Molecular detection and identification of zygomycetes species from paraffin-embedded tissues in a murine model of disseminated zygomycosis: a collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) evaluation.

The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.

Microbial diversity in the municipal composting process and development of detection methods

Composting refers to aerobic degradation of organic material and is one of the main waste treatment methods used in Finland for treating separated organic waste. The composting process allows converting organic waste to a humus-like end product which can be used to increase the organic matter in agricultural soils, in gardening, or in landscaping. Microbes play a key role as degraders during the composting-process, and the microbiology of composting has been studied for decades, but there are still open questions regarding the microbiota in industrial composting processes. It is known that with the traditional, culturing-based methods only a small fraction, below 1%, of the species in a sample is normally detected. In recent years an immense diversity of bacteria, fungi and archaea has been found to occupy many different environments. Therefore the methods of characterising microbes constantly need to be developed further. In this thesis the presence of fungi and bacteria in full-scale and pilot-scale composting processes was characterised with cloning and sequencing. Several clone libraries were constructed and altogether nearly 6000 clones were sequenced. The microbial communities detected in this study were found to differ from the compost microbes observed in previous research with cultivation based methods or with molecular methods from processes of smaller scale, although there were similarities as well. The bacterial diversity was high. Based on the non-parametric coverage estimations, the number of bacterial operational taxonomic units (OTU) in certain stages of composting was over 500. Sequences similar to Lactobacillus and Acetobacteria were frequently detected in the early stages of drum composting. In tunnel stages of composting the bacterial community comprised of Bacillus, Thermoactinomyces, Actinobacteria and Lactobacillus. The fungal diversity was found to be high and phylotypes similar to yeasts were abundantly found in the full-scale drum and tunnel processes. In addition to phylotypes similar to Candida, Pichia and Geotrichum moulds from genus Thermomyces and Penicillium were observed in tunnel stages of composting. Zygomycetes were detected in the pilot-scale composting processes and in the compost piles. In some of the samples there were a few abundant phylotypes present in the clone libraries that masked the rare ones. The rare phylotypes were of interest and a method for collecting them from clone libraries for sequencing was developed. With negative selection of the abundant phylotyps the rare ones were picked from the clone libraries. Thus 41% of the clones in the studied clone libraries were sequenced. Since microbes play a central role in composting and in many other biotechnological processes, rapid methods for characterization of microbial diversity would be of value, both scientifically and commercially. Current methods, however, lack sensitivity and specificity and are therefore under development. Microarrays have been used in microbial ecology for a decade to study the presence or absence of certain microbes of interest in a multiplex manner. The sequence database collected in this thesis was used as basis for probe design and microarray development. The enzyme assisted detection method, ligation-detection-reaction (LDR) based microarray, was adapted for species-level detection of microbes characteristic of each stage of the composting process. With the use of a specially designed control probe it was established that a species specific probe can detect target DNA representing as little as 0.04% of total DNA in a sample. The developed microarray can be used to monitor composting processes or the hygienisation of the compost end product. A large compost microbe sequence dataset was collected and analysed in this thesis. The results provide valuable information on microbial community composition during industrial scale composting processes. The microarray method was developed based on the sequence database collected in this study. The method can be utilised in following the fate of interesting microbes during composting process in an extremely sensitive and specific manner. The platform for the microarray is universal and the method can easily be adapted for studying microbes from environments other than compost.

Ferrochelatase is a conserved downstream target of the blue light-sensing White collar complex in fungi.

Light is a universal signal perceived by organisms, including fungi, in which light regulates common and unique biological processes depending on the species. Previous research has established that conserved proteins, originally called White collar 1 and 2 from the ascomycete Neurospora crassa, regulate UV/blue light sensing. Homologous proteins function in distant relatives of N. crassa, including the basidiomycetes and zygomycetes, which diverged as long as a billion years ago. Here we conducted microarray experiments on the basidiomycete fungus Cryptococcus neoformans to identify light-regulated genes. Surprisingly, only a single gene was induced by light above the commonly used twofold threshold. This gene, HEM15, is predicted to encode a ferrochelatase that catalyses the final step in haem biosynthesis from highly photoreactive porphyrins. The C. neoformans gene complements a Saccharomyces cerevisiae hem15Delta strain and is essential for viability, and the Hem15 protein localizes to mitochondria, three lines of evidence that the gene encodes ferrochelatase. Regulation of HEM15 by light suggests a mechanism by which bwc1/bwc2 mutants are photosensitive and exhibit reduced virulence. We show that ferrochelatase is also light-regulated in a white collar-dependent fashion in N. crassa and the zygomycete Phycomyces blakesleeanus, indicating that ferrochelatase is an ancient target of photoregulation in the fungal kingdom.

Evolution of the sex-related locus and genomic features shared in microsporidia and fungi.

BACKGROUND: Microsporidia are obligate intracellular, eukaryotic pathogens that infect a wide range of animals from nematodes to humans, and in some cases, protists. The preponderance of evidence as to the origin of the microsporidia reveals a close relationship with the fungi, either within the kingdom or as a sister group to it. Recent phylogenetic studies and gene order analysis suggest that microsporidia share a particularly close evolutionary relationship with the zygomycetes. METHODOLOGY/PRINCIPAL FINDINGS: Here we expanded this analysis and also examined a putative sex-locus for variability between microsporidian populations. Whole genome inspection reveals a unique syntenic gene pair (RPS9-RPL21) present in the vast majority of fungi and the microsporidians but not in other eukaryotic lineages. Two other unique gene fusions (glutamyl-prolyl tRNA synthetase and ubiquitin-ribosomal subunit S30) that are present in metazoans, choanoflagellates, and filasterean opisthokonts are unfused in the fungi and microsporidians. One locus previously found to be conserved in many microsporidian genomes is similar to the sex locus of zygomycetes in gene order and architecture. Both sex-related and sex loci harbor TPT, HMG, and RNA helicase genes forming a syntenic gene cluster. We sequenced and analyzed the sex-related locus in 11 different Encephalitozoon cuniculi isolates and the sibling species E. intestinalis (3 isolates) and E. hellem (1 isolate). There was no evidence for an idiomorphic sex-related locus in this Encephalitozoon species sample. According to sequence-based phylogenetic analyses, the TPT and RNA helicase genes flanking the HMG genes are paralogous rather than orthologous between zygomycetes and microsporidians. CONCLUSION/SIGNIFICANCE: The unique genomic hallmarks between microsporidia and fungi are independent of sequence based phylogenetic comparisons and further contribute to define the borders of the fungal kingdom and support the classification of microsporidia as unusual derived fungi. And the sex/sex-related loci appear to have been subject to frequent gene conversion and translocations in microsporidia and zygomycetes.

A preliminary survey of micro-organisms in the gut and pellets of a tropical millipede Doratogonus uncinatus Attems (Diplopoda, Spirostreptida, Spirostreptidae).

Millipede gut microbiology and decomposition of faecal pellets over a period of eight weeks were studied in the laboratory. Bacterial numbers, carbon and nitrogen content, pH and weight loss were monitored. Heterotrophic bacteria were the most abundant and reached a peak in the first two weeks of decomposition. The amount of carbon was constant while ammonium nitrogen decreased from 1.51 % to 0.03 % after eight weeksThe pH of the pellets was slightly acidic and did not change much during the course of decomposition. A succession of micro-organisms was observed on decomposing pellets. Zygomycetes were replaced by Ascomycetes after 20 days of decomposition. Decomposition was significantly affected by temperature. The rate of decomposition was highest at 35[degree]C .

Cloning, sequencing and characterization of a chitin synthase gene fragment from the fungus Phascolomyces articulosus /

Phascolomyces articulosus genomic DNA was isolated from 48 h old hyphae and was used for amplification of a chitin synthase fragment by the polymerase chain reaction method. The primers used in the amplification corresponded to two widely conserved amino acid regions found in chitin synthases of many fimgi. Amphfication resulted in four bands (820, 900, 1000 and 1500 bp, approximately) as visualized in a 1.2% agarose gel. The lowest band (820 bp) was selected as a candidate for chitin synthase because most amplified regions from other fimgi so far exhibited similar sizes (600-750 bp). The selected fragment was extracted from the gel and cloned in the Hinc n site of pUC19. The derived plasmid and insert were designated ^\5C\9'PaCHS and PaCHS respectively. The plasmid pUC19-PaC/fS was digested by several restriction enzymes and was found to contain BamHl and HincU sites. Sequencing of PaCHS revealed two intron sequences and a total open reading frame of 200 amino acids. The derived polypeptide was compared with other related sequences from the EMBL database (Heidelberg, Germany) and was matched to 36 other fiilly or partially sequenced fimgal chitin synthase genes. The closest resemblance was with two genes (74.5% and 73.1% identity) from Rhizopus oligosporus. Southern hybridization with the cloned fragment as a probe to the PCR reaction showed a strong signal at the fragment selected for cloning and weaker signals at the other two fragments. Southern hybridization with partially digested Phascolomyces articulosus genomic DNA showed a single band. The amino acid sequence was compared with sequences from other chitin synthase gene classes using the CLUSTALW program. The chitin synthase fragment from Phascolomyces articulosus was initially grouped in class n along with chitin synthase fragments from Rhizopus oligosporus and Phycomyces blakesleeanus which also belong to the same class, Zygomycetes. Bootstrap analysis using the neighbor-joining method available by CLUSTALW verified such classification. Comparison of PaCHS revealed conservation of intron positions that are characteristic of chitin synthase gene fragments of zygomycetous fungi.

Immuno-gluorescence study of five zygomycetous fungi with two chitin-binding probes

A polyclonal antiserum was prepared against a purified microsomal chitinase isolated from the fungus Choanephora cucurbitarum. Indirect immunofluorescence was used to localize chitinase at various developmental stages of five zygomycetous fungi and during abiotrophic mycoparasite interaction with a susceptible and resistant host. This was compared to localization of oligomers of N-acetylglucosamine with the lectin wheat germ agglutinin (WGA). Dotimmunoblot and Western blot techniques revealed that the anti-serum reacted strongly with the antigen from which it was derived. Cross reactivity of the antiserum was found with WGA and another chitin binding lectin, Phyto/acca americana agglutinin (PAA). Immuno-fluorescence results showed the direct involvement of chitinase in spore swelling, germination, sporangium development and response during mechanical injury. There appeared to be no involvement of chitinase during apical hyphal growth or new branch initiation in any of the fungi tested despite mild proteolysis and permeabilization of the cell surface prior to labelling. Binding with WGA revealed similar patterns of fluorescence to that of chitinase localization but differed by showing fluorescence and therefore chitin localization at the apex and new branch initiation when tested at different developmental stages. There was no difference between chitinase localization and binding with WGA in a susceptible host and resistant host challenged with the mycoparasite, Piptocephalis virginiana. Differences in binding ability of antichitinase and lectin WGA suggests that the latter is not a suitable indicator for indirect localization of the lytic enzyme, chitinase.

A phylogenomics approach to resolving fungal evolution, and phylogenetic method development

Bien que les champignons soient régulièrement utilisés comme modèle d'étude des systèmes eucaryotes, leurs relations phylogénétiques soulèvent encore des questions controversées. Parmi celles-ci, la classification des zygomycètes reste inconsistante. Ils sont potentiellement paraphylétiques, i.e. regroupent de lignées fongiques non directement affiliées. La position phylogénétique du genre Schizosaccharomyces est aussi controversée: appartient-il aux Taphrinomycotina (précédemment connus comme archiascomycetes) comme prédit par l'analyse de gènes nucléaires, ou est-il plutôt relié aux Saccharomycotina (levures bourgeonnantes) tel que le suggère la phylogénie mitochondriale? Une autre question concerne la position phylogénétique des nucléariides, un groupe d'eucaryotes amiboïdes que l'on suppose étroitement relié aux champignons. Des analyses multi-gènes réalisées antérieurement n'ont pu conclure, étant donné le choix d'un nombre réduit de taxons et l'utilisation de six gènes nucléaires seulement. Nous avons abordé ces questions par le biais d'inférences phylogénétiques et tests statistiques appliqués à des assemblages de données phylogénomiques nucléaires et mitochondriales. D'après nos résultats, les zygomycètes sont paraphylétiques (Chapitre 2) bien que le signal phylogénétique issu du jeu de données mitochondriales disponibles est insuffisant pour résoudre l'ordre de cet embranchement avec une confiance statistique significative. Dans le Chapitre 3, nous montrons à l'aide d'un jeu de données nucléaires important (plus de cent protéines) et avec supports statistiques concluants, que le genre Schizosaccharomyces appartient aux Taphrinomycotina. De plus, nous démontrons que le regroupement conflictuel des Schizosaccharomyces avec les Saccharomycotina, venant des données mitochondriales, est le résultat d'un type d'erreur phylogénétique connu: l'attraction des longues branches (ALB), un artéfact menant au regroupement d'espèces dont le taux d'évolution rapide n'est pas représentatif de leur véritable position dans l'arbre phylogénétique. Dans le Chapitre 4, en utilisant encore un important jeu de données nucléaires, nous démontrons avec support statistique significatif que les nucleariides constituent le groupe lié de plus près aux champignons. Nous confirmons aussi la paraphylie des zygomycètes traditionnels tel que suggéré précédemment, avec support statistique significatif, bien que ne pouvant placer tous les membres du groupe avec confiance. Nos résultats remettent en cause des aspects d'une récente reclassification taxonomique des zygomycètes et de leurs voisins, les chytridiomycètes. Contrer ou minimiser les artéfacts phylogénétiques telle l'attraction des longues branches (ALB) constitue une question récurrente majeure. Dans ce sens, nous avons développé une nouvelle méthode (Chapitre 5) qui identifie et élimine dans une séquence les sites présentant une grande variation du taux d'évolution (sites fortement hétérotaches - sites HH); ces sites sont connus comme contribuant significativement au phénomène d'ALB. Notre méthode est basée sur un test de rapport de vraisemblance (likelihood ratio test, LRT). Deux jeux de données publiés précédemment sont utilisés pour démontrer que le retrait graduel des sites HH chez les espèces à évolution accélérée (sensibles à l'ALB) augmente significativement le support pour la topologie « vraie » attendue, et ce, de façon plus efficace comparée à d'autres méthodes publiées de retrait de sites de séquences. Néanmoins, et de façon générale, la manipulation de données préalable à l'analyse est loin d’être idéale. Les développements futurs devront viser l'intégration de l'identification et la pondération des sites HH au processus d'inférence phylogénétique lui-même.

Species 2000 & ITIS Catalogue of Life, 2013 Annual Checklist

This release of the Catalogue of Life contains contributions from 132 databases with information on 1,352,112 species, 114,069 infraspecific taxa and also includes 928,147 synonyms and 408,689 common names covering the following groups: Viruses • Viruses and Subviral agents from ICTV_MSL UPDATED! Bacteria and Archaea from BIOS Chromista • Chromistan fungi from Species Fungorum Protozoa • Major groups from ITIS Regional, • Ciliates from CilCat, • Polycystines from WoRMS Polycystina UPDATED!, • Protozoan fungi from Species Fungorum and Trichomycetes database • Slime moulds from Nomen.eumycetozoa.com Fungi • Various taxa in whole or in part from CABI Bioservices databases (Species Fungorum, Phyllachorales, Rhytismatales, Saccharomycetes and Zygomycetes databases) and from three other databases covering Xylariaceae, Glomeromycota, Trichomycetes, Dothideomycetes • Lichens from LIAS UPDATED! Plantae (Plants) • Mosses from MOST • Liverworts and hornworts from ELPT • Conifers from Conifer Database • Cycads and 6 flowering plant families from IOPI-GPC, and 99 families from WCSP • Plus individual flowering plants families from AnnonBase, Brassicaceae, ChenoBase, Droseraceae Database, EbenaBase, GCC UPDATED!, ILDIS UPDATED!, LecyPages, LHD, MELnet UPDATED!, RJB Geranium, Solanaceae Source, Umbellifers. Animalia (Animals) • Marine groups from URMO, ITIS Global, Hexacorals, ETI WBD (Euphausiacea), WoRMS: WoRMS Asteroidea UPDATED!, WoRMS Bochusacea UPDATED!, WoRMS Brachiopoda UPDATED!, WoRMS Brachypoda UPDATED!, WoRMS Brachyura UPDATED!, WoRMS Bryozoa UPDATED!, WoRMS Cestoda NEW!, WoRMS Chaetognatha UPDATED!, WoRMS Cumacea UPDATED!, WoRMS Echinoidea UPDATED!, WoRMS Gastrotricha NEW!, WoRMS Gnathostomulida NEW!, WoRMS Holothuroidea UPDATED!, WoRMS Hydrozoa UPDATED!, WoRMS Isopoda UPDATED!, WoRMS Leptostraca UPDATED!, WoRMS Monogenea NEW!, WoRMS Mystacocarida UPDATED!, WoRMS Myxozoa NEW!, WoRMS Nemertea UPDATED!, WoRMS Oligochaeta UPDATED!, WoRMS Ophiuroidea UPDATED!, WoRMS Phoronida UPDATED!, WoRMS Placozoa NEW!, WoRMS Polychaeta UPDATED!, WoRMS Polycystina UPDATED!, WoRMS Porifera UPDATED!, WoRMS Priapulida NEW!, WoRMS Proseriata and Kalyptorhynchia UPDATED!, WoRMS Remipedia UPDATED!, WoRMS Scaphopoda UPDATED!, WoRMS Tanaidacea UPDATED!, WoRMS Tantulocarida UPDATED!, WoRMS Thermosbaenacea UPDATED!, WoRMS Trematoda NEW!, WoRMS Xenoturbellida UPDATED! • Rotifers, mayflies, freshwater hairworms, planarians from FADA databases: FADA Rotifera UPDATED!, FADA Ephemeroptera NEW!, FADA Nematomorpha NEW! & FADA Turbellaria NEW! • Entoprocts, water bears from ITIS Global • Spiders, scorpions, ticks & mites from SpidCat via ITIS UPDATED!, SalticidDB , ITIS Global, TicksBase, SpmWeb BdelloideaBase UPDATED! & Mites GSDs: OlogamasidBase, PhytoseiidBase, RhodacaridBase & TenuipalpidBase • Diplopods, centipedes, pauropods and symphylans from SysMyr UPDATED! & ChiloBase • Dragonflies and damselflies from Odonata database • Stoneflies from PlecopteraSF UPDATED! • Cockroaches from BlattodeaSF UPDATED! • Praying mantids from MantodeaSF UPDATED! • Stick and leaf insects from PhasmidaSF UPDATED! • Grasshoppers, locusts, katydids and crickets from OrthopteraSF UPDATED! • Webspinners from EmbiopteraSF UPDATED! • Bark & parasitic lices from PsocodeaSF NEW! • Some groups of true bugs from ScaleNet, FLOW, COOL, Psyllist, AphidSF UPDATED! , MBB, 3i Cicadellinae, 3i Typhlocybinae, MOWD & CoreoideaSF NEW!• Twisted-wing parasites from Strepsiptera Database UPDATED! • Lacewings, antlions, owlflies, fishflies, dobsonflies & snakeflies from LDL Neuropterida • Some beetle groups from the Scarabs UPDATED!, TITAN, WTaxa & ITIS Global • Fleas from Parhost • Flies, mosquitoes, bots, midges and gnats from Systema Dipterorum, CCW & CIPA • Butterflies and moths from LepIndex UPDATED!, GloBIS (GART) UPDATED!, Tineidae NHM, World Gracillariidae • Bees & wasps from ITIS Bees, Taxapad Ichneumonoidea, UCD, ZOBODAT Vespoidea & HymIS Rhopalosomatidae NEW!• Molluscs from WoRMS Mollusca NEW!, FADA Bivalvia NEW!, MolluscaFW NEW! & AFD (Pulmonata) • Fishes from FishBase UPDATED! • Reptiles from TIGR Reptiles • Amphibians, birds and mammals from ITIS Global PLUS additional species of many groups from ITIS Regional, NZIB and CoL China NEW!

Evaluating glucose and xylose as cosubstrates for lipid accumulation and c-linolenic acid biosynthesis of Thamnidium elegans

Aims: To study the biotechnological production of lipids containing rich amounts of the medically and nutritionally important c-linolenic acid (GLA), during cultivation of the Zygomycetes Thamnidium elegans, on mixtures of glucose and xylose, abundant sugars of lignocellulosic biomass. Methods and Results: Glucose and xylose were utilized as carbon sources, solely or in mixtures, under nitrogen-limited conditions, in batch-flask or bioreactor cultures. On glucose, T. elegans produced 31.9 g/L of biomass containing 15.0 g/L lipid with significantly high GLA content (1014 mg/L). Xylose was proved to be an adequate substrate for growth and lipid production. Additionally, xylitol secretion occurred when xylose was utilized as carbon source, solely or in mixtures with glucose. Batch-bioreactor trials on glucose yielded satisfactory lipid production, with rapid substrate consumption rates. Analysis of intracellular lipids showed that the highest GLA content was observed in early stationary growth phase, while the phospholipid fraction was the most unsaturated fraction of T. elegans. Conclusions: Thamnidium elegans represents a promising fungus for the successful valorization of sugar-based lignocellulosic residues into microbial lipids of high nutritional and pharmaceutical interest.

The mitochondrial view of Blastocladiella emersonii

The mitochondrial genome of the chytrid Blastocladiella emersonii was sequenced and annotated, revealing the complete set of oxidative phosphorylation genes and tRNAs/rRNAs necessary for the translation process. Phylogenetic reconstructions reinforce the proposal of the new phylum Blastocladiomycota. Evidences of gene duplication due to inserted elements suggest shared susceptibility to gene invasion/exchange between chytrids and zygomycetes. The gene content of B. emersonii is very similar to Allomyces macrogynus but the content of intronic and changeable elements is much lower suggesting a stronger resistance to this kind of exchange. in addition, a total of 401 potential nuclear transcripts encoding mitochondrial proteins were obtained after B. emersonii EST database scanning using Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana data as probes and TargetP tool to find N-terminal mitochondrial signal in translated sequences. (c) 2008 Elsevier B.V. All rights reserved.

Use of Bioscreen C for growth of Mucor hiemalis in different carbon and nitrogen sources

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mycoses associated with AIDS in the Third World

Despite advances in diagnosis and treatment, the epidemiological status of the human immunodeficiency virus (HIV) infection is far from under control in most of the developing world. Sub-Saharan Africa, Southeast Asia and India show increased rates of new infections. In Latin America and the Caribbean there were 1.6 million estimated cases of HIV-infected patients at the end of 1997. Fungal diseases have been one of the most relevant diagnoses in relation to the acquired immunodeficiency syndrome (AIDS), Infections due to Candida species and Cryptococcus neoformans var, neoformans are common worldwide. Histoplasma capsulatum, Coccidioides immitis and Penicillium marneffei are important causes of disease in endemic areas. Infection due to Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis are uncommon even where they are endemic. Phaeohyphomycetes, hyalohyphomycetes and zygomycetes are still rare as a cause of disease among AIDS patients, However, agents pertaining to these groups, such as Aspergillus spp., have an increasing incidence. Superficial mycoses due to dermatophytes have special features from epidemiological, clinical and therapeutic points of view.

Morphological and molecular characterization of an equine isolate of Pythium insidiosum and comparison with the first human isolate from the same geographic region

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)