Isolation and identification of Wickerhamiella tropicalis from blood culture by MALDI-MS.

Wickerhamiella is a genus of budding yeast that is mainly isolated from environmental samples, and 40 species have been detected. The yeast isolated from human clinical samples usually only contain three species: W. infanticola, W. pararugosa and W. sorbophila. In this study, we isolated W. tropicalis from a blood sample of a six-year-old female with a history of B-cell precursor lymphoblastic leukemia in Japan in 2022. Though the strain was morphologically identified as Candida species by routine microbiological examinations, it was subsequently identified as W. tropicalis by sequencing the internal transcribed spacer (ITS) of ribosomal DNA (rDNA). The isolate had amino acid substitutions in ERG11 and FKS1 associated with azole and echinocandin resistance, respectively, in Candida species and showed intermediate-resistant to fluconazole and micafungin. The patient was successfully treated with micafungin. Furthermore, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) detected three novel peaks that are specific for W. tropicalis, indicating that MALDI-MS analysis is useful for rapid detection of Wickerhamiella species in routine microbiological examinations.

Two novel species of rapidly growing mycobacteria: Mycobacterium lehmannii sp. nov. and Mycobacterium neumannii sp. nov.

Two rapidly growing mycobacteria with identical 16S rRNA gene sequences were the subject of a polyphasic taxonomic study. The strains formed a well-supported subclade in the mycobacterial 16S rRNA gene tree and were most closely associated with the type strain of Mycobacterium novocastrense. Single and multilocus sequence analyses based on hsp65, rpoB and 16S rRNA gene sequences showed that strains SN 1900T and SN 1904T are phylogenetically distinct but share several chemotaxonomic and phenotypic features that are are consistent with their classification in the genus Mycobacterium. The two strains were distinguished by their different fatty acid and mycolic acid profiles, and by a combination of phenotypic features. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values for strains SN 1900T and SN 1904T were 61.0 % and 94.7 %, respectively; in turn, the corresponding dDDH and ANI values with M. novocastrense DSM 44203T were 41.4 % and 42.8 % and 89.3 % and 89.5 %, respectively. These results show that strains SN1900T and SN 1904T form new centres of taxonomic variation within the genus Mycobacterium. Consequently, strains SN 1900T (40T=CECT 8763T=DSM 43219T) and SN 1904T (2409T=CECT 8766T=DSM 43532T) are considered to represent novel species, for which the names Mycobacteriumlehmannii sp. nov. and Mycobacteriumneumannii sp. nov. are proposed. A strain designated as 'Mycobacteriumacapulsensis' was shown to be a bona fide member of the putative novel species, M. lehmannii.

Mycobacterium eburneum sp. nov., a non-chromogenic, fast-growing strain isolated from sputum.

A polyphasic study was undertaken to establish the taxonomic position of a non-chromogenic, rapidly growing Mycobacterium strain that had been isolated from sputum. The strain, CECT 8775T, has chemotaxonomic and cultural properties consistent with its classification in the genus Mycobacterium and was distinguished from the type strains of closely related mycobacterial species, notably from Mycobacterium paraense DSM 46749T, its nearest phylogenetic neighbour, based on 16S rRNA, hsp65 and rpoB gene sequence data. These organisms were also distinguished by a broad range of chemotaxonomic and phenotypic features and by a digital DNA-DNA relatedness value of 22.8 %. Consequently, the strain is considered to represent a novel species of Mycobacterium for which the name Mycobacterium eburneum sp. nov is proposed; the type strain is X82T (CECT 8775T=DSM 44358T).

Reclassification of Amycolicicoccus subflavus as Hoyosella subflava comb. nov. and emended descriptions of the genus Hoyosella and Hoyosella altamirensis.

16S rRNA gene sequences of two type strains belonging to different genera within the suborder Corynebacterineae, namely Hoyosella altamirensis and Amycolicicoccus subflavus, show a similarity of 99.8 %. Therefore, in order to clarify their taxonomic relationship, a polyphasic recharacterization under the same conditions was carried out. The peptidoglycan of H. altamirensis NBRC 109631T and A. subflavus NBRC 109087T was of A1γ type with meso-diaminopimelic acid as their diagnostic diamino acid. Both strains contained MK-8 as the only detected menaquinone, C16 : 0 and C18 : 1ω9c as the major fatty acids, and diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol as the principal polar lipids. The coincidences of these chemotaxonomic features suggested that H. altamirensis and A. subflavus should be assigned to the same genus. Meanwhile, the average nucleotide identity value between both strains and the results of physiological and biochemical tests indicated that H. altamirensis and A. subflavus should be affiliated to different species. Therefore, according to Rules 38 and 41a of the Bacteriological Code, it is proposed that Amycolicicoccus subflavus Wang et al. 2010 be reclassified as Hoyosella subflava comb. nov. (type strain DQS3-9A1T=CGMCC 4.3532T=DSM 45089T=JCM 17490T=NBRC 109087T) and the descriptions of the genus HoyosellaJurado et al. 2009 and Hoyosella altamirensisJurado et al. 2009 are emended accordingly.

Positively charged nanogold label allows the observation of fine cell filopodia and flagella in solution by atmospheric scanning electron microscopy.

Optical microscopy is generally the first choice to observe microbes and cells. However, its resolution is not always sufficient to reveal specific target structures, such as flagella and pili, which are only nanometers wide. ASEM is an attractive higher resolution alternative, as the sample is observed in aqueous solution at atmospheric pressure. Sample pretreatment for ASEM only comprises simple tasks including fixation, gold labeling, and reagent exchange, taking less than 1 h in total. The lengthy sample pretreatments often required for more classical electron microscopies, such as embedding and dehydration, are unnecessary, and native morphology is preserved. In this study, positively charged nanogold particles were used to label the surfaces of bacteria and cultured animal cells, exploiting their net negative surface charge. After gold enhancement to increase the size of the nanogold particles, ASEM imaging of the bacteria in aqueous solution revealed pili and delicate spiral flagella. This natural shape contrasts starkly with images of dried flagella recorded by standard SEM. Positively charged nanogold labeled the plasma membrane of cultured COS7 cells, and after enhancement allowed filopodia as thin as 100 nm in diameter to be clearly visualized. Based on these studies, ASEM combined with positively charged nanogold labeling promises to become an important tool for the study of cell morphology and dynamics in the near future.

Simple and rapid characterization of mycolic acids from Dietzia strains by using MALDI spiral-TOFMS with ultra high mass-resolving power.

Mycolic acids have been used as important chemotaxonomic markers. In this study, a newly developed matrix-assisted laser desorption/ionization time-of-flight mass spectrometer with a spiral ion trajectory (MALDI spiral-TOFMS) was applied to the characterization of mycolic acids of three type strains of validated species belonging to the genus Dietzia (D. papillomatosis 105045(T), D. kunjamensis NBRC 105042(T) and D. timorensis NBRC 104184(T)), by analysis of total fatty acid methyl ester fractions. In addition, owing to the high mass-resolving power of MALDI spiral-TOFMS, adjacent peaks (0.036 Da mass differences) were successfully separated, and weak peaks corresponding to oxygenated mycolic acids were detected. For all samples, the distributions of carbon-chain lengths were mainly in the range of C30-C42 and the average number of carbon-chain lengths was about 37, which agreed reasonably well with reported results for the genus Dietzia. The number of double bonds and/or cyclopropane rings was 0-2. Relative peak intensities of each mycolic acid methyl ester were used to compare the mycolic acids of the three strains. The mycolic acids of D. papillomatosis and D. kunjamensis were characterized by a high content of mycolic acids with 0-1 double bond or cyclopropane ring and an almost equal content of mycolic acids with odd- and even-numbered carbon-chain lengths. In contrast, mycolic acids of D. timorensis were characterized by a high content of mycolic acids with 1-2 double bonds and/or cyclopropane rings with an even-numbered carbon-chain length. By using MALDI spiral-TOFMS, mycolic acids from three type strains of the genus Dietzia were characterized easily and rapidly.

Purification and characterization of polyphenols from chestnut astringent skin.

Polyphenolic compounds from chestnut astringent skin (CAS) were purified by dialysis, using Diaion HP-20 and Sephadex LH-20 columns. During purification, specific α-amylase inhibitory activities were increased about 3.4-fold, and the 50% inhibition value was 5.71 µg/mL in the Sephadex LH-20 fraction (SE-fraction). The SE-fraction contained about 67% of the total polyphenols, 57.3% of the flavanol-type tannins, and 51.3% of the procyanidins. Strong antioxidant activity was observed in the SE-fraction. Oral administration of the SE-fraction in rats fed corn starch significantly suppressed an increase in blood glucose levels. The SE-fraction contained gallic acid and ellagic acid. The MALDI-TOF spectrum showed a peak series exhibiting a mass increment of 288 Da, reflecting the variation in the number of catechin/epicatechin units. Our results suggest CAS contains polyphenols with strong α-amylase inhibitory activity. The data also suggest CAS polyphenols might be oligomeric proanthocyanidins with gallic acid and ellagic acid.