Water Extracts from Industrial Hemp Waste Inhibit the Adhesion and Development of Candida Biofilm and Showed Antioxidant Activity on HT-29 Colon Cancer Cells.

The evolution of regulatory perspectives regarding the health and nutritional properties of industrial hemp-based products (Cannabis sativa L.) has pushed research to focus on the development of new methods for both the extraction and formulation of the bioactive compounds present in hemp extracts. While the psychoactive and medicinal properties of hemp-derived cannabinoid extracts are well known, much less has been investigated on the functional and antimicrobial properties of hemp extracts. Within the hemp value chain, various agricultural wastes and by-products are generated. These materials can be valorised through eco-innovations, ultimately promoting sustainable economic development. In this study, we explored the use of waste from industrial light cannabis production for the extraction of bioactive compounds without the addition of chemicals. The five extracts obtained were tested for their antimicrobial activity on both planktonic and sessile cells of pathogenic strains of the Candida albicans, Candida parapsilosis, and Candida tropicalis species and for their antioxidant activity on HT-29 colon cancer cells under oxidative stress. Our results demonstrated that these extracts display interesting properties both as antioxidants and in hindering the development of fungal biofilm, paving the way for further investigations into the sustainable valorisation of hemp waste for different biomedical applications.

MinION Sequencing of Yeast Mock Communities To Assess the Effect of Databases and ITS-LSU Markers on the Reliability of Metabarcoding Analysis.

Microbial communities play key roles both for humans and the environment. They are involved in ecosystem functions, maintaining their stability, and provide important services, such as carbon cycle and nitrogen cycle. Acting both as symbionts and as pathogens, description of the structure and composition of these communities is important. Metabarcoding uses ribosomal DNA (rDNA) (eukaryotic) or rRNA gene (prokaryotic) sequences for identification of species present in a site and measuring their abundance. This procedure requires several technical steps that could be source of bias producing a distorted view of the real community composition. In this work, we took advantage of an innovative "long-read" next-generation sequencing (NGS) technology (MinION) amplifying the DNA spanning from the internal transcribed spacer (ITS) to large subunit (LSU) that can be read simultaneously in this platform, providing more information than "short-read" systems. The experimental system consisted of six fungal mock communities composed of species present at various relative amounts to mimic natural situations characterized by predominant and low-frequency species. The influence of the sequencing platform (MinION and Illumina MiSeq) and the effect of different reference databases and marker sequences on metagenomic identification of species were evaluated. The results showed that the ITS-based database provided more accurate species identification than LSU. Furthermore, a procedure based on a preliminary identification with standard reference databases followed by the production of custom databases, including only the best outputs of the first step, is proposed. This additional step improved the estimate of species proportion of the mock communities and reduced the number of ghost species not really present in the simulated communities. IMPORTANCE Metagenomic analyses are fundamental in many research areas; therefore, improvement of methods and protocols for the description of microbial communities becomes more and more necessary. Long-read sequencing could be used for reducing biases due to the multicopy nature of rDNA sequences and short-read limitations. However, these novel technologies need to be assessed and standardized with controlled experiments, such as mock communities. The interest behind this work was to evaluate how long reads performed identification and quantification of species mixed in precise proportions and how the choice of database affects such analyses. Development of a pipeline that mitigates the effect of the barcoding sequences and the impact of the reference database on metagenomic analyses can help microbiome studies go one step further.

How does temperature trigger biofilm adhesion and growth in Candida albicans and two non-Candida albicans Candida species?

BACKGROUND: Biofilm formation on biotic and abiotic surfaces is finely regulated by genetic factors but also by oxygen concentration, pH, temperature and other environmental factors, already extensively explored for bacterial biofilms. Much less is known about fungal biofilm, that is considered a virulence factor for Candida pathogenic species among the few fungal species able to grow and survive at high temperatures such as 37°C as well as those induced by fever. The resistance to high temperatures coupled with the ability to form biofilm are threatening factors of these fungal species that could severely impact at an epidemiological level. OBJECTIVES: In this framework, we decided to study the thermal tolerance of biofilms formed by three medical relevant species such as Candida albicans and two non-Candida albicans Candida species. METHODS: Thirty nosocomial strains were investigated for their ability to adhere and grow in proximity and over body temperature (from 31 to 43°C), mimicking different environmental conditions or severe febrile-like reactions. RESULTS: Candida sessile cells reacted to different temperatures showing a strain-specific response. It was observed that the attachment and growth respond differently to the temperature and that mechanism of adhesion has different outputs at high temperature than the growth. CONCLUSIONS: This strain-dependent response is probably instrumental to guarantee the best success to cells for the infection, attachment and growth to occur. These observations reinforce the concept of temperature as a major trigger in the evolution of these species especially in this period of increasing environmental temperatures and excessive domestic heating.

Homoplasy as an Auxiliary Criterion for Species Delimitation.

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 ITS and LSU 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.

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

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 MeTRe (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 Candida that was characterized by relatively well-separated taxa, whereas the other, comprising some relevant species of the sensu stricto group of the genus Saccharomyces, included close species and interspecific hybrids. The results showed the ability of MeTRe to evaluate marker efficacy in general and genome-derived markers specifically.

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

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 Candida. 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.

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

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 Saccharomyces sensu stricto 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.

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

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.

Meso-Raman approach for rapid yeast cells identification.

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.

Biofilm Specific Activity: A Measure to Quantify Microbial Biofilm.

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)-2H-tetrazolium-5-carboxanilide) method. The strains of four pathogenic species of the genus Candida (C. albicans, C. glabrata, C. parapsilosis and C. tropicalis) and of Staphylococcus aureus 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.

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

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 Candida albicans, C. tropicalis and C. parapsilosis. 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-1. The entire analytical part of the procedure can be accomplished with a MS Excel macro provided free of charge.

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

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.