Publications by authors named "Gianluigi Cardinali"

68 Publications

Single Strain High-Depth NGS Reveals High rDNA (ITS-LSU) Variability in the Four Prevalent Pathogenic Species of the Genus .

Microorganisms 2021 Feb 2;9(2). Epub 2021 Feb 2.

Department of Pharmaceutical Sciences, University of Perugia, 06121 Perugia, Italy.

Ribosomal RNA in fungi is encoded by a series of genes and spacers included in a large operon present in 100 tandem repeats, normally in a single locus. The multigene nature of this locus was somehow masked by Sanger sequencing, which produces a single sequence reporting the prevalent nucleotide of each site. The introduction of next generation sequencing led to deeper knowledge of the individual sequences (reads) and therefore of the variants between the same DNA sequences located in different tandem repeats. In this framework, NGS sequencing of the rDNA region was used to elucidate the extent of intra- and inter-genomic variation at both the strain and species level. Specifically, the use of an innovative NGS technique allowed the high-throughput high-depth sequencing of the ITS1-LSU D1/D2 amplicons of 252 strains belonging to four opportunistic yeast species of the genus . Results showed the presence of a large extent of variability among strains and species. These variants were differently distributed throughout the analyzed regions with a higher concentration within the Internally Transcribed Spacer (ITS) region, suggesting that concerted evolution was not able to totally homogenize these sequences. Both the internal variability and the SNPs between strain can be used for a deep typing of the strains and to study their ecology.
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http://dx.doi.org/10.3390/microorganisms9020302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912828PMC
February 2021

What Is the Best Lens? Comparing the Resolution Power of Genome-Derived Markers and Standard Barcodes.

Microorganisms 2021 Feb 2;9(2). Epub 2021 Feb 2.

Department of Pharmaceutical Sciences, University of Perugia, 06121 Perugia, Italy.

Fungal species delimitation was traditionally carried out with multicopy ribosomal RNA (rRNA) genes, principally for their ease of amplification. Since the efficacy of these markers has been questioned, single-copy protein-encoding genes have been proposed alone or in combination for Multi-Locus Sequence Typing (MLST). In this context, the role of the many sequences obtained with Next-Generation Sequencing (NGS) techniques, in both genomics and metagenomics, further pushes toward an analysis of the efficacy of NGS-derived markers and of the metrics to evaluate the marker efficacy in discriminating fungal species. This paper aims at proposing (Mean Taxonomic Resolution), a novel index that could be used both for measuring marker efficacy and for assessing the actual resolution (i.e., the level of separation) between species obtained with different markers or their combinations. In this paper, we described and then employed this index to compare the efficacy of two rRNAs and four single-copy markers obtained from public databases as both an amplicon-based approach and genome-derived sequences. Two different groups of species were used, one with a pathogenic species of that was characterized by relatively well-separated taxa, whereas the other, comprising some relevant species of the group of the genus , included close species and interspecific hybrids. The results showed the ability of to evaluate marker efficacy in general and genome-derived markers specifically.
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http://dx.doi.org/10.3390/microorganisms9020299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912933PMC
February 2021

Homoplasy as an Auxiliary Criterion for Species Delimitation.

Microorganisms 2021 Jan 28;9(2). Epub 2021 Jan 28.

Department of Pharmaceutical Sciences, University of Perugia, 06121 Perugia, Italy.

Homoplasy is a sort of noise in phylogenetic reconstructions, due to the accumulation of backmutations, convergent evolution and horizontal gene transfer (HGT), which is considered the major trigger of homoplasy in microorganism for its massive presence. It is also known that homoplasy increases with the complexity of the tree with both real and simulated data. In this paper, we analyzed the variation of homoplasy with the two widely used taxonomic markers and in four taxonomic models characterized by differences in the intra-specific distances. An algorithm (HomoDist) was developed to analyze the homoplasy index (HI) variation upon addition of a single element (strain or species) in increasing distance from a starting element. This algorithm allows to follow changes of the consistency index (CI), complementary to the HI, with the increase of the number of taxa and with the increase of the distance among elements. Results show that homoplasy increases-as expected-with the number of taxa, but also as a function of the overall distance among species, often with an almost linear relationship between distance and HI. No HI change was observed in trees with few taxa spanning through short distances, indicating that this noise is not prohibitive in this context, although the analysis of the ratio between HI and distance can be recommended as a criterion for tree acceptance. The absence of large changes of the HI within the species, and its increase when new species are added by HomoDist, suggest that homoplasy variation can be used as an auxiliary test in distance-based species delimitation with any type of marker.
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http://dx.doi.org/10.3390/microorganisms9020273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7911335PMC
January 2021

Do Metabolomics and Taxonomic Barcode Markers Tell the Same Story about the Evolution of Complex in Fermentative Environments?

Microorganisms 2020 Aug 15;8(8). Epub 2020 Aug 15.

Department of Pharmaceutical Sciences, University of Perugia, 06121 Perugia, Italy.

Yeast taxonomy was introduced based on the idea that physiological properties would help discriminate species, thus assuming a strong link between physiology and taxonomy. However, the instability of physiological characteristics within species configured them as not ideal markers for species delimitation, shading the importance of physiology and paving the way to the DNA-based taxonomy. The hypothesis of reconnecting taxonomy with specific traits from phylogenies has been successfully explored for Bacteria and Archaea, suggesting that a similar route can be traveled for yeasts. In this framework, thirteen single copy loci were used to investigate the predictability of complex Fourier Transform InfaRed spectroscopy (FTIR) and High-performance Liquid Chromatography-Mass Spectrometry (LC-MS) profiles of the four historical species of the group, both on resting cells and under short-term ethanol stress. Our data show a significant connection between the taxonomy and physiology of these strains. Eight markers out of the thirteen tested displayed high correlation values with LC-MS profiles of cells in resting condition, confirming the low efficacy of FTIR in the identification of strains of closely related species. Conversely, most genetic markers displayed increasing trends of correlation with FTIR profiles as the ethanol concentration increased, according to their role in the cellular response to different type of stress.
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http://dx.doi.org/10.3390/microorganisms8081242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463906PMC
August 2020

Personalized machine learning approach to predict candidemia in medical wards.

Infection 2020 Oct 1;48(5):749-759. Epub 2020 Aug 1.

First Division of Infectious Diseases, Cotugno Hospital, Azienda Ospedaliera Dei Colli, Napoli, Italy.

Purpose: Candidemia is a highly lethal infection; several scores have been developed to assist the diagnosis process and recently different models have been proposed. Aim of this work was to assess predictive performance of a Random Forest (RF) algorithm for early detection of candidemia in the internal medical wards (IMWs).

Methods: A set of 42 potential predictors was acquired in a sample of 295 patients (male: 142, age: 72 ± 15 years; candidemia: 157/295; bacteremia: 138/295). Using tenfold cross-validation, a RF algorithm was compared with a classic stepwise multivariable logistic regression model; discriminative performance was assessed by C-statistics, sensitivity and specificity, while calibration was evaluated by Hosmer-Lemeshow test.

Results: The best tuned RF algorithm demonstrated excellent discrimination (C-statistics = 0.874 ± 0.003, sensitivity = 84.24% ± 0.67%, specificity = 91% ± 2.63%) and calibration (Hosmer-Lemeshow statistics = 12.779 ± 1.369, p = 0.120), markedly greater than the ones guaranteed by the classic stepwise logistic regression (C-statistics = 0.829 ± 0.011, sensitivity = 80.21% ± 1.67%, specificity = 84.81% ± 2.68%; Hosmer-Lemeshow statistics = 38.182 ± 15.983, p < 0.001). In addition, RF suggests a major role of in-hospital antibiotic treatment with microbioma highly impacting antimicrobials (MHIA) that are found as a fundamental risk of candidemia, further enhanced by TPN. When in-hospital MHIA therapy is not performed, PICC is the dominant risk factor for candidemia, again enhanced by TPN. When PICC is not used and MHIA therapy is not performed, the risk of candidemia is minimum, slightly increased by in-hospital antibiotic therapy.

Conclusion: RF accurately estimates the risk of candidemia in patients admitted to IMWs. Machine learning technique might help to identify patients at high risk of candidemia, reduce the delay in empirical treatment and improve appropriateness in antifungal prescription.
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http://dx.doi.org/10.1007/s15010-020-01488-3DOI Listing
October 2020

Unambiguous identification of fungi: where do we stand and how accurate and precise is fungal DNA barcoding?

IMA Fungus 2020 10;11:14. Epub 2020 Jul 10.

International Commission on the Taxonomy of Fungi, Champaign, IL USA.

True fungi () and fungus-like organisms (e.g. , ) constitute the second largest group of organisms based on global richness estimates, with around 3 million predicted species. Compared to plants and animals, fungi have simple body plans with often morphologically and ecologically obscure structures. This poses challenges for accurate and precise identifications. Here we provide a conceptual framework for the identification of fungi, encouraging the approach of integrative (polyphasic) taxonomy for species delimitation, i.e. the combination of genealogy (phylogeny), phenotype (including autecology), and reproductive biology (when feasible). This allows objective evaluation of diagnostic characters, either phenotypic or molecular or both. Verification of identifications is crucial but often neglected. Because of clade-specific evolutionary histories, there is currently no single tool for the identification of fungi, although DNA barcoding using the internal transcribed spacer (ITS) remains a first diagnosis, particularly in metabarcoding studies. Secondary DNA barcodes are increasingly implemented for groups where ITS does not provide sufficient precision. Issues of pairwise sequence similarity-based identifications and OTU clustering are discussed, and multiple sequence alignment-based phylogenetic approaches with subsequent verification are recommended as more accurate alternatives. In metabarcoding approaches, the trade-off between speed and accuracy and precision of molecular identifications must be carefully considered. Intragenomic variation of the ITS and other barcoding markers should be properly documented, as phylotype diversity is not necessarily a proxy of species richness. Important strategies to improve molecular identification of fungi are: (1) broadly document intraspecific and intragenomic variation of barcoding markers; (2) substantially expand sequence repositories, focusing on undersampled clades and missing taxa; (3) improve curation of sequence labels in primary repositories and substantially increase the number of sequences based on verified material; (4) link sequence data to digital information of voucher specimens including imagery. In parallel, technological improvements to genome sequencing offer promising alternatives to DNA barcoding in the future. Despite the prevalence of DNA-based fungal taxonomy, phenotype-based approaches remain an important strategy to catalog the global diversity of fungi and establish initial species hypotheses.
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http://dx.doi.org/10.1186/s43008-020-00033-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7353689PMC
July 2020

Delta-Integration of Single Gene Shapes the Whole Metabolomic Short-Term Response to Ethanol of Recombinant Strains.

Metabolites 2020 Apr 3;10(4). Epub 2020 Apr 3.

Department of Pharmaceutical Sciences, University of Perugia, 06121 Perugia, Italy.

In yeast engineering, metabolic burden is often linked to the reprogramming of resources from regular cellular activities to guarantee recombinant protein(s) production. Therefore, growth parameters can be significantly influenced. Two recombinant strains, previously developed by the multiple δ-integration of a glucoamylase in the industrial 27P, did not display any detectable metabolic burden. In this study, a Fourier Transform InfraRed Spectroscopy (FTIR)-based assay was employed to investigate the effect of δ-integration on yeast strains' tolerance to the increasing ethanol levels typical of the starch-to-ethanol industry. FTIR fingerprint, indeed, offers a holistic view of the metabolome and is a well-established method to assess the stress response of microorganisms. Cell viability and metabolomic fingerprints have been considered as parameters to detecting any physiological and/or metabolomic perturbations. Quite surprisingly, the three strains did not show any difference in cell viability but metabolomic profiles were significantly altered and different when the strains were incubated both with and without ethanol. A LC/MS untargeted workflow was applied to assess the metabolites and pathways mostly involved in these strain-specific ethanol responses, further confirming the FTIR fingerprinting of the parental and recombinant strains. These results indicated that the multiple δ-integration prompted huge metabolomic changes in response to short-term ethanol exposure, calling for deeper metabolomic and genomic insights to understand how and, to what extent, genetic engineering could affect the yeast metabolome.
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http://dx.doi.org/10.3390/metabo10040140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241245PMC
April 2020

Ecological interactions are a primary driver of population dynamics in wine yeast microbiota during fermentation.

Sci Rep 2020 03 18;10(1):4911. Epub 2020 Mar 18.

South African Grape and Wine Research Institute, Department of Viticulture and Oenology, Stellenbosch University, Stellenbosch, ZA-7600, South Africa.

Spontaneous wine fermentation is characterized by yeast population evolution, modulated by complex physical and metabolic interactions amongst various species. The contribution of any given species to the final wine character and aroma will depend on its numerical persistence during the fermentation process. Studies have primarily evaluated the effect of physical and chemical factors such as osmotic pressure, pH, temperature and nutrient availability on mono- or mixed-cultures comprising 2-3 species, but information about how interspecies ecological interactions in the wine fermentation ecosystem contribute to population dynamics remains scant. Therefore, in the current study, the effect of temperature and sulphur dioxide (SO) on the dynamics of a multi-species yeast consortium was evaluated in three different matrices including synthetic grape juice, Chenin blanc and Grechetto bianco. The population dynamics were affected by temperature and SO, reflecting differences in stress resistance and habitat preferences of the different species and influencing the production of most volatile aroma compounds. Evidently at 15 °C and in the absence of SO non-Saccharomyces species were dominant, whereas at 25 °C and when 30 mg/L SO was added S. cerevisiae dominated. Population growth followed similar patterns in the three matrices independently of the conditions. The data show that fermentation stresses lead to an individual response of each species, but that this response is strongly influenced by the interactions between species within the ecosystem. Thus, our data suggest that ecological interactions, and not only physico-chemical conditions, are a dominant factor in determining the contribution of individual species to the outcome of the fermentation.
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http://dx.doi.org/10.1038/s41598-020-61690-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7080794PMC
March 2020

Metabolomic Alterations Do Not Induce Metabolic Burden in the Industrial Yeast M2n[pBKD2-]-C1 Engineered by Multiple δ-Integration of a Fungal β-Glucosidase Gene.

Front Bioeng Biotechnol 2019 28;7:376. Epub 2019 Nov 28.

Department of Pharmaceutical Sciences-Microbiology, University of Perugia, Perugia, Italy.

In the lignocellulosic yeast development, metabolic burden relates to redirection of resources from regular cellular activities toward the needs created by recombinant protein production. As a result, growth parameters may be greatly affected. Noteworthy, M2n[pBKD2-]-C1, previously developed by multiple δ-integration of the β-glucosidase , did not show any detectable metabolic burden. This work aims to test the hypothesis that the metabolic burden and the metabolomic perturbation induced by the δ-integration of a yeast strain, could differ significantly. The engineered strain was evaluated in terms of metabolic performances and metabolomic alterations in different conditions typical of the bioethanol industry. Results indicate that the multiple δ-integration did not affect the ability of the engineered strain to grow on different carbon sources and to tolerate increasing concentrations of ethanol and inhibitory compounds. Conversely, metabolomic profiles were significantly altered both under growing and stressing conditions, indicating a large extent of metabolic reshuffling involved in the maintenance of the metabolic homeostasis. Considering that four copies of gene have been integrated without affecting any parental genes or promoter sequences, deeper studies are needed to unveil the mechanisms implied in these metabolomic changes, thus supporting the optimization of protein production in engineered strains.
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http://dx.doi.org/10.3389/fbioe.2019.00376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893308PMC
November 2019

Candida palmioleophila isolation in Italy from two cases of systemic infection, after a CHROMagar and Vitek system mis-identification as C. albicans.

New Microbiol 2020 Jan 9;43(1):47-50. Epub 2019 Dec 9.

University of Perugia - Department of Pharmaceutical Sciences.

A correct, fast, reliable identification method is pivotal in nosocomial environments to guide treatment strategies, whereas misidentification might lead to treatment failure. For routine identifications the Vitek system and CHROMagar are widely used but not always reliable, especially now with an increasing number of new emerging fungal pathogens that need careful identification. Here we describe two cases of candidemia, due to Candida palmioleophila previously misidentified as Candida albicans by using the Vitek2 system and CHROMagar. The first case is a 54-year-old man with an infected ulcer in the lower right limb, treated with a targeted therapy using a central venous catheter (CVC). After two months he developed a CVC-related candidemia MDR identified as C. albicans. The second case is a 2-month-old male baby that was admitted to the neonatal unit with acute respiratory failure due to a severe community-acquired bilateral pneumonia; blood cultures were all positive for C. albicans MDR. The isolated strains where re-identified with Maldi-Tof and DNA sequencing as C. palmioleophila. From the identification point of view, CHROMagar can be clearly misleading, especially because CHROMagar types currently available are not designed to discriminate new emerging species, suggesting that systems other than MALDI-TOF and marker sequencing may be inadequate even for routine identification and could contribute to producing misleading identifications and therapeutically wrong practices, leading to failures and patient death.
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January 2020

Nanostructured zinc oxide on silica surface: Preparation, physicochemical characterization and antimicrobial activity.

Mater Sci Eng C Mater Biol Appl 2019 Nov 17;104:109977. Epub 2019 Jul 17.

Department of Pharmaceutical Sciences, University of Perugia, via del Liceo 1, 06123 Perugia, Italy.

Zinc oxide nanoparticles were synthesized using two silica supports largely used in pharmaceutical field as excipients, Cab-O-Sil-H5 and Syloid 244 FP characterized by high surface area and different porosity. In order to evaluate the effects of different silica on nanoparticle chemical physical properties, composites (ZnO-SiO) containing different amounts of ZnO nanoparticles were obtained and characterized by X-ray Powder Diffraction (XRPD), Transmission Electron Microscopy (TEM), Attenuated Transmission Reflectance (ATR), UV-vis spectroscopy and finally Photoluminescence (PL). Composites showed the presence of quite uniformly distributed zinc nanostructures on the silica surface with size in the range of 30-50 nm with an estimated specific surface area ranged from ca. 20 to 70 m/g. The formation of a Zn-O-Si interface in ZnO-SiO was observed as well. Photoluminescence studies revealed that ZnO-SiO samples based on Cab-O-Sil present a higher contribution of oxygen vacancies per unit volume. Finally, the resulting composites were tested for antibacterial and antifungal activities. Whereas silica supports did not show any antibacterial and antifungal activities, most of the prepared composites, both with Cab-O-Sil-5H and Syloid 244 FP supports, resulted active against both bacteria and fungi. In particular the contingency analysis showed that the amount of zinc oxide in the composites was partly related to MIC results in bacteria (p = 0.059), whereas it showed an interesting p = 0.022 in yeast in the case of low amount of ZnO (10%). Thus, the described ZnO-SiO composites can be proposed for the preparation of both pharmaceutical formulations and medical disposals with antibacterial and antifungal activities.
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http://dx.doi.org/10.1016/j.msec.2019.109977DOI Listing
November 2019

Meso-Raman approach for rapid yeast cells identification.

Biophys Chem 2019 11 14;254:106249. Epub 2019 Aug 14.

Department of Pharmaceutical Sciences, University of Perugia, via del Liceo 1, I-06123 Perugia, Italy; CEMIN-Excellence Research Center, University of Perugia, via Pascoli, I-06123 Perugia, Italy.

An increasing effort is currently devoted to developing Raman spectroscopy for identification of microorganisms. Micro-Raman setups are typically used for this purpose with the limit that the intra-species and inter-species spectral variability are comparable, thus limiting the identification capability. To overcome this limit a meso-Raman approach is here implemented. Thin films of planktonic cells are analyzed throughout the collection of back-scattered light providing a Raman signal already averaged over tens of cells. The collecting of unpolarized (VU) and depolarized (HV) Raman signals increased the spectral information obtainable from the data, demonstrating the ability of the principal component analysis to differentiate the most common Candida species, namely C. glabrata, C. albicans, C. parapsilosis and C. tropicalis. The proposed method can contribute to bring Raman spectroscopy closer to its potential clinical use for fast identification of yeast cells.
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http://dx.doi.org/10.1016/j.bpc.2019.106249DOI Listing
November 2019

Biofilm Specific Activity: A Measure to Quantify Microbial Biofilm.

Microorganisms 2019 Mar 7;7(3). Epub 2019 Mar 7.

Department of Pharmaceutical Sciences⁻Microbiology, University of Perugia, 06123 Perugia, Italy.

Microbes growing onto solid surfaces form complex 3-D biofilm structures characterized by the production of extracellular polymeric compounds and an increased resistance to drugs. The quantification of biofilm relays currently on a number of different approaches and techniques, often leading to different evaluations of the ability to form biofilms of the studied microbial strains. Measures of biofilm biomass were carried out with crystal violet (CV) and a direct reading at 405 nm, whereas the activity was assessed with the XTT ((2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2-tetrazolium-5-carboxanilide) method. The strains of four pathogenic species of the genus (, , and ) and of were employed to determine the effective relatedness among techniques and the specific activity of the biofilm, as a ratio between the XTT and the CV outcomes. Since the ability to form biomass and to be metabolically active are not highly related, their simultaneous use allowed for a categorization of the strains. This classification is putatively amenable of further study by comparing the biofilm type and the medical behavior of the strains.
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http://dx.doi.org/10.3390/microorganisms7030073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463164PMC
March 2019

A yeast metabolome-based model for an ecotoxicological approach in the management of lignocellulosic ethanol stillage.

R Soc Open Sci 2019 Jan 16;6(1):180718. Epub 2019 Jan 16.

Department of Agronomy Food Natural resources Animals and Environment (DAFNAE), University of Padova, Legnaro, Italy.

Lignocellulosic bioethanol production results in huge amounts of stillage, a potentially polluting by-product. Stillage, rich in heavy metals and, mainly, inhibitors, requires specific toxicity studies to be adequately managed. To this purpose, we applied an FTIR ecotoxicological bioassay to evaluate the toxicity of lignocellulosic stillage. Two weak acids and furans, most frequently found in lignocellulosic stillage, have been tested in different mixtures against three strains. The metabolomic reaction of the test microbes and the mortality induced at various levels of inhibitor concentration showed that the strains are representative of three different types of response. Furthermore, the relationship between concentrations and FTIR synthetic stress indexes has been studied, with the aim of defining a model able to predict the concentrations of inhibitors in stillage, resulting in an optimized predictive model for all the strains. This approach represents a promising tool to support the ecotoxicological management of lignocellulosic stillage.
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http://dx.doi.org/10.1098/rsos.180718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366221PMC
January 2019

High-Throughput Rapid and Inexpensive Assay for Quantitative Determination of Low Cell-Density Yeast Cultures.

Microorganisms 2019 Jan 24;7(2). Epub 2019 Jan 24.

Department of Pharmaceutical Sciences, University of Perugia, 06123 Perugia, Italy.

A procedure for microbial cell density determination with a high-throughput densitometric assay was developed to allow a precise quantification of both free and sessile cells, such as those of a biofilm, with a large range from low to high cell densities. Densitometry was chosen because it allows fast, rapid and cost-effective measures; it is non-disruptive; and has an easy learning curve. The method setup, and the further validation, was carried out with strains of and Equations were developed at the level of the single strains, of the three species and finally a general one applicable to all three species. In the cross validation, with strains absent from the training set, the method was shown to be robust and flexible. The best results were obtained with species specific equations, although the global equation performed almost as well in terms of correlation between real and estimated density values. In all cases, a correlation around 0.98 between effective and predicted density was obtained with figures ranging from 10² to 10⁸ cells mL. The entire analytical part of the procedure can be accomplished with a MS Excel macro provided free of charge.
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http://dx.doi.org/10.3390/microorganisms7020032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406537PMC
January 2019

Early Ongoing Speciation of Within the Grape Ecosystem Revealed by the Internal Variability Among the rDNA Operon Repeats.

Front Microbiol 2018 3;9:1687. Epub 2018 Aug 3.

Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Lecce, Italy.

A yeast strain was isolated during a study on vineyard-associated yeast strains from Apulia in Southern Italy. ITS and LSU D1/D2 rDNA sequences showed this strain not to belong to any known species and was described as the type strain of , a close relative of . Several secondary peaks appeared in the sequences, suggesting internal heterogeneity among the copies of the rDNA. This hypothesis was tested by sequencing single clones of the marker region. The analyses showed different levels of variability throughout the operon with differences between the rRNA encoding genes and the internally transcribed regions. and share high frequency variants, i.e., variants frequently found in many clones, whereas there is a large variability of the low frequency polymorphisms, suggesting that the mechanism of homogenization is more active with the former than with the latter type of variation. These findings indicate that low frequency variants are detected in Sanger sequencing as secondary peaks whereas in Next Generation Sequencing (NGS) of metagenomics DNA would lead to an overestimate of the alpha diversity. For the first time in our knowledge, this investigation shed light on the variation of the copy number of the rDNA cistron during the yeast speciation process. These polymorphisms can be used to investigate on the processes occurring in these taxonomic markers during the separation of fungal species, it being a genetic process highly frequent in the complex microbial ecosystem existing in grape, must and wine.
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http://dx.doi.org/10.3389/fmicb.2018.01687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085423PMC
August 2018

Impact of Yeast Pigmentation on Heat Capture and Latitudinal Distribution.

Curr Biol 2018 08 2;28(16):2657-2664.e3. Epub 2018 Aug 2.

Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205, USA. Electronic address:

Pigmentation is a fundamental characteristic of living organisms that is used to absorb radiation energy and to regulate temperature. Since darker pigments absorb more radiation than lighter ones, they stream more heat, which can provide an adaptive advantage at higher latitudes and a disadvantage near the Tropics, because of the risk of overheating. This intuitive process of color-mediated thermoregulation, also known as the theory of thermal melanism (TTM), has been only tested in ectothermic animal models [1-8]. Here, we report an association between yeast pigmentation and their latitude of isolation, with dark-pigmented isolates being more frequent away from the Tropics. To measure the impact of microbial pigmentation in energy capture from radiation, we generated 20 pigmented variants of Cryptococcus neoformans and Candida spp. Infrared thermography revealed that dark-pigmented yeasts heated up faster and reached higher temperatures (up to 2-fold) than lighter ones following irradiation. Melanin-pigmented C. neoformans exhibited a growth advantage relative to non-melanized yeasts when incubated under the light at 4°C but increased thermal susceptibility at 25°C ambient temperatures. Our results extend the TTM to microbiology and suggest pigmentation as an ancient adaptation mechanism for gaining thermal energy from radiation. The contribution of microbial pigmentation in heat absorption is relevant to microbial ecology and for estimating global temperatures. The color variations available in yeasts provide new opportunities in chromatology to quantify radiative heat transfer and validate biophysical models of heat flow [9] that are not possible with plants or animals.
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http://dx.doi.org/10.1016/j.cub.2018.06.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6245944PMC
August 2018

NGS barcode sequencing in taxonomy and diagnostics, an application in "" pathogenic yeasts with a metagenomic perspective.

IMA Fungus 2018 Jun 22;9(1):91-105. Epub 2018 May 22.

Microbiology Section, Department of Pharmaceutical Sciences, University of Perugia, 06121, Italy.

Species identification of yeasts and other Fungi is currently carried out with Sanger sequences of selected molecular markers, mainly from the ribosomal DNA operon, characterized by hundreds of tandem repeats of the 18S, ITS1, 5.8S, ITS2 and LSU . The ITS region has been recently proposed as a primary barcode marker making this region the most used one in taxonomy, phylogeny and diagnostics. The introduction of NGS is providing tools of high efficacy and relatively low cost to amplify two or more markers simultaneously with great sequencing depth. However, the presence of intra-genomic variability between the repeats requires specific analytical procedures and pipelines. In this study, 286 strains belonging to 11 pathogenic yeasts species were analysed with NGS of the region spanning from ITS1 to the D1/D2 domain of the LSU encoding ribosomal DNA. Results showed that relatively high heterogeneity can hamper the use of these sequences for the identification of single strains and even more of complex microbial mixtures. These observations point out that the metagenomics studies could be affected by species inflection at levels higher than currently expected.
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http://dx.doi.org/10.5598/imafungus.2018.09.01.07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048569PMC
June 2018

Yeast Biofilm as a Bridge Between Medical and Environmental Microbiology Across Different Detection Techniques.

Infect Dis Ther 2018 Mar 16;7(Suppl 1):27-34. Epub 2018 Mar 16.

Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy.

Medical and environmental microbiology have two distinct, although very short, histories stemming, the first from the pioneering works of Sommelweiss, Pasteur, Lister and Koch, the second mainly from the studies of Bejerink and Winogradsky. These two branches of microbiology evolved and specialized separately producing distinct communities and evolving rather different approaches and techniques. The evidence accumulated in recent decades indicate that indeed most of the medically relevant microorganisms have a short circulation within the nosocomial environment and a larger one involving the external, i.e. non-nosocomial, and the hospital environments. This evidence suggests that the differences between approaches should yield to a convergent approach aimed at solving the increasing problem represented by infectious diseases for the increasingly less resistant human communities. Microbial biofilm is one of the major systems used by these microbes to resist the harsh conditions of the natural and anthropic environment, and the even worse ones related to medical settings. This paper presents a brief outline of the converging interest of both environmental and medical microbiology toward a better understanding of microbial biofilm and of the various innovative techniques that can be employed to characterize, in a timely and quantitative manner, these complex structures. Among these, micro-Raman along with micro-Brillouin offer high hopes of describing biofilms both at the subcellular and supercellular level, with the possibility of characterizing the various landscapes of the different biofilms. The possibility of adding a taxonomic identification of the cells comprising the biofilm is a complex aspect presenting several technical issues that will require further studies in the years to come.
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http://dx.doi.org/10.1007/s40121-018-0191-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856731PMC
March 2018

The H3 Haplotype of the EPCR Gene Determines High sEPCR Levels in Critically Ill Septic Patients.

Infect Dis Ther 2018 Mar 16;7(Suppl 1):3-14. Epub 2018 Mar 16.

First Department of Critical Care Medicine and Pulmonary Services, GP Livanos and M Simou Laboratories, Evangelismos Hospital, Medical School of the National and Kapodistrian University of Athens, Athens, Greece.

Introduction: A soluble (s) form of the endothelial protein C receptor (EPCR) circulates in plasma and inhibits activated protein C (APC) activities. The clinical impact of sEPCR and its involvement in the septic process is under investigation. This study determined the frequencies of EPCR haplotypes H1 and H3 to investigate possible associations with plasma admission levels of sEPCR in an intensive care unit (ICU) cohort of septic patients.

Methods: Three polymorphisms in the EPCR gene were genotyped in 239 Caucasian critically ill patients, and their plasma sEPCR levels were also measured at the time of admission to the ICU. Multivariate logistic regression analysis controlling for sepsis severity, age, acute physiology and chronic health evaluation (APACHE II) and sequential organ failure assessment (SOFA) scores, lactate level, sex, diagnostic category, length of ICU stay and hospital mortality was performed to determine the effect of EPCR haplotypes H1 and H3 on the levels of sEPCR.

Results: Individuals carrying at least one H3 allele had significantly higher levels of sEPCR than individuals with no H3 alleles (p < 0.001). No differences were found in the distribution of the H3 allele in the patient groups categorized using the pre-existing and current sepsis-3 definitions.

Conclusion: Using the preceding and current sepsis definitions, sEPCR levels and the H3 haplotype were not associated with sepsis severity and the risk of poor outcomes in septic patients; however, the EPCR H3 allele contributed to higher levels of sEPCR.
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http://dx.doi.org/10.1007/s40121-018-0193-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856733PMC
March 2018

Merging FT-IR and NGS for simultaneous phenotypic and genotypic identification of pathogenic Candida species.

PLoS One 2017 4;12(12):e0188104. Epub 2017 Dec 4.

Department of Pharmaceutical Sciences-Microbiology, University of Perugia, Perugia (Italy).

The rapid and accurate identification of pathogen yeast species is crucial for clinical diagnosis due to the high level of mortality and morbidity induced, even after antifungal therapy. For this purpose, new rapid, high-throughput and reliable identification methods are required. In this work we described a combined approach based on two high-throughput techniques in order to improve the identification of pathogenic yeast strains. Next Generation Sequencing (NGS) of ITS and D1/D2 LSU marker regions together with FTIR spectroscopy were applied to identify 256 strains belonging to Candida genus isolated in nosocomial environments. Multivariate data analysis (MVA) was carried out on NGS and FT-IR data-sets, separately. Strains of Candida albicans, C. parapsilosis, C. glabrata and C. tropicalis, were identified with high-throughput NGS sequencing of ITS and LSU markers and then with FTIR. Inter- and intra-species variability was investigated by consensus principal component analysis (CPCA) which combines high-dimensional data of the two complementary analytical approaches in concatenated PCA blocks normalized to the same weight. The total percentage of correct identification reached around 97.4% for C. albicans and 74% for C. parapsilosis while the other two species showed lower identification rates. Results suggested that the identification success increases with the increasing number of strains actually used in the PLS analysis. The absence of reliable FT-IR libraries in the current scenario is the major limitation in FTIR-based identification of strains, although this metabolomics fingerprint represents a valid and affordable aid to rapid and high-throughput to clinical diagnosis. According to our data, FT-IR libraries should include some tens of certified strains per species, possibly over 50, deriving from diverse sources and collected over an extensive time period. This implies a multidisciplinary effort of specialists working in strain isolation and maintenance, molecular taxonomy, FT-IR technique and chemo-metrics, data management and data basing.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188104PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5714347PMC
December 2017

The role of biofilm forming on mortality in patients with candidemia: a study derived from real world data.

Infect Dis (Lond) 2018 03 9;50(3):214-219. Epub 2017 Oct 9.

i Infectious Diseases Clinic , Nuovo Santa Chiara University Hospital, Azienda Ospedaliera Universitaria Pisana , Pisa , Italy.

Background: Evaluation of the role on patient mortality exerted by biofilm forming (BF) Candida strains, by using predictive clinical data.

Methods: Eighty-nine strains isolated from Candida bloodstream infection, occurring in two Italian University Hospitals, were employed in this study. A random forest (RF) model was built with a procedure of iterative selection of the risk factors potentially able to predict the probability of death. The similarity between patient conditions and Bayesian clustering was calculated in order to evaluate the role of predictors in the stratification of the death risk.

Results: Three different groups of patients with different probability of death were obtained with a RF approach: Group 1 (mortality in 33.3% of cases), Group 2 (death in 50% of cases), and Group 3 (mortality in 76.9% of cases). The comparison between these three groups showed that BF correlated well with increased mortality in patients, admitted for medical diagnosis, with high APACHE II score and treated with azoles. Early treatment within 24 h between candidemia diagnosis and the beginning of antifungal therapy was associated with the lowest of BF rate and mortality.

Conclusions: BF by Candida spp. seems to be clinically associated with increased mortality especially in medical patients with higher Apache II score or treated with azoles.
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http://dx.doi.org/10.1080/23744235.2017.1384956DOI Listing
March 2018

Invasive listeriosis in a patient with several episodes of antibiotic associated colitis presumably due to Clostridium difficile.

Infection 2017 Jun 1;45(3):381-383. Epub 2017 Apr 1.

Department of Pharmaceutical Sciences-Microbiology, University of Perugia, Perugia, Italy.

A 62-year-old man developed a blood stream infection and meningitis due to Listeria monocytogenes, 20 days after an episode of pseudo-membranous colitis. The patient, hospitalized for the first time for transurethral prostatectomy, was readmitted 20 days later with watery diarrhea. Pseudo-membranous colitis was diagnosed and treated successfully, without testing for Clostridium difficile infection (CDI). After 15 more days, the patient developed again diarrhea, fever and confusion. Hospitalized again, blood and cerebrospinal fluid cultures resulted positive for L. monocytogenes. The patient was treated successfully and a diagnosis of recurrent CDI was confirmed following culture and nucleic acid amplification assays both positive for C. difficile. This is the first report of an invasive listeriosis after CDI underlines the importance of taking greater awareness in complicated blood stream infections that may arise after CDI.
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http://dx.doi.org/10.1007/s15010-017-1013-4DOI Listing
June 2017

Exploring ecological modelling to investigate factors governing the colonization success in nosocomial environment of Candida albicans and other pathogenic yeasts.

Sci Rep 2016 06 1;6:26860. Epub 2016 Jun 1.

Infectious Diseases Clinic, Santa Maria Misericordia University Hospital, Piazzale Santa Maria della Misericordia, 15, 33100 Udine, Italy.

Two hundred seventy seven strains from eleven opportunistic species of the genus Candida, isolated from two Italian hospitals, were identified and analyzed for their ability to form biofilm in laboratory conditions. The majority of Candida albicans strains formed biofilm while among the NCAC species there were different level of biofilm forming ability, in accordance with the current literature. The relation between the variables considered, i.e. the departments and the hospitals or the species and their ability to form biofilm, was tested with the assessment of the probability associated to each combination. Species and biofilm forming ability appeared to be distributed almost randomly, although some combinations suggest a potential preference of some species or of biofilm forming strains for specific wards. On the contrary, the relation between biofilm formation and species isolation frequency was highly significant (R(2) around 0.98). Interestingly, the regression analyses carried out on the data of the two hospitals separately were rather different and the analysis on the data merged together gave a much lower correlation. These findings suggest that, harsh environments shape the composition of microbial species significantly and that each environment should be considered per se to avoid less significant statistical treatments.
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http://dx.doi.org/10.1038/srep26860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887984PMC
June 2016

Ionic Conductivity as a Tool To Study Biocidal Activity of Sulfobetaine Micelles against Saccharomyces cerevisiae Model Cells.

Langmuir 2016 Feb 25;32(4):1101-10. Epub 2016 Jan 25.

Department of Pharmaceutical Sciences-Microbiology, University of Perugia , Borgo XX Giugno 74, I-06121 Perugia, Italy.

Zwitterionic sulfobetaine surfactants are used in pharmaceutical or biomedical applications for the solubilization and delivery of hydrophobic molecules in aqueous medium or in biological environments. In a screening on the biocidal activity of synthetic surfactants on microbial cells, remarkable results have emerged with sulfobetaine amphiphiles. The interaction between eight zwitterionic sulfobetaine amphiphiles and Saccharomyces cerevisiae model cells was therefore analyzed. S. cerevisiae yeast cells were chosen, as they are a widely used unicellular eukaryotic model organism in cell biology. Conductivity measurements were used to investigate the interaction between surfactant solution and cells. Viable counts measurements were performed, and the mortality data correlated with the conductivity profiles very well, in terms of the inflection points (IPs) observed in the curves and in terms of supramolecular properties of the aggregates. A Fourier transform infrared (FTIR)-based bioassay was then performed to determine the metabolomic stress-response of the cells subjected to the action of zwitterionic surfactants. The surfactants showed nodal concentration (IPs) with all the techniques in their activities, corresponding to the critical micellar concentrations of the amphiphiles. This is due to the pseudocationic behavior of sulfobetaine micelles, because of their charge distribution and charge densities. This behavior permits the interaction of the micellar aggregates with the cells, and the structure of the surfactant monomers has impact on the mortality and the metabolomic response data observed. On the other hand, the concentrations that are necessary to provoke a biocidal activity do not promote these amphiphiles as potential antimicrobial agents. In fact, they are much higher than the ones of cationic surfactants.
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http://dx.doi.org/10.1021/acs.langmuir.5b04077DOI Listing
February 2016

First Case of Trichoderma longibrachiatum CIED (Cardiac Implantable Electronic Device)-Associated Endocarditis in a Non-immunocompromised Host: Biofilm Removal and Diagnostic Problems in the Light of the Current Literature.

Mycopathologia 2016 Apr 20;181(3-4):297-303. Epub 2015 Nov 20.

Cardiovascular Medicine Unit 2, Azienda Ospedaliera Universitaria Pisana, Via Paradisa 2, Cisanello, 56100, Pisa, Italy.

Background: Trichoderma species are saprophytic filamentous fungi producing localized and invasive infections that are cause of morbidity and mortality, especially in immunocompromised patients, causing up to 53% mortality. Non-immunocompromised patients, undergoing continuous ambulatory peritoneal dialysis, are other targets of this fungus. Current molecular diagnostic tools, based on the barcode marker ITS, fail to discriminate these fungi at the species level, further increasing the difficulty associated with these infections and their generally poor prognosis.

Case Report: We report on the first case of endocarditis infection caused by Trichoderma longibrachiatum in a 30-year-old man. This patient underwent the implantation of an implantable cardioverter defibrillator in 2006, replaced in 2012. Two years later, the patient developed fever, treated successfully with amoxicillin followed by ciprofloxacin, but an echocardiogram showed large vegetation onto the ventricular lead. After CIED extraction, the patient had high-grade fever. The culturing of the catheter tip was positive only in samples deriving from sonication according to the 2014 ESCMID guidelines, whereas the simple washing failed to remove the biofilm cells from the plastic surface. Subsequent molecular (ITS sequencing) and microbiological (macromorphology) analyses showed that the vegetation was due to T. longibrachiatum.

Conclusions: This report showed that T. longibrachiatum is an effective threat and that sonication is necessary for the culturing of vegetations from plastic surfaces. Limitations of the current barcode marker ITS, and the long procedures required by a multistep approach, call for the development of rapid monophasic tests.
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http://dx.doi.org/10.1007/s11046-015-9961-7DOI Listing
April 2016

Microbial cell-free extracts as sources of enzyme activities to be used for enhancement flavor development of ewe milk cheese.

J Dairy Sci 2015 Sep 2;98(9):5874-89. Epub 2015 Jul 2.

Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, 70126 Bari, Italy.

Freeze-dried cell-free extracts (CFE) from Lactobacillus casei LC01, Weissella cibaria 1XF5, Hafnia alvei Moller ATCC 51815, and Debaryomyces hansenii LCF-558 were used as sources of enzyme activities for conditioning the ripening of ewe milk cheese. Compared with control cheese (CC), CFE did not affect the gross composition and the growth of the main microbial groups of the cheeses. As shown through urea-PAGE electrophoresis of the pH 4.6-soluble nitrogen fraction and the analysis of free AA, the secondary proteolysis of the cheeses with CFE added was markedly differed from that of the CC. Compared with CC, several enzyme activities were higher in the water-soluble extracts from cheeses made with CFE. In agreement, the levels of 49 volatile compounds significantly differentiated CC from the cheeses made with CFE. The level of some alcohols, ketones, sulfur compounds, and furans were the lowest in the CC, whereas most aldehydes were the highest. Each CFE seemed to affect a specific class of chemical compounds (e.g., the CFE from H. alvei ATCC 51815 mainly influenced the synthesis of sulfur compounds). Apart from the microbial source used, the cheeses with the addition of CFE showed higher score for acceptability than the control cheese. Cheese ripening was accelerated or conditioned using CFE as sources of tailored enzyme activities.
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http://dx.doi.org/10.3168/jds.2015-9362DOI Listing
September 2015

Fecal Microbiota in Healthy Subjects Following Omnivore, Vegetarian and Vegan Diets: Culturable Populations and rRNA DGGE Profiling.

PLoS One 2015 2;10(6):e0128669. Epub 2015 Jun 2.

Department of Agricultural, Forest and Food Science, University of Turin, Largo P. Braccini n°2, 10095, Grugliasco, Italy.

In this study, the fecal microbiota of 153 healthy volunteers, recruited from four different locations in Italy, has been studied by coupling viable counts, on different microbiological media, with ribosomal RNA Denaturing Gradient Gel Electrophoresis (rRNA-DGGE). The volunteers followed three different diets, namely omnivore, ovo-lacto-vegetarian and vegan. The results obtained from culture-dependent and -independent methods have underlined a high level of similarity of the viable fecal microbiota for the three investigated diets. The rRNA DGGE profiles were very complex and comprised a total number of bands that varied from 67 to 64 for the V3 and V9 regions of the 16S rRNA gene, respectively. Only a few bands were specific in/of all three diets, and the presence of common taxa associated with the dietary habits was found. As far as the viable counts are concerned, the high similarity of the fecal microbiota was once again confirmed, with only a few of the investigated groups showing significant differences. Interestingly, the samples grouped differently, according to the recruitment site, thus highlighting a higher impact of the food consumed by the volunteers in the specific geographical locations than that of the type of diet. Lastly, it should be mentioned that the fecal microbiota DGGE profiles obtained from the DNA were clearly separated from those produced using RNA, thus underlining a difference between the total and viable populations in the fecal samples.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0128669PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4452701PMC
May 2016

International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database--the quality controlled standard tool for routine identification of human and animal pathogenic fungi.

Med Mycol 2015 May 22;53(4):313-37. Epub 2015 Mar 22.

Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Sydney Medical School-Westmead Hospital, Marie Bashir Institute for Infectious Diseases and Bioscurity, University of Sydney, Westmead Millennium Institute, Sydney, Australia

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.
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http://dx.doi.org/10.1093/mmy/myv008DOI Listing
May 2015