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Background:In an era in which antimicrobial resistance is increasing at an alarming pace, it is very important to find new antimicrobial agents effective against pathogenic microrganisms resistant to traditional treatments. Among the notable breakthroughs in the past years of research in natural-drug discovery, there is the identification and testing of flavonoids, a group of plant-derived substances capable of promoting many beneficial effects on humans. These compounds show different biological activities such as inhibition of neuroinflammation and tumor growth as well as antimicrobial activity against many microbial pathogens.Methods:We undertook a review of protocols and standard strains used in studies reporting the inhibitory effects of flavonoids against Candida albicans by focusing our attention on genetic characterization of the strains examined. Moreover, using the C. albicans MLST-database, we performed a phylogenetic analysis showing the genetic variation occurring in this species.Results:Today, we have enough information to estimate genetic diversity within microbial species and recent data revealed that most of fungal pathogens show complex population structures in which not a single isolate can be designated as representative of the entire taxon. This is especially true for the highly divergent fungal pathogen C. albicans, in which the assumption that one or few “standard strains” can represent the whole species is overly unrealistic and should be laid to rest.Conclusion:The goal of this article is to shed light on the extent of genetic variation in C. albicans and how this phenomenon can largely influence the activity of flavonoids against this species.

Fungi have always attracted a lot of attention as they are able to produce a vast repertoire of enzymes that find a broad spectrum of uses in biotechnological and industrial fields. Undoubtedly, one of the most promising biocatalysts is the lipase, which has been widely used for the biotransformation of a number of commercial products due to its high stability, high catalytic efficiency, versatility and selectivity, making it one of the most attractive and best-studied enzymes. In this study we report the isolation and molecular identification of new lipase-producing fungi from different environmental samples from Morocco. The production and activity of extracellular lipases, at different parameters, was evaluated using the Rhodamine B agar, submerged fermentation and biochemical methods. Two fungal strains Arthrographis curvata and Rhodosporidium babjevae, were isolated and found to produce large amounts of lipases. The optimal activity of the extracellular lipase was detected at 40°C and pH 9.0 for A. curvata and at 40 °C and pH 8.0 for R. babjevae. This study add new information at the growing list of fungal species producing lipases with improved physicochemical proprieties which could constitute a new line of research for further studies and to be exploited for industrial or bioremediation purposes.

The isolation of patulin-producing Penicillia in apples collected in different markets in four localities in Morocco is reported. Fungi were identified by β-tubulin sequencing and further characterized using a specific PCR-based method targeting the isoepoxydon dehydrogenase (IDH) gene to discriminate between patulin-producing and non-producing strains. Production of patulin was also evaluated using standard cultural and biochemical methods. Results showed that 79.5% of contaminant fungi belonged to the genus Penicillium and that Penicillium expansum was the most isolated species (83.9%) followed by Penicillium chrysogenum (~9.7%) and Penicillium crustosum (~6.4%). Molecular analysis revealed that 64.5% of the Penicillium species produced the expected IDH-amplicon denoting patulin production in these strains. However, patulin production was not chemically confirmed in all P. expansum strains. The isolation of IDH(-)/patulin(+) strains poses the hypothesis that gentisylaldehyde is not a direct patulin precursor, supporting previous observations that highlighted the importance of the gentisyl alcohol in the production of this mycotoxin. Total agreement between IDH-gene detection and cultural/chemical methods employed was observed in 58% of P. expansum strains and for 100% of the other species isolated. Overall the data reported here showed a substantial genetic variability within P. expansum population from Morocco.

Although the epidemiology of pathogenic Candida species causing invasive human diseases is changing, Candida albicans still remains the most common cause of bloodstream infections worldwide. The propensity of this pathogen to cause infections is undoubtedly the result of its unique genetic plasticity that allow it to adapt and respond quickly to a myriad of changing conditions both in the host and in the environment. For this reason, we decided to investigate the genetic diversity of this important fungal pathogen in a particular category of patients with severe neurological deficits including the hospital environments where they are hospitalized. Genetic diversity of 21 C. albicans isolates recovered from blood, hands of healthcare workers and hospital environments was evaluated by using multilocus sequence typing (MLST) which revealed a high genetic heterogeneity with a set of 18 diploid sequence types (DSTs) recovered among 21 isolates investigated. Interestingly, 13 of these 18 MLST genotypes were completely new and added to the C. albicans MLST central database. Six eBURST clonal complexes (CC-1, CC-2, CC-6, CC-9, CC-27 and CC-42) and three singletons contained all DSTs found in this study. Among all the new DSTs identified, DST3388 was the most intriguing as this genotype was recovered from a typical C. albicans isolate clustering within the MLST-Clade 13, the most divergent evolutionary lineage within C. albicans population containing only isolates with unusual phenotypes originally known as Candida africana. In conclusion, the results of this study expand our understanding of the molecular epidemiology and global population structure of C. albicans suggesting that further studies on different categories of patients and hospital environments are needed to better understand how the population of this species adapts and evolves in heterogeneous hosts and changing environments.

Candida albicans is the most common cause of life-threatening fungal infections in humans, especially in immunocompromised individuals. Crucial to its success as an opportunistic pathogen is the considerable dynamism of its genome, which readily undergoes genetic changes generating new phenotypes and shaping the evolution of new strains. Candida africana is an intriguing C. albicans biovariant strain that exhibits remarkable genetic and phenotypic differences when compared with standard C. albicans isolates. Candida africana is well-known for its low degree of virulence compared with C. albicans and for its inability to produce chlamydospores that C. albicans, characteristically, produces under certain environmental conditions. Chlamydospores are large, spherical structures, whose biological function is still unknown. For this reason, we have sequenced, assembled, and annotated the whole transcriptomes obtained from an efficient C. albicans chlamydospore-producing clinical strain (GE1), compared with the natural chlamydospore-negative C. africana clinical strain (CBS 11016). The transcriptomes of both C. albicans (GE1) and C. africana (CBS 11016) clinical strains, grown under chlamydospore-inducing conditions, were sequenced and assembled into 7,442 (GE1 strain) and 8,370 (CBS 11016 strain) high quality transcripts, respectively. The release of the first assembly of the C. africana transcriptome will allow future comparative studies to better understand the biology and evolution of this important human fungal pathogen.

Sporothrix globosa is a thermo-dimorphic fungus belonging to a pathogenic clade that also includes Sporothrix schenckii, which causes human and animal sporotrichosis. Here, we present the first genome assemblies of two S. globosa strains providing data for future comparative genomic studies in pathogenic Sporothrix species.

This study aimed to elucidate the genetic relatedness and epidemiology of 127 clinical and environmental Candida glabrata isolates from Europe and Africa using multilocus microsatellite analysis. Each isolate was first identified using phenotypic and molecular methods and subsequently, six unlinked microsatellite loci were analyzed using automated fluorescent genotyping. Genetic relationships were estimated using the minimum-spanning tree (MStree) method. Microsatellite analyses revealed the existence of 47 different genotypes. The fungal population showed an irregular distribution owing to the over-representation of genetically different infectious haplotypes. The most common genotype was MG-9, which was frequently found in both European and African isolates. In conclusion, the data reported here emphasize the role of specific C. glabrata genotypes in human infections for at least some decades and highlight the widespread distribution of some isolates, which seem to be more able to cause disease than others., This research was supported in part by the EU Mare Nostrum (EUMN-III Call) program of the European Union, grant agreement number 2011-4050/001-EMA2. Dr Sanae Rharmitt was the recipient of a scholarship (10 months) signed within the EUMN program for PhD students (F.S. 1.04.11.01 UORI) under the supervision of Prof Orazio Romeo., info:eu-repo/semantics/publishedVersion

Sporothrix pallida is considered to be a mostly avirulent environmental fungus, phylogenetically closely related to the well-known pathogen Sporothrix schenckii . Here, we present the first assembly of its genome, which provides a valuable resource for future comparative genomic studies between nonpathogenic and pathogenic Sporothrix spp.

Cryptococcus neoformans and Cryptococcus gattii are encapsulated yeasts able to cause fatal neurological infections in both human and other mammals. Cryptococcosis is the most common fungal infection of the central nervous system and has a huge burden in sub-Saharan Africa and South East Asia. Bird excreta are considered an environmental reservoir for C. neoformans in urban areas, therefore a study aimed at isolating and characterizing this yeast is important in disease management. In this study, one hundred samples of pigeon droppings were collected in Jos, Plateau State, Nigeria. C. neoformans was isolated from three samples and initially identified using standard phenotypic and biochemical tests. Molecular analysis revealed that all three isolates belonged to C. neoformans genotype VNII, mating type α and were assigned to the sequence type ST43 by multilocus sequence typing analysis. This study reports, for the first time, the molecular characterization of C. neoformans in Nigeria, where little is still known about the environmental distribution of the genotypes, serotypes and mating types of this important human pathogen.

RESEARCH ARTICLE Molecular Characterization of the N- Acetylglucosamine Catabolic Genes in Candida africana, a Natural N-Acetylglucosamine Kinase (HXK1) Mutant Maria Rosa Felice 1 , Megha Gulati 2 , Letterio Giuffre 1 , Domenico Giosa 1 , Luca Marco Di Bella 1 , Giuseppe Criseo 1 , Clarissa J. Nobile 2 , Orazio Romeo 1,3 *, Fabio Scordino 3 1 Department of Biological and Environmental Sciences, University of Messina, Messina, Italy, 2 Department of Molecular and Cell Biology, University of California Merced, Merced, California, United States of America, 3 Scientific Institute for Research, Hospitalization and Health Care (IRCCS)—Centro Neurolesi Bonino-Pulejo , Messina, Italy * oromeo@unime.it Abstract Background OPEN ACCESS Citation: Felice MR, Gulati M, Giuffre L, Giosa D, Di Bella LM, Criseo G, et al. (2016) Molecular Characterization of the N-Acetylglucosamine Catabolic Genes in Candida africana, a Natural N- Acetylglucosamine Kinase (HXK1) Mutant. PLoS ONE 11(1): e0147902. doi:10.1371/journal.pone.0147902 Editor: Valerio Pazienza, IRCCS Casa Sollievo della Sofferenza Hospital, ITALY Received: August 12, 2015 Accepted: January 11, 2016 Published: January 25, 2016 Copyright: © 2016 Felice et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. In this study we report the genetic characterization, including expression analysis, of the genes involved in the uptake (NGT1) and catabolism (HXK1/NAG5, DAC1/NAG2, NAG1) of the aminosugar N-acetylglucosamine (GlcNAc) in Candida africana, a pathogenic biovar- iant of Candida albicans that is naturally unable to assimilate the GlcNAc. Results DNA sequence analysis of these genes revealed a number of characteristic nucleotide sub- stitutions including a unique and distinctive guanine insertion that shifts the reading frame and generates a premature stop codon (TGA) 154 bp downstream of the ATG start codon of the HXK1 gene encoding the GlcNAc-kinase, a key enzyme of the GlcNAc catabolic path- way. However, all examined genes produced transcripts even though different levels of expression were observed among the Candida isolates examined. In particular, we found an HXK1-idependent relationship of the NGT1 gene and a considerable influence of the GlcNAc-kinase functionality on the transcription of the DAC1 and NAG1 genes. Additional phenotypic analysis revealed that C. africana isolates are hyperfilamentous in the first 24- 48h of growth on filament-inducing media and revert to the yeast morphological form after 72h of incubation on these media. Data Availability Statement: All relevant data are within the paper. Funding: The authors received no specific funding for this work. Competing Interests: The authors have declared that no competing interests exist. Conclusions Our results show that C. africana is a natural HXK1 mutant, displaying a number of pheno- typic characteristics distinct from typical C. albicans isolates. PLOS ONE | DOI:10.1371/journal.pone.0147902 January 25, 2016

Given the lack of comprehensive molecular epidemiology studies in Reggio Calabria and Messina, Italy, we decided to perform an extensive environmental sampling to describe the current molecular epidemiology of C. neoformans/C. gattii species complex in southern Italy. In this study, we report the occurrence of serotypes, genotypes and mating-types of isolates of the C. neoformans/C. gattii species complex recovered from environmental sources. In addition, a number of environmental C. neoformans var. grubii strains, isolated in 1997 by our laboratory, were also retrospectively examined in order to compare their genotypes with those recently found and to infer the possible epidemiological changes in our country. One hundred and twenty-two isolates were identified as being C. neoformans, whereas only one was found to belong to C. gattii serotype B, genotype VGI and mating-type alpha. Our data revealed that all environmental isolates of C. neoformans recovered here as well as those previously isolated in 1997 belong to serotype A and genotype VNI and posses a mating-type alpha allele.

Introduction: Moesziomyces spp. are connected with poaceous plants (Grass family), and Moesziomyces bullatus is commonly associated with smut in pearl millet. Currently, the scientific knowledge of this pathogen is limited to only a few taxonomic studies and there are no clinical reports that describe the isolation of M. bullatus from humans. Case presentation: A female neonate born prematurely at 32 weeks of gestation was referred to the University Teaching Hospital in Jos, Nigeria, with a provisional diagnosis of preterm low birth weight at risk for sepsis. The birth weight of the newborn was 2000 g and her body temperature on admission was 34.3 °C. Blood cultures revealed the presence of a fungal isolate that was identified as M. bullatus by molecular methods. This fungus showed high MIC values for anidulafungin, caspofungin and micafungin, as well as fluconazole and 5‐flucytosine, and exhibited varying degrees of susceptibility to itraconazole, amphotericin B, posaconazole and voriconazole. To the best of our knowledge, this is the first case of a human M. bullatus bloodstream infection. Conclusion: Here, we report the first case of an unusual human infection caused by the fungal plant pathogen M. bullatus and highlight a high level of resistance to classical and modern antifungal drugs.

In recent years, the taxonomy of the most important pathogenic Candida species (Candida albicans, Candida parapsilosis and Candida glabrata) has undergone profound changes due to the description of new closely-related species. This has resulted in the establishment of cryptic species complexes difficult to recognize in clinical diagnostic laboratories. The identification of these novel Candida species seems to be clinically relevant because it is likely that they differ in virulence and drug resistance. Nevertheless, current phenotypic methods are not suitable to accurately distinguish all the species belonging to a specific cryptic complex and therefore their recognition still requires molecular methods. Since traditional mycological techniques have not been useful, a number of molecular based methods have recently been developed. These range from simple PCR-based methods to more sophisticated real-time PCR and/or MALDI-TOF methods. In this article, we review the current methods designed for discriminating among closely related Candida species by highlighting, in particular, the limits of the existing phenotypic tests and the development of rapid and specific molecular tools for their proper identification.

Objective To investigate the fluconazole susceptibility of the genotypes of Candida albicans ( C. albicans ) strains isolated from patients with vulvovaginal candidiasis (VVC) in Jos, Plateau State, Nigeria. Methods A total of one hundred and seventy seven (177) Candida isolates were examined. The strains were obtained from 320 female patients with VVC and were identified using both phenotypic and molecular methods. Their genotypes were determined, based on the presence or absence of a transposable intron in the 25S rDNA. Results Eighty four (84) strains were recognized as being C. albicans and all the 84 C. albicans strains resulted to be genotype A. Antifungal susceptibility testing showed that 13 of those isolates (15.48%) were resistant to fluconazole. Conclusions Based on these data, we concluded that C. albicans genotype A was common among VVC patients in Jos, and resistance to fluconazole is quite high. To our knowledge, this is the first study that reports C. albicans genotypes in Jos and its resistance to fluconazole.

Fungal nosocomial infections continue to be a serious problem among hospitalized patients, decreasing quality of life and adding millions of euros to healthcare costs. The aim of this study was to describe the pattern of fungi associated with the hands of healthcare workers and to genotype Candida parapsilosis isolates in order to understand whether their high clinical prevalence stems from endemic nosocomial genotypes or from the real emergence of epidemiologically-unrelated strains. Approximately 39% (50/129) of healthcare workers were positive for yeasts and among 77 different fungal isolates recovered, C. parapsilosis was the most frequent (44/77; 57%). Twenty-seven diverse genotypes were obtained by microsatellite analysis of 42 selected blood and hand isolates. Most of the isolates from hands showed a new, unrelated, genotype, whereas a particular group of closely related genotypes prevailed in blood samples. Some of the latter genotypes were also found on the hands of healthcare workers, indicating a persistence of these clones within our hospital. C. parapsilosis genotypes from the hands were much more heterogeneous than clinical ones, thus reflecting a high genetic diversity among isolates, which is notably unusual and unexpected for this species.

During a survey of the prevalence of Candida spp. in Jos, Plateau State, Nigeria, two atypical C. albicans isolates were recovered. These two yeasts were germ tube positive, chlamydospore-negative and gave a green color on CHROMagar Candida. Molecular analysis performed by amplification of the hwp1 gene showed that these two isolates belonged to C. africana, a newly proposed Candida species closely related to C. albicans. Based on the presence or absence of an intron in DNA sequences encoding rRNA, the two C. africana, including all C. albicans isolates examined, were found to belong to genotype A and no other genotypes or species such as C. dubliniensis were found. To our knowledge, this is the first isolation of C. africana in Nigeria.

Until recently, Cryptococcus gattii was believed to be endemic in tropical and subtropical regions. To date, it has unexpectedly emerged as primary pathogen in temperate climate indicating that it has evolved and adapted to new environmental conditions including those existing in the Mediterranean area. Earlier attempts to isolate C. gattii from our environment were unsuccessful but this time, 18 years after the last environmental screening for C. neoformans, we isolated C. gattii from Eucalyptus camaldulensis in Reggio Calabria, Italy. The strains were serotype B, mating type α and were assigned to the molecular type VGI. In this study, we reported the first real environmental isolation of C. gattii in southern Italy that emphasized the observed global expansion of this yeast.

In this study, we investigated phylogenetic relationships among Italian Sporothrix schenckii isolates, by comparing their partial calmodulin sequences. In this analysis, we used 26 environmental strains of S. schenckii, plus two autochthonous clinical isolates. The results showed that our clinical strains grouped with S. schenckii sensu stricto isolates, whereas all 26 environmental isolates co-clustered with Sporothrix albicans (now regarded as a synonym of Sporothrix pallida), a non-pathogenic species closely related to S. schenckii. Furthermore, the group of environmental strains was found to be quite heterogeneous and further subdivided into two subgroups. The data reported here also showed that molecular methods, for specific identification of S. schenckii, developed before the description of its closely related species should be used with caution because of the possibility of false positive results, which could lead to inappropriate antifungal therapy. This study improves our understanding of the distribution of these new closely related Sporothrix species which also showed significant differences in antifungal susceptibilities.

We have developed a multiplex PCR protocol for the detection of Candida glabrata and its closely related species Candida nivariensis and Candida bracarensis. The method uses four PCR primers, targeting the ITS1 region and the 5.8S ribosomal RNA gene. The combination of these primers yielded unique results to all Candida species tested. The PCR assay we developed was found to be a rapid, specific and easy to perform method and it will be useful for characterizing large numbers of isolates for epidemiological studies.