First report of isolation of Cryptococcus neoformans var. neoformans from avian excreta in Kathmandu, Nepal

This paper delineates the first report on the saprophytic distribution of Cryptococcus neoformans var. neoformans in the city of Kathmandu, Nepal. Twenty-eight samples of old and dry pigeon droppings collected from different sites in Kathmandu were investigated for the presence of C. neoformans by employing a dilution technique. The organism was isolated from seven (25%) of the specimens, representing four of the ten collection sites. All of the isolates were recovered on Pal's medium (sunflower seed agar) by observing light to dark brown coloured colonies of C. neoformans. However, no isolation could be achieved on Sabouraud medium as all the plates were badly contaminated with rapidly growing moulds. The microscopic morphology of the cultures in PHOL stain revealed circular to val, single or budding yeast cells with thin capsules. Detailed typing of all environmental strains indicated that they belonged to the variety neoformans and a mating type of Filobasidiella neoformans. The results of this study demonstrated that Pal's medium is an excellent differential medium for the screening of environmental specimens and C. neoformans var neoformans is prevalent in the environment of Kathmandu.     

Evaluation of chromogenic medium (ORSAB) for routine identification of methicillin resistant Staphylococcus aureus (MRSA)

Performance of chromogenic medium (ORSAB) for routine detection of methicillin resistant S. aureus (MRSA) was evaluated on 510 specimens collected from patients suspected of MRSA infection or colonization. Addition of ORSAB plates to the routine protocol allowed MRSA identification in 24 hours from samples plating. In 18 samples MRSA colonies were identified only on ORSAB plates, those cases would have been missed by routine protocol alone.     

一株尿素酶阴性的新生隐球菌

本文报告从鸽粪标本中分离出的一株尿素酶试验阴性、咖啡酸玉米吐温琼脂培养呈棕色菌落的酵母样真菌。根据该菌袜的生理、生化特性及致病力,认为是尿素酶阴性的新生隐球菌。此外,应注意这种菌在自然界中的存在和对人类造成感染的可能。这是关于从自然环境中分离出尿素酶阴性新生隐球菌的首次发现和报告。     

Effect of different K+ concentrations on Cryptococcus neoformans phenoloxidase activity

Melanin synthesis in Cryptococcus neoformans, catalyzed by phenoloxidase activity, is one of the oldest virulence factors known. However, until now, the relationship between melanin production in C. neoformans and its virulence has been poorly understood. Among different chemical compounds only Fe3+ and Cu2+ cations enhance the phenoloxidase activity in C. neoformans. A few reports in the literature describe the influence of different cations on C. neoformans phenoloxidase activity, excluding iron. In this study, 13 C. neoformans strains isolated from AIDS patients and 7 from bird droppings (B.D.), were examined in order to clarify the effect of different K+ concentrations on phenoloxidase activity. A new solid and liquid caffeic acid minimal synthetic medium (MSM-CAF) containing only caffeic acid and ferric citrate with different potassium concentrations was used to evaluate C. neoformans phenoloxidase activity. In the MSM-CAF solid medium the degree of brown pigmentation on the agar plates was read on days 1, 2 and 3 of incubation, and the pigmentation of the C. neoformans strains was classed into 5 categories. The brown pigment of the liquid MSM-CAF test tubes were checked after 24 hours of incubation by measuring the optical density (O.D.) at 480 nm. Three C. neoformans AIDS and B.D. strains, randomly chosen, were tested for phenoloxidase activity, according to the modified protocols of Polacheck et al., Torres-Guerrero et al. and Rhodes. According to the results obtained, it has been observed that K+ does not activate the phenoloxidase activity in the C. neoformans AIDS and B.D. strains. In particular, with an increase in potassium concentrations in the MSM-CAF solid and liquid medium, there was a corresponding inhibition of the phenoloxidase activity on both the C. neoformans AIDS and B.D. strains.     

新构建的含DF3启动子和白喉毒素A片段的重组表达载体对人乳腺癌细胞的特异性杀伤作用

目的研究新构建的含人乳腺癌DF3启动子和白喉毒素A片段的重组表达载体PGL3-DF3-DTA对人乳腺癌细胞的影响。方法构建重组表达载体PGL3-DF3-DTA,并用其转染DF3阳性和阴性的乳腺癌细胞MCF-7和MDA-MB-231。通过MTT法测定PGL3-DF3-DTA在体外对乳腺癌细胞生长的影响,建立裸鼠动物模型观察PGL3-DF3-DTA在体内对乳腺癌细胞的杀伤效应。结果成功构建出重组表达载体PGL3-DF3-DTA并建立了人乳腺癌裸鼠动物模型,经重组表达载体PGL3-DF3-DTA作用后的DF3阳性人乳腺癌细胞出现明显的凋亡现象,Ki-67、bcl-2基因表达水平降低,bax基因表达水平升高。结论重组表达载体PGL3-DF3-DTA能对DF3阳性的乳腺癌细胞产生特异性杀伤作用。     

Modified Littman Oxgall Agar to Isolate Cryptococcus neoformans

Littman Oxgall Agar was modified by adding an extract of Guizotia abyssinica seeds with a water diluent for rehydration. In this medium, colonies of Cryptococcus neoformans became brown, but the color failed to develop in eight other yeasts and yeast phases of diphasic organisms. The depression of saprophytic fungi noted on Littman Oxgall Agar was not lost in the modification.     

Medium Containing Trypan Blue and Antibiotics for the Detection of Cryptococcus neoformans in Clinical Samples

A medium containing trypan blue, gentamicin, and chloramphenicol is introduced for the detection of Cryptococcus neoformans and Cryptococcus species from clinical samples. Ten recently isolated strains of Cryptococcus species as well as several clinical isolates of C. neoformans incorporated trypan blue and produced dark blue colonies on this mycological medium, whereas other common yeasts were light blue. The laboratory diagnosis of two cases of cryptococcosis was accomplished by the isolation of C. neoformans on the antibiotic-dye-containing medium. Compared to conventional media supporting large numbers of Pseudomonas aeruginosa and other gram-negative bacilli, the new medium was selective for yeasts. In one instance, the colonization of the respiratory tract by C. neoformans which led to fungemia was traced by the use of the antibiotic-dye medium. The antibiotic mixture, utilized herein, was more effective in suppressing bacteria contained in samples from patients than a medium containing cycloheximide and chloramphenicol.     

A Replica Plating Method for Plant Cells

A replica plating method is described for plant cells growing in Petri dishes. The method involved a uniform application of plant cells (Morinda citrifolia L.) by spraying cells evenly on agar plates containing 60% conditioned medium. Subsequently the cells were allowed to grow through a nylon net. The net was removed from the master plate and placed upside down on replica plates. Cells from colonies adhering to the threads of the net were thus transferred to the replica plate and yielded colonies that, after a growth period of about 10–20 days, corresponded in position to the colonies on the master plate. An 80% transfer of colonies from the master plate to the copy plate was possible.     

New Quantitative, Qualitative, and Confirmatory Media for Rapid Analysis of Food for Clostridium perfringens

A selective and differential medium, Shahidi-Ferguson Perfringens agar (SFP agar), and a confirmatory medium, lactose-motility agar (LM agar), were developed for the enumeration and identification of Clostridium perfringens in foods. These media provide a rapid, specific, and direct diagnosis of C. perfringens. SFP agar contains sodium metabisulfite and ferric ammonium citrate to demonstrate H(2)S production and egg yolk to demonstrate lecithinase production by C. perfringens. On SFP agar, C. perfringens produces black colonies, 2 to 3 mm in diameter, surrounded by zones of opaque precipitate. The typical colonies are confirmed on LM agar. Enumeration and identification are completed within 48 hr. All of the ingredients of SFP agar are stable to heat and storage conditions. SFP agar also contains two antibiotics, kanamycin and polymyxin B, which are inhibitory to many bacteria commonly occurring in foods. A comparative study of SFP agar and noninhibitory media showed that SFP agar did not inhibit any of the 16 strains of C. perfringens tested. Recovery of C. perfringens added to foods averaged 90.6% for SFP agar as compared with 69.8% for sulfite polymyxin-sulfadiazine (SPS) agar (BBL) and 60.2% for SPS agar (Difco). The colonies on the SFP agar, were much larger and were consistently black. Of 464 food samples tested, C. perfringens was found in 27 samples with SFP agar and in 5 samples with SPS agar (Difco), with a recovery ratio considerably higher on SFP agar. SFP agar is a more specific presumptive medium for the enumeration of C. perfringens and in conjunction with LM agar should save considerable time, effort, and materials toward the final identification of the species.     

Analysis of recombinant protein expression by MALDI-TOF mass spectrometry of bacterial colonies

E. coli expressing soluble recombinant HIV antigens were analyzed directly by MALDI-TOF mass spectrometry (MS) from bacterial colonies picked from agar plates. An HIV envelope (ENV) antigen construct, penvA, was expressed in E. coli by transformation of the plasmid pPL/penvA-M. The plasmid was co-transformed into E. coli DH5 alpha cells with an equal quantity of the plasmid pKRR826, the parent vector without the penvA insert, and plated at medium density on L-agar plus ampicillin plates. A total of 24 colonies from four agar plates (six colonies per plate) were picked and transferred into 50% acetonitrile--0.1% trifluoroacetic acid aliquots for analysis by MALDI-TOF MS. The MS analysis detected 10 of 24 colonies expressing the recombinant protein; one colony expressed a mutant penvA protein; eleven of 24 colonies showed ions only from E. coli; and two of 24 colonies showed no detectable proteins. When E. coli transformed only with plasmid pPL/penvA-M were examined, all (10 of 10) colonies showed the penv insert by the MALDI-TOF MS method. The method is fast (less than 1.5 h for 24 colonies) and allows identification of colonies expressing intact or mutant proteins directly from culture plates without sample purification.     

Urease testing and yeast taxonomy

When urease production was assayed by the hydrolysis of [14C]urea, all basidiomycetous yeasts tested, including the Cryptococcus vishniacii complex (previously reported urease negative), produced significant amounts of 14CO2. The Schizosaccharomycetaceae were the only urease-positive ascomycetous yeasts tested. Yarrowia lipolytica was urease negative. The stoichiometry of [14C]urea hydrolysis paralleled by Roberts' rapid urea hydrolysis (RUH) test indicated that causes of anomalous results in conventional urease testing include acidification and alkalinization of the test medium by products of endogenous metabolism and autolysis rather than urease activity. Anomalous results also occurred when cells were grown on media containing the chelating agent ethylenediaminetetraacetic acid (EDTA) prior to RUH. The addition of EDTA to a complex natural medium inhibited urease production in all yeasts reportedly growing at 35 degrees C (and all other yeasts tested), except Filobasidiella (Cr.) neoformans var. neoformans (NIH 12). The RUH test could differentiate at the varietal level: Fil. (Cr.) neoformans var. neoformans was about 10 times more resistant to EDTA in media used for the growth of cells prior to RUH testing than was Fil. neoformans var. bacillispora (Cr. neoformans var. gattii) (NIH 191). Urease production by Fil. neoformans var. bacillispora was specifically restored to half maximal activity by the addition of 22 microM Ni+2 (as NiCl2) to a growth medium containing 0.100 mM EDTA.     

Induction by Klebsiella aerogenes of a melanin-like pigment in Cryptococcus neoformans

While studying the interaction of Cryptococcus neoformans with Dictyostelium discoideum, we noticed that yeast colonies in agar with a feeder lawn of Klebsiella aerogenes were brown. This finding was intriguing because C. neoformans colonies are not pigmented unless they are provided with precursors for melanization. Strains of all C. neoformans serotypes produced brown pigment in response to K. aerogenes at 22, 30, and 37 degrees C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when C. neoformans was exposed to live Escherichia coli or heat-killed K. aerogenes. Analysis of K. aerogenes supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by C. neoformans. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for C. neoformans melanization in the environment.     

Isolation Method for Bacterial Isolates Capable of Growth on p-Chlorobiphenyl

A method is reported for screening for p-chlorobiphenyl (pCB)-degrading bacteria from various environments. A solid medium was inoculated with the sample to be analyzed, colonies were allowed to develop, and the plates were then sprayed with a pCB solution in ether. The positive colonies were recorded as those surrounded with a clear zone in the film of pCB. That these colonies were able to degrade pCB was shown by their ability to grow on pCB in liquid medium with concomitant disappearance of the substrate and by the appearance of colored compounds in cultures grown on pCB.     

Identification of non-mutans streptococci organisms in dental plaques recovering on mitis-salivarius bacitracin agar medium

The objective of this study was to both isolate and identify non-mutans streptococci organisms (non-MSO) from dental plaques recovered on mitis-salivarius sucrose bacitracin agar (MSB) plates. The dental plaque samples, which had been collected from 63 human subjects, were diluted and plated on MSB. The bacteria growing on the MSB plates were then identified with biochemical tests, as well as with 16S rDNA cloning and sequencing techniques. Our data indicated that bacteria from 30 subjects had been recovered on the MSB plates. Among the 21 typical colonies selected from the 30 subjects, 12 colonies, derived from 10 subjects, were identified as non-MSO. These 12 colonies were determined to be Streptococcus anginosus (8 colonies), S. sanguinis (1 colony), and Pantoea agglomerans (3 colonies). These results strongly suggest that a new selective medium will be required for the reliable isolation of mutans streptococci.     

Mechanical stretch upregulates IGFBP-1 secretion from decidualized endometrial stromal cells

Decidualization is an essential process of endometrial differentiation for embryo implantation and maintenance of pregnancy. Recently, uterine movement-induced mechanical stress was noticed to have possible effects on endometrial functions. In this study, we addressed the possible effect of mechanical stress on the process of decidualization of endometrial stromal cells (ESC). ESC were cultured on flexible-bottomed culture plates. After decidualization was achieved with estradiol and progesterone for 12 days, cultures were continued for 24 h with or without cyclic stretch (25% elongation) in serum-free conditions at a rate of 2 cycles/min using a computer-operated cell tension system. Concentrations of insulin-like growth factor-binding protein-1 (IGFBP-1), a marker of decidualization, in the conditioned medium were measured by specific ELISA, and IGFBP-1 mRNA expression in the ESC was measured by RT-PCR. Cyclic stretch remarkably increased IGFBP-1 secretion from decidualized ESC. It also increased IGFBP-1 mRNA in decidualized ESC. The increase in IGFBP-1 secretion was inhibited by actinomycin D but not by indomethacin, PD-98059, or H-89. Conditioned medium of decidualized ESC cultured with cyclic stretch increased IGFBP-1 secretion from decidualized ESC cultured under stationary conditions. These findings imply that uterine movement modulates decidualization of the endometrium and has a regulatory effect on reproduction.     

Factors from Human Embryonic Stem Cell-derived Fibroblast-like Cells Promote Topology-dependent Hepatic Differentiation in Primate Embryonic and Induced Pluripotent Stem Cells

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.     

对两种新生隐球菌选择性培养基的比较研究

比较鸟籽琼脂（GASA,Guizotia abyssinica seed agar）和咖啡酸玉米琼脂（CACA,Caffeic acidcommeal agar）对新生变种和格特变种的培养效果。再同时用两种培养基分离命粪和澳洲赤桉标本中的新生隐球菌。结果表明,CACA对新生隐球菌的培养和选择性分离效果与GASA相同,能够用于新生隐球菌的选择性分离。     

Evaluation of a new chromogenic medium (Candida ID) for the isolation and presumptive identification of Candida albicans and other medically important yeasts

Candidiasis is a frequent human infection caused mainly by Candida albicans. However, other species are emerging as important pathogens, as Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei or Candida guilliermondii. Rapid identification of clinical isolates could facilitate diagnosis and treatment. Candida ID (bioMerieux, Spain) is a new medium for the isolation and presumptive identification of yeasts: C. albicans grows as blue colonies, and C. tropicalis, C. guilliermondii, Candida kefyr and Candida lusitaniae as pink ones. The utility of Candida ID was evaluated with more than 700 clinical isolates and type culture collection strains from different genera including Candida, Cryptococcus, Saccharomyces, and Rhodotorula. Presumptive identification was confirmed by germ tube test, microscopic morphology and chlamydoconidia production on corn meal agar and carbohydrate assimilation on API-ATB ID 32C or Vitek (bioMerieux). Growth on Candida ID was rapid (18-24 h) for most of the yeast strains tested. Sensitivity and specificity of identification of C. albicans was significantly high (>98%), since a very low number of isolates were found to be false negative or false positive. A better result was obtained for species growing as pink colonies (>99.5%). Detection of different species of medical important yeasts was easy with Candida ID, as perfectly distinct colors and textures of colonies were observed on this medium. Candida ID allowed the discrimination between C. glabrata (creamy and smooth) and C. krusei (rough and white) colonies. Other species showed different colony textures and colours, white being the predominant colour. Candida ID was very useful for the presumptive identification C. albicans isolates.     

The influence of newly synthesised fenporpimorph derivatives on some pathogen yeasts

The effect of minimum inhibitory concentrations (MICs) of six novel fenpropimorph derivatives on lipid and sterol composition of Candida albicans, Cryptococcus neoformans, Malassezia pachydermatis and Malassezia furfur was investigated. The MICs for the most effective derivatives were found in the range from 3.7 to 56.7 microM and were 2-3 times lower compared to the commercial fungicide bifonazol. The more efficient fenpropimorph derivatives were the piperidine derivative for C. albicans and the allylamine derivative for Cr. neoformans, M. pachydermatis and M. furfur. The inhibitor in the growth medium reduced the unsaturation index of the total lipid content in M. furfur and C. albicans.     

Membrane filter procedure for enumeration of Aeromonas hydrophila in fresh waters.

A membrane filter method (mA) for the enumeration of Aeromonas hydrophila in natural water samples was developed. The complex, primary medium employs trehalose as a fermentable carbohydrate and ampicillin and ethanol as selective inhibitors. After 20 h of incubation at 37 degrees C, an in situ mannitol fermentation test followed by an in situ oxidase test is used to further differentiate A. hydrophila from other aquatic and terrestrial microorganisms present in freshwaters. The primary medium decreases background microbial growth by about two orders of magnitude. The recoveries on mA medium from suspensions of A. hydrophila prepared from pure cultures and held for 24 h at 15 degrees C exceeded 95% of the recoveries on brain-heart infusion agar spread plates. The confirmation rate for colonies designated A. hydrophila was 98%, whereas 11% of the presumptively negative colonies were, in fact, A. hydrophila. Recoveries of A. hydrophila from fresh, surface water samples exceeded recoveries by the other methods examined.