Abstracts

Twenty-third Annual Conference on Yeasts Organized by the Commission on Yeasts of the Czechoslovak Society for Microbiology Smolenice (Slovak Republic) March 2–4, 1994

Abstracts     

Application of a wide-range yeast vector (CoMed?) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts

Background  

Yeasts provide attractive expression platforms in combining ease of genetic manipulation and fermentation of a microbial organism with the capability to secrete and to modify proteins according to a general eukaryotic scheme. However, early restriction to a single yeast platform can result in costly and time-consuming failures. It is therefore advisable to assess several selected systems in parallel for the capability to produce a particular protein in desired amounts and quality. A suitable vector must contain a targeting sequence, a promoter element and a selection marker that function in all selected organisms. These criteria are fulfilled by a wide-range integrative yeast expression vector (CoMed™) system based on A. adeninivorans- and H. polymorpha-derived elements that can be introduced in a modular way.     

Frecuencia de Candida en conductos radiculares de dientes con infección endodóntica primaria y persistente

Background

Microbiological identification in endodontic infections has focused mainly on bacteria without giving much attention to yeasts, which, due to their virulence factors, can affect the outcomes of root canal treatment.

Polyamines in growth and dimorphism ofParacoccidioides brasiliensis

Putrescine and spermidine were the only polyamines found inParacoccidioides brasiliensis, a dimorphic fungus pathogenic for humans. Free polyamines (putrescine>spermidine) increased during the first 24 h of yeast growth, with a second peak at 42 h, and also during the first 12 h of mycelium-to-yeast transition (spermidine>putrescine). Conjugated and bound polyamines were also quantified. 1,4-Diamino-2-butanone decreased free putrescine and spermidine accumulation by inhibiting the activity of ornithine decarboxylase. The increase in free polyamines corresponds to bud emergence in yeast growth and to the mycelium-to-yeast transition ofP. brasiliensis.Abbreviations DAB 1,4-Diamino-2-butanone - Y Yeasts - M Mycelia - ODC Ornithine decarboxylase     

Fermentation of maltotriose by brewer's and baker's yeasts

Two brewer's yeasts and one baker's yeast grew with 95% (w/w) pure maltotriose as carbon source in the presence of antimycin A to block respiration. Biomass yields (0.15 and 0.24 g dry yeast g^–1 sugar, respectively, with and without antimycin A) were similar for growth on maltose and maltotriose, and yields of ethanol were 80% of stoichiometric. Yeasts harvested during growth on glucose and containing low maltose transport activity did not begin to use maltotriose in the presence of antimycin A until after a long lag phase (up to 50 h), but yeast harvested during growth on maltose, and containing high maltose transport activity, began to use maltotriose after about 25 h. Much shorter lags were observed before growth started in the absence of antimycin A.     

Cassava starch effluent treatment with concomitant SCP production

Yeasts and yeast-like organisms were chosen for the aerobic treatment of cassava starch factory effluent. A mixed culture of Candida utills and Endomycopsis fibuliger efficiently and rapidly utilized both starch and free sugars. After 28 h fermentation the protein content of the biomass was 22% (w/w), which remained unchanged during the remainder of the fermentation (60 h). This treatment removed 94% of the COD and 91% of the BOD.     

Vulvovaginal candidiasis is associated with the production of germ tubes by <Emphasis Type="Italic">Candida albicans</Emphasis>

Twenty Candida albicans strains isolated from women attended at the Teaching and Research in the Laboratory of Teaching and Research in Clinical Analysis of the State University of Maringa, Paraná, Brazil, have been analyzed. Yeasts were identified by classical methods and patients subdivided into asymptomatic, vulvovaginal candidiasis(VVC) and recurrent vulvovaginal candidiasis (RVVC) groups. Yeasts were incubated in RPMI + fetal calf serum to analyze germ tubes every two hours, up to 10 h. In vitro sensitivity to fluconazole, itraconazole, ketoconazole, amphotericin B and nystatin was analyzed according to NCCLS-M27-A microdilution assay. Yeast isolated from symptomatic women produced significantly more germ tubes than asymptomatic women (P < 0.05). However, no significant difference between yeasts from VVC and RVVC occurred (P > 0.05). Variation between MIC₅₀ and MIC₉₀ of tested antifungal agents was slight among isolated yeasts, while no resistant yeasts were detected. Nevertheless, VVC yeasts were more DDS (reduced dose-dependent susceptibility) for nystatin and RVVC were more DDS for ketoconazole. Results suggest that colonization by yeast in the vagina and lack of symptoms may be partially explained by the yeast’s sparse capacity to form germ tubes, On the other hand, RVVC was not associated with antimicrobial resistance. DDS high frequency for nystatin and ketoconazole indicates that identification, and susceptibility of antifungals tests are important to management of VVC.     

Yeasts in floral nectar: a quantitative survey

Background and Aims

One peculiarity of floral nectar that remains relatively unexplored from an ecological perspective is its role as a natural habitat for micro-organisms. This study assesses the frequency of occurrence and abundance of yeast cells in floral nectar of insect-pollinated plants from three contrasting plant communities on two continents. Possible correlations between interspecific differences in yeast incidence and pollinator composition are also explored.

Methods

The study was conducted at three widely separated areas, two in the Iberian Peninsula (Spain) and one in the Yucatán Peninsula (Mexico). Floral nectar samples from 130 species (37–63 species per region) in 44 families were examined microscopically for the presence of yeast cells. For one of the Spanish sites, the relationship across species between incidence of yeasts in nectar and the proportion of flowers visited by each of five major pollinator categories was also investigated.

Key Results

Yeasts occurred regularly in the floral nectar of many species, where they sometimes reached extraordinary densities (up to 4 × 10⁵ cells mm⁻³). Depending on the region, between 32 and 44 % of all nectar samples contained yeasts. Yeast cell densities in the order of 10⁴ cells mm⁻³ were commonplace, and densities >10⁵ cells mm⁻³ were not rare. About one-fifth of species at each site had mean yeast cell densities >10⁴ cells mm⁻³. Across species, yeast frequency and abundance were directly correlated with the proportion of floral visits by bumble-bees, and inversely with the proportion of visits by solitary bees.

Conclusions

Incorporating nectar yeasts into the scenario of plant–pollinator interactions opens up a number of intriguing avenues for research. In addition, with yeasts being as ubiquitous and abundant in floral nectars as revealed by this study, and given their astounding metabolic versatility, studies focusing on nectar chemical features should carefully control for the presence of yeasts in nectar samples.Key words: Bumble-bees, floral nectar, microbial communities, pollination, yeasts

‘Il y a évidemment un intérêt très grand, dans une question encore peu connue, à recenser exactement et cela dans diverse régions, les organismes qu''on rencontre.’Schoellhorn (1919, p. 155)

     

Species delineation in the genus Nadsonia Sydow

The genus Nadsonia Sydow is revised on the basis of morphology, physiology, amino acid and fatty acid composition, electrophoretic patterns of some enzymes and DNA relatedness. Two species, N. commutata (type CBS 6640) and N. fulvescens, with two varieties, N. fulvescens var. fulvescens (type CBS 2596) and N. fulvescens var. elongata (type CBS 2594) nov. comb. are recognized. A modified diagnosis of the genus and a key are given.Parts of this work were presented at the IXth International Specialized Symposium on Yeasts (Smolenice, SSR, April 18–22, 1983); VIth General Symposium on Yeasts (Montpellier, France, July 9–13, 1984) and International Symposium The Expanding Realm of Yeast-like Fungi (Amersfoort, The Netherlands, August 3–7, 1987).     

<Emphasis Type="Italic">Brettanomyces bruxellensis</Emphasis> yeasts: impact on wine and winemaking

Yeasts belonging to the Brettanomyces/Dekkera genus are non-conventional yeasts, which affect winemaking by causing wine spoilage all over the world. This mini-review focuses on recent results concerning the presence of Brettanomyces bruxellensis throughout the wine processing chain. Here, culture-dependent and independent methods to detect this yeast on grapes and at the very early stage of wine production are encompassed. Chemical, physical and biological tools, devised for the prevention and control of such a detrimental species during winemaking are also presented. Finally, the mini-review identifies future research areas relevant to the improvement of wine safety and sensory profiles.     

Book reviews

Book reviewed in this article:
Ecology of Protozoa (1987). Tom Fenchel
Cheesemaking (1987). Edited by A. Eck
Microcomputer Applications in the Clinical Laboratory (1987). Michael D. McNeely
Recognition of Mycobacterium leprae Antigens (1987). Tom Ottenhoff & R. de Vries
Frontiers in Microbiology (1987). Edited by Erik de Clercq
Process Development in Antibiotic Fermentations (1987). C.T. Calam
The Yeasts. Vol. 1 Biology of Yeasts; Vol. 2 Yeasts and the Environment (1987) Edited by A.H. Rose & J.S. Harrison     

西藏拉鲁湿地水体酵母菌多样性及其与理化因子相关性

【目的】开展拉鲁湿地水体酵母菌多样性研究,探究理化因子与酵母菌群落结构的相互关系。【方法】采用原位培养法从拉鲁湿地11个水样中分离酵母菌,应用26S r RNA D1/D2区域序列分析,并结合经典分类法对获得的菌株进行分类鉴定,运用SPSS和CANOCO软件分析酵母菌多样性及其与环境因子相关性。【结果】从拉鲁湿地水体中分离得到169株酵母菌,鉴定分属为15个属31个种。优势种为Ustilentyloma graminis和Filobasidium magnum,优势属为Naganishia、Ustilentyloma、Filobasidium和Cystofibasidium。统计分析结果表明,化学需氧量(COD)是影响拉鲁湿地水体酵母菌数量的显著因素,另外,此理化因子是影响Ustilentyloma分布的重要环境条件。【结论】西藏拉鲁湿地酵母菌资源比较丰富,且存在明显的空间异质性。     

Matrix-assisted laser desorption ionization-time of flight mass spectrometry identification of yeasts is contingent on robust reference spectra

Background

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for yeast identification is limited by the requirement for protein extraction and for robust reference spectra across yeast species in databases. We evaluated its ability to identify a range of yeasts in comparison with phenotypic methods.

Methods

MALDI-TOF MS was performed on 30 reference and 167 clinical isolates followed by prospective examination of 67 clinical strains in parallel with biochemical testing (total n = 264). Discordant/unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene cluster.

Principal Findings

Twenty (67%; 16 species), and 24 (80%) of 30 reference strains were identified to species, (spectral score ≥2.0) and genus (score ≥1.70)-level, respectively. Of clinical isolates, 140/167 (84%) strains were correctly identified with scores of ≥2.0 and 160/167 (96%) with scores of ≥1.70; amongst Candida spp. (n = 148), correct species assignment at scores of ≥2.0, and ≥1.70 was obtained for 86% and 96% isolates, respectively (vs. 76.4% by biochemical methods). Prospectively, species-level identification was achieved for 79% of isolates, whilst 91% and 94% of strains yielded scores of ≥1.90 and ≥1.70, respectively (100% isolates identified by biochemical methods). All test scores of 1.70–1.90 provided correct species assignment despite being identified to “genus-level”. MALDI-TOF MS identified uncommon Candida spp., differentiated Candida parapsilosis from C. orthopsilosis and C. metapsilosis and distinguished between C. glabrata, C. nivariensis and C. bracarensis. Yeasts with scores of <1.70 were rare species such as C. nivariensis (3/10 strains) and C. bracarensis (n = 1) but included 4/12 Cryptococcus neoformans. There were no misidentifications. Four novel species-specific spectra were obtained. Protein extraction was essential for reliable results.

Conclusions

MALDI-TOF MS enabled rapid, reliable identification of clinically-important yeasts. The addition of spectra to databases and reduction in identification scores required for species-level identification may improve its utility.     

Comparison of conductimetric and traditional plating techniques for detecting yeasts in fruit juices

Frozen fruit juice concentrates containing an average microbial population of log₁₀ 1.54 cfu ml^-1 were examined by traditional plating techniques and direct and indirect conductimetry. The initial populations in diluted (1:4) concentrates increased to an average of log₁₀ 3.82 cfu ml^-1 during incubation at 25°C for 24 h. Incubation before plating and subjecting to conductimetric tests also facilitated the resuscitation of cells that may have been freeze-injured. Yeasts were recovered in equal numbers on acidified (pH 3.5) potato dextrose agar and dichloran rose bengal chloramphenicol agar (pH 5.6). Yeasts and bacteria were recovered on orange serum agar. Detection times determined by indirect conductimetry correlated fairly well ( r = -0.73) with populations (cfu ml^-1) detected on traditional plating media. Populations in diluted concentrates which were not incubated before examination were detected conductimetrically in an average of 48.9 h, whereas detection times for diluted concentrates incubated for 24 h at 25°C before testing were reduced to an average of 14.1 h. Examination by conventional (direct) conductimetry required an additional 10–20 h to reach changes in conductance of 5 μS h^-1.     

Modification of Cycloserine Cefoxitin Fructose Agar to Suppress Growth of Yeasts from Stool Specimens

Cycloserine cefoxitin fructose agar is a widely used selective isolation medium for Clostridium difficile from stool specimens. Yeasts often colonize in the intestine of C. difficile disease patients and, if colonized heavily, pure culture of C. difficile can be delayed. The aim of this study was to modify cycloserine cefoxitin fructose agar to suppress the growth of yeasts. Antimicrobial activities of three commonly available antifungal agents were tested against recent clinical isolates of Candida species. Amphotericin B was most active in inhibiting all isolates by ≤0.5 mg/L concentration. Cycloserine cefoxitin fructose agar was modified by adding 2 mg/L of amphotericin B. Serial ten-fold dilution of stool specimens from 126 suspected C. difficile -associated diarrhea patients were cultured both on cycloserine cefoxitin fructose agar plates and modified agar plates. Yeasts grew from 60 specimens on cycloserine cefoxitin fructose agar, but none grew on the modified medium. Growth of C. difficile was detected from 37 and 39 of 126 specimens on cycloserine cefoxitin fructose agar and modified medium, respectively. The number of C. difficile colonies was similar on both media. In conclusion, 2 mg/L of amphotericin B supplementation to cycloserine cefoxitin fructose agar can facilitate the isolation of C. difficile from stool specimens which are densely colonized with yeasts.     

肠道菌群正常参考值的检测

目的定性定量检测并比较杭州地区健康青壮年及老年人群肠道主要菌群(双歧杆菌属、类杆菌属、消化链球菌属、产气荚膜梭菌、乳杆菌属、肠杆菌科、肠球菌属及酵母菌等)的正常参考值,同时计算并比较双歧杆菌属细菌数量与肠杆菌科细菌数量的对数值比值(B/E值).方法采用光冈法 .结果老年组肠道双歧杆菌数量及B/E值较青壮年组均显著减少(P＜0.05, P＜0.001);消化链球菌、肠杆菌科及肠球菌细菌数量显著增加(P＜0.01,P ＜0 .001,P＜0.05).结论在获得肠道菌群正常参考值的同时,发现随着年龄的增加,老年人肠道双歧杆菌数量明显减少,肠杆菌科及肠球菌细菌数量显著增加.     

The biosorption of cadmium and cobalt and iron ions by yeast <Emphasis Type="Italic">Cryptococcus humicola</Emphasis> at nitrogen starvation

Yeasts Cryptococcus humicola accumulated cadmium, cobalt, and iron (~?50, 17, and 4% of the content in the medium, respectively) from the medium containing glucose, phosphate, and 2 mmol/L of metal salts. The effects of metal absorption on the levels of orthophosphate (Pi) and inorganic polyphosphate (polyP) varied for the metals under study. The levels of Pi and polyP increased in the case of cadmium and cobalt, respectively. In the case of iron, no changes in the levels of Pi and polyP were observed. Multiple DAPI-stained polyP inclusions were observed in the cytoplasm of cadmium-containing cells. The intensity of DAPI staining of the cell wall especially increased in case of cobalt and iron accumulation.     

Distribution and antifungal susceptibility of yeasts isolates from intensive care unit patients

Yeasts frequently colonize non-sterile sites in the body. The aim of the study was to determine distribution in clinical samples and antifungal susceptibility to five antifungals. From January 2013 through June 2015, 800 isolates were obtained from intensive care unit patients. Candida albicans (58.9%), Candida glabrata (20.4%), Candida krusei (8.6%), and Candida parapsilosis (3.6%) were the leading species. Majority of the C. albicans isolates were susceptible to the fluconazole. Elevated voriconazole minimal inhibitory concentrations (MICs) were observed in isolates exhibiting high fluconazole MICs, most frequently in C. glabrata. Isolates with echinocandins MICs suggesting reduced susceptibility were only sporadic cases with the exception of Trichosporon spp. The amphotericin B MICs were slightly higher for some C. krusei.