Inhibition of specific amino acid uptake in Candida albicans by lysosomal extracts from rabbit alveolar macrophages.

Lysosomal-rich fractions, obtained from normal rabbit alveolar macrophages, were extracted and tested for their effects on Candida albicans. The uptake and incorporation of various compounds (amino acids, uridine, 2-deoxy-D-glucose, and Rb+) by C. albicans were measured in the presence and absence of extract. These studies demonstrated that the extract had a specific effect on the uptake of certain amino acids by C. albicans. Of the amino acids tested, the uptake of methionine valine, lysine, phenylalanine, and leucine was drastically reduced in the presence of extract, whereas proline and glutamic acid uptake was unaffected. Those amino acids whose uptake was inhibited have been shown to be transported in other yeasts by a general amino acid permease. The existence of a general amino acid permease in C. albicans is compatible with our data. Additionally, the extract had no effect on the uptake of uridine, 2-deoxy-D-glucose, and Rb+. Leakage of 86Rb by C. albicans was detected in the presence of the extract. Viability of Candida, as measured by colony-forming ability, decreased after a 16-h incubation of C. albicans with the extract.     

Factors affecting filamentation in Candida albicans: relationship of the uptake and distribution of proline to morphogenesis.

When glucose was present in high concentration, Candida albicans formed filaments in a phosphate-buffered medium, regardless of the nitrogen source. In lower concentrations of glucose, filamentation occurred only when various members of the glutamate, succinyl, or acetoacetyl-coenzyme A families of amino acids were used as sole nitrogen sources. Yeast morphology could be maintained either by replacing the amino acids in the medium with ammonium chloride or by making the medium high in phosphate or biotin. Studies using [U-14C]proline indicated that proline was catabolized in a manner consistent with the generation of increased cellular reducing potential and that the proline label entered into the Kreb's cycle. A reduction in Kreb's cycle activity was evidenced by an initial increase and then a rapid drop of the total organic acid content of the cells as well as in specific Kreb's cycle intermediates. Filamentation under conditions of low phosphate, high glucose, and increased cellular reduction potential, accompanied by a decrease in Kreb's cycle activity, suggests that morphogenesis in C. albicans is correlated with a Crabtree-like effect, i.e., repression of mitochondrial activity.     

Naturally occurring auxotrophs of Campylobacter jejuni and Campylobacter coli.

The nutritional requirements for 439 Campylobacter jejuni isolates and 46 Campylobacter coli isolates were determined by using a previously described chemically defined medium, campylobacter defined medium. With this medium, 45% of both human and nonhuman C. jejuni isolates demonstrated auxotrophic requirements. None of the 46 C. coli isolates studied demonstrated requirements for amino acids on campylobacter defined medium. The most common auxotrophic requirement among C. jejuni isolates was for methionine, which was present as a single requirement or in combination with other markers in 21% of human and 28% of nonhuman isolates. There was no correlation between plasmid carriage and auxotype, and a comparison of the Lior serotypes of 472 of the strains showed a correlation only between proline auxotrophs and Lior serotype 11 for strains isolated in the Seattle-King County region.     

Regulation of Candida albicans morphogenesis by fatty acid metabolites

Candida albicans is an opportunistic dimorphic fungus that inhabits various host mucosal sites. Conversion from the yeast to the hyphal form has been associated with increased virulence and mucosal invasiveness. C. albicans morphogenesis is regulated by multiple signals and signaling pathways. However, signals that control morphogenesis in vivo are unknown. We investigated the effects of host long chain fatty acids, eicosanoids, and bacterial short chain fatty acids on control of germination. None of the C18 or C20 fatty acids tested had an effect on enhancing germ tube formation (arachidonic acid, oleic acid, linolenic acid, or gamma-linolenic acid). Among the different eicosanoids, both prostaglandin E2 and thromboxane B2 significantly enhanced serum-induced germination by C. albicans. Addition of antiprostaglandin or antithromboxane antibodies to serum alone inhibited germ tube formation by almost 30%, while control antibody had no effect, indicating that these eicosanoids are major morphogenic factors in the serum. Since these molecules also bind to albumin, this may also explain the hyphal transforming activity in serum that associates with albumin. Interestingly, short chain fatty acids (butyric acid), the product of lactic acid bacteria (LAB), inhibited germination. In addition, LAB culture supernatants as well as live LAB also inhibited C. albicans morphogenesis. Overall, these results indicate that fatty acid metabolites and fatty acid pathways can up-regulate and down-regulate germination in C. albicans.     

Tobacco agar, a new medium for differentiating Candida dubliniensis from Candida albicans

Isolates of Candida dubliniensis may be misidentified as Candida albicans in microbiological laboratories if only the germ tube and/or the chlamydospore test is used for identification to the species level. In this study, we have evaluated the efficacy of tobacco agar for the differentiation of C. dubliniensis from C. albicans. On this medium at 28 degrees C, all 30 C. dubliniensis isolates produced yellowish-brown colonies with hyphal fringes and abundant chlamydospores, whereas 54 C. albicans isolates formed smooth, white-to-cream-colored colonies with no chlamydospore production. This medium provides a simple tool for presumptive differentiation of C. dubliniensis from C. albicans.     

The presumptive identification of Candida albicans with germ tube induced by high temperature

For direct identification of Candida albicans from other Candida species, the chlamydospore formation and the mycelial transition induced by high temperature and by sera were examined in 198 Candida isolates. The germ tubes of C. albicans developed early at 30 min in high temperature-induction, but at 60 min in serum-induction. C. albicans generated germ tubes well at concentrations lower than 2 x 10(7) cells/ml, but the germ tube formation was markedly restrained at concentrations higher than 4 x 10(7) cells/ml. In a serum-free, yeast extract-peptone-dextrose (YEPD) medium, C. albicans grew as a yeast form at 30 degrees C and as a mycelial form at 35-42 degrees C. Mycelial development was maximal at 37 degrees C in serum and at 39 degrees C in YEPD. Germ tubes were formed within 30 min in YEPD at 39 degrees C, but after 60 min in serum at 37 degrees C. Our examination showed that the 39 degrees C-induced germ tube formation tests were very reliable (sensitivity 100%, specificity 100%) at discerning C. albicans from other Candida species. These results suggest that the high temperature-induced germ tube formation testing could be a useful identification method of C. albicans in clinical laboratories.     

Comparison of rapid testing methods for enzyme production with the germ tube method for presumptive identification of Candida albicans.

The observation of germ tube production as a method for the presumptive identification of Candida albicans has been in use for many years. Methods have recently been developed for detecting the production of the enzymes L-proline aminopeptidase and beta-galactosaminidase by yeast isolates grown in culture. Both enzymes are produced by C. albicans; other yeasts may produce either L-proline aminopeptidase or beta-galactosaminidase but not both enzymes. One hundred thirty-three clinical yeast isolates, including 55 C. albicans, 27 Candida tropicalis, 22 Torulopsis (Candida) glabrata, and 29 other yeast isolates were tested by the germ tube production method and three tests for enzyme production, with the API 20C method used as a "gold standard." All three enzymatic methods evaluated provided more objective and rapid nonmicroscopic alternatives to the germ tube test and may be used to accurately distinguish C. albicans from other yeasts.     

Phenotypic and genotypic characterization of unusual vaginal isolates of Candida albicans from Africa.

As expected by its global prevalence, the most frequently isolated species of yeast from vaginal swabs obtained from patients in Africa was Candida albicans, which accounted for 53 of 85 (62.4%) of the isolates from women in Madagascar and 35 of 54 (64.8%) of the culture-positive women in Angola. However, 40% of the Madagascan and 23% of the isolates from Angola, as well as two isolates obtained from one German patient, were not able to utilize the amino sugars glucosamine and N-acetylglucosamine as the sole carbon source. These isolates were able to form germ tubes but did not form chlamydospores. The correct identification as C. albicans was made possible only by using a PCR-based method of DNA fingerprinting. Only minor phenotypic and genotypic variation was observed among these strains. Whether they represent a distinct clone that is found mainly in Africa is not clear. The relevance of the amino sugar catabolic pathway in C. albicans is discussed in view of these results.     

Comparison of the MUREX C. albicans, Albicans-Sure, and BactiCard Candida test kits with the germ tube test for presumptive identification of Candida albicans.

The MUREX C. albicans (MC)(Murex Diagnostics), Albicans-Sure (AS) (Clinical Standards Laboratories), and BactiCard Candida (BC) (Remel) test kits were compared with the germ tube (GT) test for the rapid, presumptive identification of Candida albicans. All three test kits detect the enzymes L-proline aminopeptidase and beta-galactosaminidase in yeast cells grown on culture media and are based on the principle that C. albicans produces both enzymes whereas other yeasts produce only one or neither of the enzymes. The organisms evaluated were fresh clinical isolates identified by methods routinely used in our laboratory (API 20C system and conventional methods) and included 303 C. albicans isolates, 153 Candida glabrata isolates, 70 Candida tropicalis isolates, 36 Candida parapsilosis isolates, 13 isolates of other Candida spp., 5 Cryptococcus neoformans isolates, and 3 Saccharomyces cerevisiae isolates. The MC, AS, BC, and GT tests detected 299 (98.7%), 300 (99.0%), 301 (99.3%), and 287 (94.7%) C. albicans isolates, respectively. There was one false-positive result with both the MC and BC kits and two false-positive results with the GT test. The enzymatic methods evaluated in this study provide rapid and accurate alternatives to the GT test for the presumptive identification of C. albicans.     

Inductions of germ tube and hyphal formations are controlled by mRNA synthesis inhibitor in Candida albicans.

Candida albicans is a pathogenic dimorphic fungus. When yeast cells were pre-incubated in YPD medium at 25degreesC and released into HFM7 medium containing 4% serum at 37degreesC, germ tubes emerged within 0.5 h. To determine whether mRNA or protein synthesis was necessary for germ tube formation, we examined the effects of mRNA and protein syntheses inhibitors on this formation. In the presence of cycloheximide, cells were unbudded and no germ tube was observed. However, in the presence of actinomycin D, germ tube formation was observed while budding growth and true hyphae elongation were blocked. Next, we measured mRNA or protein accumulation during induction of germ tube formation in the presence of the inhibitors. In the presence of cycloheximide, protein was not synthesized, while in the presence of actinomycin D, mRNA synthesis decreased to 6.3% and protein synthesis to 37.7%. The condition we found which allows only germination but not budding or filamentation might be convenient to use in screening genes involved in the initial stage of morphological change in C. albicans.     

New aniline blue dye medium for rapid identification and isolation of Candida albicans.

Organic dyes have long been used in diagnostic microbiology to differentiate species by color reactions. We studied the ability of a new noninhibitory medium, YM agar containing 0.01% aniline blue WS dye, Colour Index 42780 (YMAB), to identify Candida albicans among 1,554 yeast specimens obtained from seven clinical laboratories. Appropriate American Type Culture Collection and other characterized strains served as controls. A total of 487 of the clinical strains were identified as C. albicans. The remainder were other Candida species and non-Candida yeasts. Clinical isolates and controls were grown on Sabouraud agar for 18 h at 30 degrees C and then transferred to YMAB. Plates were incubated for 12 to 18 h at 30 degrees C, and colonies were observed for yellow-green fluorescence under long-wave UV light (A365). All control strains of C. albicans and Candida stellatoidea fluoresced, as did 480 of the 490 isolates designated as C. albicans (which included 3 strains of C. stellatoidea). Cells of C. albicans grown on YMAB produced germ tubes in serum. Only five of the other 1,062 non-C. albicans yeasts fluoresced. The sensitivity and specificity were 98.0 and 99.5%, respectively, with a predictive value of 99.1%. A fluorescent metabolite was found in cell wall particulate fractions of C. albicans sonic extracts grown on YMAB but not in non-C. albicans yeasts. This metabolite showed the same spectral curve as those of metabolites from whole cells in a recording spectrofluorometer when it was excited at 400 nm and scanned from 420 to 550 nm. Thus, growth on YMAB generates the production of a fluorescent moiety that can be used to specifically identify C. albicans within 12 to 18 h.     

In vitro effect of fibrinogen on Candida albicans germ tube formation

Pseudohyphae formation by Candida albicans blastoconidia, as seen in vaginal smears, is a phenotypical change commonly assumed to mean fungal invasiveness, i.e. not mere colonization. C. albicans forms germ tubes in vitro in the presence of serum. In our search for inhibitory components of germ tube formation, we decided to study fibrinogen. The inhibition of germ tube formation by clinical isolates of C. albicans was evaluated in the presence of serial concentrations of fraction I, type IV and fraction I, type Is of fibrinogen from bovine plasma. Fibrinogen showed a dose-dependent, pH-independent inhibitory effect on the germ tube formation by C. albicans.     

Isolation and molecular characterization of Candida africana from Jos, Nigeria

During a survey of the prevalence of Candida spp. in Jos, Plateau State, Nigeria, two atypical C. albicans isolates were recovered. These two yeasts were germ tube positive, chlamydospore-negative and gave a green color on CHROMagar Candida. Molecular analysis performed by amplification of the hwp1 gene showed that these two isolates belonged to C. africana, a newly proposed Candida species closely related to C. albicans. Based on the presence or absence of an intron in DNA sequences encoding rRNA, the two C. africana, including all C. albicans isolates examined, were found to belong to genotype A and no other genotypes or species such as C. dubliniensis were found. To our knowledge, this is the first isolation of C. africana in Nigeria.     

Discrimination between Candida albicans and other pathogenic species of the genus Candida by their differential sensitivities to toxins of a panel of killer yeasts

The differential sensitivities to toxins produced by a short panel of four killer yeasts allowed discrimination between 91 strains of the yeast Candida albicans and 223 non-C. albicans Candida strains. One hundred percent of C. albicans isolates exhibited negative results to the toxin panel, while 100% of non-C. albicans cultures gave well-defined and reproducible positive results to at least one of the four killer toxins. Among C. albicans strains only 96 and 87% gave germ tube (GT)- and chlamydospore-positive results, respectively. In addition a few GT-false-positive strains were detected among non-C. albicans isolates. Susceptibility to the toxin panel is apparently expressed more consistently than either GT or chlamydospore production and may constitute a promising basis for a new simple and easy-to-use procedure for routine discrimination between the species C. albicans and other species of the genus Candida.     

Rapid identification of Candida albicans: evaluation of "Rapidec albicans". Study of 444 yeast strains]

Rapid identification of Candida albicans is of great importance as it is the most frequently isolated yeast pathogen. Rapidec albicans, a new 2-h micromethod, performs two fluorescent enzymatic activities: hexosaminidase and proline arylamidase. A total of 444 yeast strains (334 from type culture collections and 110 from recent clinical isolates) were tested. The sensitivity was 98.5% and the specificity 95.8%. When only considering the clinical strains, 47/47 Candida albicans were identified by Rapidec albicans (sensitivity 100%) but only 43/47 by the germ tube test (sensitivity 91.5%). The specificities of the two tests were respectively 98.2% and 100%. This new system is therefore very efficient for the routine diagnosis of Candida albicans in the clinical field. It is easier and quicker than the germ tube test.     

Biofilm production by isolates of Candida species recovered from nonneutropenic patients: comparison of bloodstream isolates with isolates from other sources

Biofilm production has been implicated as a potential virulence factor of some Candida species responsible for catheter-related fungemia in patients receiving parenteral nutrition. We therefore compared clinical bloodstream isolates representing seven different Candida species to each other and to those from other anatomical sites for the capacity to form biofilms in glucose-containing medium. Potential associations between the capacity to form biofilms and the clinical characteristics of fungemia were also analyzed. Isolates included the following from nonneutropenic patients: 101 bloodstream isolates (35 C. parapsilosis, 30 C. albicans, 18 C. tropicalis, 8 C. glabrata, and 10 other Candida species isolates) and 259 clinical isolates from other body sites (116 C. albicans, 53 C. glabrata, 43 C. tropicalis, 17 C. parapsilosis, and 30 other Candida species isolates). Organisms were grown in Sabouraud dextrose broth (SDB) containing a final concentration of 8% glucose to induce biofilm formation, as published previously. Biofilm production was determined by both visual and spectrophotometric methods. In this medium, biofilm production by C. albicans isolates was significantly less frequent (8%) than that by non-C. albicans Candida species (61%; P < 0.0001). The overall proportion of non-C. albicans Candida species isolates from the blood that produced biofilms was significantly higher than that of non-C. albicans Candida isolates obtained from other sites (79% versus 52%; P = 0.0001). Bloodstream isolates of C. parapsilosis alone were significantly more likely to be biofilm positive than were C. parapsilosis isolates from other sites (86% versus 47%; P = 0.0032). Non-C. albicans Candida species, including C. parapsilosis, were more likely to be biofilm positive if isolates were derived from patients whose candidemia was central venous catheter (CVC) related (95%; P < 0.0001) and was associated with the use of total parenteral nutrition (TPN) (94%; P < 0.005). These data suggest that the capacity of Candida species isolates to produce biofilms in vitro in glucose-containing SDB may be a reflection of the pathogenic potential of these isolates to cause CVC-related fungemia in patients receiving TPN.     

Critical role of germ tube formation in the pathogenesis of candidal vaginitis.

A variant strain of Candida albicans incapable of hyphal production at 37 degrees C was used to study the role of germ tube formation in the pathogenesis of experimental vaginal candidiasis in rats. No difference was observed in the in vitro adherence at 25 degrees C of blastoconidia of the variant strain to vaginal epithelial cells when compared with the parent wild-type, germ tube-producing strain and multiple clinical isolates of C. albicans. However, after exposure to conditions favoring germ tube production, the adherence of the variant strain to epithelial cells was significantly less than that of germinated strains (P less than 0.01). In vivo animal studies revealed that the variant strain was less likely to result in vaginal colonization and infection than the wild-type strain and the other clinical isolates. Furthermore, infection, when established, was milder, often transient, and with significantly lower titers of cultured vaginal microorganisms obtained by lavage. Electron microscopic studies confirmed the failure of the variant strain to produce hyphae in vivo. The capacity of C. albicans to produce hyphae appears to be an important but nonessential virulence factor in the pathogenesis of candidal vaginitis.     

Prostaglandin E2 enhances and gamma interferon inhibits germ tube formation in Candida albicans.

Prostaglandin E2 (PGE2), an immunosuppressive monokine that increases intracellular cyclic AMP (cAMP) levels, stimulated Candida albicans germ tube formation. Dibutyryl cAMP (dB-cAMP) and isoproterenol, other compounds that increase cAMP levels, also stimulated germination. Gamma interferon (IFN-gamma), a product of cellular immune system activation, inhibited Candida germ tube formation, even in the presence of PGE2, dB-cAMP, and isoproterenol. Thus, PGE2 and IFN-gamma as well as having opposing roles in the suppression or activation of cell-mediated immunity, are also antagonists for the yeast-to-hyphal transition of C. albicans.     

Hemin induces germ tube formation in Candida albicans.

Hemin induced germination of Candida albicans blastoconidia when cells grown up to the early exponential phase were shifted from 28 to 37 degrees C (70 to 75% of cells exhibited germ tubes). N-Acetyl-D-glucosamine (GlcNAc), another inducer of myceliation in this fungus, caused a similar effect. The combination of hemin and GlcNAc resulted in a higher percentage (95%) of blastoconidial germination. These results suggest that in addition to temperature, hemin levels and carbon source may coordinately regulate the expression of subsets of genes involved in the yeast-to-mycelium transition in C. albicans.     

Use of auxotyping for epidemiological studies of Campylobacter jejuni and Campylobacter coli infections.

A chemically defined medium developed for Neisseria gonorrhoeae was modified to support the growth of Campylobacter jejuni and Campylobacter coli. A total of 76 isolates of C. jejuni and 14 isolates of C. coli were tested on this medium, which was designated Campylobacter defined medium (CDM), over a 3-month period. Although none of the C. coli isolates appeared to require amino acids, 51% of the C. jejuni tested required one and 7% required multiple amino acids for growth. An analysis of isolates obtained from three household outbreaks of campylobacteriosis demonstrated that auxotyping identified the epidemic strain within each outbreak. Among 70 isolates of C. jejuni examined, no correlation could be drawn between a specific serotype and auxotype or between auxotype and plasmid profile.