Virulence of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis in reconstituted human tissue models

Candida parapsilosis is an increasingly important human pathogen. To study the interactions of C. parapsilosis with human tissues, we evaluated the effects of the CBS 604 type strain and three different clinical isolates on reconstituted human oral epithelial and epidermal tissues. The newly described species Candida orthopsilosis and Candida metapsilosis were also examined in these models. Microscopy of reconstituted tissues infected with yeast cells revealed severe attenuation, morphological changes and cellular damage. C. orthopsilosis caused damage similar to C. parapsilosis isolates, whereas C. metapsilosis was less virulent. To further quantitate tissue damage, we measured lactate dehydrogenase (LDH) in the culture supernatant. The relative LDH measurements correlated with our histopathological observations. We also examined the effect of the lipase inhibitor Ebelactone B and proteinase inhibitor Pepstatin A, to establish the utility of this model for studying factors of C. parapsilosis virulence. Both Ebelactone B and Pepstatin A reduced the destruction of epidermal and epithelial tissues. Our data show that reconstituted human tissues are extremely useful for modeling host interactions with C. parapsilosis and for studying fungal virulence factors.     

Continuous cultivation of the yeast Candida utilis at different dilution rates on pineapple cannery effluent

Candida utilis was grown on a pineapple cannery effluent in a chemostat at dilution rates ranging between 0.05 and 0.65 h^–1 to establish optimal conditions for biomass production and chemical oxygen demand (COD) reduction. Sucrose, fructose and glucose were the main sugars in the effluent. Maximum value for cell yield coefficient and productivity were (0.686, g_x/g_s) and (2.96, g_x/l/h) at a dilution rate of 0.425 and 0.475 h^–1, respectively, while maximum COD reduction (98%) was attained at a dilution rate of 0.1 h^–1. The maintenance coefficient attained a value of (0.093, g_s/g_x/h). An increase in dilution rate produced a higher protein content of the biomass.     

Growth of Candida utilis on ethanol and isopropanol

Candida utilis grew on ehtanol and an ethanol-isopropanol-water (22:2:1 vols) mixture but not on isopropanol alone. Acetone accumulated in all cultures containing isopropranol but its presence in the alcohol mixture did not lower growth rate or yield significantly when compared with growth experiments on ethanol alone. Growth rate and yield declined at ethanol concentrations greater than 1% (v/v) and 0.3% (v/v) respectively. In a 0.3% (v/v) alcohol mixture, acetate was found only during the exponential growth phase. In a 3% (v/v) mixture, acetate and ethyl acetate accumulated during growth whereas acetaldehyde was present only during the exponential growth phase.     

Anti-Candida and mode of action of two newly synthesized polymers: a modified poly (methylmethacrylate-co-vinylbenzoylchloride) and a modified linear poly (chloroethylvinylether-co-vinylbenzoylchloride) with special reference to Candida albicans and Candida tropicalis

Polymeric antimicrobial agents represent a new and important direction that is developing in the field of antimicrobial agents. Antimicrobial activity of two newly synthesized polymers: a modified poly (methylmethacrylate-co-vinylbenzoylchloride) and a modified linear poly (chloroethylvinylether-co-vinylbenzoylchloride) have been investigated and found to be active. Both polymers have showed a broad antimicrobial activity against C. albicans and C. tropicalis. Minimal inhibitory concentrations (MIC's) for poly (methylmethacrylate-co-vinylbenzoyl chloride) were 100, 75 and 100 microg/ml in case of C. albicans (ATCC 2091), C. albicans (SC5314) and C. tropicalis, respectively. However, polycholoroethylvinylether-covinylbenzoylchloride inhibited C. albicans (ATCC 2091), C. albicans (SC5314) and C. tropicalis with minimum inhibitory concentration values (MIC's) of 150 microg/ml against the three tested Candida strains. Mode of action studies of both polymers on the medically important yeasts, C. albicans and C. tropicalis revealed that poly (methylmethacrylate-co-vinylbenzoylchloride) induced cytotoxicity, DNA damage, and altered cell permeability and morphology, which was manifested as aggregated and swollen yeast cells (C. albicans ATCC 2091) by fluorescent microscopy examination. Poly (chloroethylvinylether-co-vinylbenzoylchloride) increased cell permeability, and respiration for C. albicans and C. tropicalis. The tested polymers at 50 microg/ml had pronounced effects on C. albicans and C. tropicalis cell wall phosphopeptidomannane, proteins, sugars and phosphorus. Generally, the two polymers proved effective against the tested microorganisms, but growth inhibitory effect varied according to the composition of the polymer active group. Many investigators consider polymeric antimicrobial agents as a potential new approach for enhancing the efficiency of some existing antimicrobial agents, including prolonged activity, reduce their toxicity, as well as reduce the environmental issues associated with product use.     

Hypertonic Sabouraud Dextrose Agar as a Substrate for Differentiation of Candida dubliniensis

In this study, we aimed to detect the proportion of Candida dubliniensis among yeast strains previously identified as C. albicans by using several phenotypic methods and PCR. For this purpose, we screened 300 strains by using phenotypic tests suggested for the identification of C. dubliniensis in the literature, but we detected high proportion of false-positive reactions. Only two strains (0.6%) were detected as true C. dubliniensis by PCR and API ID 32C methods. Moreover, these two strains gave the expected results with all the phenotypic tests, including modified salt tolerance test for C. dubliniensis. In conclusion, none of the phenotypic methods, except for the modified salt tolerance test, revealed 100% successful results in discrimination of C. albicans and C. dubliniensis species. However, in the tobacco agar test, the rate of false positivity was as low as 0.6%. We suggest that in the case of absence of PCR and other automatized identification systems, these two phenotypic tests can be used in routine laboratories to obtain a presumptive result.     

Candida alocasiicola sp. nov., Candida hainanensis sp. nov., Candida heveicola sp. nov. and Candida musiphila sp. nov., novel anamorphic, ascomycetous yeast species isolated from plants

In a taxonomic study on the ascomycetous yeasts isolated from plant materials collected in tropical forests in Yunnan and Hainan Provinces, southern China, four strains isolated from tree sap (YJ2E(T)) and flowers (YF9E(T), YWZH3C(T) and YYF2A(T)) were revealed to represent four undescribed yeast species. Molecular phylogenetic analysis based on the large subunit (26S) rRNA gene D1/D2 domain sequences showed that strain YJ2E(T) was located in a clade together with Candida haemulonii and C. pseudohaemulonii. Strain YF9E(T) was most closely related to C. azyma and strain YWZH3C(T) to C. sorbophila and C. spandovensis. Strain YYF2A(T) was clustered in a clade containing small-spored Metschnikowia species and related anamorphic Candida species. The new strains differed from their closely related described species by more than 10% mismatches in the D1/D2 domain. No sexual states were observed for the four strains on various sporulation media. The new species are therefore assigned to the genus Candida and described as Candida alocasiicola sp. nov. (type strain, YF9E(T) = AS 2.3484(T) = CBS 10702(T)), Candida hainanensis sp. nov. (type strain, YYF2A(T) = AS 2.3478(T) = CBS 10696(T)), Candida heveicola sp. nov. (type strain, YJ2E(T) = AS 2.3483(T) = CBS 10701(T)) and Candida musiphila sp. nov. (type strain, YWZH3C(T) = AS 2.3479(T) = CBS 10697(T)).     

Utilization of siderophores by Candida albicans

Candida albicans secretes both hydroxamate and phenolate-type siderophores when grown under iron-restricted conditions. The inhibition of candidal growth by iron limitation was reversed by the addition of supplemental hydroxamate on phenolate siderophores. Both siderophores produced equal stimulation of growth suggesting that C. albicans could utilize both siderophores with equal efficiency. Addition of heterologous siderophores from both bacteria and fungi also supported growth of the yeast in a deferrated medium. These results suggest that C. albicans has an iron-uptake mechanism which enables it to obtain iron by utilizing candidal and non-candidal siderophores.     

Enhancement of xylitol productivity and yield using a xylitol dehydrogenase gene-disrupted mutant of Candida tropicalis under fully aerobic conditions

The effects of glycerol and the oxygen transfer rate on the xylitol production rate by a xylitol dehydrogenase gene (XYL2)-disrupted mutant of Candida tropicalis were investigated. The mutant produced xylitol near the almost yield of 100% from d-xylose using glycerol as a co-substrate for cell growth and NADPH regeneration: 50 g d-xylose l⁻¹ was completely converted into xylitol when at least 20 g glycerol l⁻¹ was used as a co-substrate. The xylitol production rate increased with the O₂ transfer rate until saturation and it was not necessary to control the dissolved O₂ tension precisely. Under the optimum conditions, the volumetric productivity and xylitol yield were 3.2 g l⁻¹ h⁻¹ and 97% (w/w), respectively.     

Growth and physiology of Candida utilis NCYC 321 in potassium-limited chemostat culture

When grown in a defined simple salts medium, plus vitamins, Candida utilis displayed an absolute requirement for potassium. But the potassium content of this yeast was exceedingly variable and, with aerobic chemostat cultures (grown at a dilution rate of 0.1 h^-1; 30° C; pH 5.5), was low (< 0.2%, w/w) when they were potassium-limited and high (> 2%, w/2) when glucose-limited. With potassium-limited cultures, the cell-bound potassium content also varied markedly with growth rate, though hardly at all with glucose-limited cultures; magnesium- and phosphate-limited cultures gave intermediate responses.Changes in cell-bound potassium content correlated only weakly with changes in the cellular contents of magnesium, phosphate and RNA, but strongly with changes in both the Y _glucose and Y _O values, indicating an involvement of potassium in the generation of energy by oxidative phosphorylation reactions and/or the utilization of this energy for growth processes.Studies with isolated mitochondria revealed that potassium-limited organisms had a changed content of cytochrome b relative to cytochrome a, and lacked coupling at either site 2 or site 3 of the respiratory chain.These results are discussed in relation to the reported functions of potassium in the growth of micro-organisms, and the organizational differences between prokaryotic and eukaryotic cells.     

Characterization of a new xylitol-producer Candida tropicalis strain

A xylitol-producer yeast isolated from corn silage and designated as ASM III was selected based on its outstanding biotechnological potential. When cultivated in batch culture mode and keeping the dissolved oxygen at 40% saturation, xylitol production was as high as 130 g l(-1) with a yield of 0.93 g xylitol g(-1) xylose consumed. A preliminary identification of the yeast was performed according to conventional fermentation and assimilation physiological tests. These studies were complemented by using molecular approaches based on PCR amplification, restriction-fragment length polymorphism analysis and sequencing of the rDNA segments: intergenic transcribed spacer (ITS) 1-5.8S rDNA-ITS 2, and D1/D2 domain of the 26S rRNA gene. Results from both the conventional protocols and the molecular characterization, and proper comparisons with the reference strains Candida tropicalis ATCC 20311 and NRRL Y-1367, led to the identification of the isolate as a new strain of C. tropicalis.     

Effect of Candida albicans dsDNA in Gastrointestinal Candida Infection

Neonates are highly sensitive to infections because they are biased to develop Th2 immune responses. When exposed to certain agents, such as DNA vaccines or CpG DNA motifs, neonates are capable to mount adult-like Th1 protective responses. This study investigates the capacity of Candida albicans (C. albicans) dsDNA to induce host resistance in newborn mice against gastrointestinal C. albicans infection. The protective properties of dsDNA are related to an increased number of spleen CD4+ T cells secreting IFN-γ. In infected DNA-treated mice, an enhanced production of IFN-γ by Peyer’s patch cells was observed together with reduced colonization and histopathological changes in the stomach. Our results indicated that C. albicans dsDNA administration in neonates elicited the protective immune response against gastrointestinal Candida infection.     

RTA2, a novel gene involved in azole resistance in Candida albicans

Widespread and repeated use of azoles, particularly fluconazole, has led to the rapid development of azole resistance in Candida albicans. Overexpression of CDR1, CDR2, and CaMDR1 has been reported contributing to azole resistance in C. albicans. In this study, hyper-resistant C. albicans mutant, with the above three genes deleted, was obtained by exposure to fluconazole and fluphenezine for 28 passages. Thirty-five differentially expressed genes were identified in the hyper-resistant mutant by microarray analysis; among the 13 up-regulated genes, we successfully constructed the rta2 and ipf14030 null mutants in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1. Using spot dilution assay, we demonstrated that the disruption of RTA2 increased the susceptibility of C. albicans to azoles while the disruption of IPF14030 did not influence the sensitivity of C. albicans to azoles. Meanwhile, we found that ectopic overexpression of RTA2 in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1 conferred resistance to azoles. RTA2 expression was found elevated in clinical azole-resistant isolates of C. albicans. In conclusion, our findings suggest that RTA2 is involved in the development of azole resistance in C. albicans.     

Production of mannitol by a novel strain of Candida magnoliae

A novel microorganism was isolated which is able to produce mannitol when grown in the presence of fructose and glucose as carbon sources. In flask culture in a medium containing 150 g fructose l^–1, it yielded 67 g mannitol l^–1 after 168 h. In fed-batch culture with 3–12% (w/v) fructose, production reached a maximum of 209 g mannitol l^–1 after 200 h, corresponding to an 83% yield and a 1.03 g l^–1 h^–1 productivity. The isolated strain was identified as Candida magnoliae based on identical sequences in the D1/D2 domain of its 26S rDNA and a similar carbon source utilization pattern with C. magnoliae reference strains.     

Molecular subtyping of clinical isolates of <Emphasis Type="Italic">Candida albicans</Emphasis> and identification of <Emphasis Type="Italic">Candida dubliniensis</Emphasis>in Malaysia

The genotypes of 221 recent isolates of Candida albicans from various clinical specimens of 213 patients admitted to the University Malaya Medical Centre, Malaysia was determined based on the amplification of a transposable intron region in the 25 S rRNA gene. The analyses of 178 C. albicansisolated from nonsterile clinical specimens showed that they could be classified into three genotypes: genotype A (138 isolates), genotype B (38 isolates) and genotype C (2 isolates). The genotyping of 43 clinical isolates from sterile specimens showed that they belonged to genotype A (29 isolates), genotype B (10 isolates), genotype C (2 isolates) and genotype D (2 isolates). The overall distribution of C. albicans genotypes in sterile and nonsterile specimens appeared similar, with genotype A being the most predominant type. This study reported the identification of C. dubliniensis (genotype D) in 2 HIV-negative patients with systemic candidiasis, which were missed by the routine mycological procedure. The study demonstrated the genetic diversity of clinical isolates of C. albicans in Malaysia.     

Recombinagenicity of caffeine for Candida albicans

Caffeine at concentrations of 0.5 × 10^–2 M or higher inhibited cell replication and induced gene segregations in Candida albicans cultured on defined complete medium. Both responses increased incrementally with increasing caffeine concentrations, and were more severe during incubation at 37 °C than 25 °C; at 37 °C, caffeine levels above 1.5 × 10^–2 M caused cellular inactivation. Caffeine effects occurred only under conditions permitting cell growth, and their magnitudes were greater for unbudded than budding cells, were influenced by cellular genetic backgrounds, and were unaffected by the presence of adenine in the medium. Evaluations of segregations for recessive auxotrophic markers of a four member linkage group carried heterozygously in a cis arrangement in treated cells established that induced segregants arise through either reciprocal or nonreciprocal recombinations. The frequency distributions of classes of reciprocal and nonreciprocal recombinants for these markers conformed with those previously obtained following induction by ultraviolet radiation, indicating that the probabilities of recombinational events within the chromosomal regions defined by the markers are not biased by the differences in kinds of initial DNA lesions caused by the two recombinagens. A panel of four protoplast fusion hybrids considered deficient for DNA repair because of enhanced susceptibilities to UV induced cellular inactivation and mitotic recombination exhibited corresponding increased sensitivities to caffeine, signifying that DNA damage induced by caffeine is subject to repair. Caffeine did not affect behavior of a variant strain exhibiting high frequency phenotypic switching between minute smooth and large rough colonial forms, and no evidence for mutagenicity of the drug was obtained with systems for detection of forward or reverse mutations. The mechanism of caffeine's recombinagenicity, and the implications of that property for genetic studies of C. albicans are discussed.     

A case of recurrent episodes of Candida parapsilosis fungemia

Candida species is the fourth most commonly isolated organism in blood stream infections in the hospital setting. Patients with candidemia frequently succumb to this infection. For those that survive an initial candidemia, an increasing number of cases of breakthrough or recurrent candidemias have been reported in the literature. We report three episodes of C. parapsilosis fungemia in a cancer patient. Molecular testing was performed and confirmed that all these episodes occurring within an eight-month period were caused by the same organism. The incidence of recurrent candidemia is likely to increase in the near future. Studies and therapeutic interventions for patients at risk for recurrent candidemias are warranted.     

Production of single cell protein from rice polishings using Candida utilis

Microbial protein was produced from defatted rice polishings using Candida utilis in shake-flasks and a 14-l fermentor to optimize fermentation conditions before producing biomass in a 50-l fermentor. The organism supported maximum values of 0.224 h⁻¹, 0.94, 1.35, 1.75, 2.12 g l⁻¹ h⁻¹, 0.62 g cells g⁻¹ substrate utilized and 0.38 g g⁻¹ for specific growth rate, true protein productivity, crude protein productivity, cell mass productivity, substrate consumption rate, cell yield, crude protein yield, respectively in 50-l fermentor studies using optimized cultural conditions. Maximum values compared favourably or were superior to published data in literature. The biomass protein in the 50-l fermentor contained 22.3, 27.8, 19.2, 9.5, 38.12, 8.5 and 0.27% true protein, crude protein, crude fibre, ash, carbon, cellulose and RNA content, respectively. The dried biomass showed a gross metabolizable energy value of 2678 kcal kg⁻¹ and contained all essential and non-essential amino acids. Yeast biomass as animal feed may replace expensive feed ingredients currently being used in poultry feed and may improve the economics of feed produced in countries like Pakistan. This revised version was published online in August 2006 with corrections to the Cover Date.     

Specificity of drug transport mediated by CaMDR1: a major facilitator of Candida albicans

CaMDR1 encodes a major facilitator superfamily (MFS) protein inCandida albicans whose expression has been linked to azole resistance and which is frequently encountered in this human pathogenic yeast. In this report we have overexpressed CaMdr1p inSf9 insect cells and demonstrated for the first time that it can mediate methotrexate (MTX) and fluconazole (FLC) transport. MTX appeared to be a better substrate for CaMdr1p among these two tested drugs. Due to severe toxicity of these drugs to insect cells, further characterization of CaMdr1p as a drug transporter could not be done with this system. Therefore, as an alternative, CaMdr1p and Cdr1p, which is an ABC protein (ATP binding cassette) also involved in azole resistance inC. albicans, were independently expressed in a common hypersensitive host JG436 ofSaccharomyces cerevisiae. This allowed a better comparison between the functionality of the two export pumps. We observed that while both FLC and MTX are effluxed by CaMdr1p, MTX appeared to be a poor substrate for Cdr1p. JG436 cells expressing Cdr1p thus conferred resistance to other antifungal drugs but remained hypersensitive to MTX. Since MTX is preferentially transported by CaMdr1p, it can be used for studying the function of this MFS protein.     

Regio- and enantio-selective acetylation of 3,3,3-trifluoro-2-arylpropane-1,2-diols using Candida antarctica lipase

Of nine commercially available lipases, lipase SP 435 from Candida antarctica, showed moderate enantioselectivity (E=17) for acetylation of racemic 3,3,3-trifluoro-2-phenylpropane-1,2-diol, 2, with vinyl acetate in diisopropyl ether (S selectivity). The other eight had low selectivities, with E values below 10. The selectivity and reactivity of SP 435 for 2 was markedly improved in dichloroethane (E=41). Moreover, SP 435 had moderate to high selectivity for the related compounds 3,3,3-trifluoro-2-(1-naphthyl)-propane-1,2-diol, 4, (E=20), 3,3,3-trifluoro-2-(indol-3-yl)propane-1,2-diol, 6, (E=80), and 3,3,3-trifluoro-2-(pyrrol-2-yl)-propane-1,2-diol, 8, (E=17).