Publications

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repertoire of malic enzymes in yeast and fungi: insight into their evolutionary functional and structural significance.malic enzyme (me) is one of the important enzymes for furnishing the cofactor nad(p)h for the biosynthesis of fatty acids and sterols. due to the existence of multiple me isoforms in a range of oleaginous microbes, a molecular basis for the evolutionary relationships amongst the enzymes in oleaginous fungi was investigated using sequence analysis and structural modelling. evolutionary distance and structural characteristics were used to discriminate the mes of yeasts and fungi into several group ...201324065718
two new β-glucosidases from ethanol-fermenting fungus mucor circinelloides nbrc 4572: enzyme purification, functional characterization, and molecular cloning of the gene.two β-glucosidases (bgls 1 and 2) were purified to homogeneity from the extracellular enzyme preparations of the ethanol-fermenting mucor circinelloides nbrc 4572 statically grown on rice straw. bgls 1 and 2 are monomeric glycoproteins whose apparent molecular masses (ms) are around 78 kda, which decreased by approximately 10 kda upon enzymatic deglycosylation. both bgls showed similar enzyme characteristics in optimal temperature and ph, stability, and inhibitors. they were active against a wid ...201324061417
calcineurin plays key roles in the dimorphic transition and virulence of the human pathogenic zygomycete mucor circinelloides.many pathogenic fungi are dimorphic and switch between yeast and filamentous states. this switch alters host-microbe interactions and is critical for pathogenicity. however, in zygomycetes, whether dimorphism contributes to virulence is a central unanswered question. the pathogenic zygomycete mucor circinelloides exhibits hyphal growth in aerobic conditions but switches to multi-budded yeast growth under anaerobic/high co₂ conditions. we found that in the presence of the calcineurin inhibitor fk ...201324039585
genomic, proteomic, and biochemical analyses of oleaginous mucor circinelloides: evaluating its capability in utilizing cellulolytic substrates for lipid production.lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. however, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. we report the first comprehensive investigation of mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids. our genomic analysis ...201324023719
a single argonaute gene participates in exogenous and endogenous rnai and controls cellular functions in the basal fungus mucor circinelloides.the mechanism of rnai is well described in metazoans where it plays a role in diverse cellular functions. however, although different classes of endogenous small rnas (esrnas) have been identified in fungi, their biological roles are poorly described due, in part, to the lack of phenotype of mutants affected in the biogenesis of these esrnas. argonaute proteins are one of the key components of the rnai pathways, in which different members of this protein family participate in the biogenesis of a ...201323935973
effect of immobilized polygalacturonase from mucor circinelloides itcc-6025 on wine fermentation.pectinases are among the most widely distributed enzymes in bacteria, fungi, and plants. almost all the commercial preparations of pectinases are produced from fungal sources. mucor circinelloides itcc-6025 produced polygalacturonase when grown in riviere's medium containing pectin (methyl ester) as the sole source of carbon. immobilization of purified polygalacturonase was done on silica gel with 86% efficiency. the enzyme took 60 min to bind maximally on the support. the immobilized enzyme sho ...201323600573
genome-scale analysis of the metabolic networks of oleaginous zygomycete fungi.microbial lipids are becoming an attractive option for the industrial production of foods and oleochemicals. to investigate the lipid physiology of the oleaginous microorganisms, at the system level, genome-scale metabolic networks of mortierella alpina and mucor circinelloides were constructed using bioinformatics and systems biology. as scaffolds for integrated data analysis focusing on lipid production, consensus metabolic routes governing fatty acid synthesis, and lipid storage and mobilisat ...201323541380
effect of potassium ions on protoplast generation during yeast induction from mucor circinelloides tieghem.mucor circinelloides aerobically exhibits coenocytic thallic growth habit with straight and circinate sporangiophores which culminate in globose or pyriform columellae enclosed within sporangial walls. it undergoes dimorphic switch with its conversion to multipolar budding yeast-like cells or thallic conidia. this paper confirms the induction of plurality of reproductive structures of the pleomorphic microorganism in minimal medium. furthermore, construction of ph differentials at inflection poi ...201325969779
mixed production of filamentous fungal spores for preventing soil-transmitted helminth zoonoses: a preliminary analysis.helminth zoonoses are parasitic infections shared by humans and animals, being the soil-transmitted helminths (sths) mainly caused by roundworms (ascarids) and hookworms. this study was aimed to assess the individual and/or mixed production of two helminth-antagonistic fungi, one ovicide (mucor circinelloides) and other predator (duddingtonia flagrans). fungi were grown both in petri plates and in a submerged culture (composed by water, nacl, na2hpo4 · 12 h2o, and wheat (triticum aestivum)). a f ...201323710451
isolation of fungal pathogens to an edible mushroom, pleurotus eryngii, and development of specific its primers.fungal pathogens have caused severe damage to the commercial production of pleurotus eryngii, the king oyster mushroom, by reducing production yield, causing deterioration of commercial value, and shortening shelf-life. four strains of pathogenic fungi, including trichoderma koningiopsis dc3, phomopsis sp. mp4, mucor circinelloides mp5, and cladosporium bruhnei mp6, were isolated from the bottle culture of diseased p. eryngii. a species-specific primer set was designed for each fungus from the i ...201324493949
a white collar 1-like protein mediates opposite regulatory functions in mucor circinelloides.protein ubiquitylation plays a major role in the regulation of many cellular processes by altering the stability, localization or function of target proteins. crga is a protein of mucor circinelloides that shows the characteristics of ubiquitin ligases and is involved in the regulation of carotenogenesis and asexual sporulation in this fungus. crga, which belongs to a poorly characterized group of proteins present in almost all eukaryotes, represses carotenogenesis through the proteolysis-indepe ...201323357353
antioxidant and antimicrobial activities of 7-hydroxy-calamenene-rich essential oils from croton cajucara benth.croton cajucara is a shrub native to the amazon region locally known as "sacaca". two morphotypes are known: white and red "sacaca". the essential oils (eo) obtained by hydrodistillation from leaves of the red morphotype were, in general, rich in 7-hydroxycalamenene (28.4%-37.5%). the effectiveness of these eo regarding the antimicrobial activity against pathogenic microorganisms was initially investigated by the drop test method, showing significant inhibition zones. among the microorganisms te ...201323325101
cloning and expression of an active aspartic proteinase from mucor circinelloides in pichia pastoris.extracellular aspartic proteinase (mcap) produced by mucor circinelloides in solid state fermentations has been shown to possess milk clotting activity and represents a potential replacement for bovine chymosin in cheese manufacturing. despite its prospects in the dairy industry, the molecular characteristics of this enzyme remain unknown. this work focuses on mcap cloning and optimization of heterologous expression in pichia pastoris, and characterization of the enzyme.201324206750
in vitro antioxidant and antifungal properties of essential oils obtained from aromatic herbs endemic to the southeast of spain.the aim of this work was to determine (i) the antioxidant capacity of thymus moroderi, thymus piperella, santolina chamaecyparissus, and sideritis angustifolia essential oils (eos) by means of four different antioxidant tests (the 2,2'-diphenyl-1-picrylhydrazyl radical scavenging method, the ferrous ion-chelating ability assay, the ferric reducing antioxidant power test, and the thiobarbituric acid reactive species test) and (ii) the antifungal activity against molds and yeast of these eos by th ...201323834797
rnai function, diversity, and loss in the fungal kingdom.rnai is conserved and has been studied in a broad cross-section of the fungal kingdom, including neurospora crassa, schizosaccharomyces pombe, cryptococcus neoformans, and mucor circinelloides. and yet well known species, including the model yeast saccharomyces cerevisiae and the plant pathogen ustilago maydis, have lost rnai, providing insights and opportunities to illuminate benefits conferred both by the presence of rnai and its loss. some of the earliest studies of rnai were conducted in neu ...201324173579
canthaxanthin production with modified mucor circinelloides strains.canthaxanthin is a natural diketo derivative of β-carotene primarily used by the food and feed industries. mucor circinelloides is a β-carotene-accumulating zygomycete fungus and one of the model organisms to study the carotenoid biosynthesis in fungi. in this study, the β-carotene ketolase gene (crtw) of the marine bacterium paracoccus sp. n81106 fused with fungal promoter and terminator regions was integrated into the m. circinelloides genome to construct stable canthaxanthin-producing strains ...201323224586
phosphorylation of calcineurin at a novel serine-proline rich region orchestrates hyphal growth and virulence in aspergillus fumigatus.the fungus aspergillus fumigatus is a leading infectious killer in immunocompromised patients. calcineurin, a calmodulin (cam)-dependent protein phosphatase comprised of calcineurin a (cnaa) and calcineurin b (cnab) subunits, localizes at the hyphal tips and septa to direct a. fumigatus invasion and virulence. here we identified a novel serine-proline rich region (sprr) located between two conserved cnaa domains, the cnab-binding helix and the cam-binding domain, that is evolutionarily conserved ...201323990785
molecular characterization and in vitro antifungal susceptibility of 80 clinical isolates of mucormycetes in delhi, india.mucormycosis is a highly aggressive disease which is usually fatal in immunocompromised patients. the species of mucormycetes show significant differences in susceptibility to amphotericin b, azoles and terbinafine. the precise species level identification for this fungal group could be achieved by internal transcribed-spacer (its) region sequencing. herein, we present the largest series of antifungal susceptibility data of molecularly characterised isolates of mucormycetes reported so far from ...201425250768
transcription of the three hmg-coa reductase genes of mucor circinelloides.precursors of sterols, carotenoids, the prenyl groups of several proteins and other terpenoid compounds are synthesised via the acetate-mevalonate pathway. one of the key enzyme of this pathway is the 3-hydroxy-3-methylglutaryl-coa (hmg-coa) reductase, which catalyses the conversion of hmg-coa to mevalonate. hmg-coa reductase therefore affects many biological processes, such as morphogenesis, synthesis of different metabolites or adaptation to environmental changes. in this study, transcription ...201424731286
effects of post-harvest treatment using chitosan from mucor circinelloides on fungal pathogenicity and quality of table grapes during storage.the aim of this study was to extract chitosan (chi) from mucor circinelloides ucp 050 grown in a corn steep liquor (csl)-based medium under optimized conditions and to assess the efficacy of the obtained chi to inhibit the post-harvest pathogenic fungi aspergillus niger urm 5162 and rhizopus stolonifer urm 3482 in laboratory media and as a coating on table grapes (vitis labrusca l.). the effect of chi coating on some physical, physicochemical and sensory characteristics of the fruits during stor ...201425084665
the effect of chitin size, shape, source and purification method on immune recognition.the animal immune response to chitin is not well understood and needs to be investigated further. however, this is a challenging topic to study because of the technical difficulties in purifying chitin, and because this material usually comes associated with contaminating components that can activate the immune system. in this study, improvements to previously described purification protocols were investigated for chitin obtained from different sources, including commercial shellfish, candida al ...201424727416
identification and characterization of d-xylulokinase from the d-xylose-fermenting fungus, mucor circinelloides.d-xylulokinase catalyzes the phosphorylation of d-xylulose in the final step of the pentose catabolic pathway to form d-xylulose-5-phosphate. the d-xylulokinase activity was found to be induced by both d-xylose and l-arabinose, as well as some of the other enzymes involved in the pentose catabolism, in the d-xylose-fermenting zygomycetous fungus, mucor circinelloides nbrc 4572. the putative gene, xyl3, which may encode d-xylulokinase, was detected in the genome sequence of this strain. the amino ...201425163569
effects of 7-hydroxycalamenene isolated from croton cajucara essential oil on growth, lipid content and ultrastructural aspects of rhizopus oryzae.the leaves and bark of croton cajucara, a shrub from the amazon region, have been used in folk medicine to treat diabetes, malaria, and gastrointestinal and liver disorders. the essential oil from the leaves, rich in linalool, presented antileishmanial and antimicrobial activities. a chemotype of this species was found with an essential oil rich in 7-hydroxycalamenene. during our studies of the c. cajucara essential oil, we isolated 7-hydroxycalamenene at > 98 % purity. the minimum inhibitory co ...201424841967
quantitative multiplexed detection of common pulmonary fungal pathogens by labeled primer polymerase chain reaction.invasive fungal infections are an important cause of morbidity and mortality among immunocompromised patients.201425357108
rapid screening for human-pathogenic mucorales using rolling circle amplification.mucormycosis has emerged as a relatively common severe mycosis in patients with haematological and allogeneic stem cell transplantation. source of transmission is from unidentified sources in the environment. early diagnosis of infection and its source of contamination are paramount for rapid and appropriate therapy. in this study, rolling circle amplification (rca) is introduced as a sensitive, specific and reproducible isothermal dna amplification technique for rapid molecular identification o ...201425187214
identification of a β-glucosidase from the mucor circinelloides genome by peptide pattern recognition.mucor circinelloides produces plant cell wall degrading enzymes that allow it to grow on complex polysaccharides. although the genome of m. circinelloides has been sequenced, only few plant cell wall degrading enzymes are annotated in this species. we applied peptide pattern recognition, which is a non-alignment based method for sequence analysis to map conserved sequences in glycoside hydrolase families. the conserved sequences were used to identify similar genes in the m. circinelloides genome ...201425442948
comparative studies of oleaginous fungal strains (mucor circinelloides and trichoderma reesei) for effective wastewater treatment and bio-oil production.biological wastewater treatment typically requires the use of bacteria for degradation of carbonaceous and nitrogenous compounds present in wastewater. the high lipid containing biomass can be used to extract oil and the contents can be termed as bio-oil (or biodiesel or myco-diesel after transesterification). the separate experiments were conducted on actual wastewater samples with 5% v/v inoculum of mucor circinelloides mtcc1297 and trichoderma reesei ncim992 strains. the observed reductions i ...201425530884
rhinocerebral mucor circinelloides infection in immunocompromised patient following yogurt ingestion.the purpose of this case report is to illustrate the cause of this patient's headache and sinus pain in the setting of a unique environmental exposure: the patient ingested yogurt only days before presentation. this particular brand of yogurt caused controversy in early september 2013 when the manufacturer voluntarily recalled all flavors. the yogurt was found to be contaminated with mucor circinelloides. the recall was triggered by the fda, after receiving many complaints from consumers affecte ...201425252436
effects of 20 standard amino acids on the growth, total fatty acids production, and γ-linolenic acid yield in mucor circinelloides.twenty standard amino acids were examined as single nitrogen source on the growth, total fatty acids production, and yield of γ-linolenic acid (gla) in mucor circinelloides. of the amino acids, tyrosine gave the highest biomass and lipid accumulation and thus resulted in a high gla yield with respective values of 17.8 g/l, 23 % (w/w, dry cell weight, dcw), and 0.81 g/l, which were 36, 25, and 72 % higher than when the fungus was grown with ammonium tartrate. to find out the potential mechanism u ...201425117649
lipid accumulation by pelletized culture of mucor circinelloides on corn stover hydrolysate.microbial oil accumulated by fungal cells is a potential feedstock for biodiesel production, and lignocellulosic materials can serve as the carbon source to support the fungal growth. the dilute acid pretreatment of corn stover can effectively break down its lignin structure, and this process generates a hydrolysate containing mostly xylose at very dilute concentration and numerous by-products that may significantly inhibit the cell growth. this study utilized corn stover hydrolysate as the cult ...201425080382
antifungal drug resistance evoked via rnai-dependent epimutations.microorganisms evolve via a range of mechanisms that may include or involve sexual/parasexual reproduction, mutators, aneuploidy, hsp90 and even prions. mechanisms that may seem detrimental can be repurposed to generate diversity. here we show that the human fungal pathogen mucor circinelloides develops spontaneous resistance to the antifungal drug fk506 (tacrolimus) via two distinct mechanisms. one involves mendelian mutations that confer stable drug resistance; the other occurs via an epigenet ...201425079329
analysis of a food-borne fungal pathogen outbreak: virulence and genome of a mucor circinelloides isolate from yogurt.food-borne pathogens are ongoing problems, and new pathogens are emerging. the impact of fungi, however, is largely underestimated. recently, commercial yogurts contaminated with mucor circinelloides were sold, and >200 consumers became ill with nausea, vomiting, and diarrhea. mucoralean fungi cause the fatal fungal infection mucormycosis, whose incidence has been continuously increasing. in this study, we isolated an m. circinelloides strain from a yogurt container, and multilocus sequence typi ...201425006230
mucor circinelloides whole-cells as a biocatalyst for the production of ethyl esters based on babassu oil.the intracellular lipase production by mucor circinelloides urm 4182 was investigated through a step-by-step strategy to attain immobilized whole-cells with high lipase activity. physicochemical parameters, such as carbon and nitrogen sources, inoculum size and aeration, were studied to determine the optimum conditions for both lipase production and immobilization in polyurethane support. olive oil and soybean peptone were found to be the best carbon and nitrogen sources, respectively, to enhanc ...201424958521
genome sequence and transcriptome analyses of the thermophilic zygomycete fungus rhizomucor miehei.the zygomycete fungi like rhizomucor miehei have been extensively exploited for the production of various enzymes. as a thermophilic fungus, r. miehei is capable of growing at temperatures that approach the upper limits for all eukaryotes. to date, over hundreds of fungal genomes are publicly available. however, zygomycetes have been rarely investigated both genetically and genomically.201424746234
statistical analysis and modeling of pelletized cultivation of mucor circinelloides for microbial lipid accumulation.microbial oil accumulation via oleaginous fungi has some potential benefits because filamentous fungi can form pellets during cell growth and these pellets are easier to harvest from the culture broth than individual cells. this research studied the effect of various culture conditions on the pelletized cell growth of mucor circinelloides and its lipid accumulation. the results showed that cell pelletization was positively correlated to biomass accumulation; however, pellet size was negatively c ...201424549800
role of malic enzyme during fatty acid synthesis in the oleaginous fungus mortierella alpina.the generation of nadph by malic enzyme (me) was postulated to be a rate-limiting step during fatty acid synthesis in oleaginous fungi, based primarily on the results from research focusing on me in mucor circinelloides. this hypothesis is challenged by a recent study showing that leucine metabolism, rather than me, is critical for fatty acid synthesis in m. circinelloides. to clarify this, the gene encoding me isoform e from mortierella alpina was homologously expressed. me overexpression incre ...201424532075
purification and characteristics of an inducible by polycyclic aromatic hydrocarbons nadp(+)-dependent naphthalenediol dehydrogenase (ndd) in mucor circinelloides yr-1.we detected nadp(+)-dependent dihydrodiol dehydrogenase (dd) activity in a cell-free extract from mucor circinelloides yr-1, after high-speed centrifugation. we analyzed the enzymatic activity in the cytosolic fraction by zymograms, as described previously, and eight different dd activity bands were revealed. five constitutive dd activities (dd1-5) were present when glucose was used as carbon source and three inducible activities (ndd, pdd1 and pdd2) when aromatic hydrocarbon compounds were used ...201424530565
expression of xanthophyllomyces dendrorhous cytochrome-p450 hydroxylase and reductase in mucor circinelloides.carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. mucor circinelloides (mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates β-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of β-carotene (i.e. zeaxanthin and β-cryptoxanthin) in low amount. these xanthophylls, together with the ketolated derivatives of β-carotene (such as canthaxanthin, echinenone and ...201525504221
selection of reference genes for quantitative real time rt-pcr during dimorphism in the zygomycete mucor circinelloides.mucor circinelloides is a dimorphic fungal model for studying several biological processes including cell differentiation (yeast-mold transitions) as well as biodiesel and carotene production. the recent release of the first draft sequence of the m. circinelloides genome, combined with the availability of analytical methods to determine patterns of gene expression, such as quantitative reverse transcription-polymerase chain reaction (qrt-pcr), and the development of molecular genetic tools for t ...201525391770
in vitro activity of isavuconazole and comparators against clinical isolates of the mucorales order.the in vitro activity of isavuconazole against mucorales isolates measured by eucast e.def 9.2 and clsi m38-a2 methodologies was investigated in comparison with those of amphotericin b, posaconazole, and voriconazole. seventy-two isolates were included: 12 of lichtheimia corymbifera, 5 of lichtheimia ramosa, 5 of group i and 9 of group ii of mucor circinelloides, 9 of rhizomucor pusillus, 26 of rhizopus microsporus, and 6 of rhizopus oryzae. species identification was confirmed by internal trans ...201526438494
phylogenetic analysis of fungal heterotrimeric g protein-encoding genes and their expression during dimorphism in mucor circinelloides.in fungi, heterotrimeric g proteins are key regulators of biological processes such as mating, virulence, morphology, among others. mucor circinelloides is a model organism for many biological processes, and its genome contains the largest known repertoire of genes that encode putative heterotrimeric g protein subunits in the fungal kingdom: twelve gα (mcgpa1-12), three gβ (mcgpb1-3), and three gγ (mcgpg1-3). phylogenetic analysis of fungal gα showed that they are divided into four distinct grou ...201526615741
distinct rnai pathways in the regulation of physiology and development in the fungus mucor circinelloides.the basal fungus mucor circinelloides has become, in recent years, a valuable model to study rna-mediated gene silencing or rna interference (rnai). serendipitously discovered in the late 1900s, the gene silencing in m. circinelloides is a landscape of consensus and dissents. although similar to other classical fungal models in the basic design of the essential machinery that is responsible for silencing of gene expression, the existence of small rna molecules of different sizes generated during ...201526410030
a zebrafish larval model reveals early tissue-specific innate immune responses to mucor circinelloides.mucormycosis is an emerging fungal infection that is clinically difficult to manage, with increasing incidence and extremely high mortality rates. individuals with diabetes, suppressed immunity or traumatic injury are at increased risk of developing disease. these individuals often present with defects in phagocytic effector cell function. research using mammalian models and phagocytic effector cell lines has attempted to decipher the importance of the innate immune system in host defence agains ...201526398938
complete genome sequence of a high lipid-producing strain of mucor circinelloides wj11 and comparative genome analysis with a low lipid-producing strain cbs 277.49.the genome of a high lipid-producing fungus mucor circinelloides wj11 (36% w/w lipid, cell dry weight, cdw) was sequenced and compared with that of the low lipid-producing strain, cbs 277.49 (15% w/w lipid, cdw), which had been sequenced by joint genome institute. the wj11 genome assembly size was 35.4 mb with a g+c content of 39.7%. the general features of wj11 and cbs 277.49 indicated that they have close similarity at the level of gene order and gene identity. whole genome alignments with mau ...201526352831
(13)c-metabolic flux analysis of lipid accumulation in the oleaginous fungus mucor circinelloides.the oleaginous fungus mucor circinelloides is of industrial interest because it can produce high levels of polyunsaturated fatty acid γ-linolenic acid. m. circinelloides cbs 277.49 is able to accumulate less than 15% of cell dry weight as lipids, while m. circinelloides wj11 can accumulate lipid up to 36%. in order to better understand the mechanisms behind the differential lipid accumulation in these two strains, tracer experiments with (13)c-glucose were performed with the growth of m. circine ...201526318243
screening of phosphorus-accumulating fungi and their potential for phosphorus removal from waste streams.while bacteria have been primarily studied for phosphorus (p) removal in wastewater treatment, fungi and their ability to accumulate intracellular polyphosphate are less investigated. p-accumulating fungal strains were screened from soybean plants and surrounding soil by flask cultivation with potato dextrose broth and kh2po4 in this study. mucor circinelloides was selected for its high efficiency in p removal efficiency and high cellular p content. neisser staining and growth-curve analysis con ...201526280802
comparison of biochemical activities between high and low lipid-producing strains of mucor circinelloides: an explanation for the high oleaginicity of strain wj11.the oleaginous fungus, mucor circinelloides, is one of few fungi that produce high amounts of γ-linolenic acid (gla); however, it usually only produces <25% lipid. nevertheless, a new strain (wj11) isolated in this laboratory can produce lipid up to 36% (w/w) cell dry weight (cdw). we have investigated the potential mechanism of high lipid accumulation in m. circinelloides wj11 by comparative biochemical analysis with a low lipid-producing strain, m. circinelloides cbs 277.49, which accumulates ...201526046932
[isolation of filamentous fungi capable of enhancing sludge dewaterability and study of mechanisms responsible for the sludge dewaterability enhancement].to study the influence of filamentous fungi on the sludge dewaterability is very significant for the development of biological treatment methods for enhancing sludge dewaterability. in this study, filamentous fungi capable of enhancing sludge dewaterability were isolated from sewage sludge and the related mechanisms responsible for the sludge dewaterability enhancement were investigated. a filamentous fungus mucor circinelloides zg-3 was successfully isolated from sludge, and sludge dewaterabili ...201526031090
calcineurin orchestrates dimorphic transitions, antifungal drug responses and host-pathogen interactions of the pathogenic mucoralean fungus mucor circinelloides.calcineurin plays essential roles in virulence and growth of pathogenic fungi and is a target of the natural products fk506 and cyclosporine a. in the pathogenic mucoralean fungus mucor circinelloides, calcineurin mutation or inhibition confers a yeast-locked phenotype indicating that calcineurin governs the dimorphic transition. genetic analysis in this study reveals that two calcineurin a catalytic subunits (out of three) are functionally diverged. homology modeling illustrates modes of resist ...201526010100
identification of fungal ene-reductase activity by means of a functional screening.bioeconomy stresses the need of green processes promoting the development of new methods for biocatalyzed alkene reductions. a functional screening of 28 fungi belonging to ascomycota, basidiomycota, and zygomycota isolated from different habitats was performed to analyze their capability to reduce c=c double bonds towards three substrates (cyclohexenone, α-methylnitrostyrene, and α-methylcinnamaldehyde) with different electron-withdrawing groups, i.e., ketone, nitro, and aldehyde, respectively. ...201525986545
the rnai machinery controls distinct responses to environmental signals in the basal fungus mucor circinelloides.rna interference (rnai) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small rnas (esrnas). in fungi, knowledge of the functions regulated by esrnas has been hampered by lack of clear phenotypes in most mutants affected in the rnai machinery. mutants of mucor circinelloides affected in rnai genes show defects in physiological and developmental processes, thus making mucor an outstanding fungal model for studying e ...201525880254
a non-canonical rna silencing pathway promotes mrna degradation in basal fungi.the increasing knowledge on the functional relevance of endogenous small rnas (esrnas) as riboregulators has stimulated the identification and characterization of these molecules in numerous eukaryotes. in the basal fungus mucor circinelloides, an emerging opportunistic human pathogen, esrnas that regulate the expression of many protein coding genes have been described. these esrnas share common machinery for their biogenesis consisting of an rnase iii endonuclease dicer, a single argonaute prot ...201525875805
when you can't see the wood for the trees. mucor circinelloides: a rare case of primary cutaneous zygomycosis.a patient with refractory diffuse lymphoma treated for pulmonary invasive aspergillosis developed a concomitant primary cutaneous mucormycosis. the mucormycete was identified by sequencing as mucor circinelloides. this case confirms the importance of a rapid pathogen diagnosis in immunocompromised patients and the usefulness of molecular methods for identification of rare fungal species.201525840851
biosynthesis, characterization and enzymatic transesterification of single cell oil of mucor circinelloides--a sustainable pathway for biofuel production.the filamentous fungus mucor circinelloides urm 4182 was tested to determine its ability to produce single-cell oil suitable for obtaining biodiesel. cell growth and lipid accumulation were investigated in a medium containing glucose as the main carbon source. a microwave-assisted ethanol extraction technique (microwave power ⩽200 w, 50-60 °c) was established and applied to lipid extraction from the fungal hyphae to obtain high lipid concentration (44%wt) of the dry biomass, which was considerab ...201525625466
analysis of the effect of soil saprophytic fungi on the eggs of baylisascaris procyonis.baylisascaris procyonis is a soil-transmitted helminth mainly found in raccoons (procyon lotor) which can also affect other domestic and sylvatic animals, as well as humans, when the eggs released in the feces of parasitized raccoons are accidentally ingested. three assays have been conducted to assess the effect of three saprophytic fungi, mucor circinelloides, paecilomyces lilacinus, and verticillium sp., on the eggs of b. procyonis. firstly, their ovicidal effect was in vitro ascertained by p ...201525828813
efficacy of a coating composed of chitosan from mucor circinelloides and carvacrol to control aspergillus flavus and the quality of cherry tomato fruits.cherry tomato (lycopersicon esculentum mill) fruits are susceptible to contamination by aspergillus flavus, which may cause the development of fruit rot and significant postharvest losses. currently there are significant drawbacks for the use of synthetic fungicides to control pathogenic fungi in tomato fruits, and it has increased the interest in exploring new alternatives to control the occurrence of fungal infections in these fruits. this study evaluated the efficacy of chitosan (chi) from mu ...201526257717
the mycobiota of air inside and outside the meju fermentation room and the origin of meju fungi.the fungi on meju are known to play an important role as degrader of macromolecule of soybeans. in order to elucidate the origin of fungi on traditional meju, mycobiota of the air both inside and outside traditional meju fermentation rooms was examined. from 11 samples of air collected from inside and outside of 7 meju fermentation rooms, 37 genera and 90 species of fungi were identified. in outside air of the fermentation room, cladosporium sp. and cladosporium cladosporioides were the dominant ...201526539041
evaluation of aerial microbial pollutants in al-haram al-nabawi during pilgrimage of 2013.al-madinah al-munawwarah is the second holiest site in islam. the possibility of new emerging microbes is valid due to the increased number of pilgrims. the objectives of the current study were to estimate the numbers of fungi and bacteria inside and outside al-haram al-nabawi and to find whether new bacterial and fungal species have emerged compared to previous studies. air samples were collected twice a day from 12 spots and four directions during the pilgrim year of 2013 for four consecutive ...201528053593
identification of a critical determinant that enables efficient fatty acid synthesis in oleaginous fungi.microorganisms are valuable resources for lipid production. what makes one microbe but not the other able to efficiently synthesize and accumulate lipids is poorly understood. in the present study, global gene expression prior to and after the onset of lipogenesis was determined by transcriptomics using the oleaginous fungus mortierella alpina as a model system. a core of 23 lipogenesis associated genes was identified and their expression patterns shared a high similarity among oleaginous microb ...201526059272
the interaction of fungi with the environment orchestrated by rnai.the fungal kingdom has been key in the investigation of the biogenesis and function of small rnas (srnas). the discovery of phenomena such as quelling in neurospora crassa represents pioneering work in the identification of the main elements of the rna interference (rnai) machinery. recent discoveries in the regulatory mechanisms in some yeast and filamentous fungi are helping us reach a deeper understanding of the transcriptional and post-transcriptional gene-silencing mechanisms involved in ge ...201626932186
metagenomics analysis of microbial communities associated with a traditional rice wine starter culture (xaj-pitha) of assam, india.this is the first report on the microbial diversity of xaj-pitha, a rice wine fermentation starter culture through a metagenomics approach involving illumine-based whole genome shotgun (wgs) sequencing method. metagenomic dna was extracted from rice wine starter culture concocted by ahom community of assam and analyzed using a miseq(®) system. a total of 2,78,231 contigs, with an average read length of 640.13 bp, were obtained. data obtained from the use of several taxonomic profiling tools were ...201628330225
antifungal activity of rhamnolipids against dimorphic fungi.in this paper, rhamnolipids are investigated, for the first time, for their feasibility for inhibiting dimorphic fungi. rhamnolipids were found to effectively inhibit a dimorphic fungus isolated from tomato plants which was identified as mucor circinelloides according to characterizations by morphologies as well as 28s rdna sequences. rhamnolipids markedly reduced growth of this fungus in both the yeast-like form and the filamentous form. such an inhibitive effect was similarly obtained with ver ...201627666589
new insight into microbial diversity and functions in traditional vietnamese alcoholic fermentation.the roles of microorganisms in traditional alcoholic fermentation are often assumed based on abundance in the starter and activity in pure culture. there is a serious lack of hard evidence on the behavior and activity of individual microbial species during the actual fermentation process. in this study, microbial succession and metabolite changes during 7days of traditional vietnamese alcoholic fermentation were monitored. special attention was devoted to starch degradation. in total, 22 microbi ...201627236224
antifungal and cytotoxic assessment of lapachol derivatives produced by fungal biotransformation.in the screening for biological active compounds, the biotransformation processes catalyzed by filamentous fungi are useful because they can provide information about the possible appearance of toxic metabolites after oral administration and also generate new leads. in this paper, biotransformation of lapachol (1) by three fungal strains, mucor circinelloides nrrl3631, botrytis cinerea uca992 and botrytis cinerea 2100, has been investigated for the first time. lapachol (1) was biotransformed int ...201626996030
search for fungi-specific metabolites of four model drugs in postmortem blood as potential indicators of postmortem fungal metabolism.fungi colonizing cadavers are capable of drug metabolism and may thus change the metabolite pattern or concentration of drugs in forensic postmortem samples. the purpose of this study was to check for the presence of such changes by searching fungi-specific metabolites of four model drugs (amitriptyline, metoprolol, mirtazapine, and zolpidem) in decomposed postmortem blood samples from 33 cases involving these drugs. after isolation and identification of fungal strains present in the samples, ea ...201627022860
coprophilous mucorales (ex zygomycota) from three areas in the semi-arid of pernambuco, brazil.mucorales comprises fungi commonly isolated as saprobes from soil, dung, stored grains and plants. although these fungi have been studied in several countries, there are relatively a few reports of them in semi-arid areas. therefore, the aims of the present study were to assess and compare the mucorales communities in dung from different species and breeds of herbivores in the semi-arid of pernambuco, based on the frequency of occurrence and species richness of these fungi. samples of dung colle ...201627769884
multiple isoforms for the catalytic subunit of pka in the basal fungal lineage mucor circinelloides.protein kinase a (pka) activity is involved in dimorphism of the basal fungal lineage mucor. from the recently sequenced genome of mucor circinelloides we could predict ten catalytic subunits of pka. from sequence alignment and structural prediction we conclude that the catalytic core of the isoforms is conserved, and the difference between them resides in their amino termini. this high number of isoforms is maintained in the subdivision mucoromycotina. each paralogue, when compared to the ones ...201627890086
feeding horses with industrially manufactured pellets with fungal spores to promote nematode integrated control.the usefulness of pellets industrially manufactured with spores of parasiticide fungi as a contribution to integrated nematode control was assessed in grazing horses throughout sixteen months. two groups of 7 pura raza galega autochthonous horses (g-t and g-p) were dewormed pour-on (1mg ivermectin/kg bw) at the beginning of the trial, and other group (g-c) remained untreated. the g-p was provided daily with commercial pellets to which was added a mixture of fungal spores during the industrial ma ...201627809976
rna interference in fungi: retention and loss.rna interference (rnai) is a mechanism conserved in eukaryotes, including fungi, that represses gene expression by means of small noncoding rnas (srnas) of about 20 to 30 nucleotides. its discovery is one of the most important scientific breakthroughs of the past 20 years, and it has revolutionized our perception of the functioning of the cell. initially described and characterized in neurospora crassa, the rnai is widespread in fungi, suggesting that it plays important functions in the fungal k ...201628087943
fungal evolution: mucor and phycomyces see double.newly available genome sequences of two mucoralean fungi, phycomyces blakesleeanus and mucor circinelloides, provide evidence for an ancient whole-genome duplication that contributed to the generation of expanded gene families. these fungi have robust responses to light that can be correlated with the expansion of gene networks involved in light sensing and signaling.201627554660
lipase genes in mucor circinelloides: identification, sub-cellular location, phylogenetic analysis and expression profiling during growth and lipid accumulation.lipases or triacylglycerol hydrolases are widely spread in nature and are particularly common in the microbial world. the filamentous fungus mucor circinelloides is a potential lipase producer, as it grows well in triacylglycerol-contained culture media. so far only one lipase from m. circinelloides has been characterized, while the majority of lipases remain unknown in this fungus. in the present study, 47 potential lipase genes in m. circinelloides wj11 and 30 potential lipase genes in m. circ ...201627535142
[characterization of a malic enzyme isoform v from mucor circinelloides].we aimed at characterizing a malic enzyme isoform v from mucor circinelloides.201627373079
a new regulatory mechanism controlling carotenogenesis in the fungus mucor circinelloides as a target to generate β-carotene over-producing strains by genetic engineering.carotenoids are natural pigments with antioxidant properties that have important functions in human physiology and must be supplied through the diet. they also have important industrial applications as food colourants, animal feed additives and nutraceuticals. some of them, such as β-carotene, are produced on an industrial scale with the use of microorganisms, including fungi. the mucoral blakeslea trispora is used by the industry to produce β-carotene, although optimisation of production by mol ...201627266994
expansion of signal transduction pathways in fungi by extensive genome duplication.plants and fungi use light and other signals to regulate development, growth, and metabolism. the fruiting bodies of the fungus phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. the related fungus mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. understanding how these organisms respond to enviro ...201627238284
characterization and control of mucor circinelloides spoilage in yogurt.consumer confidence in the food industry is severely affected by large-scale spoilage incidents. however, relatively little research exists on spoilage potential of members of the fungal subphylum mucormycotina (e.g. mucor), which includes dimorphic spoilage organisms that can switch between a yeast-like and hyphal phase depending on environmental conditions. the presence of mucor circinelloides in yogurt may not cause spoilage, but growth and subsequent changes in quality (e.g. container bloati ...201627085035
vaginal colonisation by mucor circinelloides. case report with cytopathology, molecular sequencing and epidemiology. 201627000466
genome-scale metabolic modeling of mucor circinelloides and comparative analysis with other oleaginous species.we present a novel genome-scale metabolic model iwv1213 of mucor circinelloides, which is an oleaginous fungus for industrial applications. the model contains 1213 genes, 1413 metabolites and 1326 metabolic reactions across different compartments. we demonstrate that iwv1213 is able to accurately predict the growth rates of m. circinelloides on various nutrient sources and culture conditions using flux balance analysis and phenotypic phase plane analysis. comparative analysis of three oleaginous ...201626911256
proteomics analysis of high lipid-producing strain mucor circinelloides wj11: an explanation for the mechanism of lipid accumulation at the proteomic level.the oleaginous fungus, mucor circinelloides, is attracting considerable interest as it produces oil rich in γ-linolenic acid. nitrogen (n) deficiency is a common strategy to trigger the lipid accumulation in oleaginous microorganisms. although a simple pathway from n depletion in the medium to lipid accumulation has been elucidated at the enzymatic level, global changes at protein levels upon n depletion have not been investigated. in this study, we have systematically analyzed the changes at th ...201626867592
rnai pathways in mucor: a tale of proteins, small rnas and functional diversity.the existence of an rna-mediated silencing mechanism in the opportunistic fungal pathogen mucor circinelloides was first described in the early 2000. since then, mucor has reached an outstanding position within the fungal kingdom as a model system to achieve a deeper understanding of regulation of endogenous functions by the rna interference (rnai) machinery. m. circinelloides combines diverse components of its rnai machinery to carry out functions not only limited to the defense against invasiv ...201626593631
role of malate transporter in lipid accumulation of oleaginous fungus mucor circinelloides.fatty acid biosynthesis in oleaginous fungi requires the supply of reducing power, nadph, and the precursor of fatty acids, acetyl-coa, which is generated in the cytosol being produced by atp: citrate lyase which requires citrate to be, transported from the mitochondrion by the citrate/malate/pyruvate transporter. this transporter, which is within the mitochondrial membrane, transports cytosolic malate into the mitochondrion in exchange for mitochondrial citrate moving into the cytosol (fig. 1). ...201626512004
detection of the dimorphic phases of mucor circinelloides in blood cultures from an immunosuppressed female.mucormycosis fungemia is rarely documented since blood cultures are nearly always negative. we describe a case of mucor circinelloides fungemia in a patient with a history of a sinus infection, sarcoidosis, and igg deficiency. the identity of the isolate was supported by its microscopic morphology and its ability to convert into yeast forms under anaerobic conditions. the early detection, initiation of liposomal amphotericin b treatment, and reversal of underlying predisposing risk factors resul ...201627777804
development of a plasmid free crispr-cas9 system for the genetic modification of mucor circinelloides.mucor circinelloides and other members of mucorales are filamentous fungi, widely used as model organisms in basic and applied studies. although genetic manipulation methods have been described for some mucoral fungi, construction of stable integrative transformants by homologous recombination has remained a great challenge in these organisms. in the present study, a plasmid free crispr-cas9 system was firstly developed for the genetic modification of a mucoral fungus. the described method offer ...201729196656
intrinsic short-tailed azole resistance in mucormycetes is due to an evolutionary conserved aminoacid substitution of the lanosterol 14α-demethylase.mucormycoses are emerging and potentially lethal infections. an increase of breakthrough infections has been found in cohorts receiving short-tailed azoles prophylaxis (e.g. voriconazole (vcz)). although vcz is ineffective in vitro and in vivo, long-tailed triazoles such as posaconazole remain active against mucormycetes. our goal was to validate the molecular mechanism of resistance to short-tailed triazoles in mucorales. the paralogous cytochrome p450 genes (cyp51 f1 and cyp51 f5) of rhizopus ...201729162893
antimicrobial activity of water and acetone extracts of some eucomis taxa.to evaluate the antibacterial and antifungal properties of acetone and water extracts of selected eucomis taxa.201729080618
occurrence and species distribution of pathogenic mucorales in unselected soil samples from france.mucormycosis is a life-threatening invasive fungal disease that affects a variety of patient groups. although mucorales are mostly opportunistic pathogens originating from soil or decaying vegetation, there are currently few data on prevalence of this group of fungi in the environment. the aim of the present study was to assess the prevalence and diversity of species of mucorales from soil samples collected in france. two grams of soil were homogenized in sterile saline and plated on sabouraud d ...201728992337
biosurfactant production by mucor circinelloides on waste frying oil and possible uses in crude oil remediation.biosurfactants are biocompatible surface active agents which many microorganisms produce. this study investigated the production of biosurfactants by mucor circinelloides. the effects of different factors on biosurfactant production, including carbon sources and concentrations, nitrogen sources, and iron (ii) concentration, were studied and the optimum condition determined. finally, the strain's ability to remove the crude oil and its relationship with biosurfactant production was evaluated. the ...201728991787
antioxidants potential of the filamentous fungi (mucor circinelloides).three important strains of mucorcircinelloides grown in complete and minimal media for specified period (72 h, 120 h and 168 h) under submerged fermentation conditions were investigated for their potential antioxidants/secondary metabolite production. all mycelial extracts demonstrated effective antioxidant activities in terms of β-carotene/linoleic acid bleaching, radical scavenging, reduction of metal ions and chelating abilities against ferrous ions. different extraction methods and solvent s ...201728991177
old yellow enzyme homologues in mucor circinelloides: expression profile and biotransformation.the reduction of c=c double bond, a key reaction in organic synthesis, is mostly achieved by traditional chemical methods. therefore, the search for enzymes capable of performing this reaction is rapidly increasing. old yellow enzymes (oyes) are flavin-dependent oxidoreductases, initially isolated from saccharomyces pastorianus. in this study, the presence and activation of putative oye enzymes was investigated in the filamentous fungus mucor circinelloides, which was previously found to mediate ...201728935878
assessing the potential of fatty acids produced by filamentous fungi as feedstock for biodiesel production.increased costs and limited availability of traditional lipid sources for biodiesel production encourages researchers to find more sustainable feedstock at low prices. microbial lipid stands out as feedstock replacement for vegetable oil to convert fatty acid esters (faes). in this study, the potential of three isolates of filamentous fungi (mucor circinelloides urm 4140, m. hiemalis urm 4144 and penicillium citrinum urm 4126) has been assessed as single-cell oil (sco) producers. m. circinelloid ...201728857682
scale-up of puf-immobilized fungal chitosanase-lipase preparation production.mucor circinelloides ibt-83 mycelium that exhibits both lipolytic (al) and chitosanolytic (ach) activities was immobilized into polyurethane foam (puf) in a 30 l laboratory fermenter. the process of immobilization was investigated in terms of: the carrier porosity, its type, amount and shape, location inside the fermenter, mixing and aeration parameters during the culture, as well as downstream processing operations. the selected conditions allowed for immobilization of approx. 7 g of defatted a ...201728816606
improved γ-linolenic acid production in mucor circinelloides by homologous overexpressing of delta-12 and delta-6 desaturases.γ-linolenic acid (gla) is important because of its nutritional value and medicinal applications. although the biosynthetic pathways of some plant and microbial gla have been deciphered, current understanding of the correlation between desaturases and gla synthesis in oleaginous fungi is incomplete. in previous work, we found that a large amount of oleic acid (oa) had not been converted to linoleic acid (la) or gla in mucor circinelloides cbs 277.49, which may be due to inadequate activities of t ...201728637506
comparative proteome analysis between high lipid-producing strain mucor circinelloides wj11 and low lipid-producing strain cbs 277.49.mucor circinelloides is one of few oleaginous fungi that produces a useful oil rich in γ-linolenic acid, but it usually only produces <25% total lipid. nevertheless, we isolated a new strain wj11 that can produce up to 36% lipid of cell dry weight. in this study, we have systematically analyzed the global changes in protein levels between the high lipid-producing strain wj11 and the low lipid-producing strain cbs 277.49 (15%, lipid/cell dry weight) at lipid accumulation phase through comparative ...201728557429
construction of a recyclable genetic marker and serial gene deletions in the human pathogenic mucorales mucor circinelloides.mucor circinelloides is a human pathogen, biofuel producer, and model system that belongs to a basal fungal lineage; however, the genetics of this fungus are limited. in contrast to ascomycetes and basidiomycetes, basal fungal lineages have been understudied. this may be caused by a lack of attention given to these fungi, as well as limited tools for genetic analysis. nonetheless, the importance of these fungi as pathogens and model systems has increased. m. circinelloides is one of a few geneti ...201728476909
enhanced bioremediation of lead-contaminated soil by solanum nigrum l. with mucor circinelloides.strain selected from mine tailings in anshan for pb bioremediation was characterized at the genetic level by internal transcribed spacer (its) sequencing. results revealed that the strain belongs to mucor circinelloides. bioremediation of lead-contaminated soil was conducted using solanum nigrum l. combined with m. circinelloides. the removal efficacy was in the order microbial/phytoremediation > phytoremediation > microbial remediation > control. the bioremediation rates were 58.6, 47.2, and 40 ...201728251531
ftir spectroscopy for evaluation and monitoring of lipid extraction efficiency for oleaginous fungi.to assess whether fourier transform infrared (ftir) spectroscopy could be used to evaluate and monitor lipid extraction processes, the extraction methods of folch, bligh and lewis were used. biomass of the oleaginous fungi mucor circinelloides and mortierella alpina were employed as lipid-rich material for the lipid extraction. the presence of lipids was determined by recording infrared spectra of all components in the lipid extraction procedure, such as the biomass before and after extraction, ...201728118388
generation of lycopene-overproducing strains of the fungus mucor circinelloides reveals important aspects of lycopene formation and accumulation.to generate lycopene-overproducing strains of the fungus mucor circinelloides with interest for industrial production and to gain insight into the catalytic mechanism of lycopene cyclase and regulatory process during lycopene overaccumulation.201727909822
monotherapy or combination therapy of isavuconazole and micafungin for treating murine mucormycosis.previously we demonstrated the benefit of isavuconazole in treating murine mucormycosis due to rhizopus. we wanted to determine the efficacy of isavuconazole in treating murine mucormycosis caused by mucor, the second most common cause of the disease. furthermore, because we previously determined that rhizopus possesses the target enzyme for echinocandins and micafungin has activity against murine mucormycosis, we compared the activity of combination therapy (isavuconazole + micafungin) with pla ...201727798213
rnai-based functional genomics identifies new virulence determinants in mucormycosis.mucorales are an emerging group of human pathogens that are responsible for the lethal disease mucormycosis. unfortunately, functional studies on the genetic factors behind the virulence of these organisms are hampered by their limited genetic tractability, since they are reluctant to classical genetic tools like transposable elements or gene mapping. here, we describe an rnai-based functional genomic platform that allows the identification of new virulence factors through a forward genetic appr ...201728107502
a non-canonical rna degradation pathway suppresses rnai-dependent epimutations in the human fungal pathogen mucor circinelloides.mucorales are a group of basal fungi that includes the casual agents of the human emerging disease mucormycosis. recent studies revealed that these pathogens activate an rnai-based pathway to rapidly generate drug-resistant epimutant strains when exposed to stressful compounds such as the antifungal drug fk506. to elucidate the molecular mechanism of this epimutation pathway, we performed a genetic analysis in mucor circinelloides that revealed an inhibitory role for the non-canonical rdrp-depen ...201728339467
anthropogenic impact on environmental filamentous fungi communities along the mediterranean littoral.we hypothesised that anthropogenic influences impact the filamentous fungi community structure and that particular species or species patterns might serve as markers to characterise ecosystems. this study aimed to describe the filamentous fungi community structure in various biotopes along the mediterranean shore that were exposed to various levels of anthropogenic influence. we sampled filamentous fungi from yellow-legged gull faecal samples at five study sites along the mediterranean littoral ...201728276198
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