Distribution of epitheliocytes by the number of Candida albicans adhered to them and assessment of the intensity of the adhesion in an in vitro test

The study of the adhesion test in suspensions, carried out with 9 C. albicans strains and human buccal epithelial cells, has revealed that these cells are exponentially distributed by the number of C. albicans cells adhered to them. To characterize adhesion, the use of the index of the exponent describing the distribution of epithelial cells is proposed. With the use of this index, 5 C. albicans strains isolated from candidiasis patients have been found to possess, on the average, higher adhesiveness with respect to epithelial cells than 4 strains isolated from persons without symptoms of candidiasis.     

Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans

Candida dubliniensis is a pathogenic yeast species that was first identified as a distinct taxon in 1995. Epidemiological studies have shown that C. dubliniensis is prevalent throughout the world and that it is primarily associated with oral carriage and oropharyngeal infections in human immunodeficiency virus (HIV)-infected and acquired immune deficiency syndrome (AIDS) patients. However, unlike Candida albicans, C. dubliniensis is rarely found in the oral microflora of normal healthy individuals and is responsible for as few as 2% of cases of candidemia (compared to approximately 65% for C. albicans). The vast majority of C. dubliniensis isolates identified to date are susceptible to all of the commonly used antifungal agents, however, reduced susceptibility to azole drugs has been observed in clinical isolates and can be readily induced in vitro. The primary mechanism of fluconazole resistance in C. dubliniensis has been shown to be overexpression of the major facilitator efflux pump Mdr1p. It has also been observed that a large number of C. dubliniensis strains express a non-functional truncated form of Cdr1p, and it has been demonstrated that this protein does not play a significant role in fluconazole resistance in the majority of strains examined to date. Data from a limited number of infection models reflect findings from epidemiological studies and suggest that C. dubliniensis is less pathogenic than C. albicans. The reasons for the reduced virulence of C. dubliniensis are not clear as it has been shown that the two species express a similar range of virulence factors. However, although C. dubliniensis produces hyphae, it appears that the conditions and dynamics of induction may differ from those in C. albicans. In addition, C. dubliniensis is less tolerant of environmental stresses such as elevated temperature and NaCl and H(2)O(2) concentration, suggesting that C. albicans may have a competitive advantage when colonising and causing infection in the human body. It is our hypothesis that a genomic comparison between these two closely-related species will help to identify virulence factors responsible for the far greater virulence of C. albicans and possibly identify factors that are specifically implicated in either superficial or systemic candidal infections.     

Biotypes and randomly amplified polymorphic DNA (RAPD) profiles of subgingival Candida albicans isolates in HIV infection

A group of subgingival isolates of C. albicans recovered from Italian HIV-positive (HIV+) subjects were characterized both phenotypically and genotypically. Phenotyping of the isolates was carried out by a biotyping method based on the enzyme profiles, carbohydrate assimilation patterns and boric acid resistance of the yeasts. Genotyping was performed through randomly amplified polymorphic DNA (RAPD) analysis. Five biotypes were found among the 29 subgingival C. albicans strains examined. The predominant biotypes were A1R (55.17%), A1S (24.14%), and A2R (13.79%), while the biotypes A11R and A13R were represented by a single isolate each. RAPD profiles identified 15 genotypes among the 29 isolates. Almost every individual harboured his/her own specific isolate and in three out of the six subjects with multiple isolates (two to six each) more than one genotype (two to six) was found. The biotype distribution we found is consistent with previous reports on C. albicans isolates from other oral sources, whereas the resistance to boric acid was highly frequent in subgingival strains. RAPD analysis showed high genetic heterogeneity within subgingival isolates, also when isolates were phenotypically identical. These findings, obtained from HIV+ subjects living in Southern Italy, may be useful as baseline information on subgingival C. albicans colonization in the Mediterranean area.     

Biotypes of Candida albicans using the API 20C system

Abstract A total of 130 isolates of Candida albicans obtained from oral, vaginal and skin sites, were biotyped using the API 20C sugar assimilation system. One major biotype accounted for 75% of the isolates, while 12 minor biotypes accounted for the rest. The API 20C system may therefore be useful in supplementing other biotyping schemes of C. albicans available at present.     

Interaction of Candida albicans with macrophages

Study of the in vitro interaction of mouse peritoneal macrophages with C. albicans has revealed that these macrophages, though easily phagocytizing C. albicans blastospores, are incapable of destroying the fungus. The phagocytic, but not candidostatic, activity of these macrophages has been found to depend on the conditions on their cultivation, as well as on the age and viability of fungal cells. The representatives of 7 C. albicans strains, obtained from various sources and stored at the museum for different spans of time, have shown practically no difference in the character of their interaction with mouse peritoneal macrophages.     

Telomeric and dispersed repeat sequences in Candida yeasts and their use in strain identification.

Several different repetitive DNA sequences have been isolated from the pathogenic yeast Candida albicans. These include two families of large dispersed repeat sequences (Ca3, Ca24) and a short (23-bp) tandemly repeated element (Ca7) associated with C. albicans telomeres. In addition, a large subtelomeric repeat (WOL17) has been cloned. DNA fragments containing the telomeric repeats are highly variable among different C. albicans strains. We have shown that the Ca3 repeat is relatively more stable and is suitable for use as a species-specific and strain-specific probe for C. albicans.     

The use of killer sensitivity patterns for biotyping yeast strains: the state of the art, potentialities and limitations

In recent years molecular techniques have been the most useful tools for the unequivocal identification of undetermined strains at the species level. In many instances, however, a further discrimination at the strain level (biotyping) is required, such as during epidemiological investigations, in which the distribution of pathogenic microorganisms is studied, and for patent protection purposes. Although molecular methods are routinely used also for yeast biotyping, several nonmolecular techniques have been proposed. One of these, the determination of the killer sensitivity pattern (KSP) towards a panel of selected killer toxins has proven to be a good auxiliary method. Despite the plethora of studies published, the potential and limitations of the determination of KSPs have never been critically evaluated. In this review the use of this nonmolecular technique as a biotyping tool is discussed and compared with some currently used DNA-based procedures. In addition, methodological, mechanistic and ecological implications are evaluated.     

A simplified system for biotyping Staphylococcus aureus strains isolated from different animal species

D evriese , L.A. 1984. A simplified system for biotyping Staphylococcus aureus strains isolated from different animal species. Journal of Applied Bacteriology 56 , 215–220.
A biotyping system for Staphylococcus aureus strains is proposed which is a simplified version of biotyping procedures described in the literature. It differentiates Staph. aureus strains from man and animals into host-specific ecovars and biotypes which are not host-specific. With the help of tests for βhaemolysin, staphylokinase, coagulation of bovine plasma and the crystal-violet reaction, the origin of many but not all Staph. aureus strains can be determined: 604 of 809 strains from man. poultry, cattle, pigs, goats, rabbits and foods could be alloted to four ecovars which are typically associated with man, poultry, sheep and goats and cattle. The other strains belonged to five non-host specific biotypes.     

Identification of a cryptic gene associated with an insertion sequence not previously identified in Bacillus thuringiensis

Abstract High-level resistance to glycopeptides in Enterococcus faecium is associated with an inducible 39-kDa cytoplasmic membrane protein. The present paper shows that such glycopeptide-resistant E. faecium strains can not only be isolated in a definite clinical setting but also from waste water of sewage treatment plants. Nearer characterization of these and of clinical isolates by resistance pattern, biotyping, and genotyping (DNA-fingerprinting with pulsed-field get electrophoresis) has shown that different glycopeptide-resistant E. faecium strains have been isolated from clinical sources and from waste water.     

Phenotypic aspects of oral strains of Candida albicans in children with Down's syndrome.

The aim of this article is to characterize the biological aspects of oral strains of C. albicans in children with Down's syndrome. These yeasts were analyzed as to their macromorphological and enzymatic aspects and were tested as to their in vitro susceptibility to antifungal drugs using broth microdilution to determine the minimum inhibitory concentration (MIC). The morphotyping revealed that all oral C. albicans isolates from children with Down's syndrome promoted the formation of fringes regardless of size, while the control group presented smaller fringes. All oral C. albicans strains produced proteinase, but those with phospholipolytic activity showed greater enzyme capacity in the test group. In vitro susceptibility showed that all oral C. albicans isolates were sensitive to the drugs used.     

空肠弯曲菌生物学特性上的一些歧异

对195例不同年龄腹泻患者的粪便、690只健康和腹泻畜禽的直肠和泄殖腔拭子及108份腹泻死亡畜禽的脏器材料进行了空肠弯曲菌培养,共分离鉴定458株弯曲菌(其中空肠弯曲菌445株、结肠弯曲菌13株),就其一些生物学特性进行了观察,采用Lior生物学分型法进行了354株弯曲菌(空肠弯曲菌352株、结肠弯曲菌2株)的分型。虽然表明绝大多数生物学性状符合已有文献描述,但也发现在形态、培养和生理生化特性及抗菌药抗性上存有一些歧异,其中最为主要的是483%(215/445)的空肠弯曲菌和231%(3/13)的结肠弯曲菌有萘啶酮酸抗性,11%(5/445)的空肠弯曲菌和76%(1/13)的结肠弯曲菌有噻孢霉素抗性,抗菌药抗性与菌株来源有关(P<0005)。352株空肠弯曲菌生物学分型结果表明在这些动物体内生物型Ⅰ(409%)和Ⅱ(582%)占优势,同一动物体内可有该菌的2个生物型分布。     

Biofilm formation by and antifungal susceptibility of Candida isolates from urine

Biofilm formation (BF) in the setting of candiduria has not been well studied. We determined BF and MIC to antifungals in Candida spp. isolates grown from urine samples of patients and performed a retrospective chart review to examine the correlation with risk factors. A total of 67 Candida spp. isolates were grown from urine samples from 55 patients. The species distribution was C. albicans (54%), C. glabrata (36%), and C. tropicalis (10%). BF varied greatly among individual Candida isolates but was stable in sequential isolates during chronic infection. BF also depended on the growth medium and especially in C. albicans was significantly enhanced in artificial urine (AU) compared to RPMI medium. In nine of the C. albicans strains BF was 4- to 10-fold higher in AU, whereas in three of the C. albicans strains and two of the C. glabrata strains higher BF was measured in RPMI medium than in AU. Determination of the MICs showed that planktonic cells of all strains were susceptible to amphotericin B (AMB) and caspofungin (CASPO) and that three of the C. glabrata strains and two of the C. albicans strains were resistant to fluconazole (FLU). In contrast, all biofilm-associated adherent cells were resistant to CASPO and FLU. The biofilms of 14 strains (28%) were sensitive to AMB (MIC(50) of <1 mug/ml). Correlation between degree of BF and MIC of AMB was not seen in RPMI grown biofilms but was present when grown in AU. A retrospective chart review demonstrated no correlation of known risk factors of candiduria with BF in AU or RPMI. We conclude that BF is a stable characteristic of Candida strains that varies greatly among clinical strains and is dependent on the growth medium. Resistance to AMB is associated with higher BF in AU, which may represent the more physiologic medium to test BF. Future studies should address whether in vitro BF can predict treatment failure in vivo.     

DNA content, kinetic complexity, and the ploidy question in Candida albicans.

Candida albicans is a dimorphic fungus that is pathogenic for humans. No sexual cycle has been reported for this fungus, and earlier reports have differed on whether typical strains of C. albicans are haploid or diploid. Previous estimates of the DNA content of C. albicans varied by one order of magnitude. We used three independent methods to measure the kinetic complexity of the single-copy DNA from a typical strain of C. albicans (strain H317) to determine the DNA content per haploid genote; we obtained values of 15 and 20 fg per cell by using S1 nuclease and hydroxyapatite assays, respectively. Optical assays for DNA reassociation kinetics, although not definitive in themselves, yielded values in this range. Chemical measurements of the DNA content of several typical strains, including strain H317, yielded values clustered about a mean of 37 fg per cell. We concluded that these strains are diploid.     

Targeted gene disruption in Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by active drug efflux out of the cells. In clinical C. albicans strains, fluconazole resistance frequently correlates with constitutive activation of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not expressed detectably in fluconazole-susceptible isolates. However, the molecular changes causing MDR1 activation have not yet been elucidated, and direct proof for MDR1 expression being the cause of drug resistance in clinical C. albicans strains is lacking as a result of difficulties in the genetic manipulation of C. albicans wild-type strains. We have developed a new strategy for sequential gene disruption in C. albicans wild-type strains that is based on the repeated use of a dominant selection marker conferring resistance against mycophenolic acid upon transformants and its subsequent excision from the genome by FLP-mediated, site-specific recombination (MPAR-flipping). This mutagenesis strategy was used to generate homozygous mdr1/mdr1 mutants from two fluconazole-resistant clinical C. albicans isolates in which drug resistance correlated with stable, constitutive MDR1 activation. In both cases, disruption of the MDR1 gene resulted in enhanced susceptibility of the mutants against fluconazole, providing the first direct genetic proof that MDR1 mediates fluconazole resistance in clinical C. albicans strains. The new gene disruption strategy allows the generation of specific knock-out mutations in any C. albicans wild-type strain and therefore opens completely novel approaches for studying this most important human pathogenic fungus at the molecular level.     

Modulation of Toll-like receptor ligands and Candida albicans-induced cytokine responses by specific probiotics

Probiotics have been proposed as modulators of gut inflammation, especially in inflammatory bowel disease (IBD). In order to be able to use them in these clinical conditions, their capacity to modulate immune responses towards other stimuli or microorganisms has to be thoroughly understood. In the present study, three different potentially probiotic strains, Bifidobacterium breve (NumRes 204), Lactobacillus rhamnosus (NumRes1) and Lactobacillus casei (DN-114 001), have been studied for their potential to modulate responses to stimulation with pure pattern-recognition receptor (PRR) ligands or to the gut commensal fungus Candida albicans. Cytokine production induced by PRR ligands or C. albicans was assessed in conditions of simultaneous stimulation or preincubation of primary immune cells with Bifidobacterium or Lactobacillus spp. Results indicate that simultaneous stimulation leads to potentiation of IL-1β and IL-6 production, while the TNFα and IFN-γ production was inhibited. In settings of pre-incubation with these potentially probiotic strains, lower production of TNFα was observed in the presence of B. breve. Moreover, C. albicans-induced IL-17 production was decreased after pre-incubation with both Bifidobacterium or Lactobacillus probiotic strains. Whereas C. albicans induced cytokines are dampened by the tested probiotic strains, TNFα and IL-6 production by pure pattern-recognition receptor ligands are potentiated. Interestingly, an important role of Toll-like receptor 9 signalling that involves JNK kinase in the modulatory effects of these probiotic strains has been identified. In conclusion, specific probiotic strains exhibit cross-tolerance effects towards other inflammatory stimuli, especially C. albicans, which might have beneficial effects on gut inflammation.     

Delayed hypersensitivity to Candida albicans

Conditions and factors influencing the formation of delayed hypersensitivity (DH) to yeast-like fungi of the species C. albicans under experimental conditions have been studied. The intensity of this reaction has been found to depend on the dose and method used for infecting mice, the time of the test and the qualitative state of fungal cells. As the result of this study, the infectious model of DH to C. albicans has been proposed. This model may be used for the study of the influence of different exogenous and endogenous factors of cell-mediated immune response to candidiasis.     

The presence of fluconazole-resistant Candida dubliniensis strains among Candida albicans isolates from immunocompromised or otherwise debilitated HIV-negative Turkish patients

The newly described species Candida dubliniensis phenotipically resembles Candida albicans in many respects and so it could be easily misidentified. The present study aimed at determining the frequency at which this new Candida species was not recognized in the authors' university hospital clinical laboratory and to assess antifungal susceptibility. In this study, six identification methods based on significant phenotypic characteristics each proposed as reliable tests applicable in mycology laboratories for the differentiation of the two species were performed together to assess the clinical strains that were initially identified as C. albicans. Only the isolates which have had the parallel results in all methods were assessed as C. dubliniensis. One hundred and twenty-nine C. albicans strains isolated from deep mycosis suspected patients were further examined. Three of 129 C. albicans (2 from oral cavity, 1 from sputum) were reidentified as C. dubliniensis. One of the strains isolated from oral cavity and that from the sputum were obtained at two months intervals from the same patient with acute myeloid leukemia, while the other oral cavity strain was obtained from a patient who had previously been irradiated for a laryngeal malignancy. Isolates were all susceptible in vitro to amphotericin B, with the MIC range 0.125 to 0.5 &mgr;g/ml, resistant to fluconazole, with MICs >/=64 &mgr;g/ml, and resistant to ketoconazole, with MICs >/=16 &mgr;g/ml, dose-dependent to itraconazole with a MIC range 0.25-0.5 &mgr;g/ml, and susceptible to flucytosine, with a MIC range 1-4 &mgr;g/ml.     

Candida albicans VPS11 is required for vacuole biogenesis and germ tube formation

The Candida albicans vacuole has previously been observed to undergo rapid expansion during the emergence of a germ tube from a yeast cell, to occupy the majority of the parent yeast cell. Furthermore, the yeast-to-hypha switch has been implicated in the virulence of this organism. The class C vps (vacuolar protein sorting) mutants of Saccharomyces cerevisiae are defective in multiple protein delivery pathways to the vacuole and prevacuole compartment. In this study C. albicans homologues of the S. cerevisiae class C VPS genes have been identified. Deletion of a C. albicans VPS11 homologue resulted in a number of phenotypes that closely resemble those of the class C vps mutants of S. cerevisiae, including the absence of a vacuolar compartment. The C. albicans vps11Delta mutant also had much-reduced secreted lipase and aspartyl protease activities. Furthermore, vps11Delta strains were defective in yeast-hypha morphogenesis. Upon serum induction of filamentous growth, mutants showed delayed emergence of germ tubes, had a reduced apical extension rate compared to those of control strains, and were unable to form mature hyphae. These results suggest that Vps11p-mediated trafficking steps are necessary to support the rapid emergence and extension of the germ tube from the parent yeast cell.     

Differences in extracellular enzymatic activity between Candida dubliniensis and Candida albicans isolates

Twenty-six Candida dubliniensis and 27 Candida albicans oral strains isolated from patients infected by the human immunodeficiency virus (HIV) were tested for germ tube production and 21 extracellular enzymatic activities. Assessment of the enzymatic profile was performed by using the API-ZYM commercial kit system (bioMerieux, France), which tests 19 different enzymes. Protease activity was expressed during the first days of incubation by 100% of the strains studied and resulted higher than phospholipase activity in the C. dubliniensis and C. albicans strains tested. The API-ZYM profile of the C. dubliniensis and C. albicans strains differs with respect to the number and percentage of the enzymes considered, as well as with the intensity of the substrate metabolized by the strains, in particular for the enzymes n 8 (cystine-arylamidase), n 12 (naphtol-AS-BI-phosphohydrolase) and n 16 (alpha-glucosidase). These enzymes may be useful to differentiate C. dubliniensis and C. albicans together with other phenotypic characteristics proposed in the literature. No relationship among protease, phospholipase and other extracellular enzymatic activities was observed in C. dubliniensis. The average percentage of strains filamentation after 4 h was between 32 and 42%.