GC–MS and GC–MS/MS measurement of the cardiovascular risk factor homoarginine in biological samples

l-Homoarginine (hArg) has recently emerged as a novel cardiovascular risk factor and to herald a poor prognosis in heart failure patients. Here, we report on the development and thorough validation of gas chromatography–mass spectrometry (GC–MS) and gas chromatography–tandem mass spectrometry (GC–MS/MS) methods for the quantitative determination of hArg in biological samples, including human plasma, urine and sputum. For plasma and serum samples, ultrafiltrate (10 µL; cutoff, 10 kDa) was used. For urine samples, native urine (10 µL) was used. For sputum, protein precipitation by acetone was performed. hArg is derivatized to its methyl ester tri(N-pentafluoropropionyl) derivative; de novo synthesized trideutero-methyl ester hArg is used as the internal standard (IS). Alternatively, [guanidino-¹⁵N₂]-arginine can be used as an IS. Quantitative analyses were performed after electron-capture negative-ion chemical ionization by selected-ion monitoring in GC–MS and selected-reaction monitoring in GC–MS/MS. We obtained very similar hArg concentrations by GC–MS and GC–MS/MS, suggesting that GC–MS suffices for accurate and precise quantification of hArg in biological samples. In plasma and serum samples of the same subjects very close hArg concentrations were measured. The plasma-to-serum hArg concentration ratio was determined to be 1.12 ± 0.21 (RSD, 19 %), suggesting that blood anticoagulation is not a major preanalytical concern in hArg analysis. In healthy subjects, the creatinine-corrected urinary excretion of hArg varies considerably (0.18 ± 0.22 µmol/mmol, mean ± SD, n = 19) unlike asymmetric dimethylarginine (ADMA, 2.89 ± 0.89 µmol/mmol). In urine, hArg correlated with ADMA (r = 0.475, P = 0.040); in average, subjects excreted in the urine about 17.5 times more ADMA than hArg. In plasma of healthy humans, the concentration of hArg is of the order of 2 µM. hArg may be a low-abundance constituent of human plasma proteins. The GC–MS and GC-MS/MS methods we report in this article are useful to study the physiology and pathology of hArg in experimental and clinical settings.     

Review of the clingfish genus <Emphasis Type="Italic">Kopua</Emphasis> (Gobiesocidae: Trachelochisminae) in Japan,with description of a new species

A taxonomic review of the clingfish genus Kopua (Gobiesocidae: Trachelochisminae) in Japan recognizes three species: K. japonica Moore, Hutchins and Okamoto 2012, K. vermiculata Shinohara and Katayama 2015 and K. yoko sp. nov. Kopua japonica and K. vermiculata are redescribed with revised diagnoses on the basis of 20 specimens (10.4–30.4 mm standard length; SL) and the holotype, respectively. Kopua japonica is similar to K. vermiculata in head sensory pore characters (normally single nasal and postocular canal pores). However, the former differs distinctly from the latter as follows: 6–8 (modally 7) gill rakers (vs. 4 or 6); 31–33 (33) vertebrae (vs. 35); anus slightly closer to posterior margin of disc than to anal-fin origin (vs. much closer to posterior margin of disc); snout length 5.3–8.7 (mean 7.0) % SL (vs. 9.2 % SL); disc length 21.2–24.0 (22.8) % SL (vs. 18.8 % SL); pre-dorsal- and anal-fin lengths 72.9–78.4 (75.2) and 78.1–82.8 (80.1) % SL, respectively (vs. 67.5 and 73.6 % SL); and two stripes on cheek (vs. a triangular blotch). Kopua yoko sp. nov., based on 14 specimens (17.7–28.8 mm SL) from the Pacific coast of southern Japan, Sea of Japan and the East China Sea, is characterized by the following combination of characters: 6 or 7 (modally 6) dorsal-fin rays; 4–6 (5) anal-fin rays; 21 or 22 (21) pectoral-fin rays; 4–6 (5 or 6) gill rakers; 31–33 (31) vertebrae; a single (rarely two) nasal canal pores; two lacrimal and preopercular canal pores; snout length 6.5–7.9 (mean 7.1) % SL; gill opening depth 5.8–7.1 (6.5) % SL; least interorbital width 2.0–3.7 (2.6) % SL; disc length 20.3–25.0 (23.1) % SL, disc region D without flattened papillae; caudal-peduncle depth 8.1–10.2 (9.2) % SL; anus slightly closer to posterior margin of disc than to anal-fin origin; pre-dorsal- and anal-fin lengths 71.6–77.1 (73.9) and 77.0–83.7 (80.4) % SL, respectively; post-dorsal-caudal length 12.6–15.0 (13.8) % SL; arch-shaped blotches on lateral aspect of body; and two reddish-orange stripes on cheek. Morphological changes with growth in K. japonica and K. yoko sp. nov. are also described.     

Metabolite profiling of polar steroid constituents in the Far Eastern starfish Aphelasterias japonica using LC–ESI MS/MS

Numerous polar steroids are characteristic metabolites of starfish which determine physiological activities of their extracts. The Far Eastern starfish Aphelasterias japonica is a rich source of different steroid glycosides and polyhydroxysteroids. For detailed analysis of complicated mixture of steroids from this species, isolated by solid-phase extraction, a liquid chromatography–electrospray tandem mass spectrometry (LC–ESI MS/MS) approach was selected and applied. The characteristic fragmentations in ESI MS/MS spectra of steroid glycosides allowed determining types of aglycones, presence of sulfate groups, sugar sequences as well as branching. In addition, main structural features of polyhydroxylated polar steroids including position of hydroxylation and level of sulfation were also established. Totally, 68 metabolites, comprising of 33 asterosaponins, 28 sulfated polyhydroxysteroid mono- and biosides and 7 sulfated polyhydroxysteroid compounds were found by this method. In addition to 15 previously isolated compounds from A. japonica, many new steroid glycosides including asterosaponins with unusual carbohydrate chains were discovered and characterized by their ESI product ion mass spectra. Some details of biosynthesis of polyhydroxylated steroids and their conjugated forms in the species studied such as a role of sulfation and order of introduction of hydroxyl group in A. japonica were proposed.     

Structural analysis of cerebrosides from Aspergillus fungi: the existence of galactosylceramide in A. oryzae

Glucosylceramide and galactosylceramide were detected in three Aspergillus species: Aspergillus oryzae, Aspergillus sojae and Aspergillus. awamori, using borate-coated TLC. The cerebrosides from A. oryzae were further purified by ion exchange and iatrobeads column chromatographies with or without borate, and determined the composition of sugar, fatty acid and sphingoid base by GC/MS, MALDI-TOF/MS and ¹H-NMR. We identified them as β-glucosylceramide and β-galactosylceramide. The ceramide moiety of both cerebrosides consisted mainly of 2-hydroxystearic acid and either 9-methyl-octadeca-4, 8-sphingadienine or octadeca-4, 8-sphingadienine. To our knowledge, this is the first study to provide evidence for the presence of β-galactosylceramide in A. oryzae.     

Profiling of fatty acid methyl esters from the oleaginous diatom Fistulifera sp. strain JPCC DA0580 under nutrition-sufficient and -deficient conditions

The marine oleaginous diatom, Fistulifera sp. strain JPCC DA0580, is a promising candidate for biodiesel production due to its high lipid content. In order to truly evaluate the potential of this strain as biodiesel feedstock as well as the impact of nutrition-deficiency to this strain, the proportion of the lipid fractions and fatty acid methyl ester (FAME) derived from Fistulifera sp. cultured under nutrition-sufficient or -deficient conditions were analyzed. The nutrition deficiency led to the increase of the total lipid content in the form of neutral lipids (NLs) accumulation and the decline of polar lipids compared with nutrition-sufficiency. Meanwhile, the total lipid productivity was not significantly changed under two nutrition conditions while the NL productivity under nutrition-deficient condition was much higher than nutrition-sufficient condition. The major FAME components, C14:0, C16:0, C16:1, and C20:5, contribute to over 90 % of total FAMEs under both nutrition conditions. A lower polyunsaturated FAME level were observed in the nutrition-deficient condition (9.9?±?0.2 %) compared with the nutrition-sufficient condition (19.8?±?1.2 %), suggesting the availability of the nutrition stress on the strain JPCC DA0580 for improvement of fuel quality as well as productivity. The lipid quality estimation based on the FAME profile revealed that the nutrition-deficiency could further improve the lipid quality of both total lipids and NL fraction. In addition, direct infusion ESI-Q-TRAP-MS/MS was carried out for the fractionated NL in order to estimate triacylglycerol (TAG) composition, suggesting a crucial role of the chloroplast in TAG synthesis.     

Basic amino acids and dimethylarginines targeted metabolomics discriminates primary hepatocarcinoma from hepatic colorectal metastases

Hepatocellular carcinoma (HCC) is a very aggressive neoplasia requiring early and accurate diagnosis to improve patient outcomes with timely treatment. The liver is also very frequently colonized by metastases, and the most frequent differential diagnosis is HCC against intrahepatic cholangiocarcinoma or metastatic adenocarcinoma. Metabolomics is a powerful tool for identification of altered biomarkers in cancer, and to evaluate the efficacy of drug treatments. Here we analyzed by HILIC-MS/MS methylated arginines, basic amino acids (Arg, Cit, Orn), and their ratios in the extracts of primary HCC tissues, liver metastases from colorectal carcinoma (MET), cirrhotic related hepatitis-C-virus (CIR), and non-cirrhotic normal liver (NT) adjacent tissues. We found high levels of Arg (p < 0.0001) and Arg/Orn (p < 0.01) in MET compared to other tissues. In MET, compared to NT and CIR, Arg concentration was fivefold higher, while in HCC it was twofold higher. ADMA increased twofold compared to NT and CIR, while in HCC it was 50 % higher. Arg/Cit and ADMA/SDMA ratios were significantly higher in MET compared to NT and CIR (p < 0.005). Arg/Orn, Arg/Cit, and ADMA/SDMA ratios increased progressively from NT, CIR, HCC, to MET tissues. Arg/Cit correlated significantly with Arg/Orn ratios (r = 0.77; p < 0.0001), and discriminates tumor from non-tumor samples. In addition, the discriminant lactate/glucose ratio we previously found by NMR, also correlated significantly with the Arg levels (r = 0.64; p < 0.0001), and discriminated MET from all other tissues. The results indicated that Arg in MET is higher than other tissue classes, suggesting that, together with the lactate/glucose ratio, it can be considered a further biomarker for HCC-metastases differentiation.     

On a skull of a fossil macaque from the shikimizu limestone quarry in the Shikoku district,Japan

An almost complete calvaria of an adult macaque was found among remain from the Shikimizu limestone quarry at Hijikawa-cho, Kita-gun, Ehime Pref., Shikoku, Japan. Its exact age is unknown, but probably Pleistocene because mammalian bones from this quarry include not only recent but some Pleistocene elements. The Shikimizu skull has a somewhat different feature as compared with the skull of livingMacaca fuscata, mainly in that its facial region is much broader. In this feature, the Shikimizu skull seems to somewhat resembleMacaca robustus from China, suggesting that the older the lineage offuscata is traced back in the Pleistocene, the more its ancestral type would becomerobustus-like in the morphology.     

Phenotypic and genotypic characteristics of Arcanobacterium haemolyticum isolated from clinical samples in a Danish hospital

Six Arcanobacterium haemolyticum strains isolated from six patients of two hospitals in Denmark were identified phenotypically, also including matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, and by genotypic methods. The latter were performed by sequencing 16S rDNA and glyceraldehyde 3-phosphate dehydrogenase encoding gene gap and by amplification of an A. haemolyticum specific region of 16S–23S rDNA intergenic spacer region and 23S rDNA. The six A. haemolyticum strains were further investigated for the presence of seven potential virulence genes encoding arcanolysin, phospholipase D, hemolysin A, CAMP factor family protein, collagen binding protein, neuraminidase A and neuraminidase H which appeared to be present in two (seven virulence genes), two (six virulence genes) and two strains (four virulence genes), respectively. The phenotypic and genotypic properties described in the present study might help to reliably identify and further characterize A. haemolyticum isolated from human patients, a species which seems to be of increasing importance.     

Proteomics profiling of fiber development and domestication in upland cotton (Gossypium hirsutum L.)

Comparative proteomic analyses were performed to detail the evolutionary consequences of strong directional selection for enhanced fiber traits in modern upland cotton (Gossypium hirsutum L.). Using two complementary proteomic approaches, 2-DE and iTRAQ LC–MS/MS, fiber proteomes were examined for four representative stages of fiber development. Approximately 1,000 protein features were characterized using each strategy, collectively resulting in the identification and functional categorization of 1,223 proteins. Unequal contributions of homoeologous proteins were detected for over a third of the fiber proteome, but overall expression was balanced with respect to the genome-of-origin in the allopolyploid G. hirsutum. About 30 % of the proteins were differentially expressed during fiber development within wild and domesticated cotton. Notably, domestication was accompanied by a doubling of protein developmental dynamics for the period between 10 and 20 days following pollination. Expression levels of 240 iTRAQ proteins and 293 2-DE spots were altered by domestication, collectively representing multiple cellular and metabolic processes, including metabolism, energy, protein synthesis and destination, defense and stress response. Analyses of homoeolog-specific expression indicate that duplicated gene products in cotton fibers can be differently regulated in response to selection. These results demonstrate the power of proteomics for the analysis of crop domestication and phenotypic evolution.     

Micropropagation of Laburnum anagyroides Medic. through axillary shoot regeneration

An in vitro propagation system based on the proliferation of axillary buds has been developed for Laburnum anagyroides. Culture initiation was influenced by explanting season, with the maximum response obtained from explants harvested in spring and autumn while the lowest response was noted in summer explants. The best shoot induction was observed on Murashige and Skoog medium (MS) supplemented with 2.22 μM 6-benzylaminopurine (BAP). The basal media, type of cytokinin, and explant type were the most important factors affecting shoot multiplication of L. anagyroides. Medium comprised of ½MS salts was found to be more efficient for axillary shoot multiplication compared to full-strength MS or Wood Plant Medium (WPM) when using identical growth regulators. Shoot tip explants were more responsive than nodal segments of microshoots for micropropagation. In vitro-derived shoots, >10 mm in length, were successfully rooted in medium containing ¼MS salts and 2.68 μM α-naphtalene acetic acid (NAA). In vitro-regenerated plantlets were adapted to ex vitro conditions and transferred to a greenhouse. This is the first report for successful in vitro propagation of L. anagyroides from bud explants of mature trees.     

Copper tolerance in Frankia sp. strain EuI1c involves surface binding and copper transport

Several Frankia strains have been shown to be copper-tolerant. The mechanism of their copper tolerance was investigated for Frankia sp. strain EuI1c. Copper binding was shown by binding studies. Unusual globular structures were observed on the surface of the bacterium. These globular structures were composed of aggregates containing many relatively smaller “leaf-like” structures. Scanning electron microscopy with energy-dispersive X-ray (SEM-EDAX) analysis of these structures indicated elevated copper and phosphate levels compared to the control cells. Fourier transform infrared spectroscopy (FTIR) analysis indicated an increase in extracellular phosphate on the cell surface of copper-stressed cells. Bioinformatics’ analysis of the Frankia sp. strain EuI1c genome revealed five potential cop genes: copA, copZ, copC, copCD, and copD. Experiments with Frankia sp. strain EuI1c using qRT-PCR indicated an increase in messenger RNA (mRNA) levels of the five cop genes upon Cu²⁺ stress. After 5 days of Cu²⁺ stress, the copA, copZ, copC, copCD, and copD mRNA levels increased 25-, 8-, 18-, 18-, and 25-fold, respectively. The protein profile of Cu²⁺-stressed Frankia sp. strain EuI1c cells revealed the upregulation of a 36.7 kDa protein that was identified as FraEuI1c_1092 (sulfate-binding periplasmic transport protein). Homologues of this gene were only present in the genomes of the Cu²⁺-resistant Frankia strains (EuI1c, DC12, and CN3). These data indicate that copper tolerance by Frankia sp. strain EuI1c involved the binding of copper to the cell surface and transport proteins.     

The effect of Sa and MiniSa plasmids on the induction of plant crown galls and hairy roots byAgrobacterium tumefaciens andAgrobacterium rhizogenes

The Inc-W group plasmid Sa or its derivative MiniSa were introduced into two strains ofAgrobacterium tumefaciens with Ti plasmids, one strain ofA. tumefaciens with the Ri plasmid and oneA. rhizogenes strain with the Ri plasmid. The effect was similar in allAgrobacterium strains. The pSa suppressed fully the virulence ofAgrobacterium strains (i.e. their ability to induce tumor growths - crown galls or hairj⁷ roots) inKalanchoe plants and carrot root slices. The MiniSa plasmid caused only a slight decrease of the frequency and size of tumor growths induced. The mechanism of suppression of virulence by the Sa plasmid inAgrobacterium tumefaciens andAgrobacterium rhizogenes seems to be similar.     

Identification of Stringent Response-Related and Potential Serological Proteins Released from Bacillus anthracis Overexpressing the RelA/SpoT Homolog,Rsh Bant

RelA and SpoT synthesize ppGpp, a key effector molecule that facilitates the adaptation of bacteria to nutrient starvation and other stresses, known as the stringent response. To investigate the role of Rsh _Bant , a putative RelA/SpoT homolog (encoded by BAS4302) in Bacillus anthracis, we examined the alteration of the secretome profiles after the overexpression of a functional His-Rsh _Bant protein in the B. anthracis strain Sterne at the stationary growth phase. In the ppGpp-deficient E. coli mutant strain CF1693, overexpression of Rsh _Bant restored a ppGpp-dependent growth defect on minimal glucose media. The secretome profiles obtained using a two-dimensional electrophoresis (2-DE) analysis were altered by overexpression of Rsh _Bant in B. anthracis. Among the 66 protein spots differentially expressed >1.5-fold, the 29 proteins were abundant for further identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Functional categorization of those proteins implicated their involvement in various biological activities. Taken together, our results imply that overexpression of a functional His-Rsh _Bant can lead to the increased levels of intracellular ppGpp in B. anthracis, resulting in the significant changes in its secretome profiling. The stringent response-controlled proteins identified are likely useful as potential targets for serodiagnostic applications.     

Genetic Diversity Among Candida albicans Isolates Associated with Vertical Transmission in Preterm Triplets

We report a case of congenital candidiasis in triplets, in the context of premature labor at 25 weeks gestation, without symptomatic vaginitis or chorioamnionitis. All three infants died as a result of prematurity, aggravated by systemic candidiasis. Multi-locus sequence typing confirmed vertical transmission of Candida albicans from the mother to the triplets and revealed a slight diversity among the strains isolated from the neonates.     

Transient Facial Nerve Paralysis (Bell's Palsy) following Intranasal Delivery of a Genetically Detoxified Mutant of Escherichia coli Heat Labile Toxin

Background

An association was previously established between facial nerve paralysis (Bell''s palsy) and intranasal administration of an inactivated influenza virosome vaccine containing an enzymatically active Escherichia coli Heat Labile Toxin (LT) adjuvant. The individual component(s) responsible for paralysis were not identified, and the vaccine was withdrawn.

Methodology/Principal Findings

Subjects participating in two contemporaneous non-randomized Phase 1 clinical trials of nasal subunit vaccines against Human Immunodeficiency Virus and tuberculosis, both of which employed an enzymatically inactive non-toxic mutant LT adjuvant (LTK63), underwent active follow-up for adverse events using diary-cards and clinical examination. Two healthy subjects experienced transient peripheral facial nerve palsies 44 and 60 days after passive nasal instillation of LTK63, possibly a result of retrograde axonal transport after neuronal ganglioside binding or an inflammatory immune response, but without exaggerated immune responses to LTK63.

Conclusions/Significance

While the unique anatomical predisposition of the facial nerve to compression suggests nasal delivery of neuronal-binding LT–derived adjuvants is inadvisable, their continued investigation as topical or mucosal adjuvants and antigens appears warranted on the basis of longstanding safety via oral, percutaneous, and other mucosal routes.     

Identification of a microspordium isolated from Megacopta cribraria (Hemiptera: Plataspidae) and characterization of its pathogenicity in silkworms

A new microsporidium isolated from Megacopta cribraria was characterized by both biological characteristics and phylogenetic analysis. Moreover, its pathogenicity to silkworms was also studied. The spores are oval in shape and measured 3.64 ± 0.2 × 2.20 ± 0.2 μm in size. Its ultrastructure is characteristic of the genus Nosema: a diplokaryon, 13–14 polar filament coils and posterior vacuole. Its life cycle includes meronts, sporonts, sporoblasts and mature spores, with a typical diplokaryon in each stage and propagation in a binary fission. A phylogenetic tree based on SSU rRNA and rRNA ITS gene sequence analysis further indicated that the parasite is closely related to Nosema bombycis and should be placed in the genus Nosema and sub-group ‘true’ Nosema. Furthermore, the microsporidium heavily infects lepidopteran silkworm insect and can be transmitted per os (horizontally) and transovarially (vertically). Our findings showed that the microsporidium belongs to the ‘true’ Nosema group within the genus Nosema and heavily infects silkworms. Based on the information obtained during this study, we named this new microsporidium isolated from M. cribraria as Nosema sp. MC.     

A virus inhibitory protein isolated from Cyamopsis tetragonoloba (L.) Taub. upon induction of systemic antiviral resistance shares partial amino acid sequence homology with a lectin

Key message

Two virus inhibitory proteins were purified from Cyamopsis tetragonoloba , induced to resist virus infections by CIP-29, a systemic resistance inducing protein from Clerodendrum inerme , and characterized. One of them shared homology with a lectin.

Abstract

CIP-29, a known 29 kDa systemic antiviral resistance inducing protein isolated from Clerodendrum inerme, has been used to induce systemic resistance in Cyamopsis tetragonoloba against Sunn-hemp rosette virus (SRV). Paper reports the detection of virus inhibitory activity in induced-resistant leaf sap of C. tetragonoloba, and the purification of two virus inhibitory agents (VIAs) thereof. VIA activity was recorded as a reduction in lesion number of SRV, Tobacco mosaic virus, and Papaya ringspot virus, when they were incubated separately with resistant sap and inoculated onto susceptible C. tetragonoloba, Nicotiana tabacum cv. Xanthi-nc, and Chenopodium quinoa, respectively. The two VIAs were isolated from resistant C. tetragonoloba plant leaves using combinations of column chromatography. Both were basic proteins, and since their M _r was 32 and 62 kDa, these VIAs were called CT-VIA-32 and CT-VIA-62, respectively, on the basis of their molecular mass and the host. CT-VIA-62 displayed better activity, and was thus studied further. It tested positive for a glycoprotein, and was serologically detected only in leaf tissue post-induction. Tryptic peptides generated in-gel, post SDS-PAGE of CT-VIA-62, were sequenced through LC/MS/MS. All CT-VIA-62 peptides were found to share homologies with proteins from Medicago truncatula that possess a mannose-binding lectin domain.     

Penicillium strains isolated from Slovak grape berries taxonomy assessment by secondary metabolite profile

The secondary metabolite profiles of microfungi of the genus Penicillium isolated from samples of grape berries collected in two different phases during two vegetative seasons in Slovakia is described to assess the taxonomy. Three Slovak vine regions have been selected for this study, based on their climatic differences and national economic importance. Cultures of microfungi isolated from berries were incubated on different selective media for macro and micromorphology identification. The species Penicillium brevicompactum, Penicillium crustosum, Penicillium chrysogenum, Penicillium expansum, Penicillium palitans and Penicillium polonicum were identified according to growth and morphology. The related strains were found to produce a broad spectrum of fungal metabolites, including roquefortine C, chaetoglobosin A, penitrem A, cyclopeptin, cyclopenin, viridicatin, methylviridicatin, verrucofortine, secalonic acid D, cyclopiazonic acid, fumigaclavine and mycophenolic acid. Chemotaxonomy was performed using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Dried grape berries were also analyzed allowing to assess the presence of patulin, roquefortine C and penicillic acid; this last one has been identified in dried berries but not in vitro.     

Candida galli as a Cause of Tinea Unguium—Molecular Characterization of a Rare Clinical Fungal Entity

We present the first clinical report of an infection caused by Candida galli, an anamorphic yeast species in the Yarrowia clade. C. galli has been described in the literature only four times, but never before it has been isolated from clinical samples. The colony morphology on Sabouraud medium and morphotype on CHROMagar Candida medium were similar to C. lipolytica as well as the carbon assimilation profile. The phenotypic differences with C. lipolytica were the non-assimilation of N-acetyl glucosamine, the absence of urease activity, growth in 10 % NaCl with 5 % glucose and in vitamin-free medium. MALDI–TOF MS could not generate reliable identification of the strain. Molecular analysis based on amplification of the ITS1-5.8S-ITS2 rDNA regions confirmed the identity as C. galli. Antifungal susceptibility test clearly demonstrated high MICs to 5-fluorocytosine, amphotericin B and fluconazole, as in the species belonging to the Yarrowia clade.