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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.

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. © 2018, The Author(s).

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1-D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial beta-tubulin II (TUB2); iv) gamma-actin (ACT); v) translation elongation factor 1-alpha (TEF1 alpha); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5-6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1 alpha. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1 alpha, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.

A yeast strain isolated during a large-scale study on vineyard-associated yeast strains from Apulia (Southern Italy) was subjected to sequence analysis of the large subunit (LSU) and internal transcribed spacer (ITS) domains of its DNA operon encoding for the ribosomal RNA (rDNA). The two molecular marker sequences indicated that this strain could not be attributed to any known species and it was described as the type strain of Ogatea uvarum sp.nov. Moreover, the molecular assays showed several secondary peaks in the ITS2 sequence, but not in the LSU D1/D2. In the aim to test whether these peaks were due to the internal heterogeneity of the DNA operon encoding for the rDNA, the two domains themselves and the clones from them derived after PCR amplification were sequenced. The analyses on the internal variants of ITS and LSU showed a significant variability, although within that predictable among different strains of the same yeast species. In this Ogatea uvarum Sp. Nov., ITS was more variable than LSU especially in the ITS2 region. The heterogeneity revealed by this strain was then judged in the frame of its potential consequence in NGS-based environmental metagenomic studies, in which the variability among operons can lead to biodiversity overestimation and to incorrect identification at the species level. The above findings are discussed in the light of the diverse analytical approaches for fungi identification based on sequence similarity. The results of this study show that the internal variability of the rDNA operon requires careful consideration before being used in future NGS metagenomic investigations and emphasizes the need of specific models to interpret the concept of fungal species, when the reproductive barriers represented by exclusively sexual reproduction are not present.

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 Ogataea uvarum sp. nov., a close relative of O. philodendri. 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. O. uvarum and O. philodendri 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.