Multi-locus sequence typing and phylogenetics of Cryptococcus neoformans AD hybrids.

Hybrid AD strains of the human pathogenic Cryptococcus neoformans species complex have been reported from many parts of the world. However, their origin, diversity, and evolution are incompletely understood. In this study, we analyzed 102 AD hybrid strains representing 21 countries on five continents. For each strain, we obtained its mating type and its allelic sequences at each of the seven loci that have been used for genotyping haploid serotypes A and D strains of the species complex by the Cryptococcus research community. Our results showed that most AD hybrids exhibited loss of heterozygosity at one or more of the seven analyzed loci. Phylogenetic and population genetic analyses of the allelic sequences revealed multiple origins of the hybrids within each continent, dating back to one million years ago in Africa and up to the present in other continents. We found evidence for clonal reproduction and long-distance dispersal of these hybrids in nature. Comparisons with the global haploid serotypes A and D strains identified new alleles and new haploid multi-locus genotypes in AD hybrids, consistent with the presence of yet-to-be discovered genetic diversity in haploid populations of this species complex in nature. Together, our results indicate that AD hybrids can be effectively genotyped using the same multi-locus sequencing type approach as that established for serotypes A and D strains. Our comparisons of the AD hybrids among each other as well as with the global haploid serotypes A and D strains revealed novel genetic diversity as well as evidence for multiple origins and dynamic evolution of these hybrids in nature.

Activity of base analogues (5-fluorouracil, 5-flucytosine) against planktonic cells and mature biofilm of Candida yeast. Effect of combination with folinic acid.

BACKGROUND: The increasing number of Candida infections, especially those caused by non-C. albicans species and resistant strains, is a serious medical problem. OBJECTIVES: In this study, the antifungal activity of base analogues, 5-flucytosine (5-FC) and 5-fluorouracil (5-FU), was tested against planktonic cells as well as against mature biofilm. METHODS: Tests were performed according the EUCAST methodology. Antibiofilm effectiveness of tested drugs was determined by the crystal violet staining method. The cytotoxicity assays was performed according to the ISO 10993-5 norm. RESULTS: 5-FC and 5-FU were effective against fifteen fluconazole resistant Candida glabrata strains with an average minimal inhibitory concentration (MIC) of 0.152mg/L and 0.39mg/L, respectively. Folinic acid (folinate- e.g., leucovorin) is a common drug used in oncology simultaneously with 5-FU. In our tests folinate was able to lower MIC for 5-FC from 0.152 to 0.058mg/L (P<0.05). In the biofilm assay 5-FU and 5-FC alone did not induce any changes in the biomass of mature biofilm. Addition of folinate to each base analogue resulted in up to 90% reduction of biomass. Viability tests show that a concentration of 64mg/L of 5-FC and 5-FU supplemented with folinate can be fungicidal against mature biofilms of some Candida isolates. No cytotoxic effect was found for combination of FOL and 5-FC. CONCLUSION: Therapy of 5-FU+folinate is well known in cancer treatment, in this study we reveal the beneficial effect of folinate on antifungal activity of 5-FC as well as the antifungal potential of 5-FU+folinate.

Low frequency of itraconazole resistance found among Aspergillus fumigatus originating from poultry farms in Southwest Poland.

AIM: The aim of this study was to investigate the collection of avian Aspergillus fumigatus isolates for the presence of triazole resistance. MATERIAL AND METHOD: The study was performed on 60 A. fumigatus isolates cultured from lung tissue samples from chicken (25), geese (17), turkeys (13) and ducks (5). The samples were obtained from 40 different farms located in the Southwest Poland and were collected in the period of September 2015 to November 2016. The EUCAST microdilution method, with the use of three concentrations of itraconazole (ITR) (1, 0.5, and 0.25mg/L), was used to screen the susceptibility of all isolates. Additionally, the selected 20 isolates were tested with eleven concentrations ranging 0.015-16mg/L of ITR, voriconazole, posaconazole and isavuconazole. RESULTS: Most tested isolates (59/60) were susceptible to ITR (MIC≤0.5mg/L). One isolate showed elevated MIC for ITR (16mg/L), as well as voriconazole (4mg/L), izavuconazole (4mg/L), and posaconazole (0.5mg/L). This isolate was identified on the basis of DNA analysis as A. fumigatus carrying TR34/L98H mutation. All of the ITR-susceptible isolates under study were also susceptible to other triazoles. CONCLUSION: Obtained results indicated a low frequency (1.6%) of A. fumigatus resistant to triazoles among avian isolates from the Southwest regions of Poland.

The susceptibility of Candida albicans strains to selected anticancer drugs and flucytosine, relevance of the presence of self-splicing intron in 25S rDNA.

BACKGROUND: The presence of intron 25S allows to divide the Candida albicans species into three subclasses (A, B, C). Intronless and intron harboring strains were reported to have different susceptibility to some drugs, for example to flucytosine and bleomycin. OBJECTIVES: In this paper we tested the activity of selected antineoplastic agents, bleomycin, mitomycin C, dactinomycin and fluorouracil as well as antifungal drug flucytosine against 49 C. albicans isolates. Twenty-four strains used in this work contained intron, whereas twenty-five were intronless. METHODS: The minimal inhibitory concentrations were determined by the standard microdilution method according to EUCAST. RESULTS: All of the tested agents showed antifungal activity. Bleomycin was the strongest with an average minimal inhibitory concentration [MIC] of 15.5mg/L (range: 2-32), while the highest MIC was found for dactinomycin: 172.14mg/L (range: 128-256). Intron harboring strains seem to be more susceptible to bleomycin and flucytosine; however, differences were not statistically significant. The only two strains with elevated MICs for flucytosine were intronless. In contrast, the MIC of 5-fluorouracil was more than two times lower in intron harbouring strains comparing to intronless strains (P-value=0.0124). We found that the addition of folinate significantly increased the susceptibility of intronless strains to fluorouracil. MIC of fluorouracil decreased in this group from 58.24 (range: 16-256) to 16,78mg/L (2-64) after the supplementation of folinate. CONCLUSION: The antifungal potential of tested substances, especially the simultaneous action of fluorouracil and folinate (combination used in oncology), is encouraging further research.

Anti-candidal activity of selected analgesic drugs used alone and in combination with fluconazole, itraconazole, voriconazole, posaconazole and isavuconazole.

BACKGROUND: The antifungal activity of some popular analgesic drugs was postulated by several authors. OBJECTIVES: The aim of this study was to verify the antifungal effectiveness of acetylsalicylic acid, ibuprofen, metamizole, meloxicam and paracetamol against triazole-susceptible and triazole-resistant Candida spp isolates. METHODS: The minimal inhibitory concentration as well as fractional inhibitory concentration of selected analgesics and triazole on eighteen Candida spp isolates were determined by the standard microdilution method according to EUCAST. RESULTS: Among five tested compounds, only ibuprofen showed an antifungal effect against all tested isolates. In combination tests of analgesics with triazole, three types of interaction have been observed: indifference, synergism and antagonism. An azole-resistant strain of C. albicans demonstrated synergism with IBU and each of the 5 tested azole. Posaconazole also demonstrated synergism with IBU in two other cases, namely a C. albicans strain and with C. glabrata, which were both azole-resistant. Combination of FLU and IBU in two cases resulted in antagonism: in experiments with C. glabrata and C. tropicalis. This interaction was also observed when metamizole and fluconazole were used in combination on C. krusei. CONCLUSIONS: The results obtained by us confirmed antifungal activity of ibuprofen, what support purposefulness to continue the study on antimicrobial activity of this group of drugs.

Preliminary physiological characteristics of thermotolerant Saccharomyces cerevisiae clinical isolates identified by molecular biology techniques.

UNLABELLED: The aim of the study was a molecular identification and physiological characteristic of the five Saccharomyces cerevisiae strains isolated from patients. The tested isolates were compared with control strains (which are of laboratory or commercial origin). The relation of the isolates to baker's yeast S. cerevisiae was studied using species-specific primers in PCR analysis of the ITS-26S region of DNA. Five isolates were genetically identified as the yeast belonging to the genus S. cerevisiae. The effects of temperature and carbon sources on the growth of the yeast strains were analysed. A quantitative characterization of growth kinetics approve that some tested isolates are thermotolerant and are able to grow at range 37-39°C. Among them, one representative is characterized by the highest specific growth rate (0·637 h(-1) ). In conclusions, some strains are defined as potential candidates to use in the biotechnology due to a higher growth rate at elevated temperatures. Screening for further evaluation of biotechnological significance of the tested isolates will be done (e.g. ethanol and trehalose production at higher temperatures). The physiological characterization and confirmation of species identification by molecular methods for yeasts important in the context of biotechnology industry were demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Thermotolerant microbial strains are required in various industrial applications, for improving productivity and for decreasing the risk of undesirable contaminations when higher temperatures are used. It is important to search for such strains in extreme environments or exotic niches. In this paper, new thermotolerant strains were identified belonging to the Saccharomyces cerevisiae, but differed from typical bakers' yeast, essentially by their growth rate at higher temperature. The described yeast strains are promising for using in biotechnological industry, especially, for production of ethanol and other products at higher temperatures.

Evaluation of blood culture media for the detection of fungi.

The aim of this study was to compare the utility of BACTEC™ Mycosis-IC/F (Mycosis), BACTEC™ Plus Aerobic/F (Aerobic), and BACTEC™ Plus Anaerobic/F (Anaerobic) media in the detection of fungi from simulated (obtained by the inoculation of tested media first with sterile sheep's blood and subsequently with one of 60 clinical yeast isolates) and clinical blood samples, taken during routine diagnostic examination in two hospitals. All tested strains grew on Mycosis as well as Aerobic bottles, and the time to detection obtained for Mycosis was significantly shorter (p < 0.05). The largest differences in the time to positivity was found for Candida glabrata and Cryptococcus neoformans, when Mycosis preceded Aerobic in 20-48 h (mean 35.5 h) and 0.7-64 h (mean 24 h), respectively. On the contrary, C. krusei were detected earlier in Aerobic media. In clinical samples, the detection of C. glabrata was also significantly faster in Mycosis than in Aerobic (29.22 ± 11.48 h compared to 86 ± 40 h). The media complement each other and, in 45% of clinical examination sets, a single positive medium was noted (25% in Mycosis and 19% in Aerobic). The study proved that both Aerobic and Mycosis media serve as the correct condition for the culture of fungi and that they varied significantly in the detection time of clinically important species. This result could suggest that the simultaneous use of Aerobic as well as Mycosis media may improve the time of diagnosis in many patients, especially those infected with C. glabrata or C. neoformans.

Serum concentration of interleukin 10, anti-mannan Candida antibodies and the fungal colonization of the gastrointestinal tract in patients with ulcerative colitis.

PURPOSE: There is evidence for the immunomodulation disorders in the response to intestinal flora in inflammatory bowel disease, however, the role of yeasts in the aetiopathogenesis of ulcerative colitis has not been fully clarified. The aim of this study was to assess the serum concentration of interleukin 10 (IL-10), serum levels of anti-mannan Candida antibodies and fungal colonization of the lower part of the gastrointestinal tract in accordance with the clinical course of ulcerative colitis. MATERIAL/METHODS: In 42 consecutive patients with ulcerative colitis serum concentration of IL-10 and anti-mannan Candida antibodies serum levels were measured with ELISA and the quantitative and qualitative fungal cultures of stool samples were performed. RESULTS: In 20 patients IL-10 serum concentration was below the test sensitivity and in 11 patients it ranged between 0.78 and 9.43 (mean 3.38 +/- 2.8) pg/mL. Anti-mannan Candida antibodies were detected in 8 subjects (19.04%). Stool cultures revealed significant fungal colonization in 3 (8.33%) patients with the predominance of Candida albicans. In comparison with mild/moderate UC, IL-10 serum concentration was not higher in patients with severe course of the disease. CONCLUSIONS: The results of our study show that IL-10 serum concentration correlates neither with the disease activity nor with the levels of anti-mannan Candida antibodies and the fungal colonization of the gastrointestinal tract in ulcerative colitis. It seems that IL-10 serum concentration cannot be a universal marker for the assessment of ulcerative colitis activity. Moreover, anti-mannan Candida antibodies and significant fungal colonization are present in the minority of patients with ulcerative colitis suggesting that yeasts have minor, if any, influence on the clinical course of the disease.

Susceptibility to antifungal agents of Candida species isolated from paediatric and adult patients with haematological diseases.

Summary The susceptibility to six antifungals: amphotericin B (AMF), 5-fluorocytosine (5-F), miconazole (MIK), ketoconazole (KET), fluconazole (FLU) and itraconazole (ITR) was tested among 206 Candida spp. isolated from paediatric and adult patients with haematological malignancies. To determinate the susceptibility the commercial microdilution method Fungitest (Bio-Rad, France) was used. The strains were classified as susceptible, intermediate susceptible, or resistant on the base of the growth in following breakpoint concentrations of particular drugs: 2 and 8 microg ml(-1) for AMF, 2 and 32 microg ml(-1) for 5-F, 0.5 and 8 microg ml(-1) for MIK, 0.5 and 4 microg ml(-1) for KET and ITR, and 8 and 64 microg ml(-1) for FLU. The highest activity to overall species showed AMF (only one resistant strain) and 5-F (85% susceptible strains). Most of C. albicans isolates were susceptible to tested azoles. The percentages of C. albicans resistant to FLU, ITR, KET and MIK were 4, 11, 8, and 0.8%, respectively. The less susceptible to azoles were C. glabrata and C. krusei (14% and 44% isolates resistant to FLU). A non-albicans Candida isolated from adult patients receiving KET prophylaxis was more frequently resistant to FLU than isolates from patients without previous exposure to azoles (P < 0.05). We did not observe differences in the susceptibility of Candida strains isolated from children compared with those from adults.

Distribution of Ca.LSU intron and acid protease production by Candida albicans strains isolated from gastrointestinal tract of diabetes children.

The association between the presence of self-splicing intron Ca.LSU and proteolytic activity of Candida albicans isolates was tested. Study included 95 C. albicans strains isolated from gastrointestinal tract of diabetes children. The strains with the intron (genotype B) displayed a significantly higher proteolytic activity (385.2+/-192U/L) than did strains without intron (genotype A) (119+/-115U/L) (p = 0.0000048).

The study of cell-mediated immune response in recurrent vulvovaginal candidiasis.

The aim of this work was to examine in vitro the ability of cells from patients with recurrent vulvovaginal candidiasis (RVVC) to cell-mediated immune response. Peripheral blood mononuclear cells (PBMC) and whole blood cells (WBC) of 37 RVVC patients in acute infection and 14 in remission were examined for the ability to proliferation and cytokines production (IFN, TNF, IL-6). As a control, a group of 25 healthy women were examined. The cells were stimulated with Candida antigen (HKCA), LPS and PHA. To indicate the level of cytokines, the following cell-lines were used: A549 for IFN, WEHI 164 for TNF and 7TD1 for IL-6. The proliferation/death of cells was determined by colorimetric test using MTT. Distinct suppression of cell-mediated immune response (CMI) was shown in all patients comparing to the control. Greatest suppression was found in the acute phase of the disease. The ability of cells to proliferate and produce IFN increases only in remission. The data seem to suggest that in this phase of disease, the ability of cell-mediated immune response is restored. It was also indicated that IFN may take part in protection against Candida infection.

Bactericidal activity of normal human serum against Morganella, Proteus, and Providencia strains.

The susceptibility od Proteus, Providencia, and Morganella strains to bactericidal action of human serum was examined. The percentage of survival was determined after one and three hours incubation with 50% human serum. The susceptible strains were treated by serum preparations with blocked classical or alternative complement activation pathways as well as with lysozyme removed. Following mechanisms of the bactericidal action of serum were found: complement activated by the classical or alternative pathway with participation of lysozyme, complement activated simultaneously via both pathways-while the participation of lysozyme was necessary for killing some strains and superfluos for others and complement activated only via the classical pathway without lysozyme.