the substances induced in vitro morphological changes. Compounds 4c and 4e showed activity similar to itraconazole in treated mice, as demonstrated by their ability to reduce the number of cfu recovered from the lungs. Histopathological analysis showed that animals treated with 4c presented fewer areas containing inflammatory infiltrate and larger areas of preserved lung tissue, whereas animals treated with itraconazole showed accumulation of inflammatory infiltrate and some granulomas. Mice treated with 4e exhibited inflammation that compromised the tissue. CONCLUSIONS The results presented in this paper confirm the antifungal potential of the chalcones tested. The chalcone 4c was the more effective at controlling the disease in mice and this compound could be a candidate for future studies of the treatment of paracoccidioidomycosis.

Activity of Caspofungin and Voriconazole against Clinical Isolates of Candida and Other Medically Important Yeasts by the CLSI M-44A Disk Diffusion Method with Neo-Sensitabs Tablets

In vitro activity of caspofungin and vori conazole against 184 clinical isolates of Candida and other medically important yeasts in comparison with that of fluconazole, ketoconazole, itraconazole and amphotericin B was determined by using a disk diffusion method (Neo-Sensitabs) standardized according to the recommendations of the CLSI documents M44-A and M44–S1 (same medium: Mueller-Hinton plus methylene blue; inoculum and minimal inhibitory concentration/zone breakpoints). Seventy-two percent of clinical isolates were susceptible to caspofungin, 23.6% showed an intermediate susceptibility (most of them were Candida parapsilosis) and 4.3% were resistant (values for Candida spp. were 71.2, 23.8 and 5%, respectively). For voriconazole, 96.7% of clinical isolates were susceptible and 3.3% were resistant (Candida spp.: 96 and 3.8%, respectively). Both caspofungin and voriconazole showed high activity against a wide variety of clinically important yeasts.