Yeast Plus Research Articles

Etiology and Antifungal Sensitivity Test in Otomycosis Caused by Candida Sp

Otomycosis is a common fungal infection of the external auditory meatus frequently diagnosed in otolaryngology outpatient clinics. Resistance to antifungals is currently a significant concern, with intrinsic and acquired resistance increasing among isolates that cause fungal infections. The purpose of this research was to identify Candida species causing otomycosis and determine the pattern of antifungal susceptibility among these Candida species. A prospective study was conducted in the Margono Soekarjo General Hospital and Department of Microbiology, Faculty of Medicine, Universitas Jenderal Soedirman Purwokerto, Indonesia, from April – September 2022. Forty-seven (47) clinical samples of otomycosis were collected from 41 patients and then isolated bedside on fungal culture media and was prepared on an object glass for direct microscopic examination of the specimens. Fungal identification was performed using 10% potassium hydroxide (KOH) to observe fungal elements. Samples were cultured on Saboraud dextrose agar (SDA) media with chloramphenicol and Czapek dox agar. The Germ Tube Test was used to identify Candida while yeast-specific identification and antifungal susceptibility assay using a rapid commercial kit was applied for specific identification of the fungus. Antifungal susceptibility patterns were obtained using the Integral System Yeast Plus (ISYP) media pack. Candida parapsilosis was the most prevalent Candida species discovered in this study, accounting for approximately 41.66%, which was followed by Candida tropicalis (25%) and Candida krusei (12.5%), whereas Candida albicans only accounted for 4.1% of the specimens. All Candida species were sensitive to flucitosine and ketoconazole, whereas the voriconazole sensitivity rate reached 96%. This study concludes that Candida parapsilosis is the most prevalent species of Candida in otomycosis, and that all Candida species are sensitive to Flucitosine, ketoconazole, and voriconazole.

Assessment of virulence factors and antifungal susceptibility of Candida species isolated from catheter associated urinary tract infections

ABSTRACT Background: Catheter associated urinary tract infection (CAUTI) is a crucial attribute to virulence of organisms. Emergency of non albicans Candida (NAC) species become common due to several virulence factors, which play an important role in pathogenicity and their resistance to treatment. The aim of work: To investigate different virulence factors of Candida spp. isolated from CAUTI patients and evaluate their antifungal susceptibility. Patients and methods: Urine samples collected from 219 indwelling catheters of ICU patients. Candida isolates identified using Germ tube test, Chrom ID agar, Corn meal tween agar, VITEK 2 system and Integral System Yeast Plus (ISYP) used to detect antifungal susceptibility. Enzymatic, haemolytic activity and biofilm formation assessed by standard methods. Results: The incidence of candiduria was 50/219 (22.8%). Candida tropicalis was predominant (54.0%) followed by C. albicans (26.0%) and C. glabrata (20.0%). Females gender (64.0%), Diabetes (50.0%) associated with positive candiduria, while antibiotic therapy was predominant risk factor (78.0%). C. albicans have highest enzymatic activity for phospholipase (84.6 %), proteinase (84.6%) and haemolytic activity (100%), while C. tropicalis have highest biofilm formation (100%). NAC spp. showed higher rates of resistance to azole drugs (Clotrimazole 77.6%, Miconazole 70.3%, Econazole 63.2%, Nystatine 44.3%), while C. albicans didn’t show any resistance. Conclusion: Candiduria is prevalent among catheterized patients with shift toward NAC spp. causing nosocomial UTI. A strong relationship between host risk factors (old age, antibiotic use, catheterization, female sex, ICU stay, diabetes) and expression of various virulence factors of Candida spp. causing candiduria and resistance to antifungal drugs.

Indigenous yeast with cellulose-degrading activity in napa cabbage (Brassica pekinensis L.) waste: Characterisation and species identification

Napa cabbage waste contains an organic component, cellulose, which can be utilised as an ingredient for cellulose-degrading enzyme production with the help of indigenous yeast. The aim of the research was to identify and characterise potential indigenous yeast isolated from napa cabbage waste, which has cellulose-degrading activity. Indigenous yeast were isolated and characterised using the RapID Yeast Plus System, then turbidity was used to determine the yeast total population. Indigenous yeast was grown at napa cabbage waste at 27, 37, and 40°C for three days, and cellulose-degrading activity was determined by the Dinitrosalicylic Acid (DNS) method. The potential yeast isolate with the highest cellulose-degrading activity was identified by a sequence analysis of the rRNA gene internal transcribed spacer (ITS) region with using primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′- TCCTCCGCTTATTGATATGC-3′). The results were compared to the GenBank database using the Basic Local Alignment Search Tools/BLAST algorithm. Three species of indigenous yeast were isolated from napa cabbage waste (S2, S6, and S8). S8, incubated at 37ºC for three days, demonstrated the highest cellulose-degrading enzyme activity (1.188 U/mL), with the average activity of 0.684U/mL. Species identification results indicated that the S8 isolate had a 100% similarity to Pichia fermentans UniFGPF2 (KT029805.1).

Probiotic Candidates Yeast Isolated from Dangke - Indonesian Traditional Fermented Buffalo Milk

The aim of the study was to isolate and identify the yeast isolated from Dangke and its potential as probiotics. The purified isolates obtained were identified based on observations of macroscopic characteristics of colonies and microscopic cells. The ability as a probiotic yeast is obtained by testing the resistance towards acid conditions, bile salt resistance test and aggregation ability test against pathogenic bacteria using Salmonella sp. The yeast isolates were identified using the RapID Yeast Plus System. The isolation result was obtained D.10.3.d isolate that identified as Candida guilliermondii which showed probiotic characteristic. The yeast colony is round, cream‑colored, smooth surfaces, low convex elevations and entire edges, capable of growing on mediums with the pH of 4, containing 1 % and 5 % of bile salts and having the ability to aggregate Salmonella sp. at 15, 60, and 180 minutes.

Antibacterial Activity Test of Indigenous Yeast from Sapodilla Fruit against Staphylococcus aureus and Escherichia coli

The research aimed to identify indigenous yeast antibacterial activity from sapodilla fruit against Escherichia coli and Staphylococcus aureus, which conducted by experimental methods and followed by descriptive analysis. This study was done by the isolation of indigenous yeast, macroscopic and microscopic identification, yeast identification using RapID Yeast Plus System, antibacterial test by measuring the clear zone diameter, testing of pathogenic bacteria viability against indigenous yeast and identification of organic acid produced by yeast. The results of yeast isolation obtained 1 isolate (Saccharomyces cereviseae 1) from fruit and 3 isolates form sapodilla skin (S.cereviseae 2, Candida famata, and Pichia anomala) which had antibacterial activity against E. coli and S. aureus except C. famata isolates. Isolates with the largest antibacterial activity against E. coli and S. aureus based on the clear zone diameter were S. cerevisiae (2) isolates. The results of organic acid analysis by HPLC found that S.cerevisiae (2) isolate produced the highest organic acid namely acetic acid as much as 2.442 mg mL -1. Key words : antibacterial, organic acid, sapodilla fruit, yeast

Selection of Mozzarella Cheese Whey Native Yeasts with Ethanol and Glucose Tolerance Ability

The research aimed to determine the native yeast on mozzarella cheese whey that has glucose and ethanol tolerance ability. The research did experimentally and the data analyzed descriptively. Native yeasts isolated from 1 ml mozzarella cheese whey with using a modification of Potato Dextrose Agar/PDA (Oxoid Ltd.) with the addition of 3% Yeasts Extract/YE (Kraft Foods) and 10 ppm amoxicillin. The yeasts identified for macroscopic and microscopic characteristics then tested with RapID Yeast Plus System. The ability in tolerate ethanol and glucose contents tested by grown the yeasts on modified Nutrient Broth/NB (Oxoid Ltd.) with 3% Yeasts Extract/YE (Kraft Foods) and 10 ppm amoxicillin then added with glucose monohydrates (10%, 20%, 30%) or ethanol (10%, 20%, 30%) and incubated for 72h at room temperature (23-28°C). Optical density (OD) read for UV absorbance at 600 nm using UV-Vis spectrophotometer every 24h until 72h. Results showed that six native yeasts isolated and identified as C. tropicalis three isolate, Tri. beigelii two isolates and Blast. capitatus is one isolate. The best isolates with highest OD at 30% glucose concentration (2.215) gained by C.tropicalis (a), while the highest OD at 30% ethanol concentration (0.508) shown by C.tropicalis (f).

Yeast species associated with diseased fish: Occurrence, identification, experimental challenges and antifungal susceptibility testing

The effort to characterize yeasts in fish is still meager, and identification of yeasts from aquacultured diseased fish could be a good strategy to identify those yeast species which have a role in overall fish health. Yet, knowledge on their identification using Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is lacking. Hence, this study investigates the diversity of yeasts residing in the skin, gills, intestine and internal organs of three economically important diseased freshwater fish species. We carried out a comparative evaluation of the performance of the biochemical identification kit RapID Yeast Plus system and MALDI-TOF MS, with amplification/sequencing of Internal Transcribed Spacer (ITS1–ITS2) regions of rDNA as the reference method for yeast identification. Antifungal susceptibility pattern of yeast isolates was assessed using Vitek2 compact. Among 420 samples from Oreochromis species, Clarias gariepinus and Mugil cephalus, 215 yeast isolates (51.2%) were detected, with 7 genera: Candida, Cryptococcus, Debaryomyces, Rhodotorula, Trichosporon, Geotrichum and Pichia identified. Candida spp. accounted for 65.12% of the isolates and Rhodotorula spp. 28.84%. Overall, the yeast belonging to Ascomycota was higher than the Basidiomycota yeast (P < 0.001). There is statistically highly significant difference between incidences of yeast species in different fish. The intraperitoneal-challenged fish groups with Cryptococcus uniguttulatus, Candida lipolytica, Candida albicans and Trichosporon mucoides exhibited the highest cumulative mortality percentages (100, 80.95, 71.43 and 61.90%, respectively). C. uniguttulatus challenged group exhibited exophthalmia, opacity in the lens and yellow discoloration of the body and internal organs, meanwhile C. albicans group showed red mouth. The RapID Yeast Plus system identified the tested isolates at the species and genus level with a percentage of 30% for each. The MALDI-TOF MS accurately identified 75% of the isolates to the species level. Of the isolates (25%) not present in the MALDI-TOF MS database at the species level, 10% were ascribed to the genus level and an incorrect identification was given for 15%. Voriconazole, amphotericin B and caspofungin were the most active drugs against all the species tested, while fluconazole demonstrated poor activity. The diseased freshwater fish species may harbor potential clinically relevant yeast species, including C. albicans, non-albicans Candida species, Trichosporon, Cryptococcus and Rhodotorula species. MALDI-TOF MS offers an accurate and cost-effective alternative to the conventional and molecular methods for yeast identification. Subsequently, implementation of the VITEK MS with a more extended database is required.

Antifungal effects of Lavandula binaludensis and Cuminum cyminum essential oils against Candida albicans strains isolated from patients with recurrent vulvovaginal candidiasis

Recurrent vulvovaginal candidiasis (RVVC), which affects approximately 5% of women of reproductive age, is defined as 4 or more episodes of symptomatic Candida vaginitis within a year. The purposes of this study were to determine the chemical compositions and antifungal susceptibility of Cuminum cyminum (C.cyminum) and Lavandula binaludensis (L.binaludensis) essential oils and their combination against Candida albicans (C.albicans) strains isolated from patients with RVVC. C.albicans isolates were identified via germ tube test, CHROMagar and RapID Yeast Plus System. The essential oils were obtained by hydrodistillation in a Clevenger apparatus and analyzed by gas chromatography-mass spectroscopy (GC-MS). The broth microdilution method was used as antifungal susceptibility test (CLSI-M27-A3). The GC-MS analysis allowed 13 components to be determined; the main components of C.cyminum and L.binaludensis essential oils were γ-terpinene (21.07%) and 1,8-cineole (71.56%), respectively. L.binaludensis and C.cyminum oils were effective in inhibiting C.albicans growth at mean concentrations of 7.91±1.61μg/mL and 8.00±1.89μg/mL, respectively. In addition, the combination of C.cyminum with L.binaludensis oils were more active causing inhibition in all C.albicans isolates, with concentrations varying from 3.90 to 11.71μg/mL (mean value: 7.22±1.69μg/mL). The results suggested the potential substitution of the antifungal chemicals by C.cyminum and L.binaludensis essential oils as natural inhibitors to control the growth of the most important pathogenic Candida species and alternative therapies for RVVC.

Evaluación del rendimiento del sistema Rapid™ Yeast Plus (Remel) a partir de 2 medios de cultivo diferentes

Within the genus Candida, Candida albicans is the most commonly isolated species from clinical samples. Due to the emergence of other species which can show a higher index of antifungal resistance, a fast identification of these species is necessary. The aim of this work was to evaluate the performance of the RapID Yeast Plus system from two different subculture media formulations: Sabouraud dextrose agar adjusted by Emmons (the medium is indicated in the equipment insert) and Sabouraud glucose agar, which is the most frequently used in Buenos Aires City laboratories. One hundred and sixty-six clinical sample strains coming from different hospitals belonging to the Mycology Network of Buenos Aires City were studied. From the obtained results, we conclude that the conditions and culture medium indicated by the manufacturer should be followed.

The Isolation and Identification of Stress Tolerance Ethanol-fermenting Yeasts from Mozzarella Cheese Whey

Bioethanol conversions has long been considered as a possible solution for whey utilization. The existence of wild ethanol-fermenting yeasts that able to hydrolyze lactose become an important thing. Aims of the research was to isolate and identified the potential ethanol-fermenting yeasts from mozzarella cheese whey. Research done experimentally and analyzed descriptively. Yeast candidates isolated from mozarella cheese whey with pour plate method and purified on Potato Dextrose Agar (PDA) with the addition of 3% Yeast Extract and 10 ppm of Amoxycillin. The candidates were tested to stress tolerance towards various sugar and ethanol contents. Ethanol-fermenting yeast candidates tested in converting whey into bioethanol by inoculating 2% isolates into mozzarella cheese whey and incubated in room temperature (26-28°C) for 48 hours, then the ethanol contents tested by Gas Chromatography. The isolate that gave the highest ethanol contents identified using RapID Yeast Plus System. Results showed that there are 5.8 x 105 cfu/ml yeasts population in mozzarella cheese whey with four isolates that has different macroscopic character. Four candidate showed endurance towards 30% sugar and 20% ethanol contents and isolate W2 chosen as best isolate that produce the highest ethanol contents of 1,515.973 ppm. RapID Yeast Plus System has identified the isolate W2 has 96.43% similarity with Candida lambica .

Comparative Evaluation of the BD Phoenix Yeast ID Panel and Remel RapID Yeast Plus System for Yeast Identification.

Becton Dickinson Phoenix Yeast ID Panel was compared to the Remel RapID Yeast Plus System using 150 recent clinical yeast isolates and the API 20C AUX system to resolve discrepant results. The concordance rate between the Yeast ID Panel and the RapID Yeast Plus System (without arbitration) was 93.3% with 97.3% (146/150) and 95.3% (143/150) of the isolates correctly identified by the Becton Dickinson Phoenix and the Remel RapID, respectively, with arbitration.

Mycotic mastitis in sheep

The study was aimed to investigate the mycotic mastitis in sheep during the period from October 2011 to May 2012 in different areas of Al-Diwaniya province. 253 ewes were examined, and from which 500 milk samples were collected (495 samples from apparently healthy ewes that examined by California Mastitis Test (CMT ) in addition to five samples from sheep infected with clinical mastitis) for isolation and identification of yeasts and molds adopted the method of culturing on Seaboard Dextrose and Corn meal agar in addition to Chrome agar and biochemical tests as well as specific yeast kits (Integral system yeast plus) for diagnosing the mycotic agents. Results indicated that the incidence of mastitis in ewes was 17.8%, while the percentage of mycotic mastitis was 9.4%, (0.4% and 9% of clinical and subclinical forms respectively). Yeasts were isolated and identified grossly by colony shape, size, and color, and by biochemical testing which represents 9.61% of the fungal causes, and the yeasts isolates were Candida famata and Rhodotorula rubra from the clinical cases of mastitis only. Molds 90.38% also were isolated, as a high isolate of Aspergillus niger 28.84%, Aspergillus flavus 23%, Aspergillus fumigates 17.30%, Penicillium spp 13.33%, Aspergillus terrus 5.76%, and the least percentage of isolation 3.84% was of the Fusarium spp.

Evaluation of possibilities in identification and susceptibility testing for Candida glabrata clinical isolates with the Integral System Yeast Plus (ISYP)

The aim of this study was to evaluate possibilities of correct identification and susceptibility testing of C. glabrata clinical isolates with Integral System Yeast Plus (ISYP). For species identification, as the reference method, API Candida test and species-specific PCR reactions were used. The potential of antifungal susceptibility testing by the ISYP test was compared with the Sensititre Yeast One. Whilst the reference methods confirmed that the received population (n = 65 isolates) represented only C. glabrata, identification with the ISYP system showed correct data only in the case of 18 strains tested (27.7%). Species identification of the other 47 strains with the ISYP test was not possible at all. Significant differences were also observed for drug susceptibility testing carried out by the ISYP and the Sensititre Yeast One. The highest level of disagreement in classifying strains as resistant or susceptible estimated, as 73.9% and 40.0%, was observed for itraconazole and amphotericin B, respectively. Satisfactory results were only obtained for 5-fluorocytosine with 93.8% agreement between both methods. In our opinion the idea of the ISYP system is certainly good. The combination of identification ability and drug susceptibility testing in one test is very important, especially from a clinical point of view. However, the current version of the ISYP has many disadvantages. We would like to encourage the manufacturer to make an effort and develop a new, more accurate version of the test.

The effect of glycemic control on Candida colonization of the tongue and the subgingival plaque in patients with type II diabetes and periodontitis

Identify the relationship between the tongue and subgingival plaque Candida colonization, and its relationship to the quality of glycemic control (assessed by glycosylated hemoglobin) and other confounders, in type II diabetics who have periodontitis. Candida species were isolated from the tongue and subgingival plaque of the 2 deepest periodontal pockets from 78 patients, and were identified by RapID Yeast Plus System. Student's t test, χ(2) test or Fisher's exact test, analysis of variance, multivariate logistic regression analysis, and Spearman's correlation test were used. Candida species colonized 46 (59%) and 38 (48.7%) of the patients tongue and subgingival plaque, respectively. Subgingival Candida was more prevalent in females than males (P = .041). The quality of glycemic control correlated with the prevalence of subgingival Candida (P = .047) and the severity of gingival disease (P = .047). Poor glycemic control and female gender may be associated with increased subgingival Candida in patients with type II diabetes.

Glucose and Ethanol Tolerant Enzymes Produced byPichia(Wickerhamomyces) Isolates from Enological Ecosystems

A total of 17 <i>Pichia</i> (<i>Wickerhamomyces</i>) isolates obtained from enological ecosystems in the Utiel-Requena Spanish region were characterized by physiological (using API 20C AUX strips and ID Yeast Plus System miniaturized identification systems) and molecular (PCR-RFLP and sequencing) techniques as belonging to the species <i>P. fermentans, P. membranifaciens</i>, and <i>W. anomalus</i>. Data support the reclassification of <i>P. anomala</i> as <i>Wickerhamomyces anomalus</i>. In order to characterize their enzymatic abilities, xylanase, β-glucosidase, lipase, esterase, protease, and pectinase qualitative and quantitative assays were made. <i>Wickerhamomyces anomalus</i> and <i>P. membranifaciens</i> were the most interesting species as a source of enzymes for the winemaking industry. Glycosidase enzymes had a high degree of tolerance to high levels of glucose and ethanol, making them of great interest for enological use.

Comparison of Enzymatic Method Rapid Yeast Plus System with RFLP-PCR for Identification of Isolated Yeast from Vulvovaginal Candidiasis.

To compare two identification methods, i.e., restriction fragment length polymorphism (RFLP)-PCR analysis and enzymatic method Rapid TM Yeast Plus System to identify different species causing vulvovaginal candidiasis (VVC). Vaginal discharges of women who had attended the gynecology outpatient clinic of Mobini Hospital in Sabzevar, Iran were collected using cotton swabs and were cultured on Sabouraud dextrose agar. Isolated yeasts were identified by germ-tube testing and Rapid TM Yeast Plus System (Remel USA). For molecular identification, the isolated DNA was amplified with ITS1 and ITS4 universal primers and PCR products digested with the enzyme HpaІІ followed by agarose gel electrophoresis. Epidemiological and clinical features of women with respect to identified species were also evaluated. Out of 231 subjects enrolled, 62 VVC cases were detected. The isolated species were identified as follows: Candida albicans, 24 (38.7%), C. glabrata, 15 (24.2%), C. kefyr, 13 (21.0%) C. krusei, 9 (14.5%), and Saccharomyces cerevisiae, 1 (1.6%) by RFLP-PCR method; whereas findings by Rapid TM Yeast Plus System were C. albicans, 24 (38.7%), C. glabrata, 5 (8%), C. kefyr, 11 (17.7%) C. krusei, 2 (3.2%), S. cerevisiae, 9 (14.5%), and C. tropicalis, 6 (9.6%) as well as other nonpathogenic yeasts, 4 (6.9%). Statistical comparison showed that there is no significant difference in identification of C. albicans by the two methods; although, in this study, it was not true about other species of yeasts. A correlation between clinical and laboratory findings is important as it enables us to administer an appropriate treatment on time.

Antifungal efficacy of propolis against fluconazole-resistant Candida glabrata isolates obtained from women with recurrent vulvovaginal candidiasis

⁎ Corresponding author at: Mycology Research Center, Faculty of Veterinary Medicine, University of Tehran, Tehran, PO Box 14155 6453, Iran. Tel.: +98 2161117151; fax: +98 2166933222. E-mail address: Khosravi@ut.ac.ir (A.R. Khosravi). Recurrent vulvovaginal candidiasis (RVVC), which affects approximately 5% of women of reproductive age, is defined as 4 or more episodes of symptomatic Candida vaginitis within a year [1]. At present, there is no satisfactory treatment for RVVC, owing to difficulties in managing the high incidence of resistant nonalbicans Candida species such as Candida glabrata[2]. This organism is the second most common agent of candidiasis (occurring in 30% of cases) after Candida albicans and is of particular importance because of its innately increased resistance to fluconazole [3]. Natural products are currently considered to be an interesting alternative to synthetic antifungals for the control of resistant fungal agents, owing to their more effective, safe, and low-cost properties [4]. Propolis, a resinous wax-like substance produced by honey bees, is well known for its antibacterial, antifungal, antiviral, local anesthetic, anti-inflammatory, antioxidant, hepatoprotective, immunostimulating, and cytostatic activities [5]. Thepurposeof thepresent studywas to evaluate the effect of ethanol extract of propolis samples collected from the southern region of Iran (Hormozgan province) against different fluconazole-resistant C. glabrata isolated from women with RVVC. The chemical composition of thepropolis extractwasdeterminedusinggas chromatography–mass spectrometry. Female outpatients attending the Imam Khomeini Hospital, Tehran, Iran, who had experienced a symptomatic and culture-confirmed episode of RVVC and at least 4 documented episodes of Candida vaginitis within the previous 12 months were enrolled in the present study in 2010. The study was approved by the Iranian Ministry of Health and all women gave informed consent. Candida glabrata isolates were identified via urease test, differential culture medium (CHROMagar Candida; CHROMagar Microbiology, Paris, France), and sugar fermentation and assimilation tests (RapID Yeast Plus System; Remel, Lenexa, KS, USA). Minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations of propolis and fluconazole (Diflucan; Pfizer, New York, NY, USA) were determined via broth microdilution [4]. Stock solutions of fluconazole were prepared at 10 times the desired concentration and diluted with different amounts of yeast nitrogen base to achieve concentrations of 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 mg/L. The propolis samples were diluted (1 g of propolis, 1 mL of ethanol, and 4 mL of sterile distilled water) to concentrations of 0.25, 0.5, 1, 2, 4, 5, 8, 10, 15, and 20 mg/mL. The density of the yeast-cell suspension in the yeast nitrogen base was adjusted via spectrophotometer to up to 1×105 cells/mL. The tests were performed in sterile, flat-bottomed, 96-well microtiter plates. The fluconazole solutions and prepared C. glabrata samples (2×100 μL of each) were dispensed into the wells. For the propolis test, 100-μL aliquots of prepared C. glabrata samples were distributed to 11 microtiter plates and propolis solutions were added at different concentrations. After 48 hours, readings from both the fluconazole and the propolis samples were obtained using a microplate reader (Digiscan; Asys Hitech, Eugendorf, Austria). Data were analyzed via t test with 95% confidence intervals (CIs). Pb0.05 was considered to be statistically significant. The propolis extract was composed mainly of phenolic acid (89.8%), sugars (3.2%), and caffeic acid (2.2%). Other elements were oleic acid (0.8%), hexadecanoic acid (0.5%), cinnamic acid (0.4%), 3quinoline carboxamide (0.4%), 12β-hydroxypicrasan-3-one (0.3%), octadecanoic acid (0.2%), butanedioic acid (0.2%), ferulic acid (0.2%), levomenol (0.2%), isoferulic acid (0.1%), and unknown components (1.5%). Under mycologic analysis, 14 C. glabrata strains (C. glabrata1– C. glabrata14) were isolated and identified as being fluconazole resistant. The ethanol extract of propolis showed efficacy against all resistant isolates of C. glabrata (Table 1). The mean MIC values of antifungal drug and propolis against the yeast strains were 109.9 μg/mL (95% CI, 0.5–128.0) and 3.8 mg/mL (95% CI, 0.5–5.0), respectively (Pb0.05). At a propolis concentration of 8 mg/mL, 57.1% of the isolates did not grow at all.

An unusual case of granulomatous lymphadenitis due to Prototheca zopfii var. portoricensis in an immunocompetent man in China

Protothecosis is an uncommon human infection caused by Prototheca. Prototheca spp can be considered as saprophytes, and in spite of their frequency in the environment, they are of low virulence and may cause chronic infection with low-grade inflammation in humans. At present, only three species are recognized: Prototheca wickerhamii, Prototheca zopfii and Prototheca stagnora. Of these, the former two have been associated with human disease. This study was an investigation of the clinical and microbiological features of a case of granulomatous lymphadenitis due to P. zopfii var. portoricensis in an immunocompetent man in China. We report the case of a 39-year-old male, who presented with swollen lymph nodes, from which the organism was isolated and identified by the RapidID Yeast Plus test (Remel, Santa Fe, NM, USA) and PCR molecular analysis. The pathogenicity of the isolate was confirmed in a mouse model and antifungal drug susceptibility testing was carried out. The pathogen was identified as Prototheca zopfii. The DNA sequence of the 18S SSU rDNA regions of the isolate strain were 100% (1205/1205) identical with Prototheca zopfii var. portoricensis. Antifungal susceptibility tests revealed that it was sensitive to amphotericin B, but resistant to 5-flucytosine, fluconazole, ketoconazole, and itraconazole. The patient responded to treatment with intravenous itraconazole and amphotericin B. Based on the patient's symptoms and microscopic evaluation, cultures, and molecular analyses of the isolate, granulomatous lymphadenitis due to P. zopfii var. portoricensis was diagnosed. P. zopfii var. portoricensis as a causative agent of human lymphadenitis in an immunocompetent case has not been reported, though a few cases of protothecosis have been reported in China. The real number of protothecosis cases may be greater than that reported in the literature. Thus, clinicians should be vigilant for any unknown cause of granulomatous lymphadenitis and should undertake an intensive histopathology, mycology examination, and even molecular analysis to rule out or confirm a potential Prototheca infection.

Evaluation of VITEK 2 and RapID Yeast Plus Systems for Yeast Species Identification: Experience at a Large Clinical Microbiology Laboratory

A total of 750 clinical yeast isolates were evaluated by two identification systems, VITEK 2 and RapID Yeast Plus, using sequence analysis of the rRNA gene internal transcribed spacer regions as the reference method. The VITEK 2 and RapID systems correctly identified 737 (98.2%) and 716 (95.5%) isolates, respectively.

Multiple Reaction Monitoring to Identify Sites of Protein Phosphorylation with High Sensitivity

Phosphorylation governs the activity of many proteins. Insight into molecular mechanisms in biology would be immensely improved by robust, sensitive methods for identifying precisely sites of phosphate addition. An approach to selective mapping of protein phosphorylation sites on a specific target protein of interest using LC-MS is described here. In this approach multiple reaction monitoring is used as an extremely sensitive MS survey scan for potential phosphopeptides from a known protein. This is automatically followed by peptide sequencing and subsequent location of the phosphorylation site; both of these steps occur in a single LC-MS run, providing greater efficiency of sample use. The method is capable of detecting and sequencing phosphopeptides at low femtomole levels with high selectivity. As proof of the value of this approach in an experimental setting, a key Schizosaccharomyces pombe cell cycle regulatory protein, Cyclin B, was purified, and associated proteins were identified. Phosphorylation sites on these proteins were located. The technique, which we have called multiple reaction monitoring-initiated detection and sequencing (MIDAS), is shown to be a highly sensitive approach to the determination of protein phosphorylation.