Publications by authors named "Claudia Colabella"

13 Publications

  • Page 1 of 1

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

A reference map of the human binary protein interactome.

Nature 2020 04 8;580(7803):402-408. Epub 2020 Apr 8.

Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA, USA.

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships. Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome, transcriptome and proteome data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes.
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http://dx.doi.org/10.1038/s41586-020-2188-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7169983PMC
April 2020

Spectroscopic Characterization of Bovine, Avian and Johnin Purified Protein Derivative (PPD) with High-Throughput Fourier Transform InfraRed-Based Method.

Pathogens 2019 Aug 29;8(3). Epub 2019 Aug 29.

Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche "Togo Rosati", Via Gaetano Salvemini 1, 06126 Perugia, Italy.

Tuberculins purified protein derivatives (PPDs) are obtained by precipitation from heat treated mycobacteria. PPDs are used in diagnosis of mycobacterial infections in humans and animals. Bovine PPD (PPDB) is obtained from ( complex), while Avian PPD (PPDA) and Johnin PPD (PPDJ) are extracted, respectively, from and ( complex). PPDB and PPDA are used for bovine tuberculosis diagnosis, while PPDJ is experimentally used in the immunodiagnosis of paratuberculosis. Although PPDs date back to the 19th Century, limited knowledge about their composition is currently available. The goal of our study was to evaluate Fourier Transform InfraRed (FTIR) spectroscopy as a tool to differentiate PPDB, PPDA, and three PPDJs. The results highlighted that the three PPDs have specific profiles, correlated with phylogenetic characteristics of mycobacteria used for their production. This analysis is eligible as a specific tool for different PPDs batches characterization and for the assessment of their composition. The entire PPD production may be efficiently controlled, since the N content of each preparation is related to IR spectra, with a reference spectrum for each PPD and a standardized analysis protocol.
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http://dx.doi.org/10.3390/pathogens8030136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789744PMC
August 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

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

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

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

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

Phenotypic and molecular diversity of Meyerozyma guilliermondii strains isolated from food and other environmental niches, hints for an incipient speciation.

Food Microbiol 2015 Jun 20;48:206-15. Epub 2015 Jan 20.

Department of Pharmaceutical Sciences - Microbiology, University of Perugia, Borgo 20 Giugno 74, 06121 Perugia, Italy; CEMIN, Centre of Excellence on Nanostructured Innovative Materials, Department of Chemistry, Biology and Biotechnology, University of Perugia, via Elce di Sotto 8, I-06123 Perugia, Italy. Electronic address:

Meyerozyma guilliermondii is a yeast species widely isolated from several natural environments and from fruit; in medical microbiology it is known as the teleomorph of the opportunistic pathogen Candida guilliermondii, which causes about 2% of the human blood infections. This yeast is also promising in a variety of biotechnological applications as vitamins production and post-harvest control. The question if isolates from different sources are physiologically and genetically similar, or if the various environments induced significant differences, is crucial for the understanding of this species structure and to select strains appropriate for each application. This question was addressed using LSU and ITS sequencing for taxonomic assignment, i-SSR (GACA4) for the molecular characterization and FTIR for the metabolomic fingerprint. All data showed that fruit and environmental isolates cluster separately with a general good agreement between metabolomics and molecular analysis. An additional RAPD analysis was able to discriminate strains according to the isolation position within the pineapple fruit. Although all strains are members of the M. guilliermondii species according to the current standards, the distribution of large variability detected suggests that some specialization occurred in the niches inhabited by this yeast and that food related strains can be differentiated from the medical isolates.
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http://dx.doi.org/10.1016/j.fm.2014.12.014DOI Listing
June 2015

FTIR metabolomic fingerprint reveals different modes of action exerted by structural variants of N-alkyltropinium bromide surfactants on Escherichia coli and Listeria innocua cells.

PLoS One 2015 14;10(1):e0115275. Epub 2015 Jan 14.

Department of Pharmaceutical Sciences-Microbiology, University of Perugia, Borgo XX Giugno 74, I-06121 Perugia, Italy; CEMIN, Centre of Excellence on Nanostructured Innovative Materials, Department of Chemistry, Biology and Biotechnology, University of Perugia, via Elce di Sotto 8, I-06123 Perugia, Italy.

Surfactants are extremely important agents to clean and sanitize various environments. Their biocidal activity is a key factor determined by the interactions between amphiphile structure and the target microbial cells. The object of this study was to analyze the interactions between four structural variants of N-alkyltropinium bromide surfactants with the Gram negative Escherichia coli and the Gram positive Listeria innocua bacteria. Microbiological and conductometric methods with a previously described FTIR bioassay were used to assess the metabolomic damage exerted by these compounds. All surfactants tested showed more biocidal activity in L. innocua than in E. coli. N-tetradecyltropinium bromide was the most effective compound against both species, while all the other variants had a reduced efficacy as biocides, mainly against E. coli cells. In general, the most prominent metabolomic response was observed for the constituents of the cell envelope in the fatty acids (W1) and amides (W2) regions and at the wavenumbers referred to peptidoglycan (W2 and W3 regions). This response was particularly strong and negative in L. innocua, when cells were challenged by N-tetradecyltropinium bromide, and by the variant with a smaller head and a 12C tail (N-dodecylquinuclidinium bromide). Tail length was critical for microbial inhibition especially when acting against E. coli, maybe due the complex nature of Gram negative cell envelope. Statistical analysis allowed us to correlate the induced mortality with the metabolomic cell response, highlighting two different modes of action. In general, gaining insights in the interactions between fine structural properties of surfactants and the microbial diversity can allow tailoring these compounds for the various operative conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115275PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4294686PMC
December 2015

A novel, rapid and automated conductometric method to evaluate surfactant-cells interactions by means of critical micellar concentration analysis.

Chem Biol Interact 2014 Jul 6;218:20-7. Epub 2014 May 6.

CEMIN - Centro di Eccellenza Materiali Innovativi e Nanostrutturati, Dipartimento di Chimica, Biologia e Biotecnologie, Via Elce di Sotto n.8, Italy; Dipartimento di Biologia Applicata, Via Borgo XX Giugno, 74 Università degli Studi di Perugia, I 06100 Perugia, Italy.

Conductometry is widely used to determine critical micellar concentration and micellar aggregates surface properties of amphiphiles. Current conductivity experiments of surfactant solutions are typically carried out by manual pipetting, yielding some tens reading points within a couple of hours. In order to study the properties of surfactant-cells interactions, each amphiphile must be tested in different conditions against several types of cells. This calls for complex experimental designs making the application of current methods seriously time consuming, especially because long experiments risk to determine alterations of cells, independently of the surfactant action. In this paper we present a novel, accurate and rapid automated procedure to obtain conductometric curves with several hundreds reading points within tens of minutes. The method was validated with surfactant solutions alone and in combination with Saccharomyces cerevisiae cells. An easy-to use R script, calculates conductometric parameters and their statistical significance with a graphic interface to visualize data and results. The validations showed that indeed the procedure works in the same manner with surfactant alone or in combination with cells, yielding around 1000 reading points within 20 min and with high accuracy, as determined by the regression analysis.
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http://dx.doi.org/10.1016/j.cbi.2014.04.012DOI Listing
July 2014
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